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Toxins (Basel). 2013 May; 5(5): 895–911.
Construction, Part, and Biology of the Enterococcus faecalis Cytolysin
Received 2013 Mar 25; Revised 2013 Apr 22; Accepted 2013 April 23.
Abstruse
Enterococcus faecalis is a Gram-positive commensal fellow member of the gut microbiota of a wide range of organisms. With the advent of antibiotic therapy, it has emerged as a multidrug resistant, hospital-caused pathogen. Highly virulent strains of E. faecalis express a pore-forming exotoxin, called cytolysin, which lyses both bacterial and eukaryotic cells in response to quorum signals. Originally described in the 1930s, the cytolysin is a member of a large form of lanthionine-containing bacteriocins produced by Gram-positive leaner. While the cytolysin shares some core features with other lantibiotics, it possesses unique characteristics every bit well. The current understanding of cytolysin biosynthesis, construction/function relationships, and contribution to the biology of Eastward. faecalis are reviewed, and opportunities for using emerging technologies to advance this agreement are discussed.
Keywords: cytolysin, lantibiotic, bacteriocin
1. Introduction: The Enterococci as Emergent Hospital Pathogens
Enterococci are aboriginal members of the beast microbiome that are believed to date back at least to the last common ancestor of mammals, reptiles, birds and insects in the early Devonian period, 412 meg years agone [1]. These leaner thrive in the nutrient-rich, oxygen-depleted environment of the intestinal tract, and at least in part because of shedding from animal hosts, are readily constitute in the environment [2]. They are core members of the commensal intestinal microbiota, which is densely colonized with up to 1011 bacterial cells/gram feces [three,4]. Enterococci are the predominant Gram-positive cocci constitute within this niche, and in humans they tin can exist isolated at concentrations of 105 to 10vii CFU/gram feces [five,six]. Bacteriophage induction in response to environmental cues is emerging as i strategy for enterococcal colonization and command in the intestinal ecosystem [7]. The presence of enterococci in the circuitous ecology of the gastrointestinal tract provides an aplenty reservoir where genetic exchange and selection tin occur.
Enteroccoci are low-GC, Gram-positive, non-sporulating, facultative anaerobes that currently rank among the about prevalent multidrug resistant infirmary pathogens worldwide [1]. They are the third well-nigh commonly isolated healthcare pathogen [eight], and are capable of causing a diversity of infections including endocarditis, sepsis, surgical wound infections, and urinary tract infections [5,nine,10]. The genus Enterococcus consists of over 40 ecologically diverse species [5,11], yet more than than 90 per centum of enterococcal infections are caused by 2 species: E. faecalis and E. faecium [8,10,12]. The genomes of multidrug resistant enterococci consist of more than 25 per centum mobile elements, which reflect a rampant accumulation of drug resistance elements and virulence factors [13]. Many enterococcal mobile elements are transferable by conjugation on pheromone-responsive plasmids, wide host range conjugative plasmids, or conjugative transposons [14,15]. The production of sex pheromone peptides by plasmid-free strains allows conjugative pheromone-responsive plasmids to transfer at rates every bit high as 10−three to 10−1 per donor cell [16], efficiently disseminating virulence and antibiotic resistance genes between strains [17,18].
The horizontal transfer of mobile elements has contributed much to the evolving fitness of enterococci in infirmary settings [11,18]. Since the 1960's, infirmary-associated enterococcal infections have become increasingly antibiotic resistant [19]. Antibiotic treatment results in a loss of protection from host colonization also as reduced microbial species multifariousness amongst the intestinal microbiota. This provides an opportunity for drug resistant enterococci to invade the intestinal niche and proliferate uncontrollably [3,20]. Horizontally acquired antimicrobial resistances were commencement described in the 1970's [21]. Analysis of an outbreak of multidrug resistant enterococcal bacteremia in the mid-1980s determined that half of all isolates were from the same hemolytic clone [22]. Later on, the first vancomycin-resistant clinical isolate of E. faecalis, strain V583, was isolated in the United States from the bloodstream of an infected patient [23]. Hospital endemic and epidemic multidrug resistant enterococcal infection rates accept since continued to increase worldwide [24,25,26].
Enterococcal illness was kickoff described in item in the tardily 19th century, when an abundant Gram-positive diploccocus was isolated from patients with intestinal diseases that was similar to an organism isolated from healthy patients [27]. This saprophytic microbe, named 'Enterocoque', was initially difficult to culture, most probable due to now-appreciated nutrient auxotrophies [27,28]. Pathogenicity was reproduced in rabbit and mouse models, in which inoculation lead to severe infection and fatality [27]. A further report from Thiercelin [29] described translocation of the bacteria from the gastrointestinal tract to the bloodstream, resulting in septicemia. At about the same time, MacCallum and Hastings described a death due to enterococcal infection causing acute endocarditis [30]. Originally designated as Micrococcus zymogenes, the bacterium isolated from the claret and cardiac vegetations of the patient was used to intraperitoneally infected mice, rabbits and dogs, and was constitute to restate the aforementioned endocarditis symptoms [thirty], satisfying Koch's Postulates and establishing Enterococcus as the crusade of the patient's expiry.
The ascertainment that some E. faecalis strains produced zones of hemolysis on blood agar plates led to the showtime comprehensive study of the hemolysin molecule [31]. Afterwards, hemolysis was plant to be acquired by a unique toxin, at present termed cytolysin, as it lyses a broad range of target cells including both Gram-positive leaner and eukaryotic cells [31,32,33,34,35]. The cytolysin is now known to brand a big contribution to the pathogenicity of E. faecalis [36,37].
two. Cytolysin and Toxicity of Enterococcal Infections
The cytolysin toxin of E. faecalis, termed a "streptolysin" since it was produced past Lancefield grouping D Streptococcus and acquired a zone of hemolysis on blood agar, was first experimentally characterized in 1934 [31]. E. faecalis (so called Streptococcus faecalis) was first considered to be pseudo-hemolytic, as hemolytic activeness could rarely exist detected in liquid broth but was readily seen on blood agar [31]. A gradient of erythrocyte susceptibilities, depending on species of origin, was observed, with homo, horse, dog, rabbit and mouse erythrocytes beingness susceptible, and sheep and goose erythrocytes beingness resistant to lysis. A horse flesh infusion was derived that supported product of cytolysin in liquid civilisation, allowing for its characterization as a heat-labile, oxygen stable molecule (in contrast to the family of thiol-activated, cholesterol dependent cytolysins produced by other Gram-positive bacteria) [31]. In add-on, hemolysin-producing enterococcal strains were observed to have bacteriocin activity against streptococcal strains and other Gram-positive leaner [38,39,xl]. The bactericidal and hemolytic phenotypes were experimentally characterized to be due to a single molecule. Brock et al. [39] showed that the hemolytic and bactericidal activities were both lost after UV irradiation and that restoration of one activity reestablished the other. The molecule was termed E. faecalis cytolysin to reflect the dual bactericidal and cytolytic activities exhibited [41].
The association of cytolysin expression and increased toxicity of enterococcal infections has been studied in multiple creature models, as well as in clinical outcomes (Table one). Ike and Clewell first described enhanced virulence due to cytolysin expression in the mouse through dose-dependent intraperitoneal injection with Eastward. faecalis strains harboring the plasmid pAD1, which encodes cytolysin [42]. After 7 days of infection with cytolysin negative strains (3 × 109 CFU) all mice survived, while mice injected with cytolysin positive strains (≥x9 CFU) died within 4–v h [43]. Later, cytolysin positive variants were shown to lyse mouse erythrocytes, macrophages and polymorphonuclear neutrophils [44]. Toxicity due to cytolysin was as well determined in a rabbit endocarditis model, whereby cytolysin and aggregation substance positive strains were lethal in 55 percent of infections, versus xv percentage in animals infected with only aggregation substance positive strains [45]. In rabbit endophthalmitis, cytolytic strains readily destroyed organ function and were untreatable, compared to isogenic, non-cytolytic strains [46,47]. When C. elegans is fed on lawns of cytolysin positive Eastward. faecalis, expiry occurs faster than when fed on isogenic non-cytolytic leaner [48].
Table 1
Contribution of the Due east. faecalis cytolysin to virulence.
| Setting | Upshot of Cytolysin | Reference |
|---|---|---|
| Human bacteremia | Cytolysin makes infection five times more than acutely lethal | [22] |
| Rabbit endophthalmitis | Cytolysin makes infection acutely destructive to retina and other ocular structures, and refractory to antibiotic treatment | [46,47,49] |
| Mouse intraperitoneal infection | Cytolysin makes infection approximately one hundred times more acutely lethal | [42,50] |
| Rabbit endocarditis | Cytolysin makes infection acutely lethal in synergy with assemblage substance | [45] |
| C. elegans ingestion | Cytolysin makes infection acutely lethal following ingestion | [48] |
The cytolysin has also been shown to be associated with increased toxicity in human infection. A retrospective study analyzed 190 clinical E. faecalis isolates and establish that 45 percent of isolates were cytolysin positive. Furthermore, even afterwards decision-making for treatment modality and drug resistance, patients infected with cytolytic E. faecalis were at a five-fold increased risk of an acutely final outcome (death within three weeks of diagnosis) compared to patients infected with non-cytolytic strains [22]. E. faecalis can crusade a severe postoperative endophthalmitis, and cytolytic strains have been establish to be common in these infections [51]. Epidemiological studies from Japan found that 60 pct of E. faecalis isolates analyzed from ii hospitals were cytolysin positive [52]. Another report constitute that hemolysis was common to all clinical enterococci isolates investigated (which is not typical), while only half-dozen percent of food isolates were hemolytic [53]. In addition to causing increased toxicity of infection, the bacteriocin activity of the cytolysin may well be an important colonization cistron of E. faecalis in the intestine, prior to establishment of infection at another sterile body site. In vitro experiments showed that cytolytic strains can outcompete bacteriocin-sensitive enterococci and other Gram-positive leaner in liquid goop culture [39]. Cytolysin was also observed to be produced by E. faecalis isolated from 9 out of 31 healthy infants in Norway [54].
Although a wide understanding of the genetics and biosynthesis of cytolysin is fairly avant-garde, many of the details of its production, equally well equally the precise mechanism past which it contributes to the pathogenesis of infection, are not well known. Hypothetically, the ability to lyse intestinal epithelial cells may permit E. faecalis to access the blood stream in order to travel to and colonize distant sites, such equally the heart valve. Additionally, the ability to lyse mouse neutrophils and macrophages might contribute to allowed evasion [44]. Other E. faecalis products, such as gelatinase and capsular polysaccharides, have been shown to help the bacteria to circumvent host immunity [55,56], just the precise office that the cytolysin might play in allowed evasion is still unknown.
3. Cytolysin Structure and Office
3.i. Overview of the Cytolysin
A full general scheme for cytolysin production, processing, secretion, and regulation is shown in Effigy 1. Enterococci produce a wide assortment of bacterocins, simply the cytolysin is the simply well characterized lantibiotic produced past E. faecalis [57]. Cytolysin production is a variable trait among E. faecalis isolates [xvi,52,58]. Among cytolysin-producing strains, the operon is either chromosomally-encoded within a 150-kilobase pathogenicity island (PAI) [43,59,60], or on a conjugative, pheromone-responsive plasmid, such every bit pAD1 [61,62,63]. The cytolysin operon consists of six genes related to toxin biosynthesis, besides as two divergently transcribed genes encoding regulatory proteins [41,64,65,66,67] (Figure 1A). In the inactive state, the cytolysin repressor poly peptide CylR2 binds to the PLys (PL) promoter [68]. Depression-level transcription of the operon is believed to consequence in basal production of a small amount of the cytolysin subunits [67]. Autoinduction via quorum sensing in the presence of target cells triggers an inferred alter in the bounden of the cytolysin promoter by the CylR2 protein, resulting in loftier-level expression of the cytolysin operon [67].
Eastward. faecalis cytolysin expression. (A) Cytolysin operon in the inactive and active states. In the inactive land, CylR2 binds to the PLys (PL) promoter [68]. Autoinduction via quorum sensing triggers an inferred change in the bounden of the cytolysin promoter by the CylR2 protein, resulting in high-level expression of the cytolysin operon [67]. (B) Cytolysin processing and secretion. Large and modest subunits are postal service-translationally modified by CylM [65], secreted and trimmed by CylB [41], and further processed past CylA [64]. (C) Cytolysin activity, in the absence and presence of target cells. In the absence of target cells the subunits form inactive and insoluble multimeric complexes. In the presence of target cells they coordinate to grade a pore in the target cell membrane [71].
The functional cytolysin toxin consists of large and small subunit oligopeptides, encoded by the genes cylLFifty and cylLS , respectively [64] (Effigy iB). CylLL and CylLDue south primary translation products undergo extensive mail service-translational modification, including dehydration of serine and threonine residues, and subsequent germination of intramolecular lanthionine and methyllanthionine bridges between these dehydrated residues and nearby cysteine thiol groups within each subunit [69,70]. Dehydration of the toxin subunits in the initial modification pace is inferred to be catalyzed by the CylM protein [65]. The ATP-binding cassette (ABC) transporter CylB secretes and trims the CylM-modified peptides CylLL * and CylLS * [41], resulting in externalization of CylLL' and CylLS' subunits. These trimmed and secreted subunits are further candy past the CylA serine protease to generate the active toxin subunits CylLL" and CylLS" [64,69]. The final gene in the cytolysin operon is cylI, which encodes the CylI immunity protein, a transmembrane protein of unknown function that confers cocky-protection to cytolysin-producing cells [66].
In the absenteeism of target cells, CylLFifty" and CylLS" strongly acquaintance to form inactive and insoluble multimeric complexes. However, when target cells are nowadays the subunits collaborate, presumably to form a pore in the target cell membrane [71] (Figure aneC). The large subunit CylL50" has a greater analogousness for the target jail cell membrane than the small subunit, which in the presence of a target cell is believed to effect in a transient accumulation of excess free CylLSouthward", generating a quorum sensing autoinduction bespeak that triggers release of CylR2 and high level expression of the cytolysin operon. The CylLDue south" induction betoken is believed to be transmitted in some way via the cell surface protein CylR1 [67].
3.ii. Cytolysin Structural and Molecular Properties
The E. faecalis cytolysin components CylLL and CylLSouthward take been classified as Type-A, pore-forming lantibiotics [72], and more recently equally ii-component, Course 2 lantibiotics [70]. Lantibiotics are complex polycyclic antimicrobial peptides, which are ribosomally synthesized past Gram-positive bacteria and are characterized by the presence of lanthionine and methyllanthionine bridges between dehydrated serine and threonine residues and cysteine thiols. Lantibiotics have extremely varied structures and functions, but they are all characterized by undergoing extensive post-translational modification and possessing either antibiotic or mophogenic activities [seventy]. Cytolysin appears to be unique among lantibiotics, in that it tin lyse other bacteria as well as erythrocytes and other eukayotic cells [73]. The cytolysin subunits possess stretches of identity within the primary translation products, which probable target them through the same maturation pathways. They also show limited identity, but dissimilar bridging patterns, to beta-peptides (also called LanA2 peptides) of the two-component lantibiotics lacticin 3147 from Lactococcus lactis [74], and haloduracin from Bacillus halodurans [75].
The positions of lanthionine linkages within the CylLL" and CylLS" peptides have recently been established [76] (Figure 2). Following ribosomal synthesis, the cytolysin subunit prepropeptides of 63 (CylLSouth) and 68 (CylLL) amino acids are modified mail service-translationally in the cytoplasm through reactions which are inferred to be catalyzed by CylM [65,72]. Kickoff, dehydration yields 2,iii-didehydroalanine (Dha) from serine, and (Z)-2,iii-didehydrobutyrine (Dhb) from threonine [lxx]. Then, neighboring intrapeptide cysteine residues make a nucleophilic, Michael addition to the dehydrated side bondage, resulting in thioether bonds between the Dha (or Dhb) and cysteine side chains, creating the unusual amino acids lanthionine (when serine is the precursor) or methyllanthionine (when threonine is the precursor). Interestingly, the mature cytolysin peptides announced to prefer a unique stereochemistry, with CylLFifty" containing ii lanthionine bridges in the unusual LL configuration, and CylLSouthward" containing 1 [76]. Virtually all previously characterized lantibiotics comprise bridges in the DL configuration. The functional consequence of this stereochemistry is currently unknown. The three-dimensional structures of the cytolysin peptides are likewise currently unknown, but recent advances in heterologous production of the subunits in E. coli will probable facilitate their determination [76].
Sequences and structures of the E. faecalis cytolysin subunits. (A) Primary amino acrid sequences of the cytolysin subunits CylLL and CylLS. Arrows point sites of proteolytic cleavage by CylB and CylA [69], and brackets prove the positions of lanthionine and methyllanthionine bridges. (B) Structures of the processed mature cytolysin subunits. Image is reproduced with permission from [76].
Post-obit modification of the cytolysin prepropeptides, the CylLL * and CylLS * propeptides are secreted from the cell by the production of the cylB factor [41]. During secretion, CylB removes 24 amino acids from the amino terminus of CylLFifty * and 36 amino acids from the amino terminus of CylLSouth *. This removal is believed to be catalyzed by a cysteine protease domain within CylB [41], and cleavage occurs within a nearly identical stretch of 26 amino acids in the otherwise structurally dissimilar subunits [69]. Whether all or part of these conserved 26 amino acid leader sequences constitutes a trafficking signal for CylM-mediated modification or CylB secretion remains to be explored.
Precisely how the cytolysin modification enzymes CylM, CylB, and CylA are produced, processed and sent to their last destinations is unclear. The E. coli hemolysin A toxin (HlyA) is processed and secreted by a type i secretion organization consisting of the inner membrane protein HlyB, the membrane fusion poly peptide HlyD, and the outer membrane poly peptide TolC, which form a continuous just transient translocator from the cytosol directly out of the cell to allow for HlyA secretion [77].Complex natural products, including antibiotics, are also synthesized in processive steps by multienzyme megasynthase complexes as large equally 2 MDa [78]. Experimental bear witness suggests that the proteins involved in postal service-translational modification and secretion of the lantibiotic subtilin might also organize into a membrane bound complex [79]. Because of the need for processivity in the maturation of the cytolysin prepropeptides [65], it seems possible that CylM, CylB, and CylA may similarly be organized in a transmembrane complex that efficiently modifies, secretes and activates each subunit, only this remains to exist shown.
3.3. Cytolysin Regulation
The cytolysin operon contains 2 promoters; the PL promoter regulates transcription of genes related to toxin structure and function (cylLFifty , cylLS , cylM, cylB, cylA, and cylI), while the PReg (PR) promoter overlaps with PL and regulates transcription of the regulatory genes cylR1 and cylR2, which are transcribed in the opposite direction from the rest of the operon [67,68] (Figure 1A). In the uninduced land, the cytolysin operon is believed to be transcribed at a depression level, so that a small amount of all system components are available to respond to the presence of target cells when the need arises [67]. When target cells are present, the large subunit CylLL" preferentially binds to prison cell membranes with greater analogousness than the pocket-sized subunit CylLS", leading to a transient accumulation of complimentary small subunit in solution [71]. One time the concentration of free CylLS" exceeds a threshold, it induces transcription of the cytolysin operon from the PL promoter, presumably through altered association or dissociation of the CylR2 protein from the promoter region. The crystal construction of CylR2 was solved and the precise nature of its binding to PL has been determined in vitro [68]. It is also known that CylR1, a suspected membrane protein, is required for induction of the cytolysin operon [67]. However, the precise machinery of how the accumulation of extracellular CylLSouth" is transmitted to intracellular CylR2, as well equally the office of CylR1 in transmitting this signal, are not currently understood.
While information technology appears that the genes inside the cytolysin operon are transcribed polycistronically, and that the operon contains at least ii promoters, there is some experimental evidence to suggest that transcription may be more complex. Based on the behavior of transposon insertion mutants, the final two genes within the cytolysin operon, cylA and cylI, were originally thought to be transcribed independently from the rest of the operon [64,66]. Even so, promoter elements as well PL and PR have yet to be identified. In the active state, transcripts from cylL50 and cylLSouthward are far more abundant than transcripts of any other cytolysin components, possibly due to a stem-loop structure between cylLL and cylM that may form a conditional terminator element [80]. Prior experiments that focused on quantifying transcription of the various cytolysin operon components have relied on PCR-based approaches [fourscore], which can artificially simplify the picture through selective amplification of a preferred species. Newly developed technologies, such every bit RNA sequencing (RNA-seq), would allow more precise quantification of expression levels of all operon components simultaneously, and can distinguish the directionality of transcription too as transcription initiation from processing sites [81].
Every bit noted above, CylR1 plays a role in transmitting the induction signal or otherwise facilitates induction of cytolysin operon transcription in the presence of target cells [37,67], but the mechanism is not obvious. 1 possibility is that CylR1 and CylR2 may form a novel two-component regulatory system that lacks the phosphorelay elements common to classical bacterial ii-component systems [82]. CylR1 contains three predicted alpha-helical transmembrane domains, and is therefore believed to localize to the prison cell membrane, only this awaits verification. As a membrane protein, CylR1 could sense excess CylLS", either in the surroundings or in contact with the membrane. Previous models accept depicted CylR1 associating directly with CylR2, suggesting that a conformational change initiated by CylR1 causes CylR2 to dissociate with the PL promoter region [67,83]. Alternately, CylR1 could facilitate CylLS" internalization into the cytoplasm, perhaps in clan with a cellular oligopeptide permease, similar to the machinery of internalization in E. faecalis for pheromone signaling [84].
iii.four. Toxin Machinery of Activeness
Very footling is currently known regarding the nature of the interaction between cytolysin toxin subunits, either in the presence or the absence of target cells. The large subunit CylL50" binds to target cells with about a seven-fold greater affinity than CylLSouth" [71]. Interestingly, the firsthand precursors of the active toxin subunits, CylL50' and CylLS', are just six amino acids longer than the fully mature subunits, withal these precursors practise non detectably associate with each other, and have no detectable hemolytic activity [69]. This suggests that the amino terminus of the fully processed toxin subunits is instrumental in their association with membranes and into polymers.
Exactly how the CylLL" and CylLS" subunits compromise target cell membranes leading to lysis is unclear, but is probable to conduct some similarity to pore formation past the well-studied lantibiotics nisin and lacticin 3147, both produced by Lactococcus lactis [85,86]. Nisin forms pores via a multi-step process involving: (1) binding to the bacterial jail cell wall forerunner molecule lipid Ii; and (2) reorientation of nisin molecules from parallel to perpendicular to the membrane surface [87]. The amino-concluding rings of nisin bind to lipid II, and the carboxy-terminus interacts with the lipid bilayer of the target bacterial cell. Accumulation of lipid II and nisin in this way results in a pore formed by four lipid Ii and eight nisin molecules in an unknown structural organization [88]. Pore formation past lacticin 3147, a two-component lantibiotic, also involves multiple steps: (i) the LtnA1 subunit kickoff associates with the membrane; (2) it forms a complex with the LtnA2 subunit in a 1:1 stoichiometry; and (3) LtnA2 in the complex and then enters the membrane and forms a pore [89]. Whether the cytolysin subunits interact in a similar way, as well as their stoichiometry, remains to exist adamant.
Target cell surface receptor, or prison cell surface receptors, that enable cytolysin-mediated lysis are unknown. Cytolysin is unique among lantibiotics in its ability to lyse a broad range of cells, including leaner, diverse mammalian erythrocytes, and other eukaryotic cells [90]. If there is a specific receptor, information technology would accept to be highly conserved beyond widely divergent kingdoms. As noted above, nisin and many other lantibiotics employ lipid II as a docking molecule [91,92]. This could be a possible candidate for cytolysin targeting of leaner, but this would invoke different mechanisms for prokaryotic and eukaryotic prison cell lysis. A higher membrane phosphatidylcholine content has been found in those erythrocytes that are almost susceptible to lysis by cytolysin [34], and both sphingomyelin and phosphatidylcholine inhibit the lysis of equus caballus erythrocytes by cytolysin [44]. Finally, cytolysin action could exist due to general membrane properties, with susceptibility at least in office attributable to the absence of an inhibitor on the target cell surface, such as lecithin [39].
The way in which cytolysin-producing cells are protected from self-lysis, and how immunity is transferred betwixt cells, also are not well understood. Other lantibiotic-producing bacteria are protected from self-lysis by immunity proteins and/or ABC transporters that serve to decrease the local concentration of the lantibiotic [93,94]. In Eastward. faecalis, the immunity factor CylI, an apparent transmembrane protein with possible zinc metalloprotease activity, was shown to be necessary and sufficient to confer protection from cytolysin-mediated bacterial cell death [66]. Information technology is unknown whether CylI interacts with and/or cleaves 1 or both cytolysin subunits, but information technology seems possible that CylI could prevent pore formation by cleaving subunits that attempt to embed within the producer cell membrane, or by cleaving proteinaceous target cell receptors, should they exist. Because the cytolysin operon is encoded on transmissible plasmids and a mobile pathogenicity island, an important unanswered question remains as to how the operon encoding the cytolysin is transferred from an immune-producing cell to a susceptible recipient without first killing the recipient. 1 possible explanation for recipient cell protection might exist the need for high bacterial jail cell levels to induce cytolysin expression [67]. Perhaps the pheromone quorum signaling pathway involved in pAD1 transfer is triggered at lower jail cell densities, before the threshold for derepression of cytolysin expression is reached.
3.five. Biological Function of Cytolysin
Many studies of the E. faecalis cytolysin are motivated by findings that this molecule exacerbates infection in humans and model systems [22,43,51]. All the same, because of the relative rarity of E. faecalis infection in comparison to its abundance as a commensal in the GI tract of diverse animals, it seems likely that this toxin evolved for a more than common purpose, where positive selection is more likely to apply. For a commensal microbe that is dependent upon its host (and the microbial customs that the host supports) to fulfill its auxotrophies, it seems probable that selection for the cytolysin occurred in an environment that was mutually beneficial to both E. faecalis and its host. Possible cytolysin activities that could benefit a host might include: providing a defense against something that is more harmful (such as an intestinal parasite), acting as a colonization cistron, or facilitating nutrient acquisition from prokaryotic or eukaryotic sources. Maybe the bacteriocin activity of the cytolysin allows E. faecalis to occupy a novel host niche that non-cytolytic bacteria cannot admission. The impact that cytolysin production has on the host microbiome has non yet been investigated, although recent advances in microbial environmental and metagenomics should exist able to readily address this question in humans [95], or other natural hosts [96,97]. Additionally, E. faecalis can incorporate exogenous hemin into its cytochromes, and this was institute to provide a growth reward nether aerobic weather condition [eleven,98]. Mayhap the ability to co-opt extracellular hemin from a host or neighboring organism confers a big enough growth advantage to drive the evolution of target jail cell lysis by cytolysin.
In addition to possible roles in colonization and food acquisition, the cytolysin appears to role at to the lowest degree in part equally a signaling molecule that can monitor bacterial population size and probe the environs for target cells [83,99]. Cytolysin subunits are produced and secreted into the surroundings, but their relative abundance is too monitored by the producer jail cell and when target cells are close by, the pocket-size subunit CylLS" becomes a signaling molecule that induces a alter in factor expression, turning on product of boosted cytolysin subunits [71]. The power to recognize the presence or absence of target cells allows E. faecalis to respond to its environment in a more nuanced manner, and may contribute to the successful colonization of many unlike environmental niches.
4. Conclusions
The recent evolution of Due east. faecalis strains that are both hypervirulent and multidrug resistant underscores the demand for a better understanding of the biology of this of import pathogen. The cytolysin forms a critical function of this understanding, as information technology contributes more to infection toxicity than whatsoever other E. faecalis factor studied, and it likely also allows Due east. faecalis to colonize new ecologies. A better agreement of the East. faecalis cytolysin may help in understanding the biological mechanisms of other lantibiotics, likewise as deepen our knowledge of how Enterococcus evolved this molecule in the first place. Application of the latest genomics-age technologies will certainly shed new lite on the biology of the E. faecalis cytolysin, and volition provide a more complete understanding of the structure and function of this important molecule.
Acknowledgments
Portions of this piece of work were supported past the NIH/NIAID supported Harvard-wide Program on Antibiotic Resistance (AI083214), and by NIH research grants to examine different aspects of the pathogenesis of enterococci (AI072360, EY08289). The authors also thank current and quondam members of the Gilmore Laboratory for input and feedback during the preparation of this manuscript.
Conflict of Interest
The authors declare no conflict of involvement.
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