Dominique Wobser

Protection Against Staphylococcus aureus by Antibody to the Polyglycerolphosphate Backbone of Heterologous Lipoteichoic Acid

Type 1 lipoteichoic acid (LTA) is present in many clinically important gram-positive bacteria, including enterococci, streptococci, and staphylococci, and antibodies against LTA have been shown to opsonize nonencapsulated Enterococcus faecalis strains. In the present study, we show that antibodies against E. faecalis LTA also bind to type 1 LTA from other gram-positive species and opsonized Staphylocccus epidermidis and Staphylcoccus aureus strains as well as group B streptococci. Inhibition studies using teichoic acid oligomers indicated that cross-reactive opsonic antibodies bind to the teichoic acid backbone. Passive immunization with rabbit antibodies against E. faecalis LTA promoted the clearance of bacteremia by E. faecalis and S. epidermidis in mice. Furthermore, passive protection also reduced mortality in a murine S. aureus peritonitis model. The effectiveness of rabbit antibody against LTA suggests that this conserved bacterial structure could function as a single vaccine antigen that targets multiple gram-positive pathogens.

Role of mprF1 and mprF2 in the pathogenicity of Enterococcus faecalis.

Background Enterococcus faecalis is one of the leading causes of nosocomial infections. Due to its innate and acquired resistance to most antibiotics, identification of new targets for antimicrobial treatment of E. faecalis is a high priority. The multiple peptide resistance factor MprF, which was first described in Staphylococcus aureus, modifies phosphatidylglycerol with lysin and reduces the negative charge of the membrane, thus increasing resistance to cationic antimicrobial peptides. We studied the effect of mprF in E. faecalis regarding influence on bacterial physiology and virulence. Results Two putative mprF paralogs (mprF1 and mprF2) were identified in E. faecalis by BLAST search using the well-described S. aureus gene as a lead. Two deletion mutants in E. faecalis 12030 were created by homologous recombination. Analysis of both mutants by thin-layer chromatography showed that inactivation of mprF2 abolishes the synthesis of three distinct amino-phosphatidylglycerols (PGs). In contrast, deletion of mprF1 did not interfere with the biosynthesis of amino-PG. Inactivation of mprF2 increased susceptibility against several antimicrobial peptides and resulted in a 42% increased biofilm formation compared to wild-type mprF. However, resistance to opsonic killing was increased in the mutant, while virulence in a mouse bacteremia model was unchanged. Conclusion Our data suggest that only mprF2 is involved in the aminoacylation of PG in enterococci, and is probably responsible for synthesis of Lys-PG, Ala-PG, and Arg-PG, while mprF1 does not seem to have a role in aminoacylation. As in other Gram-positive pathogens, aminoacylation through MprF2 increases resistance against cationic antimicrobial peptides. Unlike mprF found in other bacteria, mprF2 does not seem to be a major virulence factor in enterococci.

Phage-mediated Dispersal of Biofilm and Distribution of Bacterial Virulence Genes Is Induced by Quorum Sensing

The microbiome and the phage meta-genome within the human gut are influenced by antibiotic treatments. Identifying a novel mechanism, here we demonstrate that bacteria use the universal communication molecule AI-2 to induce virulence genes and transfer them via phage release. High concentrations (i.e. 100 μM) of AI-2 promote dispersal of bacteria from already established biofilms, and is associated with release of phages capable of infecting other bacteria. Enterococcus faecalis V583ΔABC harbours 7 prophages in its genome, and a mutant deficient in one of these prophages (i.e. prophage 5) showed a greatly reduced dispersal of biofilm. Infection of a probiotic E. faecalis strain without lytic prophages with prophage 5 resulted in increased biofilm formation and also in biofilm dispersal upon induction with AI-2. Infection of the probiotic E. faecalis strain with phage-containing supernatants released through AI-2 from E. faecalis V583ΔABC resulted in a strong increase in pathogenicity of this strain. The polylysogenic probiotic strain was also more virulent in a mouse sepsis model and a rat endocarditis model. Both AI-2 and ciprofloxacin lead to phage release, indicating that conditions in the gastrointestinal tract of hospitalized patients treated with antibiotics might lead to distribution of virulence genes to apathogenic enterococci and possibly also to other commensals or even to beneficial probiotic strains.

Synthetic Teichoic Acid Conjugate Vaccine against Nosocomial Gram-Positive Bacteria

Lipoteichoic acids (LTA) are amphiphilic polymers that are important constituents of the cell wall of many Gram-positive bacteria. The chemical structures of LTA vary among organisms, albeit in the majority of Gram-positive bacteria the LTAs feature a common poly-1,3-(glycerolphosphate) backbone. Previously, the specificity of opsonic antibodies for this backbone present in some Gram-positive bacteria has been demonstrated, suggesting that this minimal structure may be sufficient for vaccine development. In the present work, we studied a well-defined synthetic LTA-fragment, which is able to inhibit opsonic killing of polyclonal rabbit sera raised against native LTA from Enterococcus faecalis 12030. This promising compound was conjugated with BSA and used to raise rabbit polyclonal antibodies. Subsequently, the opsonic activity of this serum was tested in an opsonophagocytic assay and specificity was confirmed by an opsonophagocytic inhibition assay. The conjugated LTA-fragment was able to induce specific opsonic antibodies that mediate killing of the clinical strains E. faecalis 12030, Enterococcus faecium E1162, and community-acquired Staphylococcus aureus strain MW2 (USA400). Prophylactic immunization with the teichoic acid conjugate and with the rabbit serum raised against this compound was evaluated in active and passive immunization studies in mice, and in an enterococcal endocarditis rat model. In all animal models, a statistically significant reduction of colony counts was observed indicating that the novel synthetic LTA-fragment conjugate is a promising vaccine candidate for active or passive immunotherapy against E. faecalis and other Gram-positive bacteria.

Identification of Peptidoglycan-Associated Proteins as Vaccine Candidates for Enterococcal Infections

Infections by opportunistic bacteria have significant contributions to morbidity and mortality of hospitalized patients and also lead to high expenses in healthcare. In this setting, one of the major clinical problems is caused by Gram-positive bacteria such as enterococci and staphylococci. In this study we extract, purify, identify and characterize immunogenic surface-exposed proteins present in the vancomycin resistant enterococci (VRE) strain Enterococcus faecium E155 using three different extraction methods: trypsin shaving, biotinylation and elution at high pH. Proteomic profiling was carried out by gel-free and gel-nanoLC-MS/MS analyses. The total proteins found with each method were 390 by the trypsin shaving, 329 by the elution at high pH, and 45 using biotinylation. An exclusively extracytoplasmic localization was predicted in 39 (10%) by trypsin shaving, in 47 (15%) by elution at high pH, and 27 (63%) by biotinylation. Comparison between the three extraction methods by Venn diagram and subcellular localization predictors (CELLO v.2.5 and Gpos-mPLoc) allowed us to identify six proteins that are most likely surface-exposed: the SCP-like extracellular protein, a low affinity penicillin-binding protein 5 (PBP5), a basic membrane lipoprotein, a peptidoglycan-binding protein LysM (LysM), a D-alanyl-D-alanine carboxypeptidase (DdcP) and the peptidyl-prolyl cis-trans isomerase (PpiC). Due to their close relationship with the peptidoglycan, we chose PBP5, LysM, DdcP and PpiC to test their potential as vaccine candidates. These putative surface-exposed proteins were overexpressed in Escherichia coli and purified. Rabbit polyclonal antibodies raised against the purified proteins were able to induce specific opsonic antibodies that mediated killing of the homologous strain E. faecium E155 as well as clinical strains E. faecium E1162, Enterococcus faecalis 12030, type 2 and type 5. Passive immunization with rabbit antibodies raised against these proteins reduced significantly the colony counts of E. faecium E155 in mice, indicating the effectiveness of these surface-related proteins as promising vaccine candidates to target different enterococcal pathogens.

Comprehensive molecular, genomic and phenotypic analysis of a major clone of Enterococcus faecalis MLST ST40.

BackgroundEnterococcus faecalis is a multifaceted microorganism known to act as a beneficial intestinal commensal bacterium. It is also a dreaded nosocomial pathogen causing life-threatening infections in hospitalised patients. Isolates of a distinct MLST type ST40 represent the most frequent strain type of this species, distributed worldwide and originating from various sources (animal, human, environmental) and different conditions (colonisation/infection). Since enterococci are known to be highly recombinogenic we determined to analyse the microevolution and niche adaptation of this highly distributed clonal type.ResultsWe compared a set of 42 ST40 isolates by assessing key molecular determinants, performing whole genome sequencing (WGS) and a number of phenotypic assays including resistance profiling, formation of biofilm and utilisation of carbon sources. We generated the first circular closed reference genome of an E. faecalis isolate D32 of animal origin and compared it with the genomes of other reference strains. D32 was used as a template for detailed WGS comparisons of high-quality draft genomes of 14 ST40 isolates. Genomic and phylogenetic analyses suggest a high level of similarity regarding the core genome, also demonstrated by similar carbon utilisation patterns. Distribution of known and putative virulence-associated genes did not differentiate between ST40 strains from a commensal and clinical background or an animal or human source. Further analyses of mobile genetic elements (MGE) revealed genomic diversity owed to: (1) a modularly structured pathogenicity island; (2) a site-specifically integrated and previously unknown genomic island of 138 kb in two strains putatively involved in exopolysaccharide synthesis; and (3) isolate-specific plasmid and phage patterns. Moreover, we used different cell-biological and animal experiments to compare the isolate D32 with a closely related ST40 endocarditis isolate whose draft genome sequence was also generated. D32 generally showed a greater capacity of adherence to human cell lines and an increased pathogenic potential in various animal models in combination with an even faster growth in vivo (not in vitro).ConclusionMolecular, genomic and phenotypic analysis of representative isolates of a major clone of E. faecalis MLST ST40 revealed new insights into the microbiology of a commensal bacterium which can turn into a conditional pathogen.

Identification and functional characterization of the putative polysaccharide biosynthesis protein (CapD) of Enterococcus faecium U0317

Most bacterial species produce capsular polysaccharides that contribute to disease pathogenesis through evasion of the host innate immune system and are also involved in inhibiting leukocyte killing. In the present study, we identified a gene in Enterococcus faecium U0317 with homologies to the polysaccharide biosynthesis protein CapD that is made up of 336 amino acids and putatively catalyzes N-linked glycosylation. A capD deletion mutant was constructed and complemented by homologous recombination that was confirmed by PCR and sequencing. The mutant revealed different growth behavior and morphological changes compared to wild-type by scanning electron microscopy, also the capD mutant showed a strong hydrophobicity and that was reversed in the reconstituted mutant. For further characterization and functional analyses, in-vitro cell culture and in-vivo a mouse infection models were used. Antibodies directed against alpha lipotechoic acid (αLTA) and the peptidyl-prolyl cis-trans isomerase (αPpiC), effectively mediated the opsonophagocytic killing in the capD knock-out mutant, while this activity was not observed in the wild-type and reconstituted mutant. By comparison more than 2-fold decrease was seen in mutant colonization and adherence to both T24 and Caco2 cells. However, a significant higher bacterial colonization was observed in capD mutant during bacteremia in the animal model, while virulence in a mouse UTI (urinary tract infection) model, there were no obvious differences. Further studies are needed to elucidate the function of capsular polysaccharide synthesis gene clusters and its involvement in the disease pathogenesis with the aim to develop targeted therapies to treat multidrug-resistant E. faecium infections.

A Novel Role for D-Alanylation of Lipoteichoic Acid of Enterococcus faecalis in Urinary Tract Infection

Background Enterococci are the third most common cause of healthcare-associated infections, which include urinary tract infections, bacteremia and endocarditis. Cell-surface structures such as lipoteichoic acid (LTA) have been poorly examined in E. faecalis, especially with respect to urinary tract infections (UTIs). The dlt operon is responsible for the D-alanylation of LTA and includes the gene dltA, which encodes the D-alanyl carrier protein ligase (Dcl). The involvement of LTA in UTI infection by E. faecalis has not been studied so far. Here, we examined the role of teichoic acid alanylation in the adhesion of enterococci to uroepithelial cells. Results In a mouse model of urinary tract infection, we showed that E. faecalis 12030ΔdltA mutant colonizes uroepithelial surfaces more efficiently than wild type bacteria. We also demonstrated that this mutant adhered four fold better to human bladder carcinoma cell line T24 compared to the wild type strain. Bacterial adherence could be significantly inhibited by purified lipoteichoic acid (LTA) and inhibition was specific. Conclusion In contrast to bacteraemia model and adherence to colon surfaces, E. faecalis 12030ΔdltA mutant colonized uroepithelial surfaces more efficiently than wild-type bacteria. In the case of the uroepithelial surface the adherence to specific host cells could be prevented by purified LTA. Our results therefore suggest a novel function of alanylation of LTA in E. faecalis.

Cell-Wall Glycolipid Mutations and Their Effects on Virulence of E. faecalis in a Rat Model of Infective Endocarditis

Enterococci are among the major pathogens implicated in cardiac infections and biofilm formation. E. faecalis has been shown to play an important role in infectious endocarditis. Several distinct mechanisms for biofilm formation have been identified in E. faecalis. Our group has previously characterized two distinct bacterial glucosyltransferases playing key roles in the production of the major cell wall glycolipids and leading to reduced biofilm production. To assess if this mechanism is involved in the pathogenesis of enterococcal endocarditis we compared the wild-type strain of E. faecalis 12030 with two mutants in gene EF2891 and EF2890 respectively in a rat model of infective endocarditis. The results showed less endocarditic lesions and reduced colony counts per vegetation in the two mutants. indicating that the modification of bacterial surface lipids results in significantly reduced virulence in infective endocarditis. These results underscore the important role of biofilm formation in the pathogenicity of enterococcal endocarditis and may indicate an interesting target for novel therapeutic strategies.

Role of Glycolipids in the Pathogenesis of Enterococcus faecalis Urinary Tract Infection

Background After uropathogenic Escherichia coli (UPEC), Enterococcus faecalis is the second most common pathogen causing urinary tract infections. Monoglucosyl-diacylglycerol (MGlcDAG) and diglucosyl-diacylglycerol (DGlcDAG) are the main glycolipids of the E. faecalis cell membrane. Examination of two mutants in genes bgsB and bgsA (both glycosyltransferases) showed that these genes are involved in cell membrane glycolipid biosynthesis, and that their inactivation leads to loss of glycolipids DGlcDAG (bgsA) or both MGlcDAG and DGlcDAG (bgsB). Here we investigate the function of bgsB and bgsA regarding their role in the pathogenesis in a mouse model of urinary tract infection and in bacterial adhesion to T24 bladder epithelial cells. Results In a mouse model of urinary tract infection, we showed that E. faecalis 12030ΔbgsB and E. faecalis 12030ΔbgsA mutants, colonize uroepithelial surfaces more efficiently than wild-type bacteria. We also demonstrated that these mutants showed a more than three-fold increased binding to human bladder carcinoma cells line T24 compared to the wild-type strain. Bacterial binding could be specifically inhibited by purified glycolipids. Lipoteichoic acid (LTA), wall-teichoic acid (WTA), and glycosaminoglycans (GAGs) were not significantly involved in binding of E. faecalis to the bladder epithelial cell line. Conclusions Our data show that the deletion of bgsB and bgsA and the absence of the major glycolipid diglucosyl-diacylglycerol increases colonization and binding to uroepithelial cells. We hypothesize that secreted diglucosyl-diacylglycerol blocks host binding sites, thereby preventing bacterial adhesion. Further experiments will be needed to clarify the exact mechanism underlying the adhesion through glycolipids and their cognate receptors.