[Show abstract][Hide abstract] ABSTRACT: 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 ELISA based screening method to identify mutants from a transposon insertion mutant library which exhibited distinct Ace surface expression profiles. A ccpA insertion mutant was identified which showed significantly decreased levels of Ace surface expression at early growth phase versus that of wild-type OG1RF. Confirmation of the observation was achieved through flow cytometry and complementation analysis. Consistent with the lack of Ace expression in the early growth phase, the E. faecalis ccpA mutant had an impaired ability to adhere to collagen when grown to early exponential phase compared to the wild-type. 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.
Journal of bacteriology 08/2013; · 3.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Enterococci are among the leading causes of hospital-acquired infections in the United States and Europe, with Enterococcus faecalis and Enterococcus faecium being the two most common species isolated from enterococcal infections. In the last decade, the proportion of enterococcal infections caused by E. faecium has steadily increased compared to other Enterococcus species. Although the underlying mechanism for the gradual replacement of E. faecalis by E. faecium in the hospital environment is not yet understood, many studies using genotyping and phylogenetic analysis have shown the emergence of a globally dispersed polyclonal subcluster of E. faecium strains in clinical environments. Systematic study of the molecular epidemiology and pathogenesis of E. faecium has been hindered by the lack of closed, complete E. faecium genomes that can be used as references.
In this study, we report the complete genome sequence of the E. faecium strain TX16, also known as DO, which belongs to multilocus sequence type (ST) 18, and was the first E. faecium strain ever sequenced. Whole genome comparison of the TX16 genome with 21 E. faecium draft genomes confirmed that most clinical, outbreak, and hospital-associated (HA) strains (including STs 16, 17, 18, and 78), in addition to strains of non-hospital origin, group in the same clade (referred to as the HA clade) and are evolutionally considerably more closely related to each other by phylogenetic and gene content similarity analyses than to isolates in the community-associated (CA) clade with approximately a 3-4% average nucleotide sequence difference between the two clades at the core genome level. Our study also revealed that many genomic loci in the TX16 genome are unique to the HA clade. 380 ORFs in TX16 are HA-clade specific and antibiotic resistance genes are enriched in HA-clade strains. Mobile elements such as IS16 and transposons were also found almost exclusively in HA strains, as previously reported.
Our findings along with other studies show that HA clonal lineages harbor specific genetic elements as well as sequence differences in the core genome which may confer selection advantages over the more heterogeneous CA E. faecium isolates. Which of these differences are important for the success of specific E. faecium lineages in the hospital environment remain(s) to be determined.
[Show abstract][Hide abstract] ABSTRACT: Most Enterococcus faecalis isolates carry gelE, but many are gelatinase nonproducers due to the lack of fsrC (EF_1820) to EF_1841 (fsrC-EF_1841; 23.9 kb in strain V583), including most of the locus encoding Fsr, which activates gelE expression. Analysis of 22 accessible E. faecalis genomes revealed the identity of the 53-amino-acid propeptide of fsrD across multiple MLSTs (multilocus sequence types), although 12 distinctly different variations were found in the EF_1814-to-EF_1902 region. Diversity was seen in fsrABC, in the region EF_1814 to EF_1902, and in a 700-kb region surrounding fsrC-EF_1841. However, analysis of five sequenced strains carrying the fsrC-EF_1841 deletion and the putative integrative conjugative element efaB5 showed almost identical single nucleotide polymorphisms (SNPs) in gelE and an identical junction sequence, despite their unrelated MLSTs, in contrast to those shown by strains without the deletion. Further analysis confirmed the conserved gelE SNPs in 6 additional strains (11 in total) with the deletion. While we were unable to detect evidence of spontaneous deletion using OG1RF and 8 other strains, we were able to engineer a deletion of the 37-kb fsrC-EF_1841 region of OG1RF without deleterious effects, and the 37-kb mutant showed changes in biofilm and chaining similar to those shown by fsr-gelE mutants. In conclusion, we describe the identity of fsrD despite high plasticity within the fsrC-EF_1841 region and the surrounding sequence. However, strains lacking the fsrC-EF_1841 region show a distinct conservation of the sequence surrounding this deletion and in gelE, suggesting that the deletion may result from horizontal transfer and recombination.
Applied and environmental microbiology 01/2011; 77(2):442-51. · 3.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this work we investigated the adaptive response of E. faecalis to Cu and the role of CopY, a Cu-dependent repressor, in the regulation of Cu metabolism. In doing so, we examined the whole-genome transcriptional response of E. faecalis wild-type (WT) and a ΔcopY strain exposed to non-toxic Cu excess. The results indicated that after Cu exposure, most of the genes that displayed a significant change in their expression levels in the WT strain (135 of the 145 up-regulated genes and 115 of the 142 down-regulated genes) were also differentially expressed in the E. faecalis ΔcopY strain. This extensive overlap in the transcriptional response, suggested that additional transcription factors mediate the response of E. faecalis to Cu. As a first step to analyze this possibility, we selected among the up-regulated genes five genes encoding putative transcriptional regulators and determined their expression levels at different times after Cu exposure. The temporal expression of these regulators was different from that of copY, which reached its maximum at the earliest time measured. Nevertheless, transcription elongation factor GreA, and members of Rrf2, Cro/CI and SorC/DeoR transcription factor families were induced shortly after Cu exposure, suggesting that these proteins are able to complement the role of CopY in the regulatory network activated by Cu. To our knowledge, this is the first report on the global transcriptional response to Cu in a member of this taxonomic group.
Biology of Metals 12/2010; 23(6):1105-12. · 3.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We previously identified ebpR, encoding a potential member of the AtxA/Mga transcriptional regulator family, and showed that it is important for transcriptional activation of the Enterococcus faecalis endocarditis and biofilm associated pilus operon, ebpABC. Although ebpR is not absolutely essential for ebpABC expression (100-fold reduction), its deletion led to phenotypes similar to those of an ebpABC mutant such as absence of pili at the cell surface and, consequently, reduced biofilm formation. A non-piliated ebpABC mutant has been shown to be attenuated in a rat model of endocarditis and in a murine urinary tract infection model, indicating an important participation of the ebpR-ebpABC locus in virulence. However, there is no report relating to the environmental conditions that affect expression of the ebpR-ebpABC locus.
In this study, we examined the effect of CO2/HCO3(-), pH, and the Fsr system on the ebpR-ebpABC locus expression. The presence of 5% CO2/0.1 M HCO3(-) increased ebpR-ebpABC expression, while the Fsr system was confirmed to be a weak repressor of this locus. The mechanism by which the Fsr system repressed the ebpR-ebpABC locus expression appears independent of the effects of CO2(-) bicarbonate. Furthermore, by using an ebpA::lacZ fusion as a reporter, we showed that addition of 0.1 M sodium bicarbonate to TSBG (buffered at pH 7.5), but not the presence of 5% CO2, induced ebpA expression in TSBG broth. In addition, using microarray analysis, we found 73 genes affected by the presence of sodium bicarbonate (abs(fold) > 2, P < 0.05), the majority of which belong to the PTS system and ABC transporter families. Finally, pilus production correlated with ebpA mRNA levels under the conditions tested.
This study reports that the ebp locus expression is enhanced by the presence of bicarbonate with a consequential increase in the number of cells producing pili. Although the molecular basis of the bicarbonate effect remains unclear, the pathway is independent of the Fsr system. In conclusion, E. faecalis joins the growing family of pathogens that regulates virulence gene expression in response to bicarbonate and/or CO2.
[Show abstract][Hide abstract] ABSTRACT: We previously identified a gene cluster, epa (for enterocococcal polysaccharide antigen), involved in polysaccharide biosynthesis of Enterococcus faecalis and showed that disruption of epaB and epaE resulted in attenuation in translocation, biofilm formation, resistance to polymorphonuclear leukocyte (PMN) killing, and virulence in a mouse peritonitis model. Using five additional mutant disruptions in the 26-kb region between orfde2 and OG1RF_0163, we defined the epa locus as the area from epaA to epaR. Disruption of epaA, epaM, and epaN, like prior disruption of epaB and epaE, resulted in alteration in Epa polysaccharide content, more round cells versus oval cells with OG1RF, decreased biofilm formation, attenuation in a mouse peritonitis model, and resistance to lysis by the phage NPV-1 (known to lyse OG1RF), while mutants disrupted in orfde2 and OG1RF_163 (the epa locus flanking genes) behaved like OG1RF in those assays. Analysis of the purified Epa polysaccharide from OG1RF revealed the presence of rhamnose, glucose, galactose, GalNAc, and GlcNAc in this polysaccharide, while carbohydrate preparation from the epaB mutant did not contain rhamnose, suggesting that one or more of the glycosyl transferases encoded by the epaBCD operon are necessary to transfer rhamnose to the polysaccharide. In conclusion, the epa genes, uniformly present in E. faecalis strains and involved in biosynthesis of polysaccharide in OG1RF, are also important for OG1RF shape determination, biofilm formation, and NPV-1 replication/lysis, as well as for E. faecalis virulence in a mouse peritonitis model.
Infection and immunity 08/2009; 77(9):3759-67. · 4.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Methicillin (meticillin)-susceptible Staphylococcus aureus (MSSA) strains producing large amounts of type A beta-lactamase (Bla) have been associated with cefazolin failures, but the frequency and impact of these strains have not been well studied. Here we examined 98 MSSA clinical isolates and found that 26% produced type A Bla, 15% type B, 46% type C, and none type D and that 13% lacked blaZ. The cefazolin MIC(90) was 2 microg/ml for a standard inoculum and 32 microg/ml for a high inoculum, with 19% of isolates displaying a pronounced inoculum effect (MICs of >or=16 microg/ml with 10(7) CFU/ml) (9 type A and 10 type C Bla producers). At the high inoculum, type A producers displayed higher cefazolin MICs than type B or C producers, while type B and C producers displayed higher cefamandole MICs. Among isolates from hemodialysis patients with MSSA bacteremia, three from the six patients who experienced cefazolin failure showed a cefazolin inoculum effect, while none from the six patients successfully treated with cefazolin showed an inoculum effect, suggesting an association between these strains and cefazolin failure (P = 0.09 by Fisher's exact test). In summary, 19% of MSSA clinical isolates showed a pronounced inoculum effect with cefazolin, a phenomenon that could explain the cases of cefazolin failure previously reported for hemodialysis patients with MSSA bacteremia. These results suggest that for serious MSSA infections, the presence of a significant inoculum effect with cefazolin could be associated with clinical failure in patients treated with this cephalosporin, particularly when it is used at low doses.
Antimicrobial Agents and Chemotherapy 06/2009; 53(8):3437-41. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We report that three (EF0089, EF2505 and EF1896, renamed here Fss1, Fss2 and Fss3, respectively, for Enterococcus faecalis surface protein) of the recently predicted MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) in E. faecalis strain V583 bind fibrinogen (Fg). Despite an absence of extensive primary sequence homology, the three proteins appear to be related structurally. Within the N-terminal regions of the three enterococcal proteins, we identified pairs of putative IgG-like modules with a high degree of predicted structural similarity to the Fg-binding N2 and N3 domains of the staphylococcal MSCRAMMs ClfA and SdrG. A second N2N3-like segment was predicted in Fss1. Far-UV circular dichroism spectroscopy revealed that all four predicted N2N3-like regions are composed mainly of beta-sheets with only a minor proportion of alpha-helices, which is characteristic of Ig-like folded domains. Three of the four identified enterococcal N2N3-like regions showed potent dose-dependent binding to Fg. However, the specificity of the Fg-binding MSCRAMMs differs, as indicated by far-Western blots, which showed that recombinant segments of the MSCRAMMs bound different Fg polypeptide chains. Enterococci grown in serum-supplemented broth adhere to Fg-coated surfaces, and inactivation in strain OG1RF of the gene encoding Fss2 resulted in reduced adherence, whilst complementation of the mutant restored full Fg adherence. Thus, E. faecalis contains a family of MSCRAMMs that structurally and functionally resemble the Fg-binding MSCRAMMs of staphylococci.
[Show abstract][Hide abstract] ABSTRACT: Ethanolamine, a product of the breakdown of phosphatidylethanolamine from cell membranes, is abundant in the human intestinal tract and in processed foods. Effective utilization of ethanolamine as a carbon and nitrogen source may provide a survival advantage to bacteria that inhabit the gastrointestinal tract and may influence the virulence of pathogens. In this work, we describe a unique series of posttranscriptional regulatory strategies that influence expression of ethanolamine utilization genes (eut) in Enterococcus, Clostridium, and Listeria species. One of these mechanisms requires an unusual 2-component regulatory system. Regulation involves specific sensing of ethanolamine by a sensor histidine kinase (EutW), resulting in autophosphorylation and subsequent phosphoryl transfer to a response regulator (EutV) containing a RNA-binding domain. Our data suggests that EutV is likely to affect downstream gene expression by interacting with conserved transcription termination signals located within the eut locus. Breakdown of ethanolamine requires adenosylcobalamin (AdoCbl) as a cofactor, and, intriguingly, we also identify an intercistronic AdoCbl riboswitch that has a predicted structure different from previously established AdoCbl riboswitches. We demonstrate that association of AdoCbl to this riboswitch prevents formation of an intrinsic transcription terminator element located within the intercistronic region. Together, these results suggest an intricate and carefully coordinated interplay of multiple regulatory strategies for control of ethanolamine utilization genes. Gene expression appears to be directed by overlapping posttranscriptional regulatory mechanisms, each responding to a particular metabolic signal, conceptually akin to regulation by multiple DNA-binding transcription factors.
Proceedings of the National Academy of Sciences 03/2009; 106(11):4435-40. · 9.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Enterococcus faecalis has emerged as a major hospital pathogen. To explore its diversity, we sequenced E. faecalis strain OG1RF, which is commonly used for molecular manipulation and virulence studies.
The 2,739,625 base pair chromosome of OG1RF was found to contain approximately 232 kilobases unique to this strain compared to V583, the only publicly available sequenced strain. Almost no mobile genetic elements were found in OG1RF. The 64 areas of divergence were classified into three categories. First, OG1RF carries 39 unique regions, including 2 CRISPR loci and a new WxL locus. Second, we found nine replacements where a sequence specific to V583 was substituted by a sequence specific to OG1RF. For example, the iol operon of OG1RF replaces a possible prophage and the vanB transposon in V583. Finally, we found 16 regions that were present in V583 but missing from OG1RF, including the proposed pathogenicity island, several probable prophages, and the cpsCDEFGHIJK capsular polysaccharide operon. OG1RF was more rapidly but less frequently lethal than V583 in the mouse peritonitis model and considerably outcompeted V583 in a murine model of urinary tract infections.
E. faecalis OG1RF carries a number of unique loci compared to V583, but the almost complete lack of mobile genetic elements demonstrates that this is not a defining feature of the species. Additionally, OG1RF's effects in experimental models suggest that mediators of virulence may be diverse between different E. faecalis strains and that virulence is not dependent on the presence of mobile genetic elements.
[Show abstract][Hide abstract] ABSTRACT: We identify ef1090 (renamed ebpR) and show its importance for the transcriptional regulation of expression of the Enterococcus faecalis pilus operon, ebpABC. An ebpR deletion (DeltaebpR) mutant was found to have reduced ebpABC expression with loss of pilus production and a defect in primary adherence with, as a consequence, reduced biofilm formation.
Journal of Bacteriology 10/2007; 189(17):6490-3. · 3.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The FsrABC system of Enterococcus faecalis controls the expression of gelatinase and a serine protease via a quorum-sensing mechanism, and recent studies suggest that the Fsr system may also regulate other genes important for virulence. To investigate the possibility that Fsr influences the expression of additional genes, we used transcriptional profiling, with microarrays based on the E. faecalis strain V583 sequence, to compare the E. faecalis strain OG1RF with its isogenic mutant, TX5266, an fsrB deletion mutant. We found that the presence of an intact fsrB influences expression of numerous genes throughout the growth phases tested, namely, late log to early stationary phase. In addition, the Fsr regulon is independent of the activity of the proteases, GelE and SprE, whose expression was confirmed to be activated at all three time points tested. While expression of some genes (i.e., ef1097 and ef0750 to -757, encoding hypothetical proteins) was activated in late log phase in OG1RF versus the fsrB deletion mutant, expression of ef1617 to -1634 (eut-pdu orthologues) was highly repressed by the presence of an intact Fsr at entry into stationary phase. This is the first time that Fsr has been characterized as a negative regulator. The newly recognized Fsr-regulated targets include other factors, besides gelatinase, described as important for biofilms (BopD), and genes predicted to encode the surface proteins EF0750 to -0757 and EF1097, along with proteins implicated in several metabolic pathways, indicating that the FsrABC system may be an important regulator in strain OG1RF, with both positive and negative effects.
Journal of Bacteriology 05/2006; 188(8):2875-84. · 3.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The poly-d-glutamic acid capsule of Bacillus anthracis is essential for virulence. Control of capsule synthesis occurs at the level of transcription and involves positive regulation of the capsule biosynthetic operon capBCAD by a CO2/bicarbonate signal and three plasmid-borne regulators: atxA, acpA, and acpB. Although the molecular mechanism for control of cap transcription is unknown, atxA affects cap expression via positive control of acpA and acpB, two genes with partial functional similarity. Transcriptional analyses of a genetically complete strain indicate that capB expression is several hundred-fold higher during growth in 5% CO2 compared to growth in air. atxA was expressed appreciably during growth in air and induced only 2.5-fold by CO2. In contrast, expression of acpA and acpB was induced up to 23-fold and 59-fold, respectively, by CO2. The 5'-end mapping of gene transcripts revealed atxA-regulated and atxA-independent apparent transcription start sites for capB, acpA, and acpB. Transcripts mapping to all atxA-regulated start sites were increased during growth in elevated CO2. The acpA gene has one atxA-regulated and one atxA-independent start site. acpB lies downstream of capBCAD. A single atxA-independent start site maps immediately upstream of acpB. atxA-mediated control of acpB appears to occur via transcriptional read-through from atxA-dependent start sites 5' of capB. One atxA-independent and two atxA-regulated start sites map upstream of capB. Transcription from the atxA-regulated start sites of capBCAD was reduced significantly in an acpA acpB double mutant but unaffected in mutants with deletion of only acpA or acpB, in agreement with the current model for epistatic relationships between the regulators.
Journal of Bacteriology 09/2005; 187(15):5108-14. · 3.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A minireplicon of plasmid pXO2 of Bacillus anthracis was isolated by molecular cloning in Escherichia coli and shown to replicate in B. anthracis, Bacillus cereus, and Bacillus subtilis. The pXO2 replicon included (i) an open reading frame encoding the putative RepS replication initiation protein and (ii) the putative origin of replication. The RepS protein was expressed as a fusion with the maltose binding protein (MBP) at its amino-terminal end and purified by affinity chromatography. Electrophoretic mobility shift assays showed that the purified MBP-RepS protein bound specifically to a 60-bp region corresponding to the putative origin of replication of pXO2 located immediately downstream of the RepS open reading frame. Competition DNA binding experiments showed that the 5' and central regions of the putative origin were important for RepS binding. MBP-RepS also bound nonspecifically to single-stranded DNA with a lower affinity.
Journal of Bacteriology 06/2004; 186(9):2717-23. · 3.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Two regulatory genes, acpA and atxA, have been reported to control expression of the Bacillus anthracis capsule biosynthesis operon capBCAD. The atxA gene is located on the virulence plasmid pXO1, while pXO2 carries acpA and the cap genes. acpA has been viewed as the major regulator of the cap operon because it is essential for capsule gene expression in a pXO1(-) pXO2(+) strain. atxA is essential for toxin gene transcription but has also been implicated in control of the cap genes. The molecular functions of the regulatory proteins are unknown. We examined cap gene expression in a genetically complete pXO1(+) pXO2(+) strain. Our results indicate that another pXO2 gene, acpB (previously called pXO2-53; accession no. NC002146.1:49418-50866), has a role in cap expression. The predicted amino acid sequence of AcpB is 62% similar to that of AcpA and 50% similar to that of AtxA. Assessment of cap gene transcription revealed that cap expression was not affected in a pXO1(+) pXO2(+) acpB-null mutant and was slightly reduced in an isogenic acpA mutant. However, cap gene expression was abolished in an acpA acpB double mutant. Microscopic examination of capsule synthesis by the mutants corroborated these findings. acpA and acpB expression is controlled by atxA; capsule synthesis and transcription of acpA and acpB were markedly reduced in an atxA mutant. The data suggest that, in a strain containing both virulence plasmids, atxA is the major regulator of capsule synthesis and controls capBCAD expression indirectly, via positive regulation of acpA and acpB.
Journal of Bacteriology 02/2004; 186(2):307-15. · 3.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Control of anthrax toxin and capsule synthesis, the two major virulence factors of Bacillus anthracis, has been associated with two regulatory genes, atxA and acpA, located on virulence plasmids pXO1 and pXO2, respectively. We used transcriptional profiling to determine whether atxA and/or acpA control genes other than those already described and to investigate functional similarities of the regulators. Transcription was assessed in a pXO1(+) pXO2(+) parent strain and in isogenic mutants in which one or both regulatory genes were deleted. We determined that in addition to the toxin and capsule genes, atxA controls expression of numerous other genes on both plasmids and the chromosome. Generally, plasmid-encoded genes were more highly regulated than chromosomal genes, and both positive and negative effects were observed. Certain atxA-regulated genes were affected synergistically in an atxA acpA mutant. Yet overall, acpA appears to be a minor regulator with fewer targets than atxA. In contrast to previous reports of acpA function in attenuated strains, acpA had a minimal influence on capsule gene transcription and capsule synthesis in a genetically complete strain. Surprisingly, acpA expression was positively affected by atxA, although atxA-activated capsule gene transcription is not acpA dependent. The newly discovered atxA-regulated targets include genes predicted to encode secreted proteins and proteins with roles in transcriptional regulation and signaling. Regulation of chromosomal genes by atxA is particularly intriguing, given that many of the target genes have homologues in other Bacillus species that lack atxA homologues. Given the global effect of atxA on gene expression in B. anthracis, previous assumptions regarding reduced virulence of strains harboring single plasmids must be reassessed and the potential roles of newly identified atxA-regulated genes should be investigated.
Infection and Immunity 06/2003; 71(5):2736-43. · 4.07 Impact Factor