**The Synergistic Contribution of** *Lactobacillus* **and Dietary Phytophenols in Host Health**

Danielle N. Kling, Guillermo E. Marcial, Dana N. Roberson, Graciela L. Lorca and Claudio F. Gonzalez

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63787

#### **Abstract**

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82 Probiotics and Prebiotics in Human Nutrition and Health

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e1612.

Phytophenols are found ubiquitously among all plants. They are important in diets rich in fruits and vegetables because these compounds provide health benefits to the host, ultimately decreasing the incidence of chronic diseases. These compounds act as natural antioxidants and provide anti-inflammatory, antiviral, antibiotic, and antineoplastic properties. Reactive oxygen species (ROS) are produced under normal physiological functions, and low/moderate levels are required for cellular turnover and signaling. However, when ROS levels become too high, oxidative stress can occur. Phytophenols quench ROS and ultimately avoid the damaging effects ROS elicit on the cell. The highest source of bioavailable phytophenols comes from our diet as a component usually esterified in plant fiber. For phytophenols to be absorbed by the body, they must be released by esterases, or other related enzymes. The highest amount of esterase activity comes from the gastrointestinal (GI) microbiota; therefore, the host requires the activity of mutualis‐ tic bacteria in the GI tract to release absorbable phytophenols. For this reason, mutualis‐ tic bacteria have been investigated for beneficial properties in the host. Our laboratory has begun studying the interaction of *Lactobacillus johnsonii* N6.2 with the host since it was found to be negatively correlated with type 1 diabetes (T1D). Analyses of this strain have revealed two important characteristics: (1) It has the ability to release phytophenols from dietary fiber through the secretion of two strong cinnamoyl esterases and (2) *L. johnso‐ nii* also has the ability to generate significant amounts of H2O2, controlling the activity of indoleamine 2,3-dioxygenase (IDO), an immunomodulatory enzyme.

**Keywords:** *Lactobacillus*, *Lactobacillus johnsonii* N6.2, Indolamine 2,3-dioxygenase, 5 hydroxytryptamine, reactive oxygen species, esterase

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **1. Phytophenols**

Phytophenols, also called polyphenols or simply phenols, are a unique group of monocyclic and polycyclic phytochemicals found within fruits, vegetables, and other plants as a compo‐ nent ofplantfiber.Phytophenols areubiquitously foundas secondarymetabolites inplants and are therefore consumed in relatively high quantities. They are a very diverse and multifunctional group of active plant compounds with substantial health potential in many areas, and numerous scientific studies demonstrate that increasing the intake of plant foods rich in fiber can minimize the incidence of modern diseases [1–3].

Consumption of foods and beverages containing phytophenols may impact nutrient levels in the body by preventing their oxidation. Their activity is based on functional groups' capacity to accept a free radical's negative charge [4, 5]. In order to be absorbed by intestinal epithelial cells, phytophenols attached to fiber can only be released by the enzymatic activities of the gastrointestinal (GI) microbiota [6–9] because the phenolic esterase enzymes necessary to release antioxidant phytophenols from plant fiber are not produced by the host GI system. It has been shown *in vitro* that after hydrolysis with purified enzymes, more biologically active compounds can be released, including hydroxytyrosol and elenolic acid from oleuropein [10, 11] and dihydroxyphenyllactic acid from rosmarinic and salvianolic acids [12, 13]. Neverthe‐ less, very little is known about the modifications that these natural compounds undergo after ingestion.

All phytophenols arise from a common intermediate, phenylalanine, or a close precursor, shikimic acid [14]. Often they are present in conjugated forms, with sugar residues linked to hydroxyl groups, although in some cases, direct links of the sugar to an aromatic carbon do exist. In addition, associations with other compounds are also common, including linkages with carboxylic and organic acids, amines, and lipids, as well as associations with other phenols [15].

Plants produce an impressive array of phenolic compounds, and it is thought that these plantbased constituents have a stronger biological antioxidant effect when compared to synthetic antioxidants. This is mainly because phytophenols are part of the normal function of living plants and therefore are thought to have better compatibility with the body [4, 16, 17]. Although there are more than 8000 identified polyphenolic compounds, they can be sorted into four main classes: phenolic acids, flavonoids, stilbenes, and lignans [18]. **Figure 1** illustrates the different groups, which are divided by the number of rings they contain as well as the structural elements that bind these rings together.

Phenolic acids are derivatives of either benzoic acid or cinnamic acid and can thus be divided into two classes. They make up about a third of the polyphenolic compounds found in human diets. These phenolic compounds can be found in all plant-based material, although they are most commonly found in acidic fruits [19]. Flavonoids are the most abundant polyphenolic compounds found in our diet and are also the most well-studied group. More than 4000 varieties have been accounted for, often contributing to the color of flowers, fruits, and leaves [20]. Six subclasses exist, as shown in **Figure 2**, based upon variations in structure: flavonols, flavones, flavanones, flavanols, anthocyanins, and isoflavones.

The Synergistic Contribution of *Lactobacillus* and Dietary Phytophenols in Host Health http://dx.doi.org/10.5772/63787 85

**Figure 1.** Chemical structures of the different classes of polyphenols, broadly divided into four classes [14].

**Figure 2.** Chemical structures of subclasses of flavonoids [14].

**1. Phytophenols**

84 Probiotics and Prebiotics in Human Nutrition and Health

ingestion.

phenols [15].

elements that bind these rings together.

flavones, flavanones, flavanols, anthocyanins, and isoflavones.

Phytophenols, also called polyphenols or simply phenols, are a unique group of monocyclic and polycyclic phytochemicals found within fruits, vegetables, and other plants as a compo‐ nent ofplantfiber.Phytophenols areubiquitously foundas secondarymetabolites inplants and are therefore consumed in relatively high quantities. They are a very diverse and multifunctional group of active plant compounds with substantial health potential in many areas, and numerous scientific studies demonstrate that increasing the intake of plant foods rich in

Consumption of foods and beverages containing phytophenols may impact nutrient levels in the body by preventing their oxidation. Their activity is based on functional groups' capacity to accept a free radical's negative charge [4, 5]. In order to be absorbed by intestinal epithelial cells, phytophenols attached to fiber can only be released by the enzymatic activities of the gastrointestinal (GI) microbiota [6–9] because the phenolic esterase enzymes necessary to release antioxidant phytophenols from plant fiber are not produced by the host GI system. It has been shown *in vitro* that after hydrolysis with purified enzymes, more biologically active compounds can be released, including hydroxytyrosol and elenolic acid from oleuropein [10, 11] and dihydroxyphenyllactic acid from rosmarinic and salvianolic acids [12, 13]. Neverthe‐ less, very little is known about the modifications that these natural compounds undergo after

All phytophenols arise from a common intermediate, phenylalanine, or a close precursor, shikimic acid [14]. Often they are present in conjugated forms, with sugar residues linked to hydroxyl groups, although in some cases, direct links of the sugar to an aromatic carbon do exist. In addition, associations with other compounds are also common, including linkages with carboxylic and organic acids, amines, and lipids, as well as associations with other

Plants produce an impressive array of phenolic compounds, and it is thought that these plantbased constituents have a stronger biological antioxidant effect when compared to synthetic antioxidants. This is mainly because phytophenols are part of the normal function of living plants and therefore are thought to have better compatibility with the body [4, 16, 17]. Although there are more than 8000 identified polyphenolic compounds, they can be sorted into four main classes: phenolic acids, flavonoids, stilbenes, and lignans [18]. **Figure 1** illustrates the different groups, which are divided by the number of rings they contain as well as the structural

Phenolic acids are derivatives of either benzoic acid or cinnamic acid and can thus be divided into two classes. They make up about a third of the polyphenolic compounds found in human diets. These phenolic compounds can be found in all plant-based material, although they are most commonly found in acidic fruits [19]. Flavonoids are the most abundant polyphenolic compounds found in our diet and are also the most well-studied group. More than 4000 varieties have been accounted for, often contributing to the color of flowers, fruits, and leaves [20]. Six subclasses exist, as shown in **Figure 2**, based upon variations in structure: flavonols,

fiber can minimize the incidence of modern diseases [1–3].

Stilbenes contain two phenyl moieties connected by a two-carbon methylene bridge. Their synthesis is typically initiated as a result of injury or infection in plants, and as a consequence, their occurrence in our diet is much lower than either phenolic acids or flavonoids. The best studied stilbene is resveratrol, found mainly in grapes and as a result also in red wine. Lignans are diphenolic compounds formed by the dimerization of two cinnamic acid residues, as seen in **Figure 1**.

Estimating the total polyphenol content is most accurately done through analysis of every individual phytophenolic compound. Due to the large diversity in phytophenolics, the only way to complete this task is through a compilation of the literature data. Fortunately, the USDA database contains a nearly complete source of food composition data [21–23]. This database combined with other literature sources for the remaining phytophenolic compounds was used to develop the Phenol-Explorer database. This recently developed database is the most complete source on the content of polyphenols in foods, including glycosides, esters, and aglycones of flavonoids, phenolic acids, lignans, stilbenes, and other polyphenols [24].

The occurrence of dietary phenolics in plants is not uniform, even at the cellular level. Insoluble phytophenols are often found in cells walls, while soluble phytophenols are found within the vacuoles of plant cells [25]. In many instances, plant-based foods contain a variable mixture of polyphenols. Some polyphenols, such as flavanones and isoflavones, are found only in specific foods, whereas others such as quercetin are found in nearly all plant products. Conventionally, the outer tissues of a plant contain higher levels of phenolics than the inner tissues [26].

Various other factors can affect the concentration of dietary phytophenols, including ripeness of the plant when harvested, environmental factors, storage, and processing of plant materials [14]. Before harvesting, abiotic factors such as soil type, exposure to sunlight, and amount of rainfall can alter phenolic compounds in plants. In addition, the degree of ripeness when harvested can be positively or negatively correlated with the concentration of polyphenols, depending upon which compound is under observation [27]. Storage of plant-based foods also affects polyphenol levels, and the oxidation of polyphenols over time can be beneficial (as in the case of black tea) or harmful (as in the case of browning of fruit) to polyphenolic compound concentrations [27]. Cooking also has a major effect on phytophenolic compounds, and depending on how the material is processed, cooking may account for a 30–80% loss of phenolic content [28].

Bioavailability is described as the proportion of the nutrient that follows natural pathways to be digested, absorbed, and metabolized in the body. For phytophenols, there is no relationship between the quantity of phenolic compounds found in food and their bioavailability, and every one of the numerous known polyphenols differs in its bioavailability. Furthermore, the most ubiquitous phytophenols found in plant-based foods are not necessarily the same as those that show the highest concentration of metabolites in tissues. Often, polyphenols are present in a form that cannot directly be absorbed by the body, including esters, glycosides, or polymers [29]. Due to the microbial modification of phytophenols during absorption in the intestinal cells and later in the liver, the compounds reaching the bloodstream and bodily tissues are drastically different from those originally ingested. As a consequence, identifying all the metabolites and subsequently evaluating their activity is a difficult task. It is the chemical structure of the phytophenolic compound that determines absorption rate and extent rather than the concentration of the compound found in the diet [30]. Evidence does indirectly suggest that phenols are absorbed to some extent through the gut barrier due to an increase in antioxidant capacity of plasma after ingestion of phytophenol-rich foods [31, 32].

The potential pharmacological properties of these natural plant compounds have been demonstrated *in vitro* and include anti-inflammatory [9], antioxidant [33–35], antineoplastic [10, 36], antiviral [37], and antibiotic [38] properties. Although several mechanisms of action combine to provide the widespread health benefits offered by phytophenols, their role as antioxidants is the mostly frequently studied mechanism. The intestinal inflammatory process is primarily a consequence of the overproduction of inflammatory mediators, triggered by an excess of reactive oxygen species (ROS) [39–41].

Estimating the total polyphenol content is most accurately done through analysis of every individual phytophenolic compound. Due to the large diversity in phytophenolics, the only way to complete this task is through a compilation of the literature data. Fortunately, the USDA database contains a nearly complete source of food composition data [21–23]. This database combined with other literature sources for the remaining phytophenolic compounds was used to develop the Phenol-Explorer database. This recently developed database is the most complete source on the content of polyphenols in foods, including glycosides, esters, and aglycones of flavonoids, phenolic acids, lignans, stilbenes, and other polyphenols [24].

The occurrence of dietary phenolics in plants is not uniform, even at the cellular level. Insoluble phytophenols are often found in cells walls, while soluble phytophenols are found within the vacuoles of plant cells [25]. In many instances, plant-based foods contain a variable mixture of polyphenols. Some polyphenols, such as flavanones and isoflavones, are found only in specific foods, whereas others such as quercetin are found in nearly all plant products. Conventionally, the outer tissues of a plant contain higher levels of phenolics than the inner tissues [26].

Various other factors can affect the concentration of dietary phytophenols, including ripeness of the plant when harvested, environmental factors, storage, and processing of plant materials [14]. Before harvesting, abiotic factors such as soil type, exposure to sunlight, and amount of rainfall can alter phenolic compounds in plants. In addition, the degree of ripeness when harvested can be positively or negatively correlated with the concentration of polyphenols, depending upon which compound is under observation [27]. Storage of plant-based foods also affects polyphenol levels, and the oxidation of polyphenols over time can be beneficial (as in the case of black tea) or harmful (as in the case of browning of fruit) to polyphenolic compound concentrations [27]. Cooking also has a major effect on phytophenolic compounds, and depending on how the material is processed, cooking may account for a 30–80% loss of

Bioavailability is described as the proportion of the nutrient that follows natural pathways to be digested, absorbed, and metabolized in the body. For phytophenols, there is no relationship between the quantity of phenolic compounds found in food and their bioavailability, and every one of the numerous known polyphenols differs in its bioavailability. Furthermore, the most ubiquitous phytophenols found in plant-based foods are not necessarily the same as those that show the highest concentration of metabolites in tissues. Often, polyphenols are present in a form that cannot directly be absorbed by the body, including esters, glycosides, or polymers [29]. Due to the microbial modification of phytophenols during absorption in the intestinal cells and later in the liver, the compounds reaching the bloodstream and bodily tissues are drastically different from those originally ingested. As a consequence, identifying all the metabolites and subsequently evaluating their activity is a difficult task. It is the chemical structure of the phytophenolic compound that determines absorption rate and extent rather than the concentration of the compound found in the diet [30]. Evidence does indirectly suggest that phenols are absorbed to some extent through the gut barrier due to an increase in

antioxidant capacity of plasma after ingestion of phytophenol-rich foods [31, 32].

The potential pharmacological properties of these natural plant compounds have been demonstrated *in vitro* and include anti-inflammatory [9], antioxidant [33–35], antineoplastic

phenolic content [28].

86 Probiotics and Prebiotics in Human Nutrition and Health

ROS are the by-products of cellular redox processes in the body. These free radical compounds contain one or more unpaired electrons in their outer orbit, creating instability that leads to significant reactivity. ROS species include superoxide (O2•−), hydroxyl (•OH), peroxyl (ROO•), lipid peroxyl (LOO•), and alkoxyl (RO•) radicals. Oxygen free radicals can also be converted to other non-radical reactive species, which are dangerous for health due to their tendency to lead to free radical reactions in living organisms. These species include hydrogen peroxide (H2O2), ozone (O3), singlet oxygen (1/2O2), and hypochlorous acid (HOCl). ROS are capable of modifying structural proteins or inactivating enzymes, and as a consequence disrupting normal physiologic functions in the body [42–44]. Production of free radicals is a normal part of our physiology and occurs continually to keep the body functioning properly. Processes that generate ROS include activities of the immune system, metabolism, and inflammation responses, along with stress, pollution, radiation, diet, toxins, exhaust fumes, and smoking. [4, 16, 42, 45].

Excessive production of ROS can easily overwhelm both the enzymatic and non-enzymatic antioxidant defense systems, leading to oxidative stress and inflammation. It has been widely discussed in scientific literature that increasing the intake of natural antioxidants minimizes the deleterious effects of ROS [34, 46–48]. Evidence collected from feeding assays using diets rich in antioxidant plant phenolics supports this claim [2, 7, 49]. The intake of phytophenols has been shown to minimize the production of ROS and mitigate their harmful impact on the GI system [3, 33, 50].

Oxidative stress leads to disease through four destructive pathways: membrane lipid peroxi‐ dation, protein oxidation, DNA damage, and disturbance of reducing equivalents in the cell [4]. These steps often lead to altered signaling pathways and cell destruction. Oxidative stress has been connected to various diseases such as cancer, cardiovascular diseases, neurological disorders, diabetes, and aging. Each molecule in the body is at risk of damage by ROS, and damaged molecules can impair cellular functioning and lead to cell death, which ultimately results in diseased states [43, 44, 51]. Due to the antioxidant properties of phytophenolic compounds, they are associated with the prevention of a large array of diseases, including cardiovascular disease, cancer, diabetes, rheumatoid arthritis, neurodegenerative diseases, GI diseases, renal disorders, pulmonary disorders, eye disorders, infertility, and pregnancy complications, as well as slowing the progression of aging [4].

Although reduction of ROS has been shown to decrease risk of a huge array of diseases, the classical model of ROS generation and resulting oxidative stress contrasts with some emerging scientific evidence. Benefits of ROS can in fact occur when these species are present in low/ moderate concentrations, as part of normal physiological functions [43]. The majority of cells produce superoxide and hydrogen peroxide constitutively, while other cells possess inducible ROS release systems. Beneficial effects can include defense against infectious agents by phagocytosis, killing of cancer cells by macrophages and cytotoxic lymphocytes, detoxification of xenobiotics by Cytochrome P450, generation of ATP in mitochondria (energy production), cell growth, and the induction of mitogenic responses at low concentrations. ROS also plays a role in cellular signaling, including activation of several cytokines and growth factors, nonreceptor tyrosine kinase activation, protein tyrosine phosphatase activation, release of calcium from intracellular stores, and activation of nuclear transcription factors. ROS can also initiate vital actions such as gene transcription and regulation of soluble guanylate cyclase activity in cells [44, 50].

Reactive oxygen species (ROS) are known to play a dual role in biological systems; they are well documented for playing a role as both deleterious and beneficial species [43, 44, 52]. We hypothesize that redox homeostasis in the GI tract is dependent on the dynamic interplay between the generation of ROS and the ROS quencher ability of antioxidant phytophenols released by intestinal microbes. Although there are possible benefits to maintain low levels of ROS in the proper functioning of the body, the diet and lifestyle of the majority results in increased levels of ROS in the body are known to be harmful and can lead to the progression of disease. In this way, it is critical to maintain the proper balance of ROS in the body, and phenolic compounds have been shown to reestablish a healthy level of ROS. Next, we turn to the vital interaction of phytophenols and microflora of the gut system that can lead to creation of redox balance critical to health.

### **2. Lactic acid bacteria**

The group known as lactobacilli is composed of several genera of bacteria (*Leuconostoc, Pediococcus, Lactococcus*, and *Streptococcus*)*, Lactobacillus* being the largest order in the phylum Firmicutes and the class Bacilli [53]. These free living lactic acid bacteria flourish in different biological niches such as soil, plants (fruits, beverage, and silage) and fermented foods (cheese, fermented milk, yogurt, meat products, alcoholic beverages, and pickled products). They are also associated with mammals as members of the microbial community characteristic of the oral cavity, GI system, urinary tract, skin, etc. [54–57]. The genus *Lactobacillus* is composed of nutritionally fastidious gram-positive, non-spore-forming rods or coccobacilli, catalasenegative, aerotolerant or anaerobic bacteria. The main characteristic of their homo- or heterofermentative metabolism is the production of lactic acid as the primary end fermentation product. The genus *Lactobacillus* is represented by over 212 species described to date, including several industrially relevant microorganisms such as *L. acidophilus, L. bulgaricus, L. casei, L. lactis, L. paracasei, L. plantarum, L. reuteri, L. fermentum, L. salivarius, L. rhamnosus, L. delbrueckii*, and *L. johnsonii*.

The *Lactobacillus* genus is widely studied because of the bacterias' capacity to produce lactic acid. Thus, most studies regarding their physiology were centered on acidifying bacteria such as *L. delbrueckii subsp. bulgaricus*, which in combination with *Streptococcus thermophilus* acidify milk in a few hours. This process is critical to optimize the production of fermented dairy products such as cheese and yogurt, or other non-dairy products such as pickles, sauerkraut, and sourdough bread.

The extensive use of these bacteria in food and beverage industries drove the scientific attention toward the evaluation of their impact on health, mainly on the GI system's integrity and responsiveness. Regardless, lactic acid bacteria were safely used for centuries to modify food flavor and texture, modern genomics bring back to light the scientific discussion toward their impact on human health [54, 58].

phagocytosis, killing of cancer cells by macrophages and cytotoxic lymphocytes, detoxification of xenobiotics by Cytochrome P450, generation of ATP in mitochondria (energy production), cell growth, and the induction of mitogenic responses at low concentrations. ROS also plays a role in cellular signaling, including activation of several cytokines and growth factors, nonreceptor tyrosine kinase activation, protein tyrosine phosphatase activation, release of calcium from intracellular stores, and activation of nuclear transcription factors. ROS can also initiate vital actions such as gene transcription and regulation of soluble guanylate cyclase activity in

Reactive oxygen species (ROS) are known to play a dual role in biological systems; they are well documented for playing a role as both deleterious and beneficial species [43, 44, 52]. We hypothesize that redox homeostasis in the GI tract is dependent on the dynamic interplay between the generation of ROS and the ROS quencher ability of antioxidant phytophenols released by intestinal microbes. Although there are possible benefits to maintain low levels of ROS in the proper functioning of the body, the diet and lifestyle of the majority results in increased levels of ROS in the body are known to be harmful and can lead to the progression of disease. In this way, it is critical to maintain the proper balance of ROS in the body, and phenolic compounds have been shown to reestablish a healthy level of ROS. Next, we turn to the vital interaction of phytophenols and microflora of the gut system that can lead to creation

The group known as lactobacilli is composed of several genera of bacteria (*Leuconostoc, Pediococcus, Lactococcus*, and *Streptococcus*)*, Lactobacillus* being the largest order in the phylum Firmicutes and the class Bacilli [53]. These free living lactic acid bacteria flourish in different biological niches such as soil, plants (fruits, beverage, and silage) and fermented foods (cheese, fermented milk, yogurt, meat products, alcoholic beverages, and pickled products). They are also associated with mammals as members of the microbial community characteristic of the oral cavity, GI system, urinary tract, skin, etc. [54–57]. The genus *Lactobacillus* is composed of nutritionally fastidious gram-positive, non-spore-forming rods or coccobacilli, catalasenegative, aerotolerant or anaerobic bacteria. The main characteristic of their homo- or heterofermentative metabolism is the production of lactic acid as the primary end fermentation product. The genus *Lactobacillus* is represented by over 212 species described to date, including several industrially relevant microorganisms such as *L. acidophilus, L. bulgaricus, L. casei, L. lactis, L. paracasei, L. plantarum, L. reuteri, L. fermentum, L. salivarius, L. rhamnosus, L. delbrueckii*,

The *Lactobacillus* genus is widely studied because of the bacterias' capacity to produce lactic acid. Thus, most studies regarding their physiology were centered on acidifying bacteria such as *L. delbrueckii subsp. bulgaricus*, which in combination with *Streptococcus thermophilus* acidify milk in a few hours. This process is critical to optimize the production of fermented dairy products such as cheese and yogurt, or other non-dairy products such as pickles, sauerkraut,

cells [44, 50].

of redox balance critical to health.

88 Probiotics and Prebiotics in Human Nutrition and Health

**2. Lactic acid bacteria**

and *L. johnsonii*.

and sourdough bread.

Studies of the human microbiome revealed that lactobacilli could occupy different microha‐ bitats in the human body, such as the buccal cavity and nasal fossa, but they mainly thrive in the gut and the urogenital tract [59]. In women, it was observed that variations of estrogen and glycogen stimulates the growth of lactic acid bacteria. Depletion of vaginal lactobacilli could give rise to adverse microbial flora colonization inducing urogenital infection [60]. Gustafsson *et al*. evaluated the population of lactobacilli in healthy fertile and postmenopausal women in correlation with hormone levels [57]. They demonstrated that *L. crispatus*is the most abundant bacteria and, together with *L. vaginalis, L. jensenii* and *L. gasseri* are responsible in protecting the urogenital tract against vaginal infection. This effect was associated with the capacity of these *Lactobacillus* species to produce H2O2, which negatively affects the viability of pathogenic bacteria [61, 62]. The GI system is home to many different kinds of microorganisms, which globally is referred as the gut microbiota. Among these microbes, one of the most abundant groups, in this complex microbial population, is *Lactobacillus*. Although it is not the most abundant genus in the microflora, it is considered one of the most important genus due to potential beneficial effects associated with them. Scientific studies revealed that the *Lactoba‐ cillus* abundance in the gut microbiota changes according to the portion of the GI tract. The highest presence of *Lactobacillus* sequences was found in the jejunum and ileum lumen, 16% respect to the total microbiota. Their abundance in the colon/rectal lumen decreased to 9.9%. Surprisingly, *Lactobacillus* sequences were lower than 0.5% in the fecal samples studied [63– 66]. The main *Lactobacillus* strains found in feces are *L. acidophilus, L. crispatus, L. gasseri, L. reuteri, L. brevis, L. sakei, L. curvatus, L. casei, L. paracasei, L. rhamnosus, L. delbrueckii, L. brevis, L. johnsonii, L. plantarum*, and *L. fermentum* [67]. In the GI context, these bacteria interact with other intestinal microbes, with food components and with the GI mucosa. The consequences of these interactions are endless; in addition, it is extremely difficult to isolate the effects and study them separately. Probably one of the most complex and interesting systems effected is the host immune system. Commensals can help in educating and maturing the host immune response and prompt the immunological defensive arsenal. All members of the *Lactobacillus* group are classified as GRAS (generally recognized as safe) organisms; consequently, they are considered innocuous or beneficial for health. The specific mechanisms by which these bacteria are considered beneficial are still the subject of important discrepancies and the center of scientific debates.

Lactobacilli are excellent organic acid producers, converting sugars into lactic acid and other by-products such as acetate, ethanol, CO2, butyrate, and succinate. They produce small molecules, as well, such as H2O2, or compounds such as diacetyl, or acetaldehyde [67]. Several of these metabolites are bioactive, with beneficial effects for the human GI. At the same time, they are essential for the dairy industry because they provide flavoring and display natural preservative properties [68]. They help to maintain the integrity of GI layers, favoring the renewal of the epithelium. A continuous renewal of the GI layers is critical to maintain an adequate barrier function to minimize several significant human diseases, including autoim‐ munity and cancer. According to recently published studies, the production of low amounts of H2O2 at the GI level is beneficial to the host. Besides its well-characterized antimicrobial activity, this molecule could directly down-regulate the early stages of the host inflammatory response and improve epithelial cell restitution and healing via the oxidation of cysteine residues in the host tyrosine phosphatases [62, 69].

Other important metabolites synthesized by *Lactobacillus* species are larger molecules such as polysaccharides (viscosifying agents) [70] and enzymes (proteases, bacteriocins, esterases, and lipases) [6, 71, 72], which improve dairy product quality (flavor development, texture modi‐ fication) and provide beneficial effects to boost human health [73]. *L. helveticus* is considered one of the most efficient species associated with proteolysis in cheese ripening. *L. helveticus* also produces bioactive peptides with antihypertensive and antimicrobial activity [74]. Indeed, *Lactobacillus* antimicrobial activity is directly related to its ability to secrete bacteriocins. A subset of *Lactobacillus* strains produce these kind of antimicrobial peptides such as *L. sakei* (bavaricin, sakacin) [75], *L. curvatus* (curvaticin), *L. plantarum* (pediocin), *L. salivairus* (bacter‐ iocins) [76], and *L. acidophilus* (acidocin) [77]. These antimicrobials may play an essential role in regulating the composition of the microbial communities within the GI system, influencing the host's health; however, not all of them showed promising effects on human [71].

Maintenance of the GI redox homeostasis is essential in minimizing human diseases. The production of enzymes, which could increase the amount of free and active antioxidant agents in the GI lumen, is another important characteristic associated with several *Lactobacillus* strains. These enzymes, such as esterases and/or lipases, are synthesized by the intestinal microbiota and can release redox quenchers like the above-described phytophenols that are ingested with the host diet. Thus, the ingestion of probiotic bacteria able to produce these enzymes is a healthy and natural alternative to modulate the redox status in the GI tract. Lactobacilli are excellent producers of lipases and esterases, and several of the best producing strains were selected by the dairy industry due to their contribution in cheese ripening. The esterases are active toward a wide range of ester substrates from free fatty acids to tri-, di-, and monoacyl‐ glyceride substrates. Cinnamoyl esterases (CE) are one of the most important enzymes involved in releasing antioxidant molecules from dietary fibers. These enzymes break down the ester linkages between hydroxycinnamates and sugars, commonly found in the fiber of dietary plants, releasing phenolics such as hydroxycinnamic, ferulic, coumaric, and caffeic acids with high ROS scavenging activity. Genes encoding various esterases have been described in *L. fermentum* and *L. reuteri, L. leichmanni*, and *L. farciminis*, and the first two species are frequently found in animal and human feces. These enzymes have also demonstrated to be active toward soluble polyphenols such as chlorogenic acid to release caffeic and quinic acids [78]. The accumulation of the enzymatic products released (monophenols) in *Lactobacil‐ lus* cultures suggests that these microorganisms do not (or do so extremely slowly) metabolize the phenolic acids released. The enzymatic action correlates directly with increased amounts of phenolics (i.e., caffeic acid) detected in the bloodstream of model animals fed with fibers in combination with probiotics formulated with those strains [78]. Guglielmetti *et al.* studied the activity of CE produced by *L. helveticus* MIMLh5 on soluble phenolics, such as chlorogenic acid, to enrich food with free caffeic acid [79]. *L. helveticus* enzymes are mainly intracellular, but some of them could be surface-associated as observed in *L. fermentum* [80]. *L. plantarum*, frequently found in plant-derived food products where hydroxycinnamoyl esters are abun‐ dant, produces the enzyme Lp\_0796 (esterase), which hydrolyzes the four model substrates for feruloyl esterases (methyl ferulate, methyl caffeate, methyl *p*-coumarate, and methyl sinapinate). This esterase is generally present among several *L. plantarum* strains and provides new insights into the metabolism of hydroxycinnamic compounds in this bacterial species [81]. Further studies on *L. plantarum* showed another esterase encoded by the *est\_1092* gene is able to hydrolyze hydroxycinnamic esters, such as methyl ferulate or methyl caffeate, and is active on a broad range of phenolic esters [82]. *L. acidophilus* produces a novel CE with high similarity (70%) with the main CE characterized in *L. johnsonii* LJ1228 [72]. Other *L. acidophilus* and *L. johnsonii* strains displayed, as well, high CE activity [79]. One strain of *L. johnsonii* showed high ferulic acid esterase activity, stimulates insulin production, and alleviates symptoms caused by diabetes [83]. However, there is no direct evidence to associate the ability to release phenolics with the capacity to stimulate insulin production. The strain *L. johnsonii* N6.2 presented two different proteins with ferulic acid esterase activity. These enzymes showed high affinities and catalytic efficiencies toward aromatic compounds such as ethyl ferulate and chlorogenic acid [6]. *L. johnsonii* NCC533 also hydrolyzes rosmarinic acid, the main compo‐ nents of rosemary extracts, and it is ascribed to many health benefits.

renewal of the epithelium. A continuous renewal of the GI layers is critical to maintain an adequate barrier function to minimize several significant human diseases, including autoim‐ munity and cancer. According to recently published studies, the production of low amounts of H2O2 at the GI level is beneficial to the host. Besides its well-characterized antimicrobial activity, this molecule could directly down-regulate the early stages of the host inflammatory response and improve epithelial cell restitution and healing via the oxidation of cysteine

Other important metabolites synthesized by *Lactobacillus* species are larger molecules such as polysaccharides (viscosifying agents) [70] and enzymes (proteases, bacteriocins, esterases, and lipases) [6, 71, 72], which improve dairy product quality (flavor development, texture modi‐ fication) and provide beneficial effects to boost human health [73]. *L. helveticus* is considered one of the most efficient species associated with proteolysis in cheese ripening. *L. helveticus* also produces bioactive peptides with antihypertensive and antimicrobial activity [74]. Indeed, *Lactobacillus* antimicrobial activity is directly related to its ability to secrete bacteriocins. A subset of *Lactobacillus* strains produce these kind of antimicrobial peptides such as *L. sakei* (bavaricin, sakacin) [75], *L. curvatus* (curvaticin), *L. plantarum* (pediocin), *L. salivairus* (bacter‐ iocins) [76], and *L. acidophilus* (acidocin) [77]. These antimicrobials may play an essential role in regulating the composition of the microbial communities within the GI system, influencing

the host's health; however, not all of them showed promising effects on human [71].

Maintenance of the GI redox homeostasis is essential in minimizing human diseases. The production of enzymes, which could increase the amount of free and active antioxidant agents in the GI lumen, is another important characteristic associated with several *Lactobacillus* strains. These enzymes, such as esterases and/or lipases, are synthesized by the intestinal microbiota and can release redox quenchers like the above-described phytophenols that are ingested with the host diet. Thus, the ingestion of probiotic bacteria able to produce these enzymes is a healthy and natural alternative to modulate the redox status in the GI tract. Lactobacilli are excellent producers of lipases and esterases, and several of the best producing strains were selected by the dairy industry due to their contribution in cheese ripening. The esterases are active toward a wide range of ester substrates from free fatty acids to tri-, di-, and monoacyl‐ glyceride substrates. Cinnamoyl esterases (CE) are one of the most important enzymes involved in releasing antioxidant molecules from dietary fibers. These enzymes break down the ester linkages between hydroxycinnamates and sugars, commonly found in the fiber of dietary plants, releasing phenolics such as hydroxycinnamic, ferulic, coumaric, and caffeic acids with high ROS scavenging activity. Genes encoding various esterases have been described in *L. fermentum* and *L. reuteri, L. leichmanni*, and *L. farciminis*, and the first two species are frequently found in animal and human feces. These enzymes have also demonstrated to be active toward soluble polyphenols such as chlorogenic acid to release caffeic and quinic acids [78]. The accumulation of the enzymatic products released (monophenols) in *Lactobacil‐ lus* cultures suggests that these microorganisms do not (or do so extremely slowly) metabolize the phenolic acids released. The enzymatic action correlates directly with increased amounts of phenolics (i.e., caffeic acid) detected in the bloodstream of model animals fed with fibers in combination with probiotics formulated with those strains [78]. Guglielmetti *et al.* studied the

residues in the host tyrosine phosphatases [62, 69].

90 Probiotics and Prebiotics in Human Nutrition and Health

The released monophenols (caffeic acid or other cinnamic acids) may exert its biological activities on the host, either at the level of the colonic mucosa itself, or in other tissues and organs, possibly after further modification by mammalian enzymes in the liver [80]. The release and solubilization of these phenolics, from fiber, also favor its absorption and further modifi‐ cation by other GI commensals. *In vitro* fermentation assays demonstrate that the fecal microbiota can efficiently metabolize caffeic, chlorogenic, and caftaric acids. With the use of highly sensitive analytical techniques, it was possible to identify two major metabolites: 3 hydroxyphenylpropionic (3-HPP) and benzoic acids (BA) once the original compounds were fully metabolized. Similar metabolic patterns were observed for other polyphenolic acids, suggesting a large and important metabolic flexibility of the gut microbiota [84]. Evidence for a metabolic pathway leading to the formation of BA from 3-HPP is supported by the estab‐ lished quality of intestinal microorganisms to carry out biological dehydroxylation of 3-HPP to 3-phenylpropionic acid, which can itself be further β-oxidized into BA by the colonic microbiota [85]. Alternatively, the absorbed cinnamic and phenylpropionic acids undergo βoxidation in the liver to produce BA, which is subsequently conjugated to glycine to form hippuric acid in the liver [86].

The capacity of lactic acid bacteria to transform phenolic compounds into smaller novel molecules able to be absorbed in the GI system reoriented modern research to use combinations of probiotics and prebiotic products together. A large variety of dietary fibers were used for this purpose. Yet, the microbial metabolism of the released compounds by different biocon‐ version pathways, such as glycosylation, deglycosylation, ring cleavage, methylation, glucur‐ onidation, and sulfate conjugation, depends on the microbial strains and substrates used. The results of such combinations are a large array of new metabolites, many of them recognized as bioactive molecules. This strategy demonstrates to have the potential to produce extracts with a high-added value from plant-based matrices (soybean, apple, cereals, among others).

Studies of apple juice fermentation to manage hyperglycemia, hypertension, and modulation of microbiota composition were also carried out. Apple juice, fermented by *L. acidophilus*, showed outstanding effects enhancing the free radical-scavenging activity in blood samples. *Lactobacillus* fermented samples inhibited *H. pylori in vitro*. However, the fermented extracts did not exert inhibitory effects on the beneficial intestinal species such as *Bifidobacterium longum*. Thus, these data provided biochemical rationale for the development of new ferment‐ ed food to reduce hyperglycemia (diabetes) and other chronic diseases [87]. The development of probiotics with therapeutic and preventative effects for various diseases and metabolic disorders is the trend of new healthy nutrition. The main limitation for oral probiotics is the harsh conditions of the GI system. For that, the beneficial bacteria have to reach the intestines alive, colonize, and locally release enzymes or bioactive metabolites.

The benefits of *Lactobacillus* intake is not only linked to the capacity to hydrolyze phytophenols inside the lower GI system but also to prehydrolyze those present in plant extract (juice, fruits, etc.) and increased the phenolic content in food and beverages. Predigestion will enhance their absorption once they reach the small intestine to exert their healing properties. For example, the use of three *Lactobacillus* strains (*L. johnsonii* LA1, *L. reuteri* SD2112, and *L. acidophilus* LA-5) improved the bioavailability of the dietary phenolics present in barley and oat flour by 20-fold [88]. The free ferulic acid in the pretreated cereals increased from 1 μg/g dried weight up to 39–56 μg/g dried weight. Comparing the three strains used, *L. johnsonii* demonstrate to be more active in releasing phenolic acids than the other strains. These data showed that cereal fermentation with specific probiotic strains can significantly increase the quantity of free phenolic acids, improving their bioavailability [89]. *L. johnsonii* NCC 533 synthesizes esterases and a hydroxycinnamate decarboxylase responsible for the biotransformation of chlorogenic and caffeic acids. The complete hydrolysis of 5-caffeoylquinic acid *in vitro* occurred during the first 16 h of incubation. After 48 h, caffeic acid was completely transformed to 4-vinylcatechol (4-VC). In this case, the bacteria increased the presence of caffeic acid and simultaneously generated flavor compounds from plant phytophenols [90]. These data provide solid evidence that the same microorganism is able to hydrolyze caffeoyl quinic acids into 4-VC, combining chlorogenate esterase and a hydroxycinnamate decarboxylase activity [6, 91]. Similar results have been reported in the case of some *L. brevis* strains [92].

The ability of lactic acid bacteria to metabolize dietary phytophenols prompts the use of new component combinations in fermented products. Several of these new blends were formulated with plant extracts rich in aromatic compounds. Example of this is the addition of green tea in bioyogurts fermented with selected lactic acid bacteria. Species such as *St. thermophilus, L. acidophilus* LA-5, *B. animalis* subsp. *lactis* BB-12, or acidophilus enhanced the antioxidant capacity of these preparations in dose-dependent manner. Similar studies were carried out with different tea extracts, green, white, and black tea (*Camellia sinensis*) in yogurt combined with *L. acidophilus* LA-5, *Bifidobacterium* Bb-12, *L. casei* LC-01, *S. thermophilus* Th-4, and *L. delbrueckii* ssp. *bulgaricus*. In general, the three types of tea extracts did not significantly affect the viability of the bacteria used during storage [93]. The tea extracts could be successfully used as a functional additives in fermented food, adding extra value to the known health benefits of probiotics. Others extracts prepared from olive, garlic, onion, and citrus were also evaluated using similar formulations [94].

### **3. A model case study,** *Lactobacillus johnsonii* **N6.2**

results of such combinations are a large array of new metabolites, many of them recognized as bioactive molecules. This strategy demonstrates to have the potential to produce extracts with a high-added value from plant-based matrices (soybean, apple, cereals, among others).

Studies of apple juice fermentation to manage hyperglycemia, hypertension, and modulation of microbiota composition were also carried out. Apple juice, fermented by *L. acidophilus*, showed outstanding effects enhancing the free radical-scavenging activity in blood samples. *Lactobacillus* fermented samples inhibited *H. pylori in vitro*. However, the fermented extracts did not exert inhibitory effects on the beneficial intestinal species such as *Bifidobacterium longum*. Thus, these data provided biochemical rationale for the development of new ferment‐ ed food to reduce hyperglycemia (diabetes) and other chronic diseases [87]. The development of probiotics with therapeutic and preventative effects for various diseases and metabolic disorders is the trend of new healthy nutrition. The main limitation for oral probiotics is the harsh conditions of the GI system. For that, the beneficial bacteria have to reach the intestines

The benefits of *Lactobacillus* intake is not only linked to the capacity to hydrolyze phytophenols inside the lower GI system but also to prehydrolyze those present in plant extract (juice, fruits, etc.) and increased the phenolic content in food and beverages. Predigestion will enhance their absorption once they reach the small intestine to exert their healing properties. For example, the use of three *Lactobacillus* strains (*L. johnsonii* LA1, *L. reuteri* SD2112, and *L. acidophilus* LA-5) improved the bioavailability of the dietary phenolics present in barley and oat flour by 20-fold [88]. The free ferulic acid in the pretreated cereals increased from 1 μg/g dried weight up to 39–56 μg/g dried weight. Comparing the three strains used, *L. johnsonii* demonstrate to be more active in releasing phenolic acids than the other strains. These data showed that cereal fermentation with specific probiotic strains can significantly increase the quantity of free phenolic acids, improving their bioavailability [89]. *L. johnsonii* NCC 533 synthesizes esterases and a hydroxycinnamate decarboxylase responsible for the biotransformation of chlorogenic and caffeic acids. The complete hydrolysis of 5-caffeoylquinic acid *in vitro* occurred during the first 16 h of incubation. After 48 h, caffeic acid was completely transformed to 4-vinylcatechol (4-VC). In this case, the bacteria increased the presence of caffeic acid and simultaneously generated flavor compounds from plant phytophenols [90]. These data provide solid evidence that the same microorganism is able to hydrolyze caffeoyl quinic acids into 4-VC, combining chlorogenate esterase and a hydroxycinnamate decarboxylase activity [6, 91]. Similar results

The ability of lactic acid bacteria to metabolize dietary phytophenols prompts the use of new component combinations in fermented products. Several of these new blends were formulated with plant extracts rich in aromatic compounds. Example of this is the addition of green tea in bioyogurts fermented with selected lactic acid bacteria. Species such as *St. thermophilus, L. acidophilus* LA-5, *B. animalis* subsp. *lactis* BB-12, or acidophilus enhanced the antioxidant capacity of these preparations in dose-dependent manner. Similar studies were carried out with different tea extracts, green, white, and black tea (*Camellia sinensis*) in yogurt combined with *L. acidophilus* LA-5, *Bifidobacterium* Bb-12, *L. casei* LC-01, *S. thermophilus* Th-4, and *L. delbrueckii* ssp. *bulgaricus*. In general, the three types of tea extracts did not significantly affect

alive, colonize, and locally release enzymes or bioactive metabolites.

92 Probiotics and Prebiotics in Human Nutrition and Health

have been reported in the case of some *L. brevis* strains [92].

The intestinal epithelium is one of the most immunologically active surfaces of the body due to the high abundance of microbes and food antigens that are constantly exposed to the GI system. The mucosal surface of the intestinal epithelium is the first line of defense from invading pathogens in the GI tract. Breaching this barrier and subsequently activating aberrant immune signaling have been involved in many diseases, both locally and systematically related. In this context, it has been proposed that there is a complex interplay between gut resident microbiota [95, 96], gut permeability [97], and altered immune function in the development of type 1 diabetes [98].

Currently, our scientific efforts are directed on characterizing a strain of *Lactobacillus* (*L. johnsonii* N6.2). This lactobacilli is abundant in GI microbiome in a line of animals used as a T1D model, in contrast to the scarcity observed in the counterpart diabetes prone animals. Type 1 diabetes (T1D), also referred to as diabetes mellitus type 1, is an autoimmune disease in which pancreatic β-cells produce little to no insulin due to their destruction. Its more commonly known and more prevalent counterpart, type 2 diabetes, occurs when the body becomes resistant to insulin. Both of these conditions result in increased blood glucose levels, called hyperglycemia. Insulin is the hormone responsible for absorbing sugar, in the form of glucose, from circulating blood to be stored in skeletal muscles and fat cells. Although type 1 diabetes has a genetic component and primarily occurs in adolescents and children, it is possible for adults to develop the disease too. Five to 10 percent of diabetes cases in adults are the result of T1D, and an estimated 80 people per day are newly diagnosed with T1D [99, 100]. Unfortunately, recently epidemiological studies have suggested that the incidence of T1D is increasing up to 3–4% globally every year, most notably among youths [101, 102].

*L. johnsonii* N6.2 was discovered when it was negatively correlated with diabetes development when analyzing the stool samples from BioBreeding diabetes-prone (BB-DP) and BioBreeding diabetes-resistant (BB-DR) rats. Stool embodies a representative microbiome of an individual and is a useful sample for understanding the microbial diversity of the GI tract. Currently, it is estimated that more than 1000 microbial species encompassing more than 100 trillion microorganisms colonize the GI system, collectively outnumbering human genes by 150-fold [103]. These microorganisms grow more in number and diversity progressing through the GI tract. The BioBreeding rat is popular model when studying type 1 diabetes, as it spontaneously develops this disease through its genetic predisposition. After using culture-independent methods, it was found that two genera, *Bifidobacterium* and *Lactobacillus*, showed a higher abundance in BB-DR rats [104]. Quantitative PCR of 16S rRNA revealed a higher abundance of *Bifidobacterium* and *Lactobacillus* in BB-DR samples [104]. However, it was unknown whether the higher abundance of these bacteria was just the common microflora of a "healthy" gut or if they played a part in preventing the onset of T1D. Further analyses of the *Lactobacillus* strains in the BB-DR rat model revealed that those with CE activity, such as *L. johnsonii* N6.2 and *L. reuteri* TD1, were negatively correlated with T1D development [6].

As it was described before, the release of antioxidant compounds by probiotic bacteria is relevant since an enhanced oxidative stress response triggered by the excessive production of reactive oxygen species is observed in T1D and other diseases [105–107]. This characteristic was relevant in the study because a low dosage of ferulic acid stimulates the release of insulin and alleviates symptoms common to T1D in rodents [83, 108, 109]. Therefore, it would seem plausible that orally administering lactic acid bacteria containing CE qualities would help reduce blood glucose levels and ultimately prevent the onset of diabetes. To confirm this, a feeding experiment of *L. johnsonii* N6.2 and *L. reuteri* TD1 on BioBreeding rats was conducted to determine whether these strains were responsible for the lack of T1D development. While *L. johnsonii* N6.2-fed rats were associated with reduced diabetes onset, *L. reuteri* T1D showed similar diabetes development characteristics as vehicle-fed control groups [110]. A feruloyl esterase screening assay of bacterial stool sample isolates from BB-DR rats on MRS media supplemented with feruloyl esters demonstrates that *L. johnsonii* N6.2 contained the highest feruloyl esterase activity [6]. Enzymatic screening of two purified *L. johnsonii* proteins, Lj0536 and Lj1228, showed high preference and good enzymatic activity using aromatic esters as substrates (**Figure 3**). Lj1228 displayed the best hydrolytic activity with ethyl ferulate, chloro‐ genic acid, and rosmarinic acid, while Lj0536 showed a preference to ethyl ferulate. Sequence analyses of these proteins revealed a 42% similarity and the classical serine nucleophilic motif characteristic for some feruloyl esterases [111, 112]. Biochemical analyses of these enzymes suggested that they maintain excellent activity in the presence of emulsifiers. Their activity was tested in the presence of conjugated and deconjugated bile salts of which none of the compounds assayed decreased their activity. Interestingly, with increasing concentrations of

**Figure 3.** *L. johnsonii* LJ0536 hydrolyze a wide range of substrates. The product(s) of hydrolysis for each substrate are boxed.

sodium glycocholate, Lj0536 showed increased activity. In this condition, both enzymes were active against a wide variety of substrates, showing the highest affinity toward aromatic esters.

if they played a part in preventing the onset of T1D. Further analyses of the *Lactobacillus* strains in the BB-DR rat model revealed that those with CE activity, such as *L. johnsonii* N6.2 and *L.*

As it was described before, the release of antioxidant compounds by probiotic bacteria is relevant since an enhanced oxidative stress response triggered by the excessive production of reactive oxygen species is observed in T1D and other diseases [105–107]. This characteristic was relevant in the study because a low dosage of ferulic acid stimulates the release of insulin and alleviates symptoms common to T1D in rodents [83, 108, 109]. Therefore, it would seem plausible that orally administering lactic acid bacteria containing CE qualities would help reduce blood glucose levels and ultimately prevent the onset of diabetes. To confirm this, a feeding experiment of *L. johnsonii* N6.2 and *L. reuteri* TD1 on BioBreeding rats was conducted to determine whether these strains were responsible for the lack of T1D development. While *L. johnsonii* N6.2-fed rats were associated with reduced diabetes onset, *L. reuteri* T1D showed similar diabetes development characteristics as vehicle-fed control groups [110]. A feruloyl esterase screening assay of bacterial stool sample isolates from BB-DR rats on MRS media supplemented with feruloyl esters demonstrates that *L. johnsonii* N6.2 contained the highest feruloyl esterase activity [6]. Enzymatic screening of two purified *L. johnsonii* proteins, Lj0536 and Lj1228, showed high preference and good enzymatic activity using aromatic esters as substrates (**Figure 3**). Lj1228 displayed the best hydrolytic activity with ethyl ferulate, chloro‐ genic acid, and rosmarinic acid, while Lj0536 showed a preference to ethyl ferulate. Sequence analyses of these proteins revealed a 42% similarity and the classical serine nucleophilic motif characteristic for some feruloyl esterases [111, 112]. Biochemical analyses of these enzymes suggested that they maintain excellent activity in the presence of emulsifiers. Their activity was tested in the presence of conjugated and deconjugated bile salts of which none of the compounds assayed decreased their activity. Interestingly, with increasing concentrations of

**Figure 3.** *L. johnsonii* LJ0536 hydrolyze a wide range of substrates. The product(s) of hydrolysis for each substrate are

boxed.

*reuteri* TD1, were negatively correlated with T1D development [6].

94 Probiotics and Prebiotics in Human Nutrition and Health

*L. johnsonii* post-weaning feedings has demonstrated a decreased incidence of diabetes in BB-DP rats compared to vehicle-fed controls and *L. reuteri* TD1-fed rats [110]. With this in mind, it was then determined what type of altered environment *L. johnsonii* created compared to healthy controls and diabetic animals (including those animals from *L. johnsonii* feedings and controls that developed T1D). The first thing that was noticed was the modification of the intestinal microbiota as determined by real-time quantification. While all animals showed an abundance of *Lactobacillus* in stool samples, differences in species seem to differ among feeding groups (*L. johnsonii, L. reuteri*, and vehicle control). Vehicle control animals displayed a predominance of *L. murinus* (65%), while 88% and 92% of *L. johnsonii* and *L. reuteri*, respectively, corresponded to the fed bacteria in each group. Analyses of ileal mucosa unveiled a significant increase of the *Lactobacillus* population in all rats that did not develop diabetes, and a significant increase of enterobacteria was found in all diabetic animals. Since no differences in the microbiota were obtained in stool samples, but were statistically significant in ileal mucosa, the positive effect of *L. johnsonii* N6.2 could be exhibited primarily in the intestinal mucosa [110].

As it was observed that an altered intestinal microbiota was associated with diabetes onset, as previously suggested [95, 96], gut permeability and barrier function were investigated next between *L. johnsonii-*fed, healthy controls, and diabetic animals. It was previously reported that changes in intestinal morphology and permeability, partly due to decreased levels of claudin-1, were observed before the onset of T1D [97]. Claudin-1 is an intercellular tight junction protein responsible for cell-to-cell adhesion in epithelial cell layers. This protein is important in strengthening the physical barrier that keeps the contents of gut lumen from passing into the lamina propria. It has been suggested that unregulated passage of environ‐ mental antigens through the intercellular space of the intestinal epithelial could trigger the autoimmune response that contributes to T1D. Expression analysis of the claudin-1 gene in *L. johnsonii*-fed animals exposed its higher abundance when compared to healthy controls or diabetic animals [110]. Furthermore, a significant increase in goblet cells was unveiled in healthy controls and *L. johnsonii*-fed animals compared to those that developed diabetes. Goblet cells produce mucin, the main constituent of the mucosal lining of the GI tract. This feature is important when considering the harsh environment of the GI tract and the constant exposure to potential invading pathogens and inflammatory antigens. The mucosal layer serves as one's first line of defense against these threats by acting as a physical, viscous, and continuously moving layer that rests above epithelial cells. Most harmful substances get trapped in the mucous and before even making it to the epithelial layer, get swept down the intestines. The increase in claudin-1 and goblet cell levels in *L. johnsonii*-fed animals strength‐ ens and physically protects the epithelial cell layer and undoubtedly intensifies intestinal barrier function contributing to the decrease in diabetes onset.

Among the destructive properties of reactive oxygen species (ROS) generated during early disease development is its ability to disrupt the function of epithelial tight junction proteins [113]. To determine the extent of the oxidative stress environment, ileal mucosal hexanoyllysine levels were quantified by ELISA and a significant increase of levels was observed in diabetic animals when compared to healthy controls and *L. johnsonii*-fed animals [110]. Due to the difference in the oxidative environment between diabetic and non-diabetic animals, the expression of genes involved in ROS detoxification pathways were also quantified. It was evident that *L. johnsonii* helps the host to cope with intestinal oxidative stress response as levels of superoxide dismutase 2, catalase, glutathione reductase, and glutathione peroxide were induced in diabetic animals. Meanwhile, superoxide dismutase and glutathione peroxidase were induced in healthy controls compared to *L. johnsonii*-fed groups. Taken collectively, catalase and glutathione reductase were negatively correlated with a healthy status, while superoxide dismutase 2 and glutathione peroxidase were negatively correlated with *L. johnsonii* feeding. Also among the stress response genes assayed was inducible nitric oxide synthase (iNOS), which produces nitric oxide in the presence of ROS. The mRNA levels of iNOS were significantly reduced in *L. johnsonii*-fed rats compared to healthy controls and those that developed diabetes. When further examining the iNOS protein levels via Western blot, *L. johnsonii*-fed rats and healthy controls showed similar levels of detection, suggesting that expression of iNOS is associated with healthy status. Amid the inducers of iNOS expression is INFγ, a pro-inflammatory cytokine [114, 115]. It was hypothesized that a negative correlation existed between pro-inflammatory cytokines, specifically INFγ, and the reduced stress response due to *L. johnsonii* feeding. This hypothesis was proven as diabetic animals showed a significant increase in INFγ gene expression compared to healthy animals; meanwhile, healthy controls and *L. johnsonii-*fed animals did not show any statistical differences.

Since it has been determined that *L. johnsonii* N6.2 feedings can promote a healthy gut microbiota and strengthen epithelial barrier function, it was next examined whether *L. johnsonii* could influence immune function. At the intestinal mucosal layer, resident microbiota and host cells reside in constant homeostasis, epithelial cells tightly controlled by the recog‐ nition and tolerance of local bacteria. Host cells recognize the resident microbiota or their associated components through pattern recognition receptors (Toll-like receptor, TLR) and/or by cytoplasmic nucleotide-binding oligomerization domain (NOD)-like receptors, which can subsequently initiate an immune response. Of the first things noticed with *L. johnsonii* administration was the overexpression of pro-inflammatory chemokine mRNA levels, particularly CCL20 (MIP3A), CXCL8 (IL-8), and CXCL10 (IP-10), suggesting that *L. johnsonii* may prime the innate immune system to become more resistant to a subsequent strong inflammatory response [116]. Investigation of the ability of *L. johnsonii* to activate TLR and NOD-like receptor revealed that exposure to *L. johnsonii* created a 4.2- and 10-fold increased expression of TLR7 and TLR9, respectively. Because both of these receptors are involved in nucleic acid recognition, cell free extracts and purified *L. johnsonii* nucleic acid extracts were tested on their ability to induce expression of these TLRs. In both cases, cell-free extracts and purified *L. johnsonii* nucleic acid were able to increase the mRNA levels of TLR7 and TL9, suggesting that the ability for epithelial cells to sense foreign nucleic acids may be involved in the observed increased of some chemokine levels. This also suggests that *L. johnsonii* predom‐ inantly exerts its signaling capability through RNA/DNA recognition, as opposed to other cell components, such as peptidoglycan that is sensed by TLR2 and NOD2. Lastly, consequences of TLR9 induction by *L. johnonii* were determined by exploring the expression of Frizzled 5 receptor (fzd5), which is responsible for Paneth cell maturation, and INF-α, which is secreted by TLR9 activity and induces the chemokine CXCL10 [117–119]. Paneth cells are located at the base of intestinal glands throughout the small intestines and secrete antimicrobial peptides. *L. johnsonii* administration showed higher levels of Paneth cells in agreement with the higher levels of fzd5, and a higher level of INF-α, in agreement with the observed increased levels of CXCL10 [116]. As discovered in this study, *L. johnsonii* may be able to prime the immune system by activating an innate immune response early on and therefore protecting the host from more prominent stimuli later on.

lysine levels were quantified by ELISA and a significant increase of levels was observed in diabetic animals when compared to healthy controls and *L. johnsonii*-fed animals [110]. Due to the difference in the oxidative environment between diabetic and non-diabetic animals, the expression of genes involved in ROS detoxification pathways were also quantified. It was evident that *L. johnsonii* helps the host to cope with intestinal oxidative stress response as levels of superoxide dismutase 2, catalase, glutathione reductase, and glutathione peroxide were induced in diabetic animals. Meanwhile, superoxide dismutase and glutathione peroxidase were induced in healthy controls compared to *L. johnsonii*-fed groups. Taken collectively, catalase and glutathione reductase were negatively correlated with a healthy status, while superoxide dismutase 2 and glutathione peroxidase were negatively correlated with *L. johnsonii* feeding. Also among the stress response genes assayed was inducible nitric oxide synthase (iNOS), which produces nitric oxide in the presence of ROS. The mRNA levels of iNOS were significantly reduced in *L. johnsonii*-fed rats compared to healthy controls and those that developed diabetes. When further examining the iNOS protein levels via Western blot, *L. johnsonii*-fed rats and healthy controls showed similar levels of detection, suggesting that expression of iNOS is associated with healthy status. Amid the inducers of iNOS expression is INFγ, a pro-inflammatory cytokine [114, 115]. It was hypothesized that a negative correlation existed between pro-inflammatory cytokines, specifically INFγ, and the reduced stress response due to *L. johnsonii* feeding. This hypothesis was proven as diabetic animals showed a significant increase in INFγ gene expression compared to healthy animals; meanwhile,

96 Probiotics and Prebiotics in Human Nutrition and Health

healthy controls and *L. johnsonii-*fed animals did not show any statistical differences.

Since it has been determined that *L. johnsonii* N6.2 feedings can promote a healthy gut microbiota and strengthen epithelial barrier function, it was next examined whether *L. johnsonii* could influence immune function. At the intestinal mucosal layer, resident microbiota and host cells reside in constant homeostasis, epithelial cells tightly controlled by the recog‐ nition and tolerance of local bacteria. Host cells recognize the resident microbiota or their associated components through pattern recognition receptors (Toll-like receptor, TLR) and/or by cytoplasmic nucleotide-binding oligomerization domain (NOD)-like receptors, which can subsequently initiate an immune response. Of the first things noticed with *L. johnsonii* administration was the overexpression of pro-inflammatory chemokine mRNA levels, particularly CCL20 (MIP3A), CXCL8 (IL-8), and CXCL10 (IP-10), suggesting that *L. johnsonii* may prime the innate immune system to become more resistant to a subsequent strong inflammatory response [116]. Investigation of the ability of *L. johnsonii* to activate TLR and NOD-like receptor revealed that exposure to *L. johnsonii* created a 4.2- and 10-fold increased expression of TLR7 and TLR9, respectively. Because both of these receptors are involved in nucleic acid recognition, cell free extracts and purified *L. johnsonii* nucleic acid extracts were tested on their ability to induce expression of these TLRs. In both cases, cell-free extracts and purified *L. johnsonii* nucleic acid were able to increase the mRNA levels of TLR7 and TL9, suggesting that the ability for epithelial cells to sense foreign nucleic acids may be involved in the observed increased of some chemokine levels. This also suggests that *L. johnsonii* predom‐ inantly exerts its signaling capability through RNA/DNA recognition, as opposed to other cell components, such as peptidoglycan that is sensed by TLR2 and NOD2. Lastly, consequences of TLR9 induction by *L. johnonii* were determined by exploring the expression of Frizzled 5 As more is studied about *L. johnsonii* N6.2, it has been found that its protective functions are very diverse, supporting its probiotic qualities. In addition to the activation of innate immune response, adaptive immune response stimulation was discovered when diabetes-resistant *L. johnsonii*-fed rats were correlated with a T helper 17 (Th17) cell bias [120]. Th17 cells protect the host from extracellular pathogens by recruiting neutrophils and macrophages to the site of infection. This activity could aid in defending the host from aberrant microflora that could ultimately trigger the autoimmune response leading to T1D. While experimenting with other host-effected pathways, our recent focus has been on the ability of *L. johnsonii* to modulate the tryptophan catabolism pathway. This pathway involves the rate-limiting enzyme indoleamine 2,3-dioxygenase (IDO), which is the first enzyme along the pathway that breaks down tryptophan into kynurenine. Kynurenine is a potent aryl hydrocarbon receptor (AhR) ligand; however, the largest source of AhR ligands is found in the diet among which are vegetable and fruit phytophenols [121–123]. Interestingly, IDO induction has also been linked to AhR [124–126]. AhR is a ligand-activated, basic helix-loop-helix transcriptional activator that is associated with many diseases, including autoimmunity [127–129]. Since it was previously found that *L. johnsonii* associates with the ileal mucosa and its colonization correlated with decreased expression of pro-inflammatory cytokine INFγ, the ileal tissue seemed to suggest the best site for observing *L. johnsonii* effects on host cells [110, 130]. More importantly, INFγ has been noted as a primary inducer of IDO in many cell types. Indeed, while surveying different tissues via quantification of the IDO gene expression, the ileum appeared to have a decreased appearance of IDO transcripts in *L. johnsonii*-fed animals compared to control animals [130]. Healthy control rats expressed a 4.7-fold higher level of IDO transcripts, while diabetic animals expressed 11.8-fold increase in mRNA levels compared to *L. johnsonii*-fed animals [130]. This correlated with an observed decrease in blood serum kynurenine levels through HPLC in *L. johnsonii*-fed animals compared to healthy controls and diabetic animals [130]. However, since this study was performed at 120 days, after diabetes onset, it could not reveal early developmental or physiological effects of the bacterial feeding in the host. To address this, a study was completed to evaluate the effects of *L. johnsonii* feedings in a prediabetic host. As a reliable indicator of IDO activity, systemic kynurenine: tryptophan ratios were quantified via HPLC in 30- and 60-day-old prediabetic BBDP rats. At both ages, serum kynurenine levels decreased significantly from the controls while only at 30 days of age did serum tryptophan levels show a significant increase [130]. To verify that IDO activity was responsible for the reduced systemic kynurenine: tryptophan ratios between bacterial-fed groups and controls, the activity of a related enzyme more commonly found in the liver, tryptophan 2,3-dioxygenase (TDO), was examined. TDO, unlike IDO, was previously reported to be unresponsive to inflammatory stimuli and is important in homeostatic control of tryptophan levels under normal conditions. However, recently TDO expression has been correlated to neurological diseases, such as Alzheimer's, and various cancers, such as ovarian carcinoma, breast, and gliomas [121, 131]. After examining activity levels from tissue lysates, there was not a significant difference between the TDO activities in *L. johnsonii*-fed rats and vehicle-fed controls. IDO is widely distributed throughout the human host, and therefore, significant levels can be found throughout the GI tract. Since this enzyme, and its downstream catabolite kynurenine, is activated during inflammatory conditions, it could be involved in the inflammatory response associated with diabetes. Interestingly, we have reported that *L. johnsonii* feedings can reduce the expression of INFγ, a pro-inflammatory cytokine, in the ileum of rats after diabetes onset [110]. Upon performing Western blots of lysates of the colon, cecum, duodenum, jejunum, ileum, liver, and pancreas, it was found that the colon and ileum had variable, but overall decreased, levels of IDO in *L. johnsonii*-fed animals compared to control animals [130]. This correlates well with reduced ileal INFγ expression and overall decreased inflammation in *L. johnsonii*-fed hosts. At this point, it appeared that *L. johnsonii* effected IDO activity and subsequent systemic kynurenine concentrations.

*L. johnsonii* produces an inhibitor of IDO affecting the enzymatic activity and the products synthesized downstream the pathway. Diluted cell-free supernatant (CFS) of *L. johnsonii* was incubated with purified recombinant IDO, and the resulting kynurenine concentrations were quantified. Increasing concentrations of *L. johnsonii* CFS caused an increased inhibition of IDO activity. Furthermore, CFS from *L. johnsonii* N6.2 most potently inhibited IDO when compared to other enteric *Lactobacillus* species CFS effect on IDO activity [130]. This observation stimu‐ lated the characterization of *L. johnsonii* N6.2 supernatant in order to locate the IDO inhibitor. It was found that increased concentrations of hydrogen peroxide (H2O2) correlated with increased *L. johnsonii* culture incubation time before centrifugation and collection of the CFS [130]. Upon increasing concentrations of catalase, which decreases the pool of H2O2, IDO activity increased, supporting the role of H2O2 in CFS as an inhibitor of IDO. Likewise, upon increasing concentrations of H2O2, IDO activity decreased in a dose-dependent manner. This strongly supported H2O2 as an inhibitor of IDO enzyme activity. This enzyme contains heme in the catalytic center to carry out its dioxygenase activity. When the active ferrous centers are oxidized to its inactive ferric form, the dioxygenase activity of IDO is restricted [132]. This causes an accumulation of tryptophan and a decrease in kynurenine levels that can be detected throughout the host. Hydrogen peroxide has the ability to oxidize the reactive heme ferrous centers of IDO, rendering the enzyme inactive. In this current study, the biological relevance of H2O2 was tested by measuring the levels of H2O2 in the GI tract of *L. johnsonii-*fed animals. Since ileal IDO levels were reduced, the hypothesis was that an increase of hydrogen peroxide would be found in these tissues compared to other sites of the body. This could potentially explain the difference in IDO expression of the ileum compared to other sites of the GI tract. Indeed, when measuring H2O2 levels from GI contents, the ileum contained higher levels compared to other sections of the digestive tract [130]. Since *L. johnsonii* most strongly associates with the host mucosa at this site, it further supports the hypothesis of the ability of *L. johnsonii* to produce an inhibitor of IDO [110]. Upon RNA-seq analysis of *L. johnsonii* grown under different aeration conditions, a gene (T285\_08005) regulating H2O2 production was identified. The encoding protein contained a Per-Arnst-Sim (PAS) domain and regulated the H2O2 production from heterodimeric FMN reductases, FRedA and FRedB (WP\_004898036.1 and WP\_011162530.1, respectively) [62].

to be unresponsive to inflammatory stimuli and is important in homeostatic control of tryptophan levels under normal conditions. However, recently TDO expression has been correlated to neurological diseases, such as Alzheimer's, and various cancers, such as ovarian carcinoma, breast, and gliomas [121, 131]. After examining activity levels from tissue lysates, there was not a significant difference between the TDO activities in *L. johnsonii*-fed rats and vehicle-fed controls. IDO is widely distributed throughout the human host, and therefore, significant levels can be found throughout the GI tract. Since this enzyme, and its downstream catabolite kynurenine, is activated during inflammatory conditions, it could be involved in the inflammatory response associated with diabetes. Interestingly, we have reported that *L. johnsonii* feedings can reduce the expression of INFγ, a pro-inflammatory cytokine, in the ileum of rats after diabetes onset [110]. Upon performing Western blots of lysates of the colon, cecum, duodenum, jejunum, ileum, liver, and pancreas, it was found that the colon and ileum had variable, but overall decreased, levels of IDO in *L. johnsonii*-fed animals compared to control animals [130]. This correlates well with reduced ileal INFγ expression and overall decreased inflammation in *L. johnsonii*-fed hosts. At this point, it appeared that *L. johnsonii* effected IDO

*L. johnsonii* produces an inhibitor of IDO affecting the enzymatic activity and the products synthesized downstream the pathway. Diluted cell-free supernatant (CFS) of *L. johnsonii* was incubated with purified recombinant IDO, and the resulting kynurenine concentrations were quantified. Increasing concentrations of *L. johnsonii* CFS caused an increased inhibition of IDO activity. Furthermore, CFS from *L. johnsonii* N6.2 most potently inhibited IDO when compared to other enteric *Lactobacillus* species CFS effect on IDO activity [130]. This observation stimu‐ lated the characterization of *L. johnsonii* N6.2 supernatant in order to locate the IDO inhibitor. It was found that increased concentrations of hydrogen peroxide (H2O2) correlated with increased *L. johnsonii* culture incubation time before centrifugation and collection of the CFS [130]. Upon increasing concentrations of catalase, which decreases the pool of H2O2, IDO activity increased, supporting the role of H2O2 in CFS as an inhibitor of IDO. Likewise, upon increasing concentrations of H2O2, IDO activity decreased in a dose-dependent manner. This strongly supported H2O2 as an inhibitor of IDO enzyme activity. This enzyme contains heme in the catalytic center to carry out its dioxygenase activity. When the active ferrous centers are oxidized to its inactive ferric form, the dioxygenase activity of IDO is restricted [132]. This causes an accumulation of tryptophan and a decrease in kynurenine levels that can be detected throughout the host. Hydrogen peroxide has the ability to oxidize the reactive heme ferrous centers of IDO, rendering the enzyme inactive. In this current study, the biological relevance of H2O2 was tested by measuring the levels of H2O2 in the GI tract of *L. johnsonii-*fed animals. Since ileal IDO levels were reduced, the hypothesis was that an increase of hydrogen peroxide would be found in these tissues compared to other sites of the body. This could potentially explain the difference in IDO expression of the ileum compared to other sites of the GI tract. Indeed, when measuring H2O2 levels from GI contents, the ileum contained higher levels compared to other sections of the digestive tract [130]. Since *L. johnsonii* most strongly associates with the host mucosa at this site, it further supports the hypothesis of the ability of *L. johnsonii* to produce an inhibitor of IDO [110]. Upon RNA-seq analysis of *L. johnsonii* grown under different aeration conditions, a gene (T285\_08005) regulating H2O2 production was

activity and subsequent systemic kynurenine concentrations.

98 Probiotics and Prebiotics in Human Nutrition and Health

After experiencing reduced kynurenine production and IDO inhibition in response to *L. johnsonii*, it was hypothesized that other tryptophan metabolite concentrations could be effected. Tryptophan is a precursor to the neurotransmitter 5-hydroxytryptamine (5-HT, serotonin), which is predominantly produced in enterochromaffin cells along the GI epithelium. IDO also has the enzymatic activity to catalyze 5-HT to 5-hydroxykynuramine, aiding in increased 5-HT turnover [133, 134]. Using ELISA, 5-HT levels in ileum tissue lysates and blood serum collected from 60-day-old BBDP rats were quantified. Serotonin levels were significantly elevated, both locally and peripherally, in *L. johnsonii*-fed animals [130].

**Figure 4** most accurately summarizes the work our group has done in characterizing *Lactobacillus johnsonii* N6.2, starting with its discovery in 2008 and most recently revealing its regulating effects on IDO. This bacterial genera was correlated with a reduced diabetes onset when comparing BioBreeding diabetes-prone and diabetes-resistant rats [104]. It was identified that *Lactobacillus* strains that contain CE activity were more correlated with diabetes resistance, and through subsequent feeding assays of these potential bacterial targets, *L. johnsonii* N6.2 was identified as being negatively associated with T1D [6, 110]. Since this discovery, *L. johnsonii* N6.2 has been characterized in regard to its diabetes resistance. *L. johnsonii* strengthens gut permeability through a higher abundance of the tight junction claudin-1 levels and goblet cells, and it reduces GI stress by reducing the expression of oxidative stress genes and inflammatory INF-γ levels [110]. Among the most recent and most interesting findings of the qualities of *L. johnsonii* is its ability to modulate IDO activity through its production of H2O2 [130]. However, this bacterium's esterase activity has the ability to quench its own H2O2 production through the release of phytophenols. These antioxidants have the potential to eliminate part of the pool of produced H2O2, along with other even more dangerous ROS that precede chronic diseases. In the case of *L. johnsonii* N6.2, sufficient H2O2 production is observed in the ileum, where *L. johnsonii* is localized [110, 130]. Conversely, reduced levels of oxidative stress genes are observed in the ileum of *L. johnsonii-*fed rats [6, 110]. Thus, one of the main probiotic properties of *L. johnsonii* could be its ability to maintain redox homeostasis in the GI tract. This balance is dependent on the dynamic interplay between the generation of H2O2 and the ROS quenching ability of antioxidant phytophenols released by this bacterium. The H2O2 released by this bacterium in the intestinal lumen would stimulate oxidative stress defense mechanisms in host cells, while controlling the activity of IDO [130, 132]. Meanwhile, enzymes unique to *L. johnsonii* will increase the pool of free, bioavailable antioxidant phytophenols in the intestinal lumen. The phenolic released will differentially quench the most reactive ROS.

Although *L. johnsonii* N6.2 was found and characterized in regards to its correlation with reduced T1D onset, it has the potential to expand its beneficial functions into the realm of other chronic diseases. IDO is an immunoregulatory enzyme whose altered activity has been

**Figure 4.** Gastrointestinal (GI) epithelium with proposed mechanisms of phytophenol and H2O2 action. This figure summarizes the results published by the group between 2009 and 2014.

observed in a multitude of diseases, including autoimmunity and cancer [135–137]. *L. johnso‐ nii* N6.2 has the ability to regulate IDO activity by inactivating its redox-sensitive heme centers through H2O2 production [130]. The effect of this inactivation has the potential to expand over into other chronic disease and reduce their occurrence. This makes regulators of IDO an important immunotherapy target in preventing some of today's most serious diseases. Resident microbiota provide many protective functions for the host, such as outcompeting pathogenic threats, releasing necessary resources from digested foods, and maintaining GI homeostasis. It is to no surprise that disturbances to microfloral composition could dictate disease onset. There are numerous probiotics in the market, encompassing many different genera. These probiotics exert beneficial properties through their unique enzymes, their released metabolites, or a combination of both as in the case of *L. johnsonii* N6.2. Therefore, probiotic administration serves as an important defense in preventing some of the most common and chronic diseases.

#### **Acknowledgements**

This material is based upon work that is supported by the National Institute of Food and Agriculture, US Department of Agriculture, under award number 2015-67017-23182, and Juvenile Diabetes Research Foundation under award number 1-INO-2014-176-A-V.

### **Author details**

Danielle N. Kling1 , Guillermo E. Marcial1,3, Dana N. Roberson1 , Graciela L. Lorca1,2 and Claudio F. Gonzalez1,2\*


### **References**

observed in a multitude of diseases, including autoimmunity and cancer [135–137]. *L. johnso‐ nii* N6.2 has the ability to regulate IDO activity by inactivating its redox-sensitive heme centers through H2O2 production [130]. The effect of this inactivation has the potential to expand over into other chronic disease and reduce their occurrence. This makes regulators of IDO an important immunotherapy target in preventing some of today's most serious diseases. Resident microbiota provide many protective functions for the host, such as outcompeting pathogenic threats, releasing necessary resources from digested foods, and maintaining GI homeostasis. It is to no surprise that disturbances to microfloral composition could dictate disease onset. There are numerous probiotics in the market, encompassing many different genera. These probiotics exert beneficial properties through their unique enzymes, their released metabolites, or a combination of both as in the case of *L. johnsonii* N6.2. Therefore, probiotic administration serves as an important defense in preventing some of the most

**Figure 4.** Gastrointestinal (GI) epithelium with proposed mechanisms of phytophenol and H2O2 action. This figure

summarizes the results published by the group between 2009 and 2014.

100 Probiotics and Prebiotics in Human Nutrition and Health

This material is based upon work that is supported by the National Institute of Food and Agriculture, US Department of Agriculture, under award number 2015-67017-23182, and

Juvenile Diabetes Research Foundation under award number 1-INO-2014-176-A-V.

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### **Chapter 6**

## **Pili in Probiotic Bacteria**

Vengadesan Krishnan, Priyanka Chaurasia and Abhiruchi Kant

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63087

#### **Abstract**

The ability to adhere to intestinal epithelial tissue and mucosal surfaces is a key criterion in selecting probiotics. Adhesion is considered to be a prerequisite for successful colonization and survival in the gastrointestinal tract to provide persistent beneficial effects to the host. Bacteria express a multitude of surface components that mediate adherence. Pili or fimbriae are surface adhesive components implicated in initiating bacterial adhesion and mediating interaction with the host. These nonflagellar proteina‐ ceous fiber appendages were identified and explored over several decades in pathogen‐ ic bacteria, and many distinct types are known. However, the presence of pili in probiotics and/or commensalic bacteria has only recently been recognized. Thus knowledge about pili in probiotics is relatively limited, but structural and functional data have begun to emerge. Availability of these data in the future would enable us to understand the pilimediated adhesion strategies of probiotics. This knowledge could be utilized to develop antiadhesion-based therapies against bacterial infections as well as probiotic designs for beneficial effects. This chapter will briefly summarize the current knowledge of pili in probiotics with emphasis on members of lactobacilli and bifidobacteria.

**Keywords:** Adhesion, Bifidobacteria, Lactobacilli, Pili, Probiotics

#### **1. Introduction**

Bacterial colonization of humans seems to commence at birth and evolves throughout life. It depends on several factors including mode of birth, age, geographical location, local environ‐ ment, diet, stress, illness, medications, and antibiotic treatment. Bacteria colonize all parts of the human body that are exposed to external environment. Specifically, the gastrointestinal tract (GIT) harbors more than 1000 species, and this complex microbial community is referred to as

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

the "gut microbiota" [1, 2]. The gut microbiota are well recognized because of their impact on health and disease. However, knowledge on the precise mechanism(s) by which the microbio‐ ta exerts its influence remains largely unknown. *Lactobacillus* and *Bifidobacterium* species constitute a major part of the microbiota and are believed to play an essential role in modulat‐ ing immune system, resisting pathogen colonization, metabolism, and energy balance [3, 4]. Some members of these two genera are also popular as probiotics. Though the specific contri‐ bution of these members to the beneficial effects is subject to investigation and speculation, it is widely accepted that their presence in the GIT often confers health benefits. The molecular mechanisms that allowthesemembers tocolonize theGIThavenotyetbeenelucidatedindetail, though their persistence was shown to be essential for the beneficial effects.

Most pathogenic bacteria are known to express multitude of surface components for estab‐ lishing contacts and mediating interactions with the host for bacterial colonization. Among these, long, hair‐like filamentous structures known as pili or fimbriae have been often implicated in adhesion processes and shown to be required for bacterial colonization on host tissues (for reviews, see [5–11]). Typically, these structures are made up of building blocks called pilins or fimbrilins. Genes for these pilins along with other genes required for the pilus assembly are located in the same place in the genome called pilus gene cluster or Pathogenicity Island. Distinct pilus structures (e.g., chaperone‐mediated, type IV, Curli, and CS1) are known in Gram‐negative pathogens. Their structure, function, and biogenesis have been well explored to some extent. The details of pili have begun to emerge for Gram‐positive pathogens a decade ago (for reviews, see [8, 10–15]). The sortase‐mediated pili seem to be conserved across the Gram‐positive pathogens. Some of the pilus types (e.g., type IV) exist both in the Gram‐ negative and Gram‐positive pathogens. The pilus types have been majorly categorized based on secretion systems, biogenesis, architecture, and function. The sortase‐mediated pili differ from other known types by being a covalent polymer in which pilin subunits are covalently tethered to each other by sortase‐mediated isopeptide bonds. The pili and their components in the pathogens are recognized as virulence factors as they play a key role in pathogenesis. Also, they are considered as potential vaccine candidates because of their immunogenic properties.

Although the focus is traditionally on pili in pathogenic bacteria for last few decades, they have been recently identified in many gut commensalic bacteria and often shown to be essential for their colonization and persistence in the the GIT and for immune modulation. Although the pili in pathogenic bacteria are regularly reviewed, this chapter attempts to give a brief overview of pili in beneficial bacteria, which is relatively recent.

### **2. Sortase‐mediated pili**

As demonstrated first in pathogen *Corynebacterium diptheriae* [15, 16], the sortase‐mediated pilus (SpaA‐type) model consists of three different types of pilins (one major pilin and two ancillary pilins). Typically, the loci for the pilins and at least one sortase are located together in the genome as a pilus operon or gene cluster (**Figure 1A**). Similar to microbial surface component recognizing adhesive matrix molecules (MSCRAMMs), the pilin precursors contain signal sequence at the N‐terminal and sorting signal at the C‐terminal. The C‐terminal sorting signal is composed of a conserved LPXTG (Leu-Pro-any-Thr-Gly) motif, a hydrophobic domain, and a positively charged tail (**Figure 1B**). Multiple copies of major pilin form the pilus backbone like beads on a string (**Figure 1C**). Hence, they are also referred to as backbone or shaft pilins. The major pilins often contain a conserved YPKN (Tyr-Pro-Lys-Asn)‐like motif close to the N‐terminal (**Figure 1B**). The pilin‐specific sortase, whose gene is located in the pilus gene cluster, generates the covalently cross‐linked pilus shaft as follows. Prior to polymeriza‐ tion into pilus fibers, the prepilins or pilin precursors are exported across the membrane through the Sec apparatus. These precursors are then embedded into the membrane by their C‐terminal hydrophobic domain and positively charged tail. The membrane‐bound pilin‐ specific sortase forms acyl‐enzyme intermediate by cleaving the LPXTG motif of major pilin between threonine and glycine, and creates a thioester bond between its catalytic cysteine residue and the nascent C‐terminal threonine. This intermediate receives nucleophilic attack from the lysine residue of pilin motif of another major pilin that results in an amide bond formation between the cleaved threonine and lysine side chain. The repeated reaction pro‐ motes the growth of pilus structure on the cell surface (**Figure 1**). The ancillary pilins are incorporated into the pilus structure, presumably by similar transpeptidation reaction. Ancillary pilin 1, which is larger in size, is generally located at the pilus tip. This pilin, also known as tip pilin, often plays a role in adhesion to host. Ancillary pilin 2 or basal pilin is often observed at the base of pilus and smaller in size. These basal pilins are shown to contain a pilin‐like motif for their incorporation into the pilus base [21]. A different transpeptidase known as housekeeping sortase, which is not part of the pilus gene cluster, anchors the assembled pilus structure on the cell wall. Similar to pilin‐specific sortase transpeptidase reaction, the housekeeping sortase forms acyl‐enzyme intermediate with basal pilin. This intermediate receives nucleophilic attack from the peptidoglycan cross‐bridge that results in the formation of covalent link between the carboxyl threonine in the basal pilin and the free amino group of the cell wall lipid II precursors.

the "gut microbiota" [1, 2]. The gut microbiota are well recognized because of their impact on health and disease. However, knowledge on the precise mechanism(s) by which the microbio‐ ta exerts its influence remains largely unknown. *Lactobacillus* and *Bifidobacterium* species constitute a major part of the microbiota and are believed to play an essential role in modulat‐ ing immune system, resisting pathogen colonization, metabolism, and energy balance [3, 4]. Some members of these two genera are also popular as probiotics. Though the specific contri‐ bution of these members to the beneficial effects is subject to investigation and speculation, it is widely accepted that their presence in the GIT often confers health benefits. The molecular mechanisms that allowthesemembers tocolonize theGIThavenotyetbeenelucidatedindetail,

Most pathogenic bacteria are known to express multitude of surface components for estab‐ lishing contacts and mediating interactions with the host for bacterial colonization. Among these, long, hair‐like filamentous structures known as pili or fimbriae have been often implicated in adhesion processes and shown to be required for bacterial colonization on host tissues (for reviews, see [5–11]). Typically, these structures are made up of building blocks called pilins or fimbrilins. Genes for these pilins along with other genes required for the pilus assembly are located in the same place in the genome called pilus gene cluster or Pathogenicity Island. Distinct pilus structures (e.g., chaperone‐mediated, type IV, Curli, and CS1) are known in Gram‐negative pathogens. Their structure, function, and biogenesis have been well explored to some extent. The details of pili have begun to emerge for Gram‐positive pathogens a decade ago (for reviews, see [8, 10–15]). The sortase‐mediated pili seem to be conserved across the Gram‐positive pathogens. Some of the pilus types (e.g., type IV) exist both in the Gram‐ negative and Gram‐positive pathogens. The pilus types have been majorly categorized based on secretion systems, biogenesis, architecture, and function. The sortase‐mediated pili differ from other known types by being a covalent polymer in which pilin subunits are covalently tethered to each other by sortase‐mediated isopeptide bonds. The pili and their components in the pathogens are recognized as virulence factors as they play a key role in pathogenesis. Also, they are considered as potential vaccine candidates because of their immunogenic

Although the focus is traditionally on pili in pathogenic bacteria for last few decades, they have been recently identified in many gut commensalic bacteria and often shown to be essential for their colonization and persistence in the the GIT and for immune modulation. Although the pili in pathogenic bacteria are regularly reviewed, this chapter attempts to give a brief

As demonstrated first in pathogen *Corynebacterium diptheriae* [15, 16], the sortase‐mediated pilus (SpaA‐type) model consists of three different types of pilins (one major pilin and two ancillary pilins). Typically, the loci for the pilins and at least one sortase are located together in the genome as a pilus operon or gene cluster (**Figure 1A**). Similar to microbial surface

overview of pili in beneficial bacteria, which is relatively recent.

though their persistence was shown to be essential for the beneficial effects.

116 Probiotics and Prebiotics in Human Nutrition and Health

properties.

**2. Sortase‐mediated pili**

The pilins are commonly made up of two building blocks, which are variants of immunoglo‐ bulin fold known as CnaA [17] and CnaB [18], often with intradomain isopeptide bond [19] (for reviews, see [20–22]) (**Figure 2**). In addition, the tip pilins also contain adhesin modules such as von Willebrand factor type A domain (vWFA) with two inserted arms [23, 24] and thioester containing domains [25–27] (**Figure 2**). The pilus model of *C. diptheriae* appears to be conserved across the Gram‐positive pathogenic strains (e.g., *Streptococcus agalactiae*, *Strepto‐ coccus pyogenes*, *Streptococcus pneumoniae*, *Streptococcus parasanguinis*, *Streptococcus salivarius*, *Streptococcus sanguinis*, *Enterococcus faecalis*, *Enterococcus faecium*, *Bacillus cererus*, and *Actino‐ myces naeslundi*) with some variations in number of pilus gene clusters, number of pilins, number of pilin‐specific sortases, and pilus architecture. They majorly participate in cellular adhesion and colonization processes. More than one sortase‐mediated pilus gene cluster are often present in the same bacterial strains suggesting their different cellular targets and functions.

**Figure 1. Schematic diagram of typical sortase‐mediated pili**. (A) Pilus gene cluster for sortase‐mediated pilus assem‐ bly. It encodes genes for a major (red), basal (blue), tip (green) pilins, and a pilin‐specific sortase (purple). More than one sortase (e.g., SpaD‐ and SpaH‐pilus gene cluster in *C. diphtheriae*) and less than three pilins (e.g., type 1 and 2 pilus gene clusters in *A. oris*) have also been observed. In the pilus gene cluster, differences in the order of gene's arrange‐ ment and the presence of transposon elements in the vicinity are often observed. (B) Conserved features of sortase pi‐ lins. Signal sequence (SS) and LPXTG‐containing cell wall sorting signal (CWSS) are at the N‐ and C‐terminals of all the (basal, major, and tip) pilins. In addition, the basal and major pilins have pilin motif (YPKN) in the vicinity of N‐ terminals. A conserved element called E‐box (LXET) has also been observed in the sortase pilins. The basal pilins con‐ sist of 1–3 CnaB domains (Figure 2A). The major pilins contain 2–4 CnaA/B domains. CnaB domains are often at the N‐ and C‐terminals, and CnaA at the middle (Figure 2B). The tip plins have adhesive domains (vWFA/thioester containing domains) in addition to CnaA/B domains (Figure 2C). (C) Sortase‐mediated pilus structure. The pilus is made up of three distinct types of pilins: basal (blue), major (red), and tip (green) pilin. In the pilus, the pilins are teth‐ ered to each other by sortase‐mediated covalent links (see the text for details). Multiple copies of the major pilins form the pilus shaft in a head‐to‐tail fashion like beads on a string. The tip pilin is often located at tip projecting adhesive domain for favoring adhesion. The basal pilin is often located at the base of pilus shaft and helping for anchoring the polymerized pilus on the cell wall through the housekeeping sortase.

The sortase‐mediated pili, which are being actively investigated in Gram‐positive pathogens and considered as virulence factors, have been detected in several gut commensals as men‐ tioned in the following sections. The pilus‐like gene clusters were earlier noticed in probiotic *Lactobacillus johnsonii* NCC 533 [28], but first received attention through probiotic *Lactobacillus rhamnosus* GG in 2009 [29, 30]. Since then, it has been identified in several species and strains of probiotic and other commensal bacteria by genomic analysis and shown to be essential for their adherence and colonization in GIT. Their presence was further confirmed by imaging analysis in the *L. rhamnosus* GG [29, 31], genus of *Bifidobacterium* [32, 33], *Lacococcus lactis*IL1403 and TIL448 [34, 35], and recently in *Lactobacillus ruminis* ATCC 25644 [36]. Hence, the view of surface piliation has now been expanded to include its role also as a niche‐adaptation factor.

**Figure 2. Three‐dimensional structures of sortase‐pilins from pathogenic bacteria**. (A) Basal pilin, GBS52 (PDB id: 3PHS), from *S. agalactiae*. It consists of two CnaB domains, and the lysine from the pilin motif is shown as stick (in red). A proline‐rich C‐terminal tail is shown in magenta. (B) Major pilin, SpaA (PDB id: 3HR6), from *C. diphtheriae* consists of three domains. CnaB domains (in blue) are at N‐ and C‐terminals, and CnaA (in red) at the middle. Pilin motif ly‐ sine is shown in stick (red). (C) Tip pilin, RrgA (PDB id: 2WW8), from *S. pneumoniae* contains four domains. CnaB do‐ mains (in blue) are at the terminals and CnaA (in red) at the middle. Metal (pink)‐ion‐dependent adhesion site (MIDAS) containing vWFA domain with two inserted arms are shown in green.

#### **2.1. Pili in** *L. rhamnosus* **GG**

**Figure 1. Schematic diagram of typical sortase‐mediated pili**. (A) Pilus gene cluster for sortase‐mediated pilus assem‐ bly. It encodes genes for a major (red), basal (blue), tip (green) pilins, and a pilin‐specific sortase (purple). More than one sortase (e.g., SpaD‐ and SpaH‐pilus gene cluster in *C. diphtheriae*) and less than three pilins (e.g., type 1 and 2 pilus gene clusters in *A. oris*) have also been observed. In the pilus gene cluster, differences in the order of gene's arrange‐ ment and the presence of transposon elements in the vicinity are often observed. (B) Conserved features of sortase pi‐ lins. Signal sequence (SS) and LPXTG‐containing cell wall sorting signal (CWSS) are at the N‐ and C‐terminals of all the (basal, major, and tip) pilins. In addition, the basal and major pilins have pilin motif (YPKN) in the vicinity of N‐ terminals. A conserved element called E‐box (LXET) has also been observed in the sortase pilins. The basal pilins con‐ sist of 1–3 CnaB domains (Figure 2A). The major pilins contain 2–4 CnaA/B domains. CnaB domains are often at the N‐ and C‐terminals, and CnaA at the middle (Figure 2B). The tip plins have adhesive domains (vWFA/thioester containing domains) in addition to CnaA/B domains (Figure 2C). (C) Sortase‐mediated pilus structure. The pilus is made up of three distinct types of pilins: basal (blue), major (red), and tip (green) pilin. In the pilus, the pilins are teth‐ ered to each other by sortase‐mediated covalent links (see the text for details). Multiple copies of the major pilins form the pilus shaft in a head‐to‐tail fashion like beads on a string. The tip pilin is often located at tip projecting adhesive domain for favoring adhesion. The basal pilin is often located at the base of pilus shaft and helping for anchoring the

The sortase‐mediated pili, which are being actively investigated in Gram‐positive pathogens and considered as virulence factors, have been detected in several gut commensals as men‐ tioned in the following sections. The pilus‐like gene clusters were earlier noticed in probiotic *Lactobacillus johnsonii* NCC 533 [28], but first received attention through probiotic *Lactobacillus rhamnosus* GG in 2009 [29, 30]. Since then, it has been identified in several species and strains of probiotic and other commensal bacteria by genomic analysis and shown to be essential for their adherence and colonization in GIT. Their presence was further confirmed by imaging analysis in the *L. rhamnosus* GG [29, 31], genus of *Bifidobacterium* [32, 33], *Lacococcus lactis*IL1403 and TIL448 [34, 35], and recently in *Lactobacillus ruminis* ATCC 25644 [36]. Hence, the view of surface piliation has now been expanded to include its role also as a niche‐adaptation factor.

polymerized pilus on the cell wall through the housekeeping sortase.

118 Probiotics and Prebiotics in Human Nutrition and Health

*L. rhamnosus* GG is one the of well-documented and widely used probiotic strains [37]. The pilus‐like protrusions in *L. rhamnosus* GG were initially seen in 2009 [30]. *L. rhamnosus* GG contains two pilus gene clusters *SpaCBA* and *SpaFED* as shown by comparative genomic analysis [29] (**Figure 3A**). The *SpaCBA* encodes a major pilin SpaA, two ancillary pilins SpaB and SpaC, and a pilin‐specific sortase (SrtC1). As further confirmed by western blotting and immunogold electron microscopy [29, 31], the SpaCBA pilus of *L. rhamnosus* GG has similar morphology to the three‐pilins architecture model of *C. diptheriae* [15, 16]. The repeating SpaA makes the pilus backbone. The cell wall anchoring SpaB and adhesive SpaC ancillary pilins are found at the base and tip of the pilus, respectively (**Figure 3C**). However, in contrast to the pili from most Gram‐positive pathogens, the tip pilin (SpaC) and, to a lesser extent, basal pilin (SpaB) are found sporadically throughout the SpaCBA pilus backbone. Such a distribution is thought to enhance adherence to the intestinal mucosa and epithelial layer and thereby then extend the relative longevity and transient colonization of *L. rhamnosus* GG cells in the gut. The SpaCBA pilus was demonstrated to be pivotal for efficient adherence to mucus [29, 38, 39], collagen [40], and Caco‐2 intestinal epithelial cell line and biofilm formation [41]. The immunomodulation of SpaCBA pili includes toll‐like receptor 2 (TLR2)‐dependent activation and dendritic cell cytokine production [42], dampening endogenous interleukin (IL)‐8 mRNA levels [41], eliciting macrophage‐mediated anti‐inflammatory cytokine mRNA expression [43], inducing TLR‐related gene expression in a human fetal intestine model [44], and stimulating cellular responses in intestinal epithelial cells [45]. Interestingly, the SpaC plays a role in most of the SpaCBA pili‐triggered host cell immune responses. The surface piliation apparently provides a niche‐specific fitness to *L. rhamnosus* GG cells for extending their transient coloni‐ zation in the gut [46]. Presumably, this is an advantage over nonpiliated probiotic bacteria. For example, the non‐SpaCBA piliated *L. rhamnosus* LC705, which is genetically similar to *L. rhamnosus* GG, shows decreased adherence to intestinal mucus in the comparative study [29]. More recently, the key role of *L. rhamnosus* GG pili in interaction with β‐lactoglobulin has also been demonstrated [47].

**Figure 3. Schematic diagram of sortase‐mediated pili in** *L. rhamnosus* **GG**. (A) *SpaCBA* and *SpaFED* pilus gene clus‐ ters identified in *L. rhamnosus* GG. Each cluster encodes a tip pilin (SpaC/SpaF), major pilin (SpaA/SpaD), basal pilin (SpaB/SpaE), and pilin‐specific sortase (SrtC1/SrtC2). (B) Predicted elements required for the pilus assembly in the SpaCBA pilins. The basal pilin SpaB contains a single CnaB domain with FPKN pilin motif and LPQTG‐containing CWSS at C‐terminal. Residue numbers and positions were labeled and marked by arrow. The major pilin SpaA contain two CnaB domains, and its pilin and sorting motif are marked. The tip pilin SpaC contains a vWFA domain and its MIDAS (DMSGS) motif is marked. (C) The SpaCBA pilus model consists of SpaA, SpaB, and SpaC. The possible sor‐ tase‐mediated intercovalent link is marked by arrow with details of residues involved. A possible mode of association for SpaC and SpaB along the pilus shaft other than at the tip and base of the pilus needs to be further shown by a high‐ resolution imaging technique or structural studies.

Similar to *SpaCBA*, the *SpaFED* operon encodes the pilus backbone (SpaD), the pilus tip (SpaF) and the base (SpaE) pilins, as well as a putative sortase enzyme (SrtC2) required for pilus assembly (**Figure 3A**). Though the recombinant SpaF has been shown to bind intestinal mucus [39], the genes associated with the spaFED pilus gene cluster are not constitutively expressed in the tested laboratory conditions [31]. Thus, the native form of the SpaFED pilus remains hypothetical, not only in *L. rhamnosus* GG, but also in other strains carrying the spaFED operon (e.g., *L. rhamnosus* LC705) [31, 46]. However, *L. rhamnosus* GG SpaFED pili can be readily produced as an assembled structure in recombinant *L. lactis* [48].

and dendritic cell cytokine production [42], dampening endogenous interleukin (IL)‐8 mRNA levels [41], eliciting macrophage‐mediated anti‐inflammatory cytokine mRNA expression [43], inducing TLR‐related gene expression in a human fetal intestine model [44], and stimulating cellular responses in intestinal epithelial cells [45]. Interestingly, the SpaC plays a role in most of the SpaCBA pili‐triggered host cell immune responses. The surface piliation apparently provides a niche‐specific fitness to *L. rhamnosus* GG cells for extending their transient coloni‐ zation in the gut [46]. Presumably, this is an advantage over nonpiliated probiotic bacteria. For example, the non‐SpaCBA piliated *L. rhamnosus* LC705, which is genetically similar to *L. rhamnosus* GG, shows decreased adherence to intestinal mucus in the comparative study [29]. More recently, the key role of *L. rhamnosus* GG pili in interaction with β‐lactoglobulin has also

**Figure 3. Schematic diagram of sortase‐mediated pili in** *L. rhamnosus* **GG**. (A) *SpaCBA* and *SpaFED* pilus gene clus‐ ters identified in *L. rhamnosus* GG. Each cluster encodes a tip pilin (SpaC/SpaF), major pilin (SpaA/SpaD), basal pilin (SpaB/SpaE), and pilin‐specific sortase (SrtC1/SrtC2). (B) Predicted elements required for the pilus assembly in the SpaCBA pilins. The basal pilin SpaB contains a single CnaB domain with FPKN pilin motif and LPQTG‐containing CWSS at C‐terminal. Residue numbers and positions were labeled and marked by arrow. The major pilin SpaA contain two CnaB domains, and its pilin and sorting motif are marked. The tip pilin SpaC contains a vWFA domain and its MIDAS (DMSGS) motif is marked. (C) The SpaCBA pilus model consists of SpaA, SpaB, and SpaC. The possible sor‐ tase‐mediated intercovalent link is marked by arrow with details of residues involved. A possible mode of association for SpaC and SpaB along the pilus shaft other than at the tip and base of the pilus needs to be further shown by a high‐

Similar to *SpaCBA*, the *SpaFED* operon encodes the pilus backbone (SpaD), the pilus tip (SpaF) and the base (SpaE) pilins, as well as a putative sortase enzyme (SrtC2) required for pilus assembly (**Figure 3A**). Though the recombinant SpaF has been shown to bind intestinal mucus [39], the genes associated with the spaFED pilus gene cluster are not constitutively expressed

been demonstrated [47].

120 Probiotics and Prebiotics in Human Nutrition and Health

resolution imaging technique or structural studies.

Obtaining three‐dimensional structural insights into pilus assembly and adhesion mecha‐ nisms through the structural biology techniques has been instrumental for Gram‐negative pathogens in the past (for reviews, see [5, 8, 49, 50]), and it was begun much later for Gram‐ positive pathogens in 2007 ([19, 51], for reviews, see [11, 20–22]). The structures of individ‐ ual major as well as ancillary pilins from several pathogenic strains have been determined (for recent review, see [21]) (**Figure 2**). A Cryo‐EM study on *S. pneumoniae* pili has also supported the sortase‐mediated three pilins architectural model [52]. According to current structural knowledge, the basal pilins consist of 1–3 CnaB domains often with intradomain isopeptide bonds (**Figure 2A**). Conserved proline‐rich C‐terminal tails in the known basal pilins suggest their likely role in pilus anchoring via housekeeping sortase. The presence of a pilin‐like motif with a lysine in the basal pilin indicates that they could be incorporated into the pilus base by sortase (**Figure 2**). The major pilins are made of 2–4 CnaB/A do‐ mains (**Figure 2**). The CnaB domains are at the N‐ and C‐terminals, whereas the CnaA domain is in the middle. The pilin motif is present at the C‐terminal region of N‐terminal CnaB domain (**Figure 2B**). The N‐terminal domain in many pilins seems to be flexible with no or slow forming internal isopeptide bond. In some crystal structure studies, a fiber‐like pilus arrangement in the crystal packing has been observed though the sortase‐mediated intermolecular amide bond between the backbone pilins was absent. The tip pilins contain adhesive domains at the tip in addition to CnaA and CnaB domains that form a stalk and connect adhesive domains to the pilus shaft (**Figure 2C**). These adhesive domains are often a modified vWFA domain with two inserted arms [23, 24], and thioester containing domain [25]. The complicated domains arrangement and folding in tip pilins makes difficult to predict them from their sequence.

Detailed structural knowledge is yet to emerge for pili and related components for probiot‐ ic bacteria. However, preliminary crystallographic data are available for some of the pilins (SpaA [53], SpaD [54], and SpaC [55]) in *L. rhamnosus* GG. Our initial analysis of ongoing structural investigations on pilus constituents of *L. rhamnosus* GG and comparison with their counterparts in pathogens suggest that SpaA may consist of two CnaB domains (**Fig‐ ure 3B**), and SpaD contains three domain with CnaB domains at the terminals and CnaA domain in the middle. Though it is yet to be validated, it is tempting us to describe Lys171 from the pilin motif SpaA as the possible linking lysine that could involve in the SpaA‒ SpaA and SpaA‒SpaC pilins covalent association during SpaCBA pilus shaft polymeriza‐ tion by pilin‐specific SrtC1 (**Figure 3B** and **C**). Similarly, Lys182 in SpaB seems a likely candidate for its incorporation into the pilus (**Figure 3B** and **C**). Such a linking lysine is yet to be predicted for SpaC for its incorporation other than at the pilus tip. In contrast to known pathogenic tip pilins (e.g., GBS104 [24] and RrgA [23]), but similar to eukaryotic proteins (e.g., integrins, complement C2a, and Fb), the vWFA domain predicted in SpaC [55] seems not to have the two inserted arms, suggesting both possible differences and similarities in

binding mechanism via a metal‐ion‐containing vWFA adhesin domain. Certainly, knowl‐ edge generated from our ongoing structural investigations would provide new insights into pilus assembly and adhesion mechanisms in *L. rhamnosus* GG, and serves as a model for probiotics.

#### **2.2. Pili in** *L. ruminis*

*L. ruminis*, one of the dominating *Lactobacillus* species in the mammalian intestines, is routinely isolated from the feces of human, cattle, and pigs. It is one of the few motile members known in lactobacilli. It is also recognized as an autochthonous microbiota in the GIT. The pilus gene identified in the human‐derived intestinal isolate *L. rumini* ATCC 25644 has been named as *lrpCBA* (*L*. *r*umini *p*ilus) [36] since they appear to be different from the known lactobacillar pilus types (SpaCBA and SpaFED) at the primary structural level. The *LrpCBA* pilus operon encodes tip (lrpC), basal (lrpB), and major (lrpA) pilins and a pilin‐specific sortase (SrtC). Sequence of *L. ruminis* pilins displays the common pilin features such as LPXTG‐like motifs, E‐box motif, and pilin motifs (in major and basal pilins) [36] (**Figure 2**). The expression and surface localization of *lrpCBA* pilus gene product have further confirmed by immunoblot analysis and immune‐electron microscopic visualization (for details, see [36]). Interestingly, the pilus genes have also been detected in *L. ruminis* ATCC 27782 from bovine gut origin [56], but the microarray analysis showed that the corresponding genes were upregulated in human strain compared with the bovine isolate. The ability of LrpCBA pilus to adhere to gut epithelial cells and extracellular matrix (ECM) proteins, and immune‐modulation activities has been demonstrated using recombinant‐piliated lactococci (for details, see [36]). Interestingly, the tip pilin LrpC supports *L. ruminis* binding to ECM‐related substrates but not to the mucosal surfaces.

#### **2.3. Pili in other** *Lactobacillus* **species**

The presence of sortase‐mediated pilus gene clusters has been reported in many strains of *Lactobacillus casei* [57–60] and *Lactobacillus paracasei* [61], which are members of the normal human gut microbiota and used extensively as probiotics and in the food industries. Although the pilus expression and function are yet to be studied in detail, the most analyzed strains in the *L. casei* and *L. paracasei* group show that they contain *SpaCBA* and *SpaFED* pilus gene clusters. In contrast, only few strains in *L. rhamnosus* group have *SpaCBA* cluster (e.g., *L. rhamnosus GG* and LMS2‐1 strain). However, several *L. paracasei* strains including COM0101 are shown to have truncated SpaC gene [60]. The transposon genes, which are present in the vicinity of the *SpaCBA* cluster in *L. rhamnosus*, seem to be absent in the *L. casei* suggesting that L. *rhamnosus* GG and LMS2-1 could have acquired the *SpaCBA* pilus gene cluster through horizontal gene transfer (HGT) from *L. casei* [57, 62]. This is further evi‐ denced by the presence of high nucleotide sequence identity in spaCBA cluster of *L. rhamnosus* and *L. casei* [57, 62].

### **2.4. Pili in** *L. lactis*

binding mechanism via a metal‐ion‐containing vWFA adhesin domain. Certainly, knowl‐ edge generated from our ongoing structural investigations would provide new insights into pilus assembly and adhesion mechanisms in *L. rhamnosus* GG, and serves as a model for

*L. ruminis*, one of the dominating *Lactobacillus* species in the mammalian intestines, is routinely isolated from the feces of human, cattle, and pigs. It is one of the few motile members known in lactobacilli. It is also recognized as an autochthonous microbiota in the GIT. The pilus gene identified in the human‐derived intestinal isolate *L. rumini* ATCC 25644 has been named as *lrpCBA* (*L*. *r*umini *p*ilus) [36] since they appear to be different from the known lactobacillar pilus types (SpaCBA and SpaFED) at the primary structural level. The *LrpCBA* pilus operon encodes tip (lrpC), basal (lrpB), and major (lrpA) pilins and a pilin‐specific sortase (SrtC). Sequence of *L. ruminis* pilins displays the common pilin features such as LPXTG‐like motifs, E‐box motif, and pilin motifs (in major and basal pilins) [36] (**Figure 2**). The expression and surface localization of *lrpCBA* pilus gene product have further confirmed by immunoblot analysis and immune‐electron microscopic visualization (for details, see [36]). Interestingly, the pilus genes have also been detected in *L. ruminis* ATCC 27782 from bovine gut origin [56], but the microarray analysis showed that the corresponding genes were upregulated in human strain compared with the bovine isolate. The ability of LrpCBA pilus to adhere to gut epithelial cells and extracellular matrix (ECM) proteins, and immune‐modulation activities has been demonstrated using recombinant‐piliated lactococci (for details, see [36]). Interestingly, the tip pilin LrpC supports *L. ruminis* binding to ECM‐related substrates but not to the mucosal

The presence of sortase‐mediated pilus gene clusters has been reported in many strains of *Lactobacillus casei* [57–60] and *Lactobacillus paracasei* [61], which are members of the normal human gut microbiota and used extensively as probiotics and in the food industries. Although the pilus expression and function are yet to be studied in detail, the most analyzed strains in the *L. casei* and *L. paracasei* group show that they contain *SpaCBA* and *SpaFED* pilus gene clusters. In contrast, only few strains in *L. rhamnosus* group have *SpaCBA* cluster (e.g., *L. rhamnosus GG* and LMS2‐1 strain). However, several *L. paracasei* strains including COM0101 are shown to have truncated SpaC gene [60]. The transposon genes, which are present in the vicinity of the *SpaCBA* cluster in *L. rhamnosus*, seem to be absent in the *L. casei* suggesting that L. *rhamnosus* GG and LMS2-1 could have acquired the *SpaCBA* pilus gene cluster through horizontal gene transfer (HGT) from *L. casei* [57, 62]. This is further evi‐ denced by the presence of high nucleotide sequence identity in spaCBA cluster of *L.*

probiotics.

surfaces.

**2.3. Pili in other** *Lactobacillus* **species**

*rhamnosus* and *L. casei* [57, 62].

**2.2. Pili in** *L. ruminis*

122 Probiotics and Prebiotics in Human Nutrition and Health

*L. lactis* is another widely used species as starter in dairy fermentation and best characterized strain in lactic acid bacteria (LAB). They seem to present in nutrient‐rich ecological niches (gut mucus, milk, and plants). A functional pilus operon (*pil*) has been shown to present in *L. lactis* IL1403 [34, 63]. It encodes tip (YhgD), major (YhgE), and basal (YhhB) pilins and a pilin‐ specific sortase (SrtC). The presence of pilus structures has been confirmed by immunogold electron microscopy and atomic force microscopy (AFM) analyses. The major YhgE and basal YhhB pilins display typical LPXTG motifs and pilin motifs. Additionally, the YhgE has an E‐ box. The pili were also shown to promote biofilm formation by confocal laser scanning microscopy (CLSM). The occurrence of pili in few other *L. lactis* isolates from clinical and vegetal environments was also visualized by by transmission electron microscopy (TEM) analysis [34]. Later, a proteomic analysis study has also detected pilus genes (YhgE2, YhhB2, ORF4, and SrtC2) in a vegetal isolate *L. lactis* subsp. *lactis* TIL448 [35]. The YhgE2 was shown to play a major role in intestinal epithelial Caco‐2 cells adhesion. The pilus biogenesis and morphology were further analyzed by immunoblot, electron micrograph, transcriptional, and AFM experiments [35, 64].

#### **2.5. Pili in bifidobacteria**

Bifidobacteria are the common components of the gut microbiota of a broad range of hosts [65]. Several members of bifidobacteria are typical inhabitants of the infant intestine [66], which is thought to be sterile at birth. Identification of many bifidobacterial strains in the stools of healthy infants suggests that they could be the first colonizers in the GIT subsequent to birth. Genomic analysis has revealed pilus genes cluster in several bifidobacterial strains [67]. Interestingly, many pilus gene clusters are flanked by transposon elements indicating their acquisition by HGT. The presence of pilus structures was further examined by AFM and transcription analysis in *Bifidobacterium bifidum*, *Bifidobacterium dentium*, *Bifidobacterium longum* subsp. *longum*, *Bifidobacterium adolescentis*, and *Bifidobacterium animalis* subsp. *lactis*[67]. The pilus gene clusters often found to contain one major pilin (FimA or FimP) and one or two ancillary pilins (FimB or FimQ) with a pilin‐specific sortase. Many of these pilus genes are similar to the (two‐pilins) pilus gene clusters identified in Gram‐positive pathogens such as *Actinomyces oris* [68, 69] and *Bacillus cereus* [70], which lack basal pilus genes differing from the three‐pilins architectural model of *C. diphtheriae* [15, 16]. *A. oris* encodes two different fimbriae (types 1 and 2). Type 1 fimbria, which mediates the interaction of actinomyces to tooth enamel, consists of the major pilin (FimP) and tip pilin (FimQ). Whereas, the type 2 fimbria that mediate interaction with oral streptococci and host cell for causing dental plaque is made of major pilin (FimA) and tip pilin (FimB). Similarly, *B. cereus* pili is composed of major pilin BcpA and the tip pilin (BcpB). In the two‐pilin sortase‐mediated pili model, the last major pilin may function as the pilus base. The three‐dimensional structures for major pilins for *A. oris* are available while they are yet to be elucidated for tip pilins. The major pilins of bifidobacteria have typical pilin motif and LPXTG motif required for pilus polymerization [67]. The role of pili in adherence, immunomodulation, and bacterial aggregations was further extensively explored in *B. bifidum* PRL2010, which contains three different pilus gene clusters (*pil1*, *pil2*, and *pil3*) [71]. Apart from sortase‐mediated pili, the presence of type IV pili has also been reported in bifidobacteria (e.g., *Bifidobacterium breve* UCC2003 [32]), which is described below.

### **3. Tad pili**

The Tad (tight adherence) pili, which was first described in *Aggregatibacter (Actinobacillus) actinomycetemcomitans* [72], is a specialized subtype of type IV pili (for reviews, see [5, 7, 8, 73, 74]). Tad pili in this bacterium were shown to mediate adhesion to surfaces and essential for colonization and pathogenesis. Apart from adhesion, the type IV pili have been implicated in several functions such as aggregation, biofilm formation, twitching motility, DNA uptake, and electron transfer. Type IV pili are found to be present in Gram‐negative (e.g., enteropathogenic *Escherichia coli*, *Salmonella enterica*, *Pseudomonas aeruginosa*, *Neisseria meningitides*, and *Vibrio cholerae*) as well as Gram‐positive bacteria (*Clostridium perfringens*, *Mycobacterium tuberculosis*, and *Ruminococcus albus*). Type IV pili are typically 6‒8 nm in diameter and several micrometers long. The type IV pilus is comprised of homopolymers of a single (major) pilin subunit (**Figure 4**). The major pilins in Tad pili are relatively smaller in size (∼7 kDa) compared with other known pilus types in type IV. The flexible homopolymer filaments in type IV often have tendency to form characteristic helical bundles by lateral interactions. Some pili possess an adhesive or ancillary pilins at the pilus tip or can be decorated with pseudopilins along the pilus.

**Figure 4. Schematic diagram of type IV pilus structure**. (A) Type IV (gonococcal) pilus model. Major pilins form the filament majorly by hydrophobic interactions between their N‐terminal helices in the filament core. The globular head of major pilins pack on the filament surface. (B) Type IV major pilin, PilE (PDB id: 1AY2), from *N. gonorrhoeae* showing N‐terminal helix (in red) and globular head with D‐region.

Type IV pilus assembly is a complex process, which requires protein products from multiple genes (∼14) including minor pilins, prepilin peptidase, ATPase, inner membrane core proteins, and accessory proteins. Many of the core genes are conserved across different bacterial species. Tad pili seem to differ from other type IV pilus types by lacking four core homologous minor pilins. The type IV pilins are synthesized as precursors with a leader peptide and transported across the inner membrane into the periplasmic space, where they are retained in the inner membrane through their N‐terminal hydrophobic segments. The globular domain is folded with stabilizing intramolecular disulfide bonds. A dedicated prepilin peptidase cleaves the positively charged leader sequence and methylates the N‐terminal amine to generate the mature pilin. The methylated, positively charged N‐terminal residue is thought to attract negatively charged glutamate (at fifth position) of adjacent major pilin in the growing pilus fiber. This results in vertical displacement between one pilin and the next. The assembly ATPase associated with the cytoplasmic part of the inner membrane protein undergoes conformational change during ATP hydrolysis and pushes the pilus filament out of the membrane, providing a gap for the next major pilin. Type IV pili is further complicated by divergence and divided into two classes (types IVa and IVb) based on the length of leader peptides and mature pilins. The pilins of type IVa are typically 150‒160 residues long with a short leader peptide (<10 residues), whereas the pilins of type IVb are either long (180‒200 residues) or short (40‒50 residues) with longer leader peptides (∼15‒30 residues). The Tad pili are monophyletic subclass of type IVb pili [73]. The pilins of Tad pili are short with 40‒50 residues long.

Though the sequence and structural diversity are associated with the pilins in type IV, they share a common lollipop‐like architecture consisting of an extended N‐terminal helical stick followed by a globular head containing a β‐sheet with 4–7 strands [74] (**Figure 4B**). The N‐terminal half of the helix is hydrophobic and multifunctional regulatory domain. It protrudes from the globular head and forms the central hydrophobic core of the grow‐ ing filament during the pilus assembly. Prior to assembly, it acts as transmembrane segment to retain individual pilin in the cytoplasmic membrane. The C‐terminal half of the helix is amphipathic and embedded in the globular head. For many pili, a hypervariable C‐ terminal loop known as D‐region or disulfide‐bonded loop (DSL) performs an essential role in surface adherence (**Figure 4B**). The conserved disulfide bridge in the D‐region observed in several Gram‐negative major pilins appears to be off in Gram‐positive pilins (e.g., PilA1 in *Chlostrodium difficle* [75]). The Tad genes are also widespread in the ge‐ nomes of Gram‐positive species (*C. diphtheriae*, *Thermobifida fusca*, and *Streptomyces coelicolor*). Recently, they have been identified in probiotic *B. breve.*

#### **3.1. Tad pili in** *B. breve*

[71]. Apart from sortase‐mediated pili, the presence of type IV pili has also been reported in

The Tad (tight adherence) pili, which was first described in *Aggregatibacter (Actinobacillus) actinomycetemcomitans* [72], is a specialized subtype of type IV pili (for reviews, see [5, 7, 8, 73, 74]). Tad pili in this bacterium were shown to mediate adhesion to surfaces and essential for colonization and pathogenesis. Apart from adhesion, the type IV pili have been implicated in several functions such as aggregation, biofilm formation, twitching motility, DNA uptake, and electron transfer. Type IV pili are found to be present in Gram‐negative (e.g., enteropathogenic *Escherichia coli*, *Salmonella enterica*, *Pseudomonas aeruginosa*, *Neisseria meningitides*, and *Vibrio cholerae*) as well as Gram‐positive bacteria (*Clostridium perfringens*, *Mycobacterium tuberculosis*, and *Ruminococcus albus*). Type IV pili are typically 6‒8 nm in diameter and several micrometers long. The type IV pilus is comprised of homopolymers of a single (major) pilin subunit (**Figure 4**). The major pilins in Tad pili are relatively smaller in size (∼7 kDa) compared with other known pilus types in type IV. The flexible homopolymer filaments in type IV often have tendency to form characteristic helical bundles by lateral interactions. Some pili possess an adhesive or ancillary pilins at the pilus tip or can be decorated with pseudopilins along the

**Figure 4. Schematic diagram of type IV pilus structure**. (A) Type IV (gonococcal) pilus model. Major pilins form the filament majorly by hydrophobic interactions between their N‐terminal helices in the filament core. The globular head of major pilins pack on the filament surface. (B) Type IV major pilin, PilE (PDB id: 1AY2), from *N. gonorrhoeae* showing

Type IV pilus assembly is a complex process, which requires protein products from multiple genes (∼14) including minor pilins, prepilin peptidase, ATPase, inner membrane core proteins, and accessory proteins. Many of the core genes are conserved across different bacterial species. Tad pili seem to differ from other type IV pilus types by lacking four core homologous minor

N‐terminal helix (in red) and globular head with D‐region.

bifidobacteria (e.g., *Bifidobacterium breve* UCC2003 [32]), which is described below.

**3. Tad pili**

124 Probiotics and Prebiotics in Human Nutrition and Health

pilus.

Apart from sortase‐dependent pili, *B. breve* UCC2003 was recently shown to contain the type IVb or Tad pilus gene cluster named tad<sup>2003</sup> [32]. The presence of pili was further confirmed by immunogold transmission electron microscopy and shown to be essential for efficient gut colonization in a murine model by mutational analysis [32]. Specifically, the Tad locus is highly conserved among all sequenced bifidobacterial strains supporting a ubiquitous pilus‐mediat‐ ed host colonization and persistence mechanism for intestinal bifidobacteria. The structural data are yet to come for pilins of Tad pilus from beneficial bacteria for shedding light on their structure and function.

### **4. Future perspectives**

Adhesion of bacteria to host surfaces is a prerequisite and crucial step for bacterial coloniza‐ tion, which may result in pathogenic or commensal relationship. The pili have been often implicated in initiating adhesion and mediating interaction with host. Understanding pilus structure and function, and their mediated interactions with the host has been achieved to a certain extent in pathogenic strains. The pili and their components are recognized as viru‐ lence factors in pathogenic strains, and also considered as potential vaccine candidates in combating bacterial infection. Recent identification of such surface organelles in probiotic or commensal bacteria gives a new perspective as a niche‐adaption factor as well. The sortase‐ mediated pili initially discovered in Gram‐positive pathogens appear to be widespread among commensals. The Tad pili, which are known to present in both Gram‐negative and Gram‐positive pathogens, have also been detected in some commensal strains. It may not be a surprise if additional pilus type comes in the future from the fast‐growing technology and genomes for gut microbiota. Available preliminary data suggest that the pili from pathogen‐ ic and beneficial bacteria share several sequence and structural features. The presence of transposable element in several pilus gene clusters indicates that the pathogenic and com‐ mensal bacteria may be acquired from each other during the evolution. The challenge is now to understand the differences between the (enemy) pathogenic and (friendly) beneficial bacteria in their pili‐mediated adhesion strategies and interactions with the host. This knowledge is crucial in optimizing probiotics and targeting adhesion‐based therapies for human health. The journey of pilus research in probiotics has begun with the prototype SpaCBA pili in *L. rhamnosus* GG. The ongoing and future research hopefully would shed light in this area.

### **Acknowledgments**

This work was funded by the Regional Centre for Biotechnology (RCB) and the Department of Biotechnology (DBT) (Grant No. BT/PR5891/BRB/10/1098/2012), India.

### **Author details**

Vengadesan Krishnan1\*, Priyanka Chaurasia1,2 and Abhiruchi Kant1,2

\*Address all correspondence to: kvengadesan@rcb.res.in


### **References**

**4. Future perspectives**

126 Probiotics and Prebiotics in Human Nutrition and Health

light in this area.

**Acknowledgments**

**Author details**

Adhesion of bacteria to host surfaces is a prerequisite and crucial step for bacterial coloniza‐ tion, which may result in pathogenic or commensal relationship. The pili have been often implicated in initiating adhesion and mediating interaction with host. Understanding pilus structure and function, and their mediated interactions with the host has been achieved to a certain extent in pathogenic strains. The pili and their components are recognized as viru‐ lence factors in pathogenic strains, and also considered as potential vaccine candidates in combating bacterial infection. Recent identification of such surface organelles in probiotic or commensal bacteria gives a new perspective as a niche‐adaption factor as well. The sortase‐ mediated pili initially discovered in Gram‐positive pathogens appear to be widespread among commensals. The Tad pili, which are known to present in both Gram‐negative and Gram‐positive pathogens, have also been detected in some commensal strains. It may not be a surprise if additional pilus type comes in the future from the fast‐growing technology and genomes for gut microbiota. Available preliminary data suggest that the pili from pathogen‐ ic and beneficial bacteria share several sequence and structural features. The presence of transposable element in several pilus gene clusters indicates that the pathogenic and com‐ mensal bacteria may be acquired from each other during the evolution. The challenge is now to understand the differences between the (enemy) pathogenic and (friendly) beneficial bacteria in their pili‐mediated adhesion strategies and interactions with the host. This knowledge is crucial in optimizing probiotics and targeting adhesion‐based therapies for human health. The journey of pilus research in probiotics has begun with the prototype SpaCBA pili in *L. rhamnosus* GG. The ongoing and future research hopefully would shed

This work was funded by the Regional Centre for Biotechnology (RCB) and the Department

of Biotechnology (DBT) (Grant No. BT/PR5891/BRB/10/1098/2012), India.

Vengadesan Krishnan1\*, Priyanka Chaurasia1,2 and Abhiruchi Kant1,2

1 Regional Centre for Biotechnology, NCR Biotech Science Cluster, Faridabad, India

2 Department of Biotechnology, Manipal University, Manipal, Karnataka, India

\*Address all correspondence to: kvengadesan@rcb.res.in


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