**6.1.2 Engineered lactic acid bacteria with enhanced adhesion**

Some of the adhesion factors characterized in probiotic bacteria may be targets for strain engineering aimed to enhance bacterial adhesion. In addition, heterologous expression of well characterized adhesins from different sources can be envisaged. This can be useful to increase residence times at the gastrointestinal tract, enhance interactions with the mucosal immune system and promote competitive exclusion of pathogens by probiotics. Some probiotic strains like *L. casei* Shirota have been engineered to express a fibronectin binding domain from the Sfb protein of *Streptococcus pyogenes*, allowing this strain, which barely binds fibronectin, to bind this ECM substrate, fibrinogen and human fibroblasts (Kushiro *et al.*, 2001). However, to date most genetic engineering strategies aimed to increase lactic acid bacteria adhesion have been carried out in the model *L. lactis* species. This bacterium is not a

macrophages. Therefore, EICs actively participate in the discrimination of both pathogenic and commensal bacteria, they are crucial in triggering lymphocyte differentiation, maintaining intestinal immune homeostasis and mechanisms of innate defense (Artis, 2008). As consequence, commensal bacteria and pathogens are detected at different levels, in IECs, DC and macrophages. Different receptors recognise different bacterial ligands, so that the mucosa would integrate the information to recognise the microorganisms approaching the mucosa. PRRs in IECs and DC binding to bacterial molecular patterns (PAMPs) are expressed at the cell surface (TLR2, TLR4, CD14, TLR5) or in specialised endosomes (TLR3, TLR7, TLR8, TLR9). They can recognise single bacterial ligands or act synergistically to recognise others. As a quick overview, TLR3 recognises double stranded viral RNA, TLR9 hypomethylated CpG bacterial DNA, TLR7 and TLR8 were reported to recognize small imidazoquinoline compounds and TLR4, with the aid of CD14, recognises lipopolysaccharides (LPS) and lipoarabinomannans. Peptidoglycan (PGN) of different grampositive bacteria have been shown to interact with TLR2 (Iwaki *et al.*, 2002 ), however, TLR2 recognises other PAMPs (PGN, lipoteicoic acid, mycobacterial cell walls, protozoan parasite GPI anchors, lipoproteins, glycoproteins, glycolipids, LPS, etc). Furthermore, the complex TLR2/TLR6 recognises dipalmitoylated mycoplasma lipoprotein (MALP2), phenol soluble modulin from *Staphylococcus epidermidis* and fungal zymosan, and also, TLR2 associated to TLR1 recognises triacylated lipoproteins such as *Borrelia burgdorferi* OspA (for review see (Qureshi & Medzhitov, 2003)). In addition, cell wall components in lactobacilli and firmicutes are recognized by intracellular pattern-recognition molecules members of the nucleotide-binding oligomerization domain (NOD) family. The NLR family (also called Nod-leucine-rich repeats (LRRs)) are responsible for the signalling response induced by bacterial PGN and bacterial surface components, for instance, Nod1/CARD4 receptor in macrophages recognises Meso-diaminopimelic acid (meso-DAP), Nod2/CARD15 recognises muramyl dipeptide (MDP), Nod2 acting as a general sensor for bacteria, Ipaf/CLAN/CARD12 recognises intracellular flagellin (independently of TLR5, which senses extracellular flagellin) and cryopyrin/PYPAF1/NALP3 recognises bacterial RNA (and endogenous danger signals) and among others (for review, see (Franchi *et al.*, 2006). In particular, Nod2 recognizes a PGN motif present on both Gram-positive and Gram-negative

151

**6.2.1 Mechanisms of immunomodulation and probiotic factors involved** 

Nuclear factor κB (NF-κB) is a transcriptional regulator, or rather a regulator family, that controls the expression of hundreds of genes related to different cellular processes, including innate and adaptive immune responses. NF-κB signalling is the major proinflammatory pathway controlling the expression of cytokines, chemokines, enzymes that produce secondary inflammatory mediators and inhibitors of apoptosis. It is activated by numerous proteins, among them pathogen-associated molecular patterns (PAMPs). Various lactobacilli have been described to inhibit NF-κB activity (Kim *et al.*, 2008; Petrof *et al.*, 2004). Although the initial steps of the process are not known, it was determined that *L. casei* DN-114.001 can maintain intestinal homeostasis after an inflammatory stimulus through a process that controls the ubiquitin/proteasome pathway upstream of I-kBα resulting in the stabilization of it, therefore blocking NF-κB for nuclear translocation (Tien *et al.*, 2006). Such effects possibly occur through targeting of multiple effectors and, in some cases, through complementary pathways such as NF-κB and p38 MAPK signaling pathways as shown in *L. casei* and *L. reuteri* where they could play important roles in the augmentation of innate

bacteria.

normal inhabitant of the gastrointestinal tract but it has been used as a food grade delivery vehicle for presenting bioactive molecules to mucosal surfaces, including antigens, cytokines or enzymes. Expression of a protein containing a chitin-binding domain from *L. plantarum* on the surface of *L. lactis* resulted in enhanced capacity to attach to natural compounds carrying polymers of *N*-acetylglucosamine, such as human mucins (Sanchez *et al.*, 2011). The recombinant strain also showed increased attachment to epithelial Caco-2 cells. In another approach, the receptor binding domain of FedD adhesin from *E. coli* F18 fimbriae was expressed and anchored to the bacterial surface by creating a fusion with the surface anchoring domain of the *L. lactis* autolysin AcmA. This fusion protein promoted the binding of *L. lactis* to porcine intestinal epithelial cells (Lindholm *et al.*, 2004). Finally, expression in *L. lactis* of either a fibronectin binding protein from *Staphylococcus aureus* or Internalin A from *Listeria monocytogenes* promoted its binding to human epithelial cells and bacterial internalization, providing a tool for DNA delivery into eukaryotic cells (Innocentin *et al.*, 2009).

#### **6.2 Inmunomodulation of colonic mucosa**

In the case of functional properties of lactobacilli, due to legal restrictions and public opinion attitudes against the use of genetically modified microorganisms, the most general strategy has been the selection of naturally competent probiotic strains, nevertheless, some examples of mutants and genetically engineered strains can be found with specific and improved properties (see below). The molecular mechanisms underlying this process are still unknown, however, intervention studies using probiotics in controlled placebo double blind clinical assays are very abundant and different meta-analysis confirmed that several specific beneficial effects of probiotics pass very stringent examination criteria (Williams, 2010), however, they are costly in time and resources and cannot be used to test a great number of strains. Hence, this review will initially describe the general features that characterise the recognition of bacteria by the mucosa and, then, it will focus on the characterisation of the mechanisms of action and the understanding of the effect of probiotics on model systems, as a mean efficiently select funtional strains.

As described in the introduction, the mucosal surface is continuously exposed to both potential pathogens and beneficial commensal microorganisms. This creates a requirement for a homeostatic balance between tolerance and immunity that represents a unique regulatory challenge to the mucosal immune system. Dendritic cells (DCs) in the lamina propria efficiently recognise microbial components from the intestinal lumen through PRRs, TLRs and NLRs. Then, DC migrate to draining lymph nodes, where they have the unique ability to activate and influence functional differentiation of naïve T cells. Signals from DC can determine whether tolerance or active immune responses occur to a particular antigen and furthermore influence whether a T helper (Th) cells of the type Th1 (innate immune response), Th2 (adaptive immune response and allergy), Th17 or Treg (lymphocyte differentiation) predominates. The DC subtype, whether CD11c+ (myeloid) or CD11c- (plasmacytoid), maturation status, and cytokine production contribute to the type of T cell response. For example, when DCs upregulate the coestimulatory molecules CD80 and CD86, produce IL-12 which contributes a Th1 response, but if DCs produce IL-10 and IL-4, they will promote the generation of a Th2 or regulatory T cells (Hart *et al.*, 2004).

Furthermore, intestinal epithelial cells (IECs) are not just a simple physical barrier. They express TLRs as well as intracellular NLRs and they can secrete cytokines and regulatory molecules (TSLP, TGFβ, IL-10, etc) that regulate cytokine secretion by DCs and

normal inhabitant of the gastrointestinal tract but it has been used as a food grade delivery vehicle for presenting bioactive molecules to mucosal surfaces, including antigens, cytokines or enzymes. Expression of a protein containing a chitin-binding domain from *L. plantarum* on the surface of *L. lactis* resulted in enhanced capacity to attach to natural compounds carrying polymers of *N*-acetylglucosamine, such as human mucins (Sanchez *et al.*, 2011). The recombinant strain also showed increased attachment to epithelial Caco-2 cells. In another approach, the receptor binding domain of FedD adhesin from *E. coli* F18 fimbriae was expressed and anchored to the bacterial surface by creating a fusion with the surface anchoring domain of the *L. lactis* autolysin AcmA. This fusion protein promoted the binding of *L. lactis* to porcine intestinal epithelial cells (Lindholm *et al.*, 2004). Finally, expression in *L. lactis* of either a fibronectin binding protein from *Staphylococcus aureus* or Internalin A from *Listeria monocytogenes* promoted its binding to human epithelial cells and bacterial internalization,

In the case of functional properties of lactobacilli, due to legal restrictions and public opinion attitudes against the use of genetically modified microorganisms, the most general strategy has been the selection of naturally competent probiotic strains, nevertheless, some examples of mutants and genetically engineered strains can be found with specific and improved properties (see below). The molecular mechanisms underlying this process are still unknown, however, intervention studies using probiotics in controlled placebo double blind clinical assays are very abundant and different meta-analysis confirmed that several specific beneficial effects of probiotics pass very stringent examination criteria (Williams, 2010), however, they are costly in time and resources and cannot be used to test a great number of strains. Hence, this review will initially describe the general features that characterise the recognition of bacteria by the mucosa and, then, it will focus on the characterisation of the mechanisms of action and the understanding of the effect of

As described in the introduction, the mucosal surface is continuously exposed to both potential pathogens and beneficial commensal microorganisms. This creates a requirement for a homeostatic balance between tolerance and immunity that represents a unique regulatory challenge to the mucosal immune system. Dendritic cells (DCs) in the lamina propria efficiently recognise microbial components from the intestinal lumen through PRRs, TLRs and NLRs. Then, DC migrate to draining lymph nodes, where they have the unique ability to activate and influence functional differentiation of naïve T cells. Signals from DC can determine whether tolerance or active immune responses occur to a particular antigen and furthermore influence whether a T helper (Th) cells of the type Th1 (innate immune response), Th2 (adaptive immune response and allergy), Th17 or Treg (lymphocyte differentiation) predominates. The DC subtype, whether CD11c+ (myeloid) or CD11c- (plasmacytoid), maturation status, and cytokine production contribute to the type of T cell response. For example, when DCs upregulate the coestimulatory molecules CD80 and CD86, produce IL-12 which contributes a Th1 response, but if DCs produce IL-10 and IL-4,

providing a tool for DNA delivery into eukaryotic cells (Innocentin *et al.*, 2009).

probiotics on model systems, as a mean efficiently select funtional strains.

they will promote the generation of a Th2 or regulatory T cells (Hart *et al.*, 2004).

Furthermore, intestinal epithelial cells (IECs) are not just a simple physical barrier. They express TLRs as well as intracellular NLRs and they can secrete cytokines and regulatory molecules (TSLP, TGFβ, IL-10, etc) that regulate cytokine secretion by DCs and

**6.2 Inmunomodulation of colonic mucosa** 

macrophages. Therefore, EICs actively participate in the discrimination of both pathogenic and commensal bacteria, they are crucial in triggering lymphocyte differentiation, maintaining intestinal immune homeostasis and mechanisms of innate defense (Artis, 2008). As consequence, commensal bacteria and pathogens are detected at different levels, in IECs, DC and macrophages. Different receptors recognise different bacterial ligands, so that the mucosa would integrate the information to recognise the microorganisms approaching the mucosa. PRRs in IECs and DC binding to bacterial molecular patterns (PAMPs) are expressed at the cell surface (TLR2, TLR4, CD14, TLR5) or in specialised endosomes (TLR3, TLR7, TLR8, TLR9). They can recognise single bacterial ligands or act synergistically to recognise others. As a quick overview, TLR3 recognises double stranded viral RNA, TLR9 hypomethylated CpG bacterial DNA, TLR7 and TLR8 were reported to recognize small imidazoquinoline compounds and TLR4, with the aid of CD14, recognises lipopolysaccharides (LPS) and lipoarabinomannans. Peptidoglycan (PGN) of different grampositive bacteria have been shown to interact with TLR2 (Iwaki *et al.*, 2002 ), however, TLR2 recognises other PAMPs (PGN, lipoteicoic acid, mycobacterial cell walls, protozoan parasite GPI anchors, lipoproteins, glycoproteins, glycolipids, LPS, etc). Furthermore, the complex TLR2/TLR6 recognises dipalmitoylated mycoplasma lipoprotein (MALP2), phenol soluble modulin from *Staphylococcus epidermidis* and fungal zymosan, and also, TLR2 associated to TLR1 recognises triacylated lipoproteins such as *Borrelia burgdorferi* OspA (for review see (Qureshi & Medzhitov, 2003)). In addition, cell wall components in lactobacilli and firmicutes are recognized by intracellular pattern-recognition molecules members of the nucleotide-binding oligomerization domain (NOD) family. The NLR family (also called Nod-leucine-rich repeats (LRRs)) are responsible for the signalling response induced by bacterial PGN and bacterial surface components, for instance, Nod1/CARD4 receptor in macrophages recognises Meso-diaminopimelic acid (meso-DAP), Nod2/CARD15 recognises muramyl dipeptide (MDP), Nod2 acting as a general sensor for bacteria, Ipaf/CLAN/CARD12 recognises intracellular flagellin (independently of TLR5, which senses extracellular flagellin) and cryopyrin/PYPAF1/NALP3 recognises bacterial RNA (and endogenous danger signals) and among others (for review, see (Franchi *et al.*, 2006). In particular, Nod2 recognizes a PGN motif present on both Gram-positive and Gram-negative bacteria.

#### **6.2.1 Mechanisms of immunomodulation and probiotic factors involved**

Nuclear factor κB (NF-κB) is a transcriptional regulator, or rather a regulator family, that controls the expression of hundreds of genes related to different cellular processes, including innate and adaptive immune responses. NF-κB signalling is the major proinflammatory pathway controlling the expression of cytokines, chemokines, enzymes that produce secondary inflammatory mediators and inhibitors of apoptosis. It is activated by numerous proteins, among them pathogen-associated molecular patterns (PAMPs). Various lactobacilli have been described to inhibit NF-κB activity (Kim *et al.*, 2008; Petrof *et al.*, 2004). Although the initial steps of the process are not known, it was determined that *L. casei* DN-114.001 can maintain intestinal homeostasis after an inflammatory stimulus through a process that controls the ubiquitin/proteasome pathway upstream of I-kBα resulting in the stabilization of it, therefore blocking NF-κB for nuclear translocation (Tien *et al.*, 2006). Such effects possibly occur through targeting of multiple effectors and, in some cases, through complementary pathways such as NF-κB and p38 MAPK signaling pathways as shown in *L. casei* and *L. reuteri* where they could play important roles in the augmentation of innate

In other cases mutants have been very useful to demonstrate the functional effect of certain cell components. In *L. casei* Shirota (LcS), a mutant strain was very useful to prove that inhibition of the pro-inflammatory cytokine IL-6, through Nod2/ NF-B, was due to a cell

153

The ability of lactobacilli to survive on the mucosal surfaces of humans and animals has been utilized for the delivery and presentation of bioactive molecules at these surfaces. These bacteria have several advantages, including their recognised GRAS/QPS status, their capacity to interact with the host at several levels and their public acceptance. Studies of lactobacilli as delivery vehicles have mainly focused on the development of mucosal vaccines. In addition, interleukins have been also co-expressed with antigens in lactobacilli to enhance the immune response. Other applications of *Lactobacillus* species as delivery systems include anti-infectives, therapies for allergic diseases and therapies for gastrointestinal diseases. The ability of lactobacilli and other LAB to express these antigens/bioactive molecules at mucosal surfaces have been widely reviewed (Monedero &

Some recent examples include the use of *Lactobacillus jensenii* strains isolated from human vaginal mucosa for the delivery vehicle of a surface-anchored two-domain CD4 (2D CD4) molecule for the mitigation of heterosexual transmission of HIV (Liu *et al.*, 2008). *L. casei* was the host to express the viral proteins from porcine rotavirus and porcine parvovirus fused the heat-labile toxin B subunit from *Escherichia coli*. The results showed that mice responded producing increased levels of anti-viral antibodies (Liu *et al.*, 2011). *L. casei* Zhang was engineered to stably express the p23 immunodominant surface protein of *Cryptosporidium parvum* sporozoites. Recombinant *L. casei* Zhang-p23 was able to activate the mucosal immune system and to elicit specific serum immunoglobulin G (IgG) and mucosal IgA in mice. The expression of cytokines such as IL4, IL6, and IFN-gamma was detected in splenocytes of mice by real-time PCR after oral immunization with this strain (Geriletu *et al.*, 2011). A recombinant *L. casei* strain secreting biologically active murine interleukin-1has been constructed. This strain was able to induce IL8 secretion in Caco2 cells and IL6 in vivo using a ligated-intestinal-loop assay in mice after oral administration. The increased adjuvant properties of this strain were confirmed after intragastric immunization with heat-

Another application of lactobacilli has been their use to express molecules to deliver passive immunity against pathogens, such as single-chain antibodies (lactobodies). Martin and coworkers have been able to construct a series of expression cassettes stably integrated in the chromosome to mediate the secretion or surface display of antibody fragments in *Lactobacillus paracasei*. These new constructed strains, producing surface-anchored variable domain of llama heavy chain (VHH) (ARP1) directed against rotavirus, showed efficient binding to rotavirus

Lactobacilli can also be engineered to produce an increased immune response against cancer cells. *L. rhamnosus* GG (LGG) has been used to successfully induce tumor regression in an orthotopic model of bladder cancer. The potential of LGG to induce a directed anti-tumor response was evaluated with modified LGG secreting the prostate specific antigen (PSA) or IL15 and PSA (IL-15-PSA). Recombinant LGG activated neutrophils, induced dendritic cells maturation, T cell proliferation and PSA specific cytotoxic T lymphocytes activity leading to

wall-derived polysaccharide– PGN complex (PSPG) (Matsumoto *et al.*, 2009).

**6.3 Engineered probiotics to delivery bioactive proteins** 

Pérez-Martínez, 2008; Wells & Mercenier, 2008).

killed *Salmonella enterica* serovar *Enteritidis* (Kajikawa *et al.*, 2010).

the tumor regression (Kandasamy *et al.*, 2011).

and protection in the mouse model of rotavirus infection (Martin *et al.*, 2011).

immunity (Iyer *et al.*, 2008; Kim *et al.*, 2006). Two proteins of *L. rhamnosus* GG (p40 and p75) (Yan *et al.*, 2007) have been suggested to preserve the tight junction (TJ) integrity, an effect mediated by the PKC and MAPK pathways. They display antiapoptotic activity, prevent epithelial barrier damage caused by several agents and show *in vivo* effect by decreasing the susceptibility to dextran sulphate sodium (DSS)-induced colon epithelial injury (Yan *et al.*, 2007). Interestingly, recent studies (Bäuerl *et al.*, 2010) showed that these proteins are active cell wall hydrolases present in the seven genome sequences available for strains in the *L. casei-paracasei/rhamnosus* group. PGNs are cell wall components that interact with the intracellular receptor Nod2. Their *in vivo* effect changes with their composition composition, which also varies between strains. In the case of *Lactobacillus salivarius* Ls33 the muropeptide, M-tri-Lys, acts as ligand that protected mice from colitis in a NOD2 dependent but MyD88-independent manner (Macho Fernandez *et al.*, 2011).

Two strains of *L. reuteri* and *L. casei*, but not *L. plantarum*, had the ability to prime DC to drive the development of Treg cells which produced increased levels of the antiinflammatory cytokine IL-10 (Smits *et al.*, 2005). This ability was mediated by binding to the C-type lectin DC-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN). Targeting of DC-SIGN by certain probiotic bacteria could explain their beneficial effect in the treatment of several immune diseases, including atopic dermatitis and Crohn's disease. However, a DC-SIGN ligand capacity has been found in an extracellular protein (LP\_2145) in *L. plantarum* WCFS1(patent application WO/2009/035330) and in the surfacelayer protein A (SlpA) from *L. acidophilus* NCFM, inducing IL-10, IL-4 and decreasing IL12p70 synthesis (proinflammatory) (Konstantinov *et al.*, 2008).

There are also experiments that reported the *in vivo* effect on gene transcription in human volunteers where biopsies were analysed by microarrays. Strains of *L. rhamosus* GG (Di Caro *et al.*, 2005) and *L. plantarum* WCFS1 (van Baarlen *et al.*, 2011) were used administrated and a differential expression of analysed, noticing that expression affected mainly to genes involved in the immune and inflammatory response, as well as to genes related to apoptosis, growth and cell differentiation, signalling, adhesion, etc. Remarkably, striking differences were found in the modulation of NF-κB related pathways.

#### **6.2.2 Engineered lactic acid bacteria**

An efficient expression of IL-10 was achieved in *L. lactis* , that showed a striking effect in the remediation of DSS (dextran sodium sulphate) induced colitis in IL-10-/- mice (Steidler *et al.*, 2000). Then, the genetic manipulation strategy was improved and the human IL-10 encoding gene used to replace the thymidylate synthase gene (*thy*A) in the bacterial chromosome, which achieved a stable genetic construction and a self contained recombinant strain requiring thymidine to proliferate. This strain was used in a double blind assay on Crohn's disease patients (phase I), proving that the intake of this strain significantly decreased the disease activity (Braat *et al.*, 2006). Other anti-inflammatory and epithelium repairing peptide, such as the trefoil factor, was similarly successful in mice (Vandenbroucke *et al.*, 2004).

Mutants obtained by site directed mutagenesis, holding genetic changes precisely introduced, have been tested for improved health features. Teichoic acids (TA) activate NF-B through TLR-2 binding and they are one of the main immunostimulatory components of pathogenic grampositive bacteria. This effect was also observed in *L. plantarum* NCIMB8826, however, a mutant (*dlt*) deficient in D-alanylation of TA was much more anti-inflammatory than the parental strain on peripheral blood mononuclear cells and mice (Grangette *et al.*, 2005).

In other cases mutants have been very useful to demonstrate the functional effect of certain cell components. In *L. casei* Shirota (LcS), a mutant strain was very useful to prove that inhibition of the pro-inflammatory cytokine IL-6, through Nod2/ NF-B, was due to a cell wall-derived polysaccharide– PGN complex (PSPG) (Matsumoto *et al.*, 2009).

#### **6.3 Engineered probiotics to delivery bioactive proteins**

158 Food Industrial Processes – Methods and Equipment

immunity (Iyer *et al.*, 2008; Kim *et al.*, 2006). Two proteins of *L. rhamnosus* GG (p40 and p75) (Yan *et al.*, 2007) have been suggested to preserve the tight junction (TJ) integrity, an effect mediated by the PKC and MAPK pathways. They display antiapoptotic activity, prevent epithelial barrier damage caused by several agents and show *in vivo* effect by decreasing the susceptibility to dextran sulphate sodium (DSS)-induced colon epithelial injury (Yan *et al.*, 2007). Interestingly, recent studies (Bäuerl *et al.*, 2010) showed that these proteins are active cell wall hydrolases present in the seven genome sequences available for strains in the *L. casei-paracasei/rhamnosus* group. PGNs are cell wall components that interact with the intracellular receptor Nod2. Their *in vivo* effect changes with their composition composition, which also varies between strains. In the case of *Lactobacillus salivarius* Ls33 the muropeptide, M-tri-Lys, acts as ligand that protected mice from colitis in a NOD2-

Two strains of *L. reuteri* and *L. casei*, but not *L. plantarum*, had the ability to prime DC to drive the development of Treg cells which produced increased levels of the antiinflammatory cytokine IL-10 (Smits *et al.*, 2005). This ability was mediated by binding to the C-type lectin DC-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN). Targeting of DC-SIGN by certain probiotic bacteria could explain their beneficial effect in the treatment of several immune diseases, including atopic dermatitis and Crohn's disease. However, a DC-SIGN ligand capacity has been found in an extracellular protein (LP\_2145) in *L. plantarum* WCFS1(patent application WO/2009/035330) and in the surfacelayer protein A (SlpA) from *L. acidophilus* NCFM, inducing IL-10, IL-4 and decreasing

There are also experiments that reported the *in vivo* effect on gene transcription in human volunteers where biopsies were analysed by microarrays. Strains of *L. rhamosus* GG (Di Caro *et al.*, 2005) and *L. plantarum* WCFS1 (van Baarlen *et al.*, 2011) were used administrated and a differential expression of analysed, noticing that expression affected mainly to genes involved in the immune and inflammatory response, as well as to genes related to apoptosis, growth and cell differentiation, signalling, adhesion, etc. Remarkably, striking

An efficient expression of IL-10 was achieved in *L. lactis* , that showed a striking effect in the remediation of DSS (dextran sodium sulphate) induced colitis in IL-10-/- mice (Steidler *et al.*, 2000). Then, the genetic manipulation strategy was improved and the human IL-10 encoding gene used to replace the thymidylate synthase gene (*thy*A) in the bacterial chromosome, which achieved a stable genetic construction and a self contained recombinant strain requiring thymidine to proliferate. This strain was used in a double blind assay on Crohn's disease patients (phase I), proving that the intake of this strain significantly decreased the disease activity (Braat *et al.*, 2006). Other anti-inflammatory and epithelium repairing peptide, such as

Mutants obtained by site directed mutagenesis, holding genetic changes precisely introduced, have been tested for improved health features. Teichoic acids (TA) activate NF-B through TLR-2 binding and they are one of the main immunostimulatory components of pathogenic grampositive bacteria. This effect was also observed in *L. plantarum* NCIMB8826, however, a mutant (*dlt*) deficient in D-alanylation of TA was much more anti-inflammatory than the parental strain on peripheral blood mononuclear cells

dependent but MyD88-independent manner (Macho Fernandez *et al.*, 2011).

IL12p70 synthesis (proinflammatory) (Konstantinov *et al.*, 2008).

differences were found in the modulation of NF-κB related pathways.

the trefoil factor, was similarly successful in mice (Vandenbroucke *et al.*, 2004).

**6.2.2 Engineered lactic acid bacteria** 

and mice (Grangette *et al.*, 2005).

The ability of lactobacilli to survive on the mucosal surfaces of humans and animals has been utilized for the delivery and presentation of bioactive molecules at these surfaces. These bacteria have several advantages, including their recognised GRAS/QPS status, their capacity to interact with the host at several levels and their public acceptance. Studies of lactobacilli as delivery vehicles have mainly focused on the development of mucosal vaccines. In addition, interleukins have been also co-expressed with antigens in lactobacilli to enhance the immune response. Other applications of *Lactobacillus* species as delivery systems include anti-infectives, therapies for allergic diseases and therapies for gastrointestinal diseases. The ability of lactobacilli and other LAB to express these antigens/bioactive molecules at mucosal surfaces have been widely reviewed (Monedero & Pérez-Martínez, 2008; Wells & Mercenier, 2008).

Some recent examples include the use of *Lactobacillus jensenii* strains isolated from human vaginal mucosa for the delivery vehicle of a surface-anchored two-domain CD4 (2D CD4) molecule for the mitigation of heterosexual transmission of HIV (Liu *et al.*, 2008). *L. casei* was the host to express the viral proteins from porcine rotavirus and porcine parvovirus fused the heat-labile toxin B subunit from *Escherichia coli*. The results showed that mice responded producing increased levels of anti-viral antibodies (Liu *et al.*, 2011). *L. casei* Zhang was engineered to stably express the p23 immunodominant surface protein of *Cryptosporidium parvum* sporozoites. Recombinant *L. casei* Zhang-p23 was able to activate the mucosal immune system and to elicit specific serum immunoglobulin G (IgG) and mucosal IgA in mice. The expression of cytokines such as IL4, IL6, and IFN-gamma was detected in splenocytes of mice by real-time PCR after oral immunization with this strain (Geriletu *et al.*, 2011). A recombinant *L. casei* strain secreting biologically active murine interleukin-1has been constructed. This strain was able to induce IL8 secretion in Caco2 cells and IL6 in vivo using a ligated-intestinal-loop assay in mice after oral administration. The increased adjuvant properties of this strain were confirmed after intragastric immunization with heatkilled *Salmonella enterica* serovar *Enteritidis* (Kajikawa *et al.*, 2010).

Another application of lactobacilli has been their use to express molecules to deliver passive immunity against pathogens, such as single-chain antibodies (lactobodies). Martin and coworkers have been able to construct a series of expression cassettes stably integrated in the chromosome to mediate the secretion or surface display of antibody fragments in *Lactobacillus paracasei*. These new constructed strains, producing surface-anchored variable domain of llama heavy chain (VHH) (ARP1) directed against rotavirus, showed efficient binding to rotavirus and protection in the mouse model of rotavirus infection (Martin *et al.*, 2011).

Lactobacilli can also be engineered to produce an increased immune response against cancer cells. *L. rhamnosus* GG (LGG) has been used to successfully induce tumor regression in an orthotopic model of bladder cancer. The potential of LGG to induce a directed anti-tumor response was evaluated with modified LGG secreting the prostate specific antigen (PSA) or IL15 and PSA (IL-15-PSA). Recombinant LGG activated neutrophils, induced dendritic cells maturation, T cell proliferation and PSA specific cytotoxic T lymphocytes activity leading to the tumor regression (Kandasamy *et al.*, 2011).

*lactis* due to increased levels of expression of the NIZO B40 eps gene cluster. *Appl* 

155

Transgenic Bacteria Expressing Interleukin-10 in Crohn's Disease. *Clinical* 

in *Lactobacillus delbrueckii* subsp. *bulgaricus*: glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and triosephosphate isomerase.

bacteria based on the minimal replicon of pRV500 from *Lactobacillus sakei*. *Plasmid*

infectivity by a natural human isolate of *Lactobacillus jensenii* engineered to express

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(2005). Effects of *Lactobacillus* GG on genes expression pattern in small bowel

*Lactobacillus casei* Shirota on T cell activation, natural killer cell activity and cytokine

Nelson, K. E. & Relman, D. A. (2005). Diversity of the Human Intestinal Microbial

Live Lactic Acid Bacteria *Food and Agriculture Organization of the United Nations and* 

glucose and citrate end products in an ldhL-ldhD double-knockout strain of

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shift from homolactic acid to mixed-acid fermentation in *Lactococcus lactis*:

Braat, H., Rottiers, P., Hommes, D. W. & other authors (2006). A Phase I Trial With

Branny, P., de la Torre, F. & Garel, J. R. (1998). An operon encoding three glycolytic enzymes

Crutz-Le Coq, A. M. & Zagorec, M. (2008). Vectors for Lactobacilli and other Gram-positive

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Eckburg, P. B., Bik, E. M., Bernstein, C. N., Purdom, E., Dethlefsen, L., Sargent, M., Gill, S. R.,

Falagas, M. E., Betsi, G. I. & Athanasiou, S. (2007). Probiotics for the treatment of women

Fang, F. & O'Toole, P. W. (2009). Genetic tools for investigating the biology of commensal

FAO/WHO (2001). Evaluation of Health and Nutritional Properties of Powder Milk and

Ferain, T., Schanck, A. N. & Delcour, J. (1996). 13C nuclear magnetic resonance analysis of

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**10** 

163

*Italy* 

**Mycotoxins in Food** 

*Politecnico di Torino* 

Francesca Bosco and Chiara Mollea

The term mycotoxin was used for the first time in 1961 in the aftermath of a veterinary crisis in England, during which thousands of animals died. The disease was linked to a peanut meal, incorporated in the diet, contaminated with a toxin produced by the filamentous

In general, mycotoxins are low-molecular-weight compounds that are synthetized during secondary metabolism by filamentous fungi; their chemical structure may range from

Mycotoxins are natural contaminants in raw materials, food and feeds. Mould species that produce mycotoxins are extremely common, and they can grow on a wide range of substrates under a wide range of environmental conditions; they occur in agricultural products all around the world (Bennet & Klich, 2003). Many mycotoxins may be toxic to vertebrates and other animal groups and, in low concentrations, some of them can cause autoimmune illnesses, and have allergenic properties, while others are teratogenic, carcinogenic, and mutagenic (Bennet & Klich, 2003; Council for Agricultural Science and

Apparently, mycotoxins have no biochemical significance on fungal growth; they may have developed to provide a defense system against insects, microorganisms, nematodes, animals

Exposure to mycotoxins may occur through ingestion, inhalation, and dermal contact, and it is almost always accidental. Most cases of mycotoxicoses (animals and humans) result from eating contaminated food. Human exposure can be direct *via* cereals or indirect *via* animal

Most mycotoxins are relatively heat-stable within the conventional food processing temperature range (80–121°C), therefore so little or no destruction occurs under normal cooking conditions, such as boiling and frying, or even following pasteurization (Milicevic et al., 2010). The stability of mycotoxins during food processing has been reviewed in the work by Bullerman & Bianchini (2007). In general, the application of a food process reduces mycotoxin concentrations significantly, but does not eliminate them completely. The food processes that have been examined include physical treatments (cleaning and milling) and thermal processing (e.g. cooking, baking, frying, roasting and extrusion). The different treatments have various effects on mycotoxins, and those that utilize the highest temperatures have the greatest effects: roasting or cooking at high temperatures (above 150 °C) appear to reduce mycotoxin concentrations significantly (Bullerman & Bianchini, 2007).

fungus *Aspergillus flavus* (Bennet & Klich, 2003; Richard, 2007).

simple C4 compounds to complex substances (Paterson & Lima, 2010).

**1. General principles** 

Technology [CAST], 2003).

and humans (Etzel, 2002).

products (e.g. meat, milk and eggs) (CAST, 2003).

