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ABSTRACTEbp are endocarditis- and biofilm-associated pili of Enterococcus faecalisthat are also important in experimental urinary tract infections (UTIs). Our analyses, using available genomes, found that the ebplocus is unique to enterococci. In E. faecalis, the ebplocus is very highly conserved and only 1/473 E. faecalisisolates tested lacked ebpABC, while only 1.2% had the beepilus locus. No other pilus-encoding operon was identified in 55 available genomes, indicating that the vast majority of E. faecalisstrains (unlike Enterococcus faeciumand streptococci) have a single pilus locus. Surface expression studies showed that Ebp pili were produced in vitroby 91/91 brain heart infusion (BHI) plus serum-grown E. faecalisisolates and that strain OG1RF expressed pili at even higher levels in rat endocarditis vegetations. However, Ebp expression was restricted to 30 to 72% of E. faecaliscells, consistent with a bistability mode of expression. We also evaluated E. faecalisinteractions with human platelets and found that growth of E. faecalisin BHI plus serum significantly enhanced adherence to human platelets and that sortase deletion mutants (the ΔsrtA, Δbps, and ΔbpsΔsrtAmutants) were markedly defective. Further studies identified that Ebp pili, but not the microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) Ace and Fss2, mediate adherence of E. faecalisto platelets. Taken together, our data show that the immunogenic (in human endocarditis patients) and commonly expressed Ebp pili, which are known to be important for experimental endocarditis, are highly conserved and mediate adherence to platelets, suggesting that Ebp pili may be a reasonable immunotherapeutic target for prevention or possibly treatment of endocarditis caused by this species.

ABSTRACTThe antigenicity, structural location, and function of the predicted lipoprotein TP0136 of Treponema pallidumsubsp. pallidumwere investigated based on previous screening studies indicating that anti-TP0136 antibodies are present in the sera of syphilis patients and experimentally infected rabbits. Recombinant TP0136 (rTP0136) protein was purified and shown to be strongly antigenic during human and experimental rabbit infection. The TP0136 protein was exposed on the surface of the bacterial outer membrane and bound to the host extracellular matrix glycoproteins fibronectin and laminin. In addition, the TP0136 open reading frame was shown to be highly polymorphic among T. pallidumsubspecies and strains at the nucleotide and amino acid levels. Finally, the ability of rTP0136 protein to act as a protective antigen to subsequent challenge with infectious T. pallidumin the rabbit model of infection was assessed. Immunization with rTP0136 delayed ulceration but did not prevent infection or the formation of lesions. These results demonstrate that TP0136 is expressed on the outer membrane of the treponeme during infection and may be involved in attachment to host extracellular matrix components.

ABSTRACTThe genome of Treponema paraluiscuniculistrain Cuniculi A was compared to the genome of the syphilis spirochete Treponema pallidumsubsp. pallidumstrain Nichols using DNA microarray hybridization, whole-genome fingerprinting, and DNA sequencing. A DNA microarray of T. pallidumsubsp. pallidumNichols containing all 1,039 predicted open reading frame PCR products was used to identify deletions and major sequence changes in the Cuniculi A genome. Using these approaches, deletions, insertions, and prominent sequence changes were found in 38 gene homologs and six intergenic regions of the Cuniculi A genome when it was compared to the genome of T. pallidumsubsp. pallidumNichols. Most of the observed differences were localized in tprloci and the vicinity of these loci. In addition, 14 other genes were found to contain frameshift mutations resulting in major changes in protein sequences. Analysis of restriction target sites representing 0.34% of the total genome length and DNA sequencing of three PCR products (0.46% of the total genome length) amplified from Cuniculi A chromosomal regions and comparison to the Nichols genome revealed a sequence similarity of 98.6 to 99.3%. These results are consistent with a close genetic relationship among the T. pallidumstrains and subspecies and a strong, but relatively divergent connection between the human and rabbit pathogens.

ABSTRACTA gene encoding a major secreted antigen, SagA, was identified in Enterococcus faeciumby screening an E. faeciumgenomic expression library with sera from patients with E. faecium-associated endocarditis. Recombinant SagA protein showed broad-spectrum binding to extracellular matrix (ECM) proteins, including fibrinogen, collagen type I, collagen type IV, fibronectin, and laminin. A fibrinogen-binding protein, purified from culture supernatants of an E. faeciumclinical isolate, was found to match the N-terminal sequence of the predicted SagA protein and to react with the anti-SagA antibody, confirming that it was the SagA protein; this protein appeared as an 80- to 90-kDa smear on a Western blot that was sensitive to proteinase K and resistant to periodate treatment and glycoprotein staining. When overexpressed in E. faeciumand Escherichia coli, the native and recombinant SagA proteins formed stable oligomers, apparently via their C-terminal domains. The SagA protein is composed of three domains: (i) a putative coiled-coil N-terminal domain that shows homology to the N-terminal domain of Streptococcus mutansSagA protein (42% similarity), previously shown to be involved in cell wall integrity and cell shape maintenance, and to the P45 protein of Listeria monocytogenes(41% similarity); (ii) a central domain containing direct repeats; and (iii) a C-terminal domain that is similar to that found in various proteins, including P45 (50% similarity) and P60 (52% similarity) of L. monocytogenes. The P45 and P60 proteins both have cell wall hydrolase activity, and the latter has also been shown to be involved in virulence, whereas cell wall hydrolase activity was not detected for SagA protein. The E. faecium sagAgene, like the S. mutanshomologue, is located in a cluster of genes encoding proteins that appear to be involved in cell wall metabolism and could not be disrupted unless it was first transcomplemented, suggesting that the sagAgene is essential for E. faeciumgrowth and may be involved in cell wall metabolism. In conclusion, the extracelluar E. faeciumSagA protein is apparently essential for growth, shows broad-spectrum binding to ECM proteins, forms oligomers, and is antigenic during infection.

ABSTRACTIn previous studies, we cloned a cluster of genes involved in polysaccharide biosynthesis (epa) from Enterococcus faecalisstrain OG1RF and showed that this gene cluster mediated synthesis of a polysaccharide in Escherichia coli. Disruption of two open reading frames in the epagene cluster of OG1RF generated two mutants, TX5179 and TX5180, which were attenuated in a mouse peritonitis model. In the current study, Western blotting was performed with serum from a patient with E. faecalisendocarditis and polysaccharide extracts from OG1RF and the mutants TX5179 and TX5180. OG1RF showed a smear in the high-molecular-weight region and discrete bands in the low-molecular-weight region, which were missing from the mutants; periodate treatment and carbohydrate staining confirmed the polysaccharide nature of this material. In a neutrophil killing assay using OG1RF-absorbed normal human serum, the mutants TX5179 and TX5180, respectively, were 50 and 2.4 times more susceptible to killing than wild-type OG1RF (P≤ 0.01). With a fluorescence phagocytosis assay, 2.5 to 3 times more of the mutants were taken up by neutrophils than OG1RF (P≤ 0.001). Finally, with restriction digestion and hybridization under high-stringency conditions, the epagene cluster of OG1RF (which is also present in the sequenced E. faecalisstrain V583) was detected in 12 of 12 other clonally distinct E. faecalisstrains tested: a similar polysaccharide pattern was detected for the 12 strains on Western blots using an E. faecalisendocarditis patient serum, and sera from four other patients with E. faecalisendocarditis all reacted with polysaccharide extracts of OG1RF. These results indicate that the epagene cluster is widespread among E. faecalisand confers some protection against human host defenses.

ABSTRACTOur previous work reported that most Enterococcus faecalisstrains adhered to the extracellular matrix proteins collagen types I and IV and laminin after growth at 46°C, but not 37°C, and we subsequently identified an E. faecalissequence, ace, that encodes a bacterial adhesin similar to the collagen binding protein Cna of Staphylococcus aureus. In this study, we examined the diversity of E. faecalis-specific acegene sequences among different isolates obtained from various geographic regions as well as from various clinical sources. A comparison of nucleotide and deduced amino acid sequences of Ace from nine E. faecalisstrains identified a highly conserved N-terminal A domain, followed by a variable B domain which contains two to five repeats of 47 amino acids in tandem array, preceded by a 20-amino-acid partial repeat. Using 17 other strains collected worldwide, the 5′ region of acethat encodes the A domain was sequenced, and these sequences showed ≥97.5% identity. Among the previously reported five amino acids critical for collagen binding by Cna of S. aureus, four were found to be identical in Ace from all strains tested. Polyclonal immune rabbit serum prepared against recombinant Ace A derived fromE. faecalisstrain OG1RF detected Ace in mutanolysin extracts of seven of nine E. faecalisstrains after growth at 46°C; Ace was detected in four different molecular sizes that correspond to the variation in the B repeat region. To determine if there was any evidence to indicate that Ace might be produced under physiological conditions, we quantitatively assayed sera collected from patients with enterococcal infections for the presence of anti-Ace A antibodies. Ninety percent of sera (19 of 21) from patients withE. faecalisendocarditis showed reactivity with titers from 1:32 to >1:1,024; the only 2 sera which lacked antibodies to Ace A had considerably lower titers of antibodies to other E. faecalisantigens as well. Human-derived, anti-Ace A immunoglobulins G purified from an E. faecalisendocarditis patient serum inhibited adherence of 46°C-grown E. faecalisOG1RF to collagen types I and IV and laminin. In conclusion, these results show that aceis highly conserved among isolates of E. faecalis, with at least four variants related to the differences in the B domain, is expressed by different strains during infection in humans, and human-derived antibodies can block adherence to these extracellular matrix proteins.

ABSTRACTAdhesin-mediated binding to extracellular matrix (ECM) proteins is thought to be a crucial step in the pathogenic process of many bacterial infections. We have previously reported conditional adherence of most Enterococcus faecalisisolates, after growth at 46°C, to ECM proteins collagen types I and IV and laminin; identified an E. faecalis-specific gene, ace, whose encoded protein has characteristics of a bacterial adhesin; and implicated Ace in binding to collagen type I. In this study, we constructed an acedisruption mutant from E. faecalisstrain OG1RF that showed marked reduction in adherence to collagen types I and IV and laminin when compared to the parental OG1RF strain after growth at 46°C. Polyclonal immune serum raised against the OG1RF-derived recombinant Ace A domain reacted with a single ∼105-kDa band of mutanolysin extracts from OG1RF grown at 46°C, while no band was detected in extracts from OG1RF grown at 37°C, nor from the OG1RF acemutant grown at 37 or 46°C. IgGs purified from the anti-Ace A immune serum inhibited adherence of 46°C-grown E. faecalisOG1RF to immobilized collagen type IV and laminin as well as collagen type I, at a concentration as low as 1 μg/ml, and also inhibited the 46°C-evoked adherence of two clinical isolates tested. We also showed in vitro interaction of collagen type IV with Ace from OG1RF mutanolysin extracts on a far-Western blot. Binding of recombinant Ace A to immobilized collagen types I and IV and laminin was demonstrated in an enzyme-linked immunosorbent assay and was shown to be concentration dependent. These results indicate that Ace A mediates the conditional binding of E. faecalisOG1RF to collagen type IV and laminin in addition to collagen type I.

ABSTRACTThree agr-like genes (fsrA,fsrB, and fsrC, for Enterococcus faecalisregulator) were found upstream of the previously reported gelatinase gene (gelE) and a downstream putative serine protease gene (sprE; accession number Z12296) ofEnterococcus faecalisOG1RF. The deduced amino acid sequence of fsrAshows 26% identity and 38% similarity toStaphylococcus aureusAgrA (the response regulator of the accessory gene regulator system in the agrlocus), FsrB shows 23% identity and 41% similarity to S. aureusAgrB, and FsrC shows 23% identity and 36% similarity to S. aureusAgrC (the sensor transducer of Agr system). Northern blot analysis suggested that gelEand sprEare cotranscribed and that fsrBand fsrCare also cotranscribed in OG1RF. Northern blot analysis of fsrA,fsrB, fsrC, gelE, andsprEinsertion mutants showed that fsrB,fsrC, gelE, and sprEare not expressed in fsrA, fsrB, and fsrCmutants, while insertion in an open reading frame further upstream offsrAdid not effect the expression of these genes, suggesting that agr-like genes may be autoregulated and that they regulate gelEand sprEexpression, as further confirmed by complementation of fsrgene mutations with a 6-kb fragment which contains all three fsrgenes in the shuttle vector, pAT18. Testing of 95 other isolates of E. faecalisshowed that 62% produced gelatinase (Gel+), while 91% (including all Gel+strains) hybridized to agelEprobe; 71% (including all Gel+strains) hybridized to an fsrprobe, corroborating the conclusion that both gelEand fsrare necessary for gelatinase production. Testing of fsrA, fsrB, and sprEmutants in a mouse peritonitis model showed that sprEand agr-like gene mutants resulted in highly significantly prolonged survival compared to the parent strain OG1RF, a finding similar to what we had previously shown for agelEmutant. These results suggest that sprEand agr-like genes contribute to the virulence of E. faecalisOG1RF in this model.

ABSTRACTPreviously, we described a gene cluster of Enterococcus faecalisOG1RF that produced an antigenic polysaccharide when cloned in Escherichia coli. The polysaccharide antigen was not detectable in E. faecalisstrains, however. Here, we show by reverse transcriptase-PCR that the 16 genes in this region are transcribed in OG1RF. Gene disruption of orfde4, encoding a putative glycosyl transferase, and orfde6, a putative dTDP-rhamnose biosynthesis gene, generated two OG1RF mutants. The mutants showed delayed killing and a higher 50% lethal dose in a mouse peritonitis model. In addition, two mucoid E. faecalisisolates from patients with chronic urinary tract infections were found to produce the polysaccharide antigen.