Role of the Enterococcus faecalis GelE protease in determination of cellular chain length,supernatant pheromone levels,and degradation of fibrin and misfolded surface proteins

Gelatinase (GelE), a secreted Zn-metalloprotease of Enterococcus faecalis, has been implicated as a virulence factor by both epidemiological data and animal model studies. Expression of gelE is induced at a high cell density by the fsr quorum-sensing system. In the present study, GelE was shown to be responsible for the instability of a number of Asc10 (aggregation substance) mutant proteins, implying that GelE functions to clear the bacterial cell surface of misfolded proteins. Disruption of GelE production led to increased cell chain length of E. faecalis, from a typical diplococcus morphology to chains of 5 to 10 cells. This function of GelE was also exhibited when the protein was expressed in Streptococcus pyogenes. GelE-expressing E. faecalis strains were more autolytic, suggesting that GelE affects chain length through activation of an autolysin. GelE was also essential for degradation of polymerized fibrin. GelE expression reduced the titer of cCF10, the peptide pheromone that induces conjugation of pCF10, and pCF10 had increased conjugation into non-GelE-expressing strains. These new functions attributed to GelE suggest that it acts to increase the dissemination of E. faecalis in high-density environments.     

Library Screen Identifies Enterococcus faecalis CcpA,the Catabolite Control Protein A,as an Effector of Ace,a Collagen Adhesion Protein Linked to Virulence

The Enterococcus faecalis cell wall-anchored protein Ace is an important virulence factor involved in cell adhesion and infection. Expression of Ace on the cell surface is affected by many factors, including stage of growth, culture temperature, and environmental components, such as serum, urine, and collagen. However, the mechanisms that regulate or modulate Ace display are not well understood. With interest in identifying genes associated with Ace expression, we utilized a whole-cell enzyme-linked immunosorbent assay (ELISA)-based screening method to identify mutants from a transposon insertion mutant library which exhibited distinct Ace surface expression profiles. We identified a ccpA insertion mutant which showed significantly decreased levels of Ace surface expression at early growth phase versus those of wild-type OG1RF. Confirmation of the observation was achieved through flow cytometry and complementation analysis. Compared to the wild type, the E. faecalis ccpA mutant had an impaired ability to adhere to collagen when grown to early exponential phase, consistent with the lack of Ace expression in the early growth phase. As a key component of carbon catabolite regulation, CcpA has been previously reported to play a critical role in regulating expression of proteins involved in E. faecalis carbohydrate uptake and utilization. Our discovery is the first to associate CcpA with the production of a major E. faecalis virulence factor, providing new insights into the regulation of E. faecalis pathogenesis.     

Regulation of autolysis-dependent extracellular DNA release by Enterococcus faecalis extracellular proteases influences biofilm development

Enterococci are major contributors of hospital-acquired infections and have emerged as important reservoirs for the dissemination of antibiotic resistance traits. The ability to form biofilms on medical devices is an important aspect of pathogenesis in the hospital environment. The Enterococcus faecalis Fsr quorum system has been shown to regulate biofilm formation through the production of gelatinase, but the mechanism has been hitherto unknown. Here we show that both gelatinase (GelE) and serine protease (SprE) contribute to biofilm formation by E. faecalis and provide clues to how the activity of these proteases governs this developmental process. Confocal imaging of biofilms suggested that GelE(-) mutants were significantly reduced in biofilm biomass compared to the parental strain, whereas the absence of SprE appeared to accelerate the progression of biofilm development. The phenotype observed in a SprE(-) mutant was linked to an observed increase in autolytic rate compared to the parental strain. Culture supernatant analysis and confocal microscopy confirmed the inability of mutants deficient in GelE to release extracellular DNA (eDNA) in planktonic and biofilm cultures, whereas cells deficient in SprE produced significantly more eDNA as a component of the biofilm matrix. DNase I treatment of E. faecalis biofilms reduced the accumulation of biofilm, implying a critical role for eDNA in biofilm development. In conclusion, our data suggest that the interplay of two secreted and coregulated proteases--GelE and SprE--is responsible for regulating autolysis and the release of high-molecular-weight eDNA, a critical component for the development of E. faecalis biofilms.     

Gelatinase-associated phenotypes and genotypes among clinical isolates of Enterococcus faecalis in Poland

Enterococcus faecalis is an important nosocomial pathogen causing serious invasive infections. One of the virulence factors of this pathogen, gelatinase GelE, is a protease whose gene expression is regulated by the Fsr quorum sensing system. In this study, we used a well-characterized collection of 153 clinical E. faecalis isolates to investigate the distribution of genes involved in gelatinase expression. Although 140 isolates (91% of the group) harbored the gelE gene, only 81 isolates (53%) produced active gelatinase. The gelatinase-negative phenotype was found in several unrelated clones, and appeared to be caused by various genetic events. Isolates of the hospital-adapted clonal complex 2 (CC2) and of CC40 were uniformly gelatinase-positive, while all the CC87 isolates contained the 23.9 kb deletion encompassing most of the fsr locus and were gelatinase-negative. No significant differences among isolates of different clinical origin and gelatinase activity or presence of the fsr genes were found with the exception of isolates from cerebrospinal fluid, which were more often gelatinase-positive than colonizing isolates.     

Immune evasion of Enterococcus faecalis by an extracellular gelatinase that cleaves C3 and iC3b

Enterococcus faecalis (Ef) accounts for most cases of enterococcal bacteremia, which is one of the principal causes of nosocomial bloodstream infections (BSI). Among several virulence factors associated with the pathogenesis of Ef, an extracellular gelatinase (GelE) has been known to be the most common factor, although its virulence mechanisms, especially in association with human BSI, have yet to be demonstrated. In this study, we describe the complement resistance mechanism of Ef mediated by GelE. Using purified GelE, we determined that it cleaved the C3 occurring in human serum into a C3b-like molecule, which was inactivated rapidly via reaction with water. This C3 convertase-like activity of GelE was shown to result in a consumption of C3 and thus inhibited the activation of the complement system. Also, GelE was confirmed to degrade an iC3b that was deposited on the Ag surfaces without affecting the bound C3b. This proteolytic effect of GelE against the major complement opsonin resulted in a substantial reduction in Ef phagocytosis by human polymorphonuclear leukocytes. In addition, we verified that the action of GelE against C3, which is a central component of the complement cascade, was human specific. Taken together, it was suggested that GelE may represent a promising molecule for targeting human BSI associated with Ef.     

Comparison of genotypic and phenotypic cluster analyses of virulence determinants and possible role of CRISPR elements towards their incidence in Enterococcus faecalis and Enterococcus faecium

Enterococcus faecalis and Enterococcus faecium are human commensals frequently found in fermented foods or used as probiotics, but also recognized as opportunistic pathogens. We investigated 62 Enterococcus strains isolated from clinical, food and environmental origins towards a rationale for safety evaluation of strains in food or probiotic applications. All isolates were characterised with respect to the presence of the virulence determinants fsrB, sprE, gelE, ace, efaAfs/fm, as, esp, cob and the cytolysin operon. In addition RAPD-PCR was used to obtain genomic fingerprints that were clustered and compared to phenotypic profiles generated by MALDI-TOF-MS. The gelatinase phenotype (GelE) and the haemolytic activity (β-haemolysis) were analysed. E. faecium strains contained esp and efaAfm only, and none of them contained any CRISPR elements. The amenability of E. faecalis strains to acquisition of virulence factors was investigated along the occurrence of CRISPR associated (cas) genes. While distribution of most virulence factors, and RAPD versus MALDI-TOF-MS typing patterns were unrelated, 2 out of 5 RAPD clusters almost exclusively contained clinical E. faecalis isolates, and an occurrence of CRISPR elements versus reduced number of virulence factors was observed. The presence of the cytolysin operon, cob and as encoding pheromone and aggregation substance, respectively, significantly corresponded to absence of cas. As their production promote genetic exchange, their absence limits further gene acquisition and distribution. Thus, absence of the cytolysin operon, cob and as in a cas positive environment suggests itself as promising candidate for E. faecalis evaluation towards their occurrence in food fermentation or use as probiotics.     

Esp-independent biofilm formation by Enterococcus faecalis

Enterococcus faecalis is a gram-positive opportunistic pathogen known to form biofilms in vitro. In addition, this organism is often isolated from biofilms on the surfaces of various indwelling medical devices. However, the molecular mechanisms regulating biofilm formation in these clinical isolates are largely unknown. Recent work has suggested that a specific cell surface protein (Esp) of E. faecalis is critical for biofilm formation by this organism. However, in the same study, esp-deficient strains of E. faecalis were found to be capable of biofilm formation. To test the hypothesis that Esp is dispensable for biofilm formation by E. faecalis, we used microtiter plate assays and a chemostat-based biofilm fermentor assay to examine biofilm formation by genetically well-defined, non-Esp-expressing strains. Our results demonstrate that in vitro biofilm formation occurs, not only in the absence of esp, but also in the absence of the entire pathogenicity island that harbors the esp coding sequence. Using scanning electron microscopy to evaluate biofilms of E. faecalis OG1RF grown in the fermentor system, biofilm development was observed to progress through multiple stages, including attachment of individual cells to the substratum, microcolony formation, and maturation into complex multilayered structures apparently containing water channels. Microtiter plate biofilm analyses indicated that biofilm formation or maintenance was modulated by environmental conditions. Furthermore, our results demonstrate that expression of a secreted metalloprotease, GelE, enhances biofilm formation by E. faecalis. In summary, E. faecalis forms complex biofilms by a process that is sensitive to environmental conditions and does not require the Esp surface protein.     

Different protein expression profiles in cheese and clinical isolates of Enterococcus faecalis revealed by proteomic analysis

The use of Enterococcus faecalis in the food industry has come under dispute because of the pathogenic potential of some strains of this species. In this study, we have compared the secretome and whole-cell proteome of one food isolate (E. faecalis DISAV 1022) and one clinical isolate (E. faecalis H1) by 2-DE and iTRAQ analyses, respectively. Extracellular protein patterns differed significantly, with only seven proteins common to both strains. Notably, only the clinical isolate expressed various well-characterized virulence factors such as the gelatinase coccolysin (GelE) and the extracellular serine proteinase V8 (SprE). Moreover, various other putative virulence factors, e.g. superoxide dismutase, choline- and chitin-binding proteins and potential moonlighting proteins, have been detected exclusively in the secretome of the clinical isolate, but not in the food isolate. The iTRAQ analysis of whole-cell proteins of the two strains highlighted a stronger expression of pathogenic traits such as an endocarditis-specific antigen and an adhesion lipoprotein in the pathogenic strain E. faecalis H1. Subsequently, six food isolates (including E. faecalis DISAV 1022) and six clinical isolates (including E. faecalis H1) were tested for the presence of gelatinase and protease activity in the culture supernatants. Both enzymatic activities were found in the clinical as well as the food isolates which clearly indicates that protease expression is strain specific and not representative for pathogenic isolates. Genetic analyses revealed that not only the gelatinase and serine protease genes but also the regulatory fsr genes must be present to allow protease expression.     

Heterologous inducible expression of Enterococcus faecalis pCF10 aggregation substance asc10 in Lactococcus lactis and Streptococcus gordonii contributes to cell hydrophobicity and adhesion to fibrin

Aggregation substance proteins encoded by the sex pheromone plasmid family of Enterococcus faecalis have been shown previously to contribute to the formation of a stable mating complex between donor and recipient cells and have been implicated in the virulence of this increasingly important nosocomial pathogen. In an effort to characterize the protein further, prgB, the gene encoding the aggregation substance Asc10 on pCF10, was cloned in a vector containing the nisin-inducible nisA promoter and its two-component regulatory system. Expression of aggregation substance after nisin addition to cultures of E. faecalis and the heterologous bacteria Lactococcus lactis and Streptococcus gordonii was demonstrated. Electron microscopy revealed that Asc10 was presented on the cell surfaces of E. faecalis and L. lactis but not on that of S. gordonii. The protein was also found in the cell culture supernatants of all three species. Characterization of Asc10 on the cell surfaces of E. faecalis and L. lactis revealed a significant increase in cell surface hydrophobicity upon expression of the protein. Heterologous expression of Asc10 on L. lactis also allowed the recognition of its binding ligand (EBS) on the enterococcal cell surface, as indicated by increased transfer of a conjugative transposon. We also found that adhesion of Asc10-expressing bacterial cells to fibrin was elevated, consistent with a role for the protein in the pathogenesis of enterococcal endocarditis. The data demonstrate that Asc10 expressed under the control of the nisA promoter in heterologous species will be an useful tool in the detailed characterization of this important enterococcal conjugation protein and virulence factor.     

The Enterococcus faecalis exoproteome: identification and temporal regulation by Fsr

Analysis of the culture supernatant exoproteins produced by two PFGE clusters of high-level gentamicin and ciprofloxacin-resistant clinical isolates of Enterococcus faecalis from the UK and Ireland revealed two distinct protein profiles. This grouping distinguished OG1RF and GelE metalloprotease-expressing isolates from JH2-2 and other GelE-negative isolates. The integrity of the fsrABDC operon was found to determine the exoproteome composition, since an fsrB mutant of strain OG1RF appeared very similar to that of strain JH2-2, and complementation of the latter with the fsrABDC operon produced an OG1RF-like exoproteome. The proteins present in the supernatant fraction of OG1RF were separated using 2D gels and identified by mass spectrometry and comprised many mass and pI variants of the GelE and SprE proteases. In addition cell wall synthesis and cell division proteins were identified. An OG1RF fsrB mutant had a distinct exoprotein fraction with an absence of the Fsr-regulated proteases and was characterised by general stress and glycolytic proteins. The exoproteome of the OG1RF fsrB mutant resembles that of a divIVA mutant of E. faecalis, suggestive of a stress phenotype.     

Fsr1, a striatin homologue,forms an endomembrane‐associated complex that regulates virulence in the maize pathogen Fusarium verticillioides

Fsr1, a homologue of mammalian striatin, containing multiple protein‐binding domains and a coiled‐coil (CC) domain, is critical for Fusarium verticillioides virulence. In mammals, striatin interacts with multiple proteins to form a STRIPAK (striatin‐interacting phosphatase and kinase) complex that regulates a variety of developmental processes and cellular mechanisms. In this study, we identified the homologue of a key mammalian STRIPAK component STRIP1/2 (striatin‐interacting proteins 1 and 2) in F. verticillioides, FvStp1, which interacts with Fsr1 in vivo. Gene deletion analysis indicates that FvStp1 is critical for F. verticillioides stalk rot virulence. In addition, we identified three proteins, designated FvCyp1, FvScp1 and FvSel1, which interact with the Fsr1 CC domain via a yeast two‐hybrid screen. Importantly, FvCyp1, FvScp1 and FvSel1 co‐localize to endomembrane structures, each having a preferred localization in the cell, and they are all required for F. verticillioides stalk rot virulence. Moreover, these proteins are necessary for the correct localization of Fsr1 to the endoplasmic reticulum (ER) and nuclear envelope. Thus, we identified several novel components in the STRIPAK complex that regulates F. verticillioides virulence, and propose that the correct organization and localization of Fsr1 are critical for STRIPAK complex function.     

Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates

Enterococci are used as starter and probiotic cultures in foods, and they occur as natural food contaminants. The genus Enterococcus is of increased significance as a cause of nosocomial infections, and this trend is exacerbated by the development of antibiotic resistance. In this study, we investigated the incidence of known virulence determinants in starter, food, and medical strains of Enterococcus faecalis, E. faecium, and E. durans. PCR and gene probe strategies were used to screen enterococcal isolates from both food and medical sources. Different and distinct patterns of incidence of virulence determinants were found for the E. faecalis and E. faecium strains. Medical E. faecalis strains had more virulence determinants than did food strains, which, in turn, had more than did starter strains. All of the E. faecalis strains tested possessed multiple determinants (between 6 and 11). E. faecium strains were generally free of virulence determinants, with notable exceptions. Significantly, esp and gelE determinants were identified in E. faecium medical strains. These virulence determinants have not previously been identified in E. faecium strains and may result from regional differences or the evolution of pathogenic E. faecium. Phenotypic testing revealed the existence of apparently silent gelE and cyl genes. In E. faecalis, the trend in these silent genes mirrors that of the expressed determinants. The potential for starter strains to acquire virulence determinants by natural conjugation mechanisms was investigated. Transconjugation in which starter strains acquired additional virulence determinants from medical strains was demonstrated. In addition, multiple pheromone-encoding genes were identified in both food and starter strains, indicating their potential to acquire other sex pheromone plasmids. These results suggest that the use of Enterococcus spp. in foods requires careful safety evaluation.     

Influence of culture heterogeneity in cell surface charge on adhesion and biofilm formation by Enterococcus faecalis

Biofilm formation is an increasing problem in medicine, due to the intrinsic resistance of microorganisms in the biofilm mode of growth against the host immune system and antimicrobial therapy. Adhesion is an important step in biofilm formation, influenced, among other factors, by the surface hydrophobicities and charges of both the substratum and the adhering microorganisms. Enterococcus faecalis strains generally display subpopulations with different surface charges, expressed as bimodal zeta potential distributions. Two-thirds of E. faecalis strains isolated from clogged biliary stents displayed such heterogeneity of surface charges in culture. In this study, the influence of this culture heterogeneity on initial adhesion and subsequent biofilm formation was investigated. Heterogeneous strains were retained in higher numbers on polystyrene than homogeneous strains. Also, biofilm formation was much more pronounced for heterogeneous strains than for homogeneous strains. In a population enriched to display only one subpopulation, fewer bacteria were retained than in its original heterogeneous culture. Also, the enriched subpopulation formed less biofilm than its original heterogeneous culture. The presence of ox bile during adhesion resulted in fewer retained bacteria, although heterogeneous strains were still retained in significantly higher numbers than were homogeneous strains, and, in general, the presence of ox bile reduced biofilm formation. The initial adhesion and biofilm formation were independent of the presence of the gene encoding the enterococcal surface protein (esp) or the expression of gelatinase (GelE). It is concluded that heterogeneity in cell surface charge represents an advantage for bacteria in the colonization of surfaces.     

FSR1 is essential for virulence and female fertility in Fusarium verticillioides and F. graminearum

Fusarium verticillioides (teleomorph Gibberella moniliformis) and F. graminearum (teleomorph G. zeae) are well known to cause devastating diseases on cereal crops. Despite their importance, our understanding of the molecular mechanisms involved in these host-pathogen interactions is limited. The FSR1 locus in F. verticillioides was identified by screening REMI mutants for loss of virulence in maize stalk rot inoculation studies. FSR1 encodes an 823-codon open reading frame interrupted by two introns. The Fsr1 protein shares 60% sequence identity with the Sordaria macrospora Pro11, a multimodular protein with four putative protein-protein binding domains (caveolin-binding domain, coiled-coil structure, calmodulin-binding motif, and seven-WD40 repeats), which plays a regulatory role in cell differentiation and ascocarp development. Our data demonstrate that FSR1 is essential for female fertility and virulence in F. verticillioides. Significantly, targeted disruption of the FSR1 ortholog in F. graminearum (FgFSR1) reduced virulence on barley and deterred perithecia formation. Cross-complementation experiments demonstrated that the gene function is conserved in the two Fusarium species. FSR1 is expressed constitutively, and we hypothesize that Fsr1 regulates virulence by acting as a scaffold for a signal transduction pathway. A survey of available genome databases indicates Fsr1 homologs are present in a number of filamentous fungi and animal systems but not in budding yeast or plants. A maximum likelihood analysis of this gene family reveals well-supported monophyletic clades associated with fungi and animals.     

Enterococcal surface protein Esp does not facilitate intestinal colonization or translocation of Enterococcus faecalis in clindamycin-treated mice

Enterococcal surface protein (Esp) is a cell wall-associated protein of Enterococcus faecalis that has been identified as a potential virulence factor. We used a mouse model to examine whether Esp facilitates intestinal colonization or translocation of E. faecalis to mesenteric lymph nodes. After clindamycin treatment, similar levels of high-density colonization were established after orogastric inoculation of an E. faecalis isolate containing the esp gene within a large pathogenicity island and an isogenic mutant created by allelic replacement of the esp gene with a chloramphenicol resistance cassette (P=0.7); translocation to mesenteric lymph nodes was detected in 3 of 12 (25%) mice in both groups. Isogenic mutants of FA2-2 (a plasmid-free derivative of E. faecalis strain JH2) with or without the esp gene failed to establish colonization of clindamycin-treated mice. These results suggest that Esp does not facilitate intestinal colonization or translocation of E. faecalis.     

High-resolution visualization by field emission scanning electron microscopy of Enterococcus faecalis surface proteins encoded by the pheromone-inducible conjugative plasmid pCF10.

Enterococcus faecalis can acquire antibiotic resistance and virulence genes by transfer of pheromone-inducible conjugative plasmids such as pCF10, which encodes tetracycline resistance. Two pCF10-encoded cell surface proteins, Sec10 and Asc10, have been previously shown to play an important role in the transfer of this plasmid. We used high-resolution, field emission scanning electron microscopy to visualize these proteins on the surfaces of a series of isogenic strains of E. faecalis. Immunogold labeling, using both 6- and 12-nm colloidal gold, unambiguously demonstrated the expression and distribution of Sec10 and Asc10 on the surface of the E. faecalis cells. On unlabeled E. faecalis cells which expressed either Sec10 or Asc10, the former appeared to be more readily detected. Immunogold labeling of E. faecalis cells expressing both Asc10 and Sec10 clearly demonstrated the abundance and intermixing of both proteins on the cell surface except at septal regions. Sec10 was observed to be distributed over the cell surface. At regions of cell-cell contact, fine strands representing Asc10 were observed directly attaching adjacent cells to one another.