**2. Mechanisms of cell-cell communication**

Multicellular organisms are composed by a rigidly regulated society of individual cells, organized into tissues and organs, which all together collaborate for the functioning of the individual and whose final "purpose," from the biological point of view, is to reproduce (or to allow to the reproduction of a similar genome). The coordinated work of the different cell types that leads to the formation of an adult individual, as well as the cell growth, differentiation, and organogenesis giving rise from a single fertilized cell, requires sophisticated signaling mechanisms. Thus, in the course of evolution, molecular messengers were generated, synthesized, and released in some part of the organism and then specifically recognized by the respective receptors expressed in the target cells. Complex molecular machines were simultaneously selected to transduce the activated receptor signal. While generally the term "extracellular messengers" involves those intercellular communication mechanisms taking place within a multicellular organism, it should be emphasized that even unicellular microorganisms are capable of "social" behaviors that require a coordinated response. This sophisticated cell-to-cell process of communication between microorganisms, the so-called quorum sensing (QS), consists in the synthesis by bacteria, both Gram-positive and Gram-negative, of specific molecules, which are called "autoinducers" or "bacterial pheromones." After production of such molecules, bacteria release them into the extracellular medium to be detected by specific receptors/transducers. Quorum sensing is an extremely important communication system for microorganisms. Through this system, bacteria are in fact capable to measure their concentration and to modulate gene expression in response to population density, which lead to the secretion of virulence factors, biofilm formation, competence, and bioluminescence [6, 7]. When bacteria that generate signals are in close proximity to each other, the concentration of their

**167**

*Essential Oils and Microbial Communication DOI: http://dx.doi.org/10.5772/intechopen.85638*

autoinducers are known for bacteria, archaea, and fungi.

QS signal amplifies. This event leads to a boost of the binding of the QS signal to specific receptor proteins, to a consequent activation of the specific receptor, and to the enhanced gene transcription with appropriate promoter sequences. QSs give to bacteria a great evolutionary advantage, allowing them to adapt to the change of the environment. Some authors propose these as neo-Darwinian mechanisms of evolution, which had an important function in the arrival of the first multicellular organisms [8]. The result of this "bacterial communication" can be represented by an increase in virulence (e.g., *Staphylococcus aureus*), by the formation of a biofilm (e.g., *Pseudomonas aeruginosa*), by sporulation, etc. To date, more than 100 different

Bacteria exhibit two main QS mechanisms, based on distinct signaling pathways, which present a certain analogy with the mechanisms found also in multicellular organisms. The first, generally used by Gram-negative bacteria, is based on the synthesis of a family of small molecules, the so-called AHL (acylated homoserine lactones). These molecules have a similar central structure and differ only in the length of a side chain, which specificity is determined by the length of the acyl chain and the substitution (▬H,▬OH or 〓O) on carbon. Generally, every type of bacterium can produce at least one AHL type; however, it can happen that bacteria produce more than one of them. Due to their chemical characteristics, AHL are capable to cross the bacterial membrane and spread outside; in addition, from the extracellular medium, they can freely enter within the cell and bind to specific receptors, called LuxR because the QS phenomenon was described for the first time in a microorganism, *Aliivibrio fischeri*, which is able to emit light *in vitro* only when its concentration exceeds a certain threshold [9]. In this microorganism, the AHL autoinducer is at low concentration and does not induce bioluminescence; when the bacterium is in the luminous organ of the giant squid, its cell density is high, so the transcriptional activator reaches the DNA, binds to the recognition sequence (LuxBox), and activates the transcription of genes for the enzyme luciferase, which produces bioluminescence. The advantage of this mechanism is to save energy, to ensure that bacteria become luminescent only when they are present in large numbers, and to prevent them from wasting energy when the population is toosmall to emit a visible signal. The complex AHL-LuxR in turn binds to the bacterial DNA, thus regulating the transcription of specific genes. In a situation where the concentration of bacteria is low, the level of synthesized AHL is below the threshold required for the LuxR bond. However, as the concentration of bacteria increases, the amount of AHL also increases, and the AHL-LuxR complex is formed accordingly. A gene encoding for the enzyme catalysing the AHL synthesis is present within the genes induced by the AHL-LuxR complex. This gives rise to a positive feedback leading to a rapid and synchronous answer from the whole microbial population. Signal transduction through the AHL-LuxR system, based on an extracellular messenger able to cross the membrane and on a receptor that is also a transcription factor, can be thought to obey the same logic of signaling through steroid hormones. On the other hand, Gram-positive bacteria use autoinducer molecules formed by peptides with a variable length ranging between 5 and 17 amino acids. Such molecules are produced by the processing of precursors and are often subjected to posttranslational modifications. These peptides require special transporters to be secreted in the extracellular environment that, in turn, is detected by a sensor histidine kinase [10] and transduces the signal through phosphorylation of intracellular targets. This mechanism of action is therefore similar to that used by growth factors in multicellular organisms. Not always, the nature of quorum sensing molecules (QSMs) is peptidic: for instance, some Gram-positive bacteria, such as *Streptomyces*, produce ɣ-butyrolactones as QSMs [11]. Finally, different researches report the autoinducer 2 (AI-2), with a rather unusual cyclic boronic ester, as a QS

#### *Essential Oils and Microbial Communication DOI: http://dx.doi.org/10.5772/intechopen.85638*

*Essential Oils - Oils of Nature*

microorganism and host. Microorganisms that generally do not cause diseases in their natural habitats, due to this new environmental situation, can become highly pathogenic. Normal constituents of the intestinal flora, such as *Escherichia coli*, may therefore become harmful in other districts, such as the urinary bladder, spinal cord, lungs, etc. Other microorganisms can become highly pathogenic under certain conditions: for instance, *Streptococcus viridans* physiologically present in the oropharyngeal tract, in some circumstances can invade different organs through the bloodstream, causing serious diseases (e.g., bacterial endocarditis). Today, there is much talk about the so-called "multidrug-resistant" (MDR), "extensively drugresistant" (XDR), and "pan-drug-resistant" (PDR) strains. Such microorganisms can be figuratively enclosed in a cluster comprising pathogens of infections that are today intractable [2]. Unfortunately, it is also difficult to fight those pathogens belonging to the so-called "ESKAPE" group, an acronym comprising the microbial species *Enterococcus faecium*, *Staphylococcus aureus*, *Klebsiella pneumoniae*, *Acinetobacter baumannii*, *Pseudomonas aeruginosa*, and *Enterobacter* spp. It is widely believed by the scientific community that the study of alternative strategies to the use of conventional antibiotics could represent an important way to be taken into consideration, to combat this dangerous situation. The use of bacteriophages in phage therapies, known for their high specificity, the development of new vaccines against *P. aeruginosa* [3] and *A. baumannii* [4], and the use of strategies to inhibit the bacterial virulence factors can be considered some of the solutions. Recently, research also focused on the exploitation and identification of new microorganisms, isolated, for example, from the ground, enabling to block the microbial growth of

one or more species belonging to the ESKAPE group [5].

Multicellular organisms are composed by a rigidly regulated society of individual cells, organized into tissues and organs, which all together collaborate for the functioning of the individual and whose final "purpose," from the biological point of view, is to reproduce (or to allow to the reproduction of a similar genome). The coordinated work of the different cell types that leads to the formation of an adult individual, as well as the cell growth, differentiation, and organogenesis giving rise from a single fertilized cell, requires sophisticated signaling mechanisms. Thus, in the course of evolution, molecular messengers were generated, synthesized, and released in some part of the organism and then specifically recognized by the respective receptors expressed in the target cells. Complex molecular machines were simultaneously selected to transduce the activated receptor signal. While generally the term "extracellular messengers" involves those intercellular communication mechanisms taking place within a multicellular organism, it should be emphasized that even unicellular microorganisms are capable of "social" behaviors that require a coordinated response. This sophisticated cell-to-cell process of communication between microorganisms, the so-called quorum sensing (QS), consists in the synthesis by bacteria, both Gram-positive and Gram-negative, of specific molecules, which are called "autoinducers" or "bacterial pheromones." After production of such molecules, bacteria release them into the extracellular medium to be detected by specific receptors/transducers. Quorum sensing is an extremely important communication system for microorganisms. Through this system, bacteria are in fact capable to measure their concentration and to modulate gene expression in response to population density, which lead to the secretion of virulence factors, biofilm formation, competence, and bioluminescence [6, 7]. When bacteria that generate signals are in close proximity to each other, the concentration of their

**2. Mechanisms of cell-cell communication**

**166**

QS signal amplifies. This event leads to a boost of the binding of the QS signal to specific receptor proteins, to a consequent activation of the specific receptor, and to the enhanced gene transcription with appropriate promoter sequences. QSs give to bacteria a great evolutionary advantage, allowing them to adapt to the change of the environment. Some authors propose these as neo-Darwinian mechanisms of evolution, which had an important function in the arrival of the first multicellular organisms [8]. The result of this "bacterial communication" can be represented by an increase in virulence (e.g., *Staphylococcus aureus*), by the formation of a biofilm (e.g., *Pseudomonas aeruginosa*), by sporulation, etc. To date, more than 100 different autoinducers are known for bacteria, archaea, and fungi.

Bacteria exhibit two main QS mechanisms, based on distinct signaling pathways, which present a certain analogy with the mechanisms found also in multicellular organisms. The first, generally used by Gram-negative bacteria, is based on the synthesis of a family of small molecules, the so-called AHL (acylated homoserine lactones). These molecules have a similar central structure and differ only in the length of a side chain, which specificity is determined by the length of the acyl chain and the substitution (▬H,▬OH or 〓O) on carbon. Generally, every type of bacterium can produce at least one AHL type; however, it can happen that bacteria produce more than one of them. Due to their chemical characteristics, AHL are capable to cross the bacterial membrane and spread outside; in addition, from the extracellular medium, they can freely enter within the cell and bind to specific receptors, called LuxR because the QS phenomenon was described for the first time in a microorganism, *Aliivibrio fischeri*, which is able to emit light *in vitro* only when its concentration exceeds a certain threshold [9]. In this microorganism, the AHL autoinducer is at low concentration and does not induce bioluminescence; when the bacterium is in the luminous organ of the giant squid, its cell density is high, so the transcriptional activator reaches the DNA, binds to the recognition sequence (LuxBox), and activates the transcription of genes for the enzyme luciferase, which produces bioluminescence. The advantage of this mechanism is to save energy, to ensure that bacteria become luminescent only when they are present in large numbers, and to prevent them from wasting energy when the population is toosmall to emit a visible signal. The complex AHL-LuxR in turn binds to the bacterial DNA, thus regulating the transcription of specific genes. In a situation where the concentration of bacteria is low, the level of synthesized AHL is below the threshold required for the LuxR bond. However, as the concentration of bacteria increases, the amount of AHL also increases, and the AHL-LuxR complex is formed accordingly. A gene encoding for the enzyme catalysing the AHL synthesis is present within the genes induced by the AHL-LuxR complex. This gives rise to a positive feedback leading to a rapid and synchronous answer from the whole microbial population. Signal transduction through the AHL-LuxR system, based on an extracellular messenger able to cross the membrane and on a receptor that is also a transcription factor, can be thought to obey the same logic of signaling through steroid hormones. On the other hand, Gram-positive bacteria use autoinducer molecules formed by peptides with a variable length ranging between 5 and 17 amino acids. Such molecules are produced by the processing of precursors and are often subjected to posttranslational modifications. These peptides require special transporters to be secreted in the extracellular environment that, in turn, is detected by a sensor histidine kinase [10] and transduces the signal through phosphorylation of intracellular targets. This mechanism of action is therefore similar to that used by growth factors in multicellular organisms. Not always, the nature of quorum sensing molecules (QSMs) is peptidic: for instance, some Gram-positive bacteria, such as *Streptomyces*, produce ɣ-butyrolactones as QSMs [11]. Finally, different researches report the autoinducer 2 (AI-2), with a rather unusual cyclic boronic ester, as a QS

system common to both Gram-positive and Gram-negative bacteria, although its role as a true QSM has been doubted for some microorganisms [12–15]. This system might give rise to a family of molecules that are supposed to operate as a common language for most bacteria.

The production of the AHL involved in the QS mechanism was recently discovered also in several Gram-negative bacteria, such as *Roseobacter* sp. TB60 and *Psychrobacter* sp. TB67 associated with the Antarctic sponge, *Anoxycalyx joubini* [16], indicating this a certain "universality" of the system. Dong and Zhang [17] and McDougald and co-workers [18] demonstrated the existence of other two novel signaling pathways: hydroxyl-palmitic acid methyl ester and methyl-dodecanoic acid.

## **2.1 QS in eukaryotes**

Some eukaryotic microorganisms monitor their population density through QS mechanisms [19, 20] too. This is not so surprising, taking into account that many bacteria and eukaryotic microorganisms inhabit in common ecological niches and often play similar challenges. In fungi, QS mechanisms are in charge to check and regulate processes such as sporulation and production of some molecules, such as secondary metabolites, as well as to those events giving rise to the morphological transition and enzyme secretion by the cells. Considering this and starting from the assumption that even this type of organisms is extremely varied, we can undoubtedly affirm that fungi exhibit different cell-cell communication mechanisms, using a wide variety of signal molecules [19]. Furthermore, fungi can communicate with bacteria and even with their plant or mammalian hosts. In yeasts and dimorphic fungi, aromatic alcohols originating from amino acids mediate the QS type of regulation [21]. Therefore, yeasts, through the production of tryptophol and phenylethyl alcohol, can manage the formation of pseudohyphae and biofilm [22] and probably trigger the virulence process toward some plants such as *Vitis vinifera* [23]. QSMs stimulate the exit from lag phase inducing germtube formation and hyphal development [24]. *Candida albicans* remains the most studied species from this point of view. It produces some QSMs, such as tyrosol, described also in other fungal species, such as *Saccharomyces cerevisiae* [25]. QSMs of *C. albicans* influence the formation and structure of biofilms [26, 27] as well as the dispersal of cells from a biofilm; hence, it, as well as other molecules, plays important roles in pathogenesis. E-farnesol, the other most known QSM produced by *C. albicans* [28], is an exogenous molecule that, on the contrary, inhibits biofilm formation when provided early during adherence; furthermore, it acts as an inhibitor of hyphal formation indeed [29]. Therefore, this organism can modulate its morphology (vegetative/hyphal) and, consequently, all events related, including the pathogenicity, through the modulating production mainly of these two QSMs. Dodecanol and γ-butyrolactone are other molecules identified as mediators of QS processes present in other eukaryotic organisms, such as the filamentous fungi, *Aspergillus* and *Penicillium* spp. Some species of *Penicillium*, such as *P. sclerotinum*, produce sclerotiorin, a secondary metabolite with antibiotic properties, and γ-butyrolactone-containing molecules such as multicolic acid, which act as QSMs [30]. Taking into account that Gram-negative bacteria produced lactones (AHLs) as QSMs and that filamentous fungi produce butyrolactone I [31], γ-heptalactone [32], and γ-butyrolactones [33], the discovery that γ-butyrolactones are produced also by the filamentous bacterium *Streptomyces* [11] suggests a convergent evolution or a horizontal gene transfer occurring during the evolution [30]. At the same time, different fungi, such as basidiomycete *Cryptococcus neoformans*, produce as QSMs some peptides, similar to how Gram-positive bacteria do. This means that fungi use a language "analogous" in some way to that exhibited by other phyla [10, 30].

**169**

**Figure 1.**

*Mechanism of biofilm formation.*

*Essential Oils and Microbial Communication DOI: http://dx.doi.org/10.5772/intechopen.85638*

processes of resistance to pathogens.

**2.2 Biofilm**

In some species of *Aspergillus*, such as *A. flavus*, oxylipins were identified as QSMs: these molecules modulate both the morphological differentiation and the production of either asexual spores or sclerotia. Furthermore, oxylipins regulate a QS-dependent pathway controlling development and mycotoxin production [34]. Fungi produce also other QSMs: terpenes, such as farnesol, are produced, for instance, by the dimorphic fungi *C. albicans* [28] and *Ophiostoma piceae* [35]; cyclic sesquiterpenes act as QSMs for the dimorphic fungus *Ophiostoma floccosum* [36]; QS alcohols, including tryptophol and phenylethyl alcohol, are produced by *S. cerevisiae* [21]. It is important to underline that the higher organisms evolved mechanisms with which they are capable to interfere with the quorum sensing process of the bacteria. These mechanisms could play an important role both for peaceful cohabitation of human and microorganisms, such as the case of the intestinal flora, and in

The term "biofilm" is referred to a structure, enough complex, formed by microbial cells, associated with each other that attach to a surface, which are in a certain sense kept isolated from the external environment (although they exert an important influence on this) through the formation of a sort of "dome" of polysaccharide nature [37]. Biofilm has generally a three-dimensional structure: it contains more or less channels and pores, used as a sort of intercellular communication channel and for the maintenance of the entire bacterial community [38, 39]. Biofilm is thus one of the subsequent mechanisms giving rise from the communication among bacteria, which precisely through the formation of biofilm and other microbial behaviors and social exchange (not only bioluminescence, conjugation, and virulence but also motility, sporulation, competence, etc.) form the social microbial system of interaction, the QS (**Figure 1**) [40, 41], that prokaryotes and some eukaryotes developed many millions of years before the actual human social media, which is certainly more organized and complex. The system is so organized and evolved that allows microorganisms to easily adapt to adverse environmental conditions and to use them even to switch to counterattack, with an action of growth, proliferation, and change in their metabolic pathways and morphology. Biofilms allow the survival of bacterial cells in a hostile environment; the extremely *Essential Oils and Microbial Communication DOI: http://dx.doi.org/10.5772/intechopen.85638*

In some species of *Aspergillus*, such as *A. flavus*, oxylipins were identified as QSMs: these molecules modulate both the morphological differentiation and the production of either asexual spores or sclerotia. Furthermore, oxylipins regulate a QS-dependent pathway controlling development and mycotoxin production [34]. Fungi produce also other QSMs: terpenes, such as farnesol, are produced, for instance, by the dimorphic fungi *C. albicans* [28] and *Ophiostoma piceae* [35]; cyclic sesquiterpenes act as QSMs for the dimorphic fungus *Ophiostoma floccosum* [36]; QS alcohols, including tryptophol and phenylethyl alcohol, are produced by *S. cerevisiae* [21]. It is important to underline that the higher organisms evolved mechanisms with which they are capable to interfere with the quorum sensing process of the bacteria. These mechanisms could play an important role both for peaceful cohabitation of human and microorganisms, such as the case of the intestinal flora, and in processes of resistance to pathogens.
