Quorum sensing (QS) is cell communication that is widely used by bacterial pathogens to coordinate the expression of several collective traits, including the production of multiple virulence factors, biofilm formation, and swarming motility once a population threshold is reached. Several lines of evidence indicate that QS enhances virulence of bacterial pathogens in animal models as well as in human infections; however, its relative importance for bacterial pathogenesis is still incomplete. In this review, we discuss the present evidence from in vitro and in vivo experiments in animal models, as well as from clinical studies, that link QS systems with human infections. We focus on two major QS bacterial models, the opportunistic Gram negative bacteria Pseudomonas aeruginosa and the Gram positive Staphylococcus aureus, which are also two of the main agents responsible of nosocomial and wound infections. In addition, QS communication systems in other bacterial, eukaryotic pathogens, and even immune and cancer cells are also reviewed, and finally, the new approaches proposed to combat bacterial infections by the attenuation of their QS communication systems and virulence are also discussed.

Several important bacterial pathogens, like Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus), and Vibrio cholerae, utilize quorum sensing (QS) cell communication to coordinate the expression of multiple virulence factors and associated behaviors such as swarming and biofilm formation, once a population size threshold is reached. QS systems consist of an enzyme that catalyzes the synthesis of the signal (such as acyl-homoserine lactones or cyclic peptides) and a receptor that binds the signal and reprograms the expression of several genes, including those encoding the enzyme that produces the signal, creating a positive feedback loop. In bacterial pathogens, most of the QS controlled genes codify several different virulence factors, such as proteases, toxins, and adhesins[1]. The expression of QS controlled phenotypes is energetically costly to the cells and only provides an advantage if it is expressed when cells reach high densities[2,3]; hence, in the context of bacterial infections, the expression of QS regulated virulence factors is delayed until a sufficient bacterial load is achieved and once this threshold is reached, bacteria coordinate their attack against the host, which maximizes the probability of establishing the infections and disseminating them, hence increasing the pathogen fitness. In fact, QS along with subversion of the immune system are the main factors that determine the bacterial infectious doses. Hence those bacterial pathogens that need small doses to infect, generally lack QS systems but are very effective at inactivating the immune response by killing professional phagocytes. In contrast, those bacterial pathogens that need high infectious doses rely in QS for the coordination of the expression of virulence[4]. In this review, the current knowledge about QS control of virulence factors in two main model bacterial pathogens, P. aeruginosa and S. aureus (which are also responsible for nosocomial and wound infections), will be discussed along with the relationship of their QS systems, its virulence in animal infection models, and the data available from human infections. Furthermore, the role of QS in other important infections and the role of QS in immune and cancer cells are discussed. Finally, proposed novel approaches of blocking QS/virulence as an alternative in fighting recalcitrant bacterial infections are also reviewed.

A number of animal models including the nematode (Caenorhabditis elegans), fruit fly (Drosophila melanogaster), zebrafish (Danio rerio) and mouse (Mus musculus), have been used to identify and define the role of virulence determinants in the pathogenesis of P. aeruginosa[43,44]. The attenuation of its QS is achieved by two basic strategies: (1) the utilization of mutant strains with QS genes disrupted; and (2) the quenching of QS by treatments that interfere with it; these methods have shown that QS systems as well as QS-independent virulence determinants are required for P. aeruginosa infections in animals.

The main animal model that was used to discover the relationship between QS and virulence of P. aeruginosa is the nematode C. elegans. In 1999 Tan and coworkers, first described conditions to test the role of QS in virulence using this model, showing that the reference strain PA14 kills the nematode either after days (slow killing) or quickly after a few hours (fast killing)[45]. Their evidence indicate that fast and slow killing occur by distinct mechanisms; the slow killing involves an infection-like process and correlates with accumulation of PA14 within worm intestines, while the fast killing is mediated by the production of phenazines(regulated by QS); that increase active oxygen species[45,46]. A third mode by which P. aeruginosa can kill C. elegans is lethal paralysis; this mechanism is mediated by QS since Darby and coworkers, using QS-less mutant strains of P. aeruginosa, found that the lethal effect is associated with a rapid neuromuscular paralysis, caused by the action of diffusible unidentified factors whose production requires the las and rhl genes, since the infection with a lasR mutant and with a rhlR reduces the paralysis (by 28%-100% and 100% respectively)[47]. A potential target of these diffusible factors is the EGL-9 worm protein, which is expressed in the neuronal muscle tissues[47]. In a recent study, a reduction of 83% in the death of the nematodes by the double mutant (PA14rhlRlasR) was reported; however, the analysis of individual mutants, revealed that only the rhlR mutant reduced death 69%, implying that the RhlR system is crucial for infection under their experimental conditions[48]. In addition to lethal paralysis and slow and fast killing, a fourth kind of C. elegans death induced by P. aeruginosa is the “red death”, characterized by the formation of red precipitate (PQS + Fe³⁺ complex) within the intestine of the nematodes. This mode of death is mediated by the quinolone dependant QS system Mvfr-PQS in coordination with the PhoB phosphate sensor and the pyoverdine iron acquisition system[49]. The role of QS in P. aeruginosa infectivity and virulence in C. elegans is also evidenced by the effect of QS inhibitors, since a synthetic analog of HSL, meta-bromo-thiolactone (Figure 2A) that partially inhibits in vitro the LasR and RhrlR systems also reduces the death of worms infected with PA14, to 60% at 24 h. Interestingly, the in vivo action of the quencher in the worm model occurs mainly through the RhrlR system[48]. Moreover, phenylacetic acid (Figure 2B), which is a byproduct of the degradation of antibiotics such as penicillin G and cephalosporin Gby G acylase[50], increases the survival of PAO1 infected nematodes by 53%, while untreated worms die within 72 h. This protective activity is perhaps a consequence of interfering with the LasR and RhrlR systems, due to the structural similarity of phenylacetic acid with salicylic acid, a quorum quencher[51]. Another compound,2,5-piperazinedione (Figure 2C), increases the survival of worms by 66%, compared to untreated ones, and it was shown by molecular docking that it interacts with an amino acid residue (E145) in LasR, which is required for correct binding of the natural HSL ligand[52]. Similarly cucurmin (Figure 2D), a secondary metabolite from Curcuma longa, increases the survival of worms by 28%; this compound decreases the expression of genes involved in biofilm formation and attenuates HSL production in PAO1. Thus, it was suggested that it may act as a quorum quencher, delaying the synthesis of HSL molecules or by impairing autoinducers perception[53]. Moreover, various enzymes that degrade natural autoinducers are able to decrease the pathogenicity of P. aeruginosa; for example, adding the purified acylase PvdQ to C. elegans infected with P. aeruginosa PAO1, strongly reduces their pathogenicity and increases the nematodes life span[54]. Although the utilization of C. elegans as a model for studying P. aeruginosa infections has been very fruitful, recently it was proposed to use the fruit fly (D. melanogaster) as an animal model for the study of the P. aeruginosa pathogenesis, since the fly has a higher similarity to human[55-57]. The importance of both P. aeruginosa HSL QS pathways for infection was also demonstrated in D. melanogaster using the feeding assay, in which the bacteria are ingested and a local infection type is established in the intestine. In this assay the PA103 (lasR), PDO100 (rhlI), PDO111 (rhlR), PAOR1 (lasR) and PAOJP2 (lasI/rhIl) mutant strains were avirulent with respect to wild-type PAO1 whose infected flies were killed at 14 d post-infection. Similarly, using the nicking assay (needle pricking), in which an injury is produced in the dorsum of the flies and P. aeruginosa is added to the wound, all mutant strains showed a lower death rate than wild-type, including the PDO100 mutant (rhlI) with 50% survival of the flies compared to 90% death for the PAO1 wild-type 35 d post-infection[57]. However, in contrast to the work with C. elegans, to date the effect of quorum quenching in P. aeruginosa virulence in the fly was not yet evaluated. With regard of these two infection models, Clatworthy and colleagues pointed out that a drawback to study P. aeruginosa infections using invertebrate hosts are the differences between their immune response and the one of vertebrates. For example, C. elegans and D. melanogaster do not have an adaptive immunity, or complex multilineage immune cells, such as those present in vertebrates[58]. Thus it is important to analyze the participation of QS in the pathogenesis of P. aeruginosa in vertebrate animal models, like zebrafish and mice. Specifically for zebrafish, the microinjection of PA14 QS^- mutant strains (lasR and mvfR) during two different stages of fish development [28 and 50 h post-fertilization (hpf)], revealed that the participation of these two transcriptional activators during the infections is different and is influenced by the maturity of the immune system at different stages of the embryo development, since for the lasR mutant, only a 40% decrease in the death of the embryos at 50 hpf (a developmental stage when both macrophages and neutrophils are present) was recorded, whereas the mvfR mutant showed a moderate effect by decreasing death by 20% to 28 hpf (an stage in which only macrophages are present), but a higher effect of 60% decrease at 50 hpf[58].

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Figure 2 Structures of quorum sensing inhibitors evaluated in animal models. A: Meta-bromo-thiolactone; B: Phenylacetic acid; C: 2,5-piperazinedione; D: Cucurmin; E: Furanone C-30; F: Furanone C-56; G: Ajoene 4,5,9,-trithiadodeca-1,6,11-triene-9-oxide; H: 2-amino-6-chlorobenzoic acid; I: 2-amino-6-fluorobenzoic acid; J: 2-amino-4-chlorobenzoic acid; K: AIP-II; L: RIP (H-Tyr-Ser-Pro-Trp-Thr-Asn-Phe-NH₂); M: FS8 (H-Tyr-Ser-Pro-Trp-Thr-Asn-Ala-NH₂).

For murine models, different protocols have been used to determine the participation of QS in P. aeruginosa pathogenicity[43,44]. The thermal induced injury model is frequently used and consists of producing a burn of second or third degree on the dorsal side of the mouse using water at 90 °C and subsequent inoculation of P. aeruginosa. Several experiments using this model have linked QS and virulence; for example, a PAO1-R1 (ΔlasR) mutant has a diminished ability to spread systemically, as well as lower dispersion through the lesion at early stages[59,60]. Also, mice infected with PA14 pqsA show a 75% survival rate in contrast to 10% survival with wild-type PA14[61]. Similarly, virulence is reduced in PAO1 lasR, lasI, and rhlI mutants, with the greatest effect seen for the double mutant lasI-rhlI that decreased the mortality of animals by approximately 88%, significantly reduced the number of c.f.u in the lesion, liver and spleen, and delayed the spread of the bacteria from the lesion[59]. In agreement, similar results were found using the pneumonia model in neonatal mice, in which lasR mutants showed reduced virulence and are unable to replicate efficiently in the lung tissue; as a consequence, less damage occurs and the bacterial infection does not spread[62]. A third kind of experimental infection, the foreign-body infection model, consists in introducing a fragment of P. aeruginosa infected silicone into the peritoneal cavity of mice. This model was successfully used to determine the participation of QS in biofilm formation[43]. In this system, the mutant strain lasR-rhlR disappears from the silicone fragments during the first 7 d of infection, in contrast with wild-type PAO1 cells which remain in the silicon implant for at least 14 and up to 21 d. Critically, the establishment of the PAO1 infection in the implants depends in the mouse strain that is used, since for Balb/c, bacterial counts in the implants decayed constantly from day one and several of the implants were completely cleared after 21 d, while for the NMRI strain, bacterial counts initially decreased (day 1 to 4), then remained constant and finally increased at levels similar to the initials at day 15[63].

Regarding studies testing the quorum quenching effect on virulence, by using the foreign body model, it was found that the intraperitoneal addition of furanone C-30 greatly increased the bacterial clearance rate (Figure 2E)[63]. In agreement a similar effect was observed with the lung infection model in mice in which a related compound, C-furanone 56 (Figure 2F) accelerated the bacterial clearance from the lungs, reducing the severity of the damage and significantly increasing mice survival[64]. Another quorum quencher, the synthetic molecule ajoene (ajoene 4,5,9,-trithiadodeca-1,6,11-triene-9-oxide) (Figure 2G), is able to attenuate the production of various P. aeruginosa QS-controlled virulence factors in vitro, while in the pulmonary infection model in mice infected with PAO1, its prophylactic administration from two days before the infection and during its course, reduced the bacterial c.f.u. in the lungs 500-fold relative to the non-treated mice[65]. Moreover, in the burn mice model, the intravenous administration of three related quorum quenchers, the anthranilic acid analogues: 2-amino-6-chlorobenzoic acid, 2-amino-6-fluorobenzoic acid, and 2-amino-4-chlorobenzoic acid (Figure 2H-J), that inhibit the biosynthesis of quinolone signals and disrupt the MvfR-dependant gene expression, restrict the systemic spread of the PA14 strain and decreases the animals death by 30% to 50%[61].

The importance of the QS systems of P. aeruginosa in human infections is highlighted by their presence in most clinical strains that were isolated during the moment of the infection. This was demonstrated in 2004 by Schaber et al[66], by screening 200 isolates from patients with urinary tract, lower respiratory tract, and wound infections. Of those isolates, 97.5% (195 isolates) had robust functional HSL-QS communication systems and hence were able to produce elastase (codified by the genes lasB and lasA, which expression is QS dependent through LasR) and high levels of both HSL autoinducers while only 5 isolates failed to satisfy those criteria; however, 2 isolates were identified as being the same bacteria, but isolated at two different times from the same patient, and for one isolate there was no clinical data available to support that it was implicated in an infective process[66]. Hence only approximately 1% of the isolates that were implicated in infections appeared to be QS deficient. Critically, one of these isolates had no lasR and rhlR functional genes. In addition the authors demonstrated that those isolates deficient in HSL-QS systems produced high levels of non-QS controlled virulence factors, such as the ExoS and ExoT proteins that are components of the type three secretion system. Hence, perhaps this was an adaptive response that potentially could compensate for the decrease in virulence caused by QS deficiency., Nevertheless, the production of those proteins in the QS proficient isolates was not evaluated, and the virulence of the isolates was not tested using infection models. Other studies have reported similar results;, for example, the characterization of 442 P. aeruginosa isolates colonizing the respiratory tract of 13 intubated patients identified 9 genotypically different strains and of these, 6 strains produced both HSL-autoinducers and the virulence factors: elastase, exoproteases, rhamnolipid, hydrogen cyanide, and pyocyanin in vitro, and two of them had mutations in both lasR and rhlR genes, while the third had a mutant lasR gene[67]. Another study performed with 100 isolates from patients with respiratory infections that were collected from sputum, tracheal aspirate, and bronchoalveolar lavage identified 11 HSL-QS deficient isolates, six of them with absent QS genes (one isolate negative for rhlR, two isolates negative for rhlI and rhlR, and three isolates were negative for rhlI, rhlR, lasI and lasR). Interestingly, this study found a negative correlation between the expression of QS controlled virulence factors and antibiotic resistance[68]. Furthermore, the analysis of 82 P. aeruginosa clinical strains isolated from urinary tract infections identified 6 isolates deficient in the production of both HSL autoinducers, biofilm, rhamnolipids, and elastase, correlating with the absence of the lasR gene in one isolate and the absence of lasI, lasR, rhlR in another isolate, while the other 4 isolates harbored point mutations that probably inactivated their lasI, lasR, rhlR, and rhlI genes[69].

Taken together, these independent studies indicate that about 90% of P. aeruginosa isolates that cause infection generally preserve active HSL-QS systems, although clearly a small percentage of the isolates have those systems impaired by mutations or loss of the important QS regulatory genes, nevertheless, in all these studies, the third QS system of P. aeruginosa, the quinolone dependent system, was not evaluated; hence, it is not reliable to conclude that these isolates were indeed 100% QS deficient. In addition, the existence of cell communication systems not yet described in this organism cannot be ruled out, and indeed in the reference strain PAO1, cell communication by fatty acids was recently discovered (DSF-like fatty acids, cis-2-decenoic acid) (Figure 1D)[70]. Another possibility that may explain the isolation of QS deficient strains from infections is the presence of multiple P. aeruginosa strains in the infection site and that the QS deficient isolates coexist with QS proficient strains; this was demonstrated recently in 8 patients with cystic fibrosis (CF), in which a complex mixture of QS-proficient and deficient isolates were found. Interestingly, among all the patients, the deficiency of the isolates in individual QS regulated phenotypes (LasA and LasB elastase, rhamnolipids, growth in adenosine, and HSL signals) ranged from 0 to approximately 90% and no single patient with 100% QS deficient isolates was found.

Such high diversity in isolates from the same patient likely is the result of a complex and multifactorial selective process, perhaps including social components like the advantages accrued by QS-deficient clones that use the resources made by the QS positive strains (siderophores, proteases, etc.), without contributing to the generation of the public goods; these bacteria are termed social cheaters[71]. Regarding the importance of QS for infections, these results indicate that at the population level, QS may be essential for CF infections; however, more studies increasing the number of CF patients and including other kinds of infections are necessary to better understand the importance of P. aeruginosa QS in the infective process. In addition, the elucidation of factors that shape the mosaic-like composition of isolates in patients or in animal models need to be determined in order to design better anti-QS therapies since the current ones are focused on laboratory strains with QS-proficient systems rather than clinical strains recently isolated from infections[72,73]. Although such factors are still unknown, some variables like: the severity and progression of the infection, the nutritional, health, and immunological status of the patients, the exposure of the susceptible individuals to only one, a few, or several strains and the bacterial loads during the infections could be involved. In this sense, animal models would be useful to evaluate the role of these and other valuables in the colonization diversity in the patients, for example experiments comparing the colonization of well feed animals and animals with a deficient nutrition, immune competent animals and immunosuppressed ones, or healthy animals compared to animals harboring important disorders such as the alpha-1-antitrypsin deficiency that promotes major pulmonary inflammation, degradation of lung tissue, and eventually manifestations of pulmonary emphysema, etc. using several bacterial strains (QS proficient and QS deficient) alone or in combination could be very valuable to determine the factors involved in the in vivo bacterial ecology in infections.

In addition, although P. aeruginosa virulence is multifactorial[74], the individual importance of QS controlled virulence factors in different kinds of infections is a current research area, and the role of molecules from the HSL autoinducers themselves, to extracellular factors like rhamnolipids, elastase, pyocyanin, etc. have been established. For example several independent studies have shown that the main P. aeruginosa autoinducer N-(3-oxododecanoyl)-HSL is readily detected in sputum samples collected from patients with cystic fibrosis[75-77], which correlates with a QS dependent gene expression during the infections[78-80]. However, besides its role as a signal, the autoinducer is also able to inhibit lymphocyte proliferation as well as secretion of tumor necrosis alpha by macrophages and interferon gamma by T-cells[27]. Moreover QS controlled secreted factors such as alkaline protease can interfere with the classical and the lectin pathway-mediated complement activation via cleavage of C2, blocking phagocytosis and killing of P. aeruginosa by neutrophils[81]. Also elastase, by cleaving the pulmonary surfactant protein-A, can contribute to phagocytosis evasion[82]. Furthermore, rhamnolipids are able to disrupt calcium-regulated pathways and protein kinase C activation, preventing the induction of human beta-defensin-2 in keratinocytes[83]. Remarkably, the production of rhamnolipids in mechanically ventilated patients is associated with the development of life-threatening ventilator-associated pneumonia (VAP), while elastase production and QS independent production of the cytotoxins ExoU and ExoS are not[84]. Another QS controlled virulence factor, the polysaccharide alginate, protects P. aeruginosa biofilm cells from IFN-gamma-mediated macrophage killing[85]. Surprisingly, the importance of pyocyanin, a blue redox-active compound which is one of the main P. aeruginosa virulence factors studied in vitro and one of the more notorious in infections (present in large quantities in sputum from patients with cystic fibrosis infected by P. aeruginosa) during clinical infection is still underexplored[86].

The participation of SarA and Agr S.aureus QS systems in pathogenicity has been evaluated using numerous animal models, in which the bacteria induce diseases such as osteomyelitis, septic arthritis, endocarditis, endophthalmitis and soft tissue abscesses. By using the mutant strains agr and sarA, as well as QS inhibition, the participation of these systems in the infectivity of the bacterium and the damage of tissues has been proved. Intravenous inoculation of the bacteria in mice induces the development of septic arthritis. In this model, agr mutants showed a reduced ability to induce the pathology since it is produced in only approximately 10% of the animals while the wild-type strain produces it in approximately 60% of the inoculated mice. Furthermore, in mice infected with the mutant strain, the arthritis severity is less, and only a few developed erosive arthropathy in contrast to those infected with wild-type[87]. Similarly in the endophthalmitis-rabbit model, which is established by the intraocular injection of the bacteria, agr mutants produced a smaller loss of neuroretinal function during the first 3 d of the infection, with respect to the wild-type strain. In addition, those infected with the mutant strain had normal eye histology, whereas those infected with the wild-type strain showed focal retinal destruction and mild vitritis[88]. In a subsequent study employing the same animal model, no significant differences in the rabbits eyes infected with the mutant strain (sarA) and with the wild-type were found; however, the simultaneous deletion of genes agr and sarA resulted in a near to complete attenuation of virulence[89]. Moreover, employing the model of endocarditis in rabbits, which consists in introducing a catheter into the ventricle and subsequently colonizing it by intravenous administration of the bacteria, it was observed that single mutations (sarA and agr) diminish the bacterial ability to induce the pathology, while a agr/sarA double mutant was incapable of inducing endocarditis in 100% of the animals inoculated with 10³ or 10⁴ c.f.u.[90]. Another infection model in mammals is the murine brain abscess model in which lesions are produced by embedding bacteria in agarose beads that are later inoculated in the cranial cavity. In this model, the agr/sarA double mutant, but not the single mutants, had reduced virulence, lower proliferation in the brain and poorly developed abscesses that were drastically smaller than those produced by the wild-type strain. Furthermore, the double mutation attenuates the expression of pro-inflammatory cytokines and chemokines[91]. Similarly invertebrate models such as the nematode C. elegans, which is killed by feeding on S. aureus, showed similar outcomes, since mutating sarA or agr increased the survival of the worms with respect to the wild-type strain[92].

Regarding the effect of quorum quenching in S. aureus animal infections, several inhibitory peptides have been evaluated; for example, in the murine subcutaneous abscess model, the administration of the synthetic autoinducer analog of AIP-II peptide (Figure 2K) in a single dose was able to decrease the formation of abscesses, and although AIP-II prevents expression of the S. aureus agr QS regulon for only a short time period, this transient inhibition is sufficient to achieve significant effects[93]. Other QS inhibitory peptides such as the RNAIII-inhibiting peptide (RIP) and its analogues (Figure 2K and L), that inhibit the phosphorylation of a target protein called “target of RNAIII-activating protein” (TRAP), leading to the suppression of virulence factor production in vitro[94,95], are also effective in vivo. For example, in the vascular-graft rat model, the administration of RIP (Figure 2L), both locally and systemically, is able to completely inhibit the formation of biofilms in graft and in polymethylmethacrylate beads infected with methicillin-susceptible and resistant S. aureus. Similarly, in the mouse sepsis model, administering RIP significantly reduced the bacterial load and mice mortality; this effect is potentiated by co-administration of antibiotics like cefazolin, imipenem, or vancomycin[96]. In addition, using the graft rat model, a RIP treatment increases its effectiveness in combination with antibiotics rifampin and temporin, and the complete elimination of infection is achieved by combining it with temporin A[97]. The same phenomenon has been documented with a derivative of RIP, termed FS3, which contains a substitution of alanine in the second position, since FS3 in combination with daptomycin has higher efficiency than single compounds, in the rat model of vascular graft staphylococcal infection[98]. Also in this model, a similar effect was obtained, by combining tigecycline and the RIP analogue called FS8 which contains a terminal alanine (Figure 2M)[99]. Taken together these extensive studies demonstrate the participation of S. aureus QS systems in its pathogenicity and indicate that QS inhibition in combination with antibiotics is a promising new strategy that may be effective to treat the infections produced by this important pathogen.

Although it is not always pathogenic to humans, the Gram (+) bacterium S. aureus, is frequently found in the human respiratory tract and on the skin and it is considered as a transient member of the human microbial flora[32], it is able to cause several kind of infections with a plethora of clinical manifestations. There are risk factors that complicate the infection caused by S. aureus, including the presence of prosthetic material and immunosuppression[100], and it is considered one of the three main causes of nosocomial bacterial infections. Among the many kinds of S. aureus infections, skin ones are very common; for instance, in children it is the main cause of impetigo, a superficial skin infection that according to its clinical manifestations is divided into non-bullous and bullous impetigo, the non-bullous is the most common form, the lesions begin as papules that progress to vesicles with erythema on its periphery. These become pustules that later form adherent crusts with a golden appearance and can be accompanied by regional lymphadenitis, although systemic symptoms are usually absent. Bullous impetigo is seen in young children in which the vesicles enlarge to form flaccid blisters with clear yellow liquid, which later becomes darker and turbid, leaving a thin brown crust[3,4]. Other common infections produced by S. aureus are hair follicle infection or folliculitis, furunculosis, and cellulitis. In addition to skin infections, S. aureus also causes respiratory tract infections such as nosocomial and septic pneumonia, septic pulmonary emboli and post viral empyema. It also infects the apocrine glands, causes musculoskeletal infections, produces bacteremia and its complications like sepsis, septic shock, and infective endocarditis and is able to produce toxic shock syndrome and food poisoning. Therefore, S. aureus is a major health concern worldwide.

Although the role of QS in regulating the expression of several virulence determinants including toxin production, and biofilm formation have been extensively studied in vitro and in animal models, its importance in actual human infections is yet under studied; nevertheless, it is known that as in the case of P. aeruginosa the great majority of S. aureus clinical isolates implicated in human infections possess active QS systems (agr⁺). Although some agr^- strains are also commonly found in S. aureus infections[101-103], the presence of both kind of strains during infection indicates that agr⁺ and agr^- variants may have a cooperative interaction[103] and also raises the possibility that social interactions like QS cheating may exist during the infections[39]. In addition, the link of QS and biofilm formation in S. aureus strongly suggests that this QS is important for the development and establishment of its chronic infections[32]; however, further work in this area is needed to define the importance and the specifics of QS in regulating S. aureus virulence in human infections.

In addition to P. aeruginosa and S. aureus, several other bacterial pathogens utilize QS systems to control the expression of multiple virulence factors during infection. Among the more relevant for human health are Vibrio spp, Acinetobacter spp, Burkholderia cepacea, and enteric bacteria like Escherichia spp. and Salmonella typhimurium. The following section is an overview of their known QS systems and their relationship with virulence.

QS systems have been extensively studied in bacteria and are of great interest for understanding the development of clinically-significant infections, but whether eukaryotes have cell signaling systems similar to bacterial QS mechanisms is a question that has recently drawn the attention of research worldwide. In this section, we will discuss some examples of eukaryotic microorganisms and human cells that use QS for the development of certain biological functions. The first report of a QS system in eukaryotes was carried out more than 40 years ago, when it was observed that dense cultures of the fungi Candida albicans show a reduced tendency towards the morphological transition from yeast to hypha, which is considered a key virulence factor for this opportunistic fungal pathogen[186]. To date, several compounds have been identified as responsible for this phenomenon, such as 2-phenylethanol, tryptophol, farnesol, farnesoic acid, and tyrosol[187]; these QS molecules are secreted by C. albicans and when they accumulate over a threshold level, they trigger changes in: (1) fungal dimorphisms[186]; (2) biofilm formation[188]; and (3) expression of virulence genes[189]. In addition, in other dimorphic fungi (Mucor rouxii, Histoplasma capsulatum, Ceratocystis ulmi), the “inoculum size effect” is usually observed; however, QS autoinducers in these organisms have not been identified[187]. In 1997, it was discovered that in the protozoan parasite of humans Trypanosoma cruzi, the differentiation of replicating and slender forms to non-dividing and stumpy ones is also a density-dependent (quorum) response that limits the population size[190]. This phenomenon is mediated by the soluble factor SIF (stumpy induction factor) that is released by trypanosomes, and a recent study revealed that the QS signaling in T. cruzi shares components with the quiescence pathways of mammalian stem cells, providing novel therapeutic targets via QS interference[191]. Like those parasitic species described thus far, in 2006 it was demonstrated that the budding yeast Saccharomyces cerevisiae endure morphological transition from the yeast form to a filamentous form in response to both cell density and the nutritional state of the environment. This induction is mediated by the phenylethanol and tryptophol auto signaling molecules, that regulate the transcription of a set of approximately 150 genes and which include FLO11, an essential gene for filamentous growth as well as several others genes that may play a role in the transition from the exponential to the stationary growth phase[192,193]. However, not only parasitic infections or traits present in unicellular eukaryotes are controlled by QS, since surprisingly in our body the number of cells of the immune system is maintained throughout a similar mechanism, where IL-2 is produced and secreted by activated CD4⁺ T cells and sensed with high affinity (IL-2Rα) by a population of CD4⁺ Treg, which in turn can regulate the number of total CD4⁺ T population[194] by competition for the IL-2 factor[195]. Failure of QS due to the absence of IL-2 or by defects on the sensor IL-2Rα leads to lymphoid hyperplasia and autoimmune diseases[196]. Furthermore, in 2009 Hickson et al[197] proposed that cancer cells may use QS mechanisms to operate as communities and regulate different multicellular functions as the metastatic process resembles bacterial biofilm formation and dispersion. This idea emerged based on several lines of evidence that suggested a close relationship between high cancer cell densities and high metastatic ability[197]. This relationship can be possibly explained by the fact that the cells secrete paracrine factors or autoinducers that increase their metastatic efficiency; these observations have been made since the 90’s[198] and recently by combining mathematical modeling with experimental evidence, the presence of QS systems in cancer was confirmed[199]. Such interesting findings are now opening new areas of study, including the development of future clinical applications (a summary of all the QS reviewed is provided in Table 1).