Andrea Kropec

Glycolipids are involved in biofilm accumulation and prolonged bacteraemia in Enterococcus faecalis

Biofilm production is thought to be an important step in many enterococcal infections. In several Gram‐positive bacteria, membrane glycolipids have been implicated in biofilm formation. We constructed a non‐polar deletion mutant of a putative glucosyltransferase designated biofilm‐associated glycolipid synthesis A (bgsA) in Enterococcus faecalis 12030. Analysis of major extracted glycolipids by nuclear magnetic resonance spectroscopy revealed that the cell membrane of 12030ΔbgsA was devoid of diglucosyl–diacylglycerol (DGlcDAG), while monoglucosyl–diacylglycerol was overrepresented. The cell walls of 12030ΔbgsA contained longer lipoteichoic acid molecules and were less hydrophobic than wild‐type bacteria. Inactivation of bgsA in E.u2003faecalis 12030 and E.u2003faecalis V583 led to an almost complete arrest of biofilm formation on plastic surfaces. Overexpression of bgsA, on the other hand, resulted in increased biofilm production. While initial adherence was not affected, bgsA‐deficient bacteria did not accumulate in the growing biofilm. Also, adherence of E.u2003faecalisΔbgsA to Caco‐2 cells was impaired. In a mouse bacteraemia model, E.u2003faecalis 12030ΔbgsA was cleared more rapidly from the bloodstream than the wild‐type strain. In summary, BgsA is a glycosyltransferase synthetizing DGlcDAG, a glycolipid and lipoteichoic acid precursor involved in biofilm accumulation, adherence to host cells, and virulence in vivo.

Serodiversity of Opsonic Antibodies against Enterococcus faecalis —Glycans of the Cell Wall Revisited

In a typing system based on opsonic antibodies against carbohydrate antigens of the cell envelope, 60% of Enterococcus faecalis strains can be assigned to one of four serotypes (CPS-A to CPS-D). The structural basis for enterococcal serotypes, however, is still incompletely understood. Here we demonstrate that antibodies raised against lipoteichoic acid (LTA) from a CPS-A strain are opsonic to both CPS-A and CPS-B strains. LTA-specific antibodies also bind to LTA of CPS-C and CPS-D strains, but fail to opsonize them. From CPS-C and CPS-D strains resistant to opsonization by anti-LTA, we purified a novel diheteroglycan with a repeating unit of →6)-β-Galf-(1→3)- β-D-Glcp-(1→ with O-acetylation in position 5 and lactic acid substitution at position 3 of the Galf residue. The purified diheteroglycan, but not LTA absorbed opsonic antibodies from whole cell antiserum against E. faecalis type 2 (a CPS-C strain) and type 5 (CPS-D). Rabbit antiserum raised against purified diheteroglycan opsonized CPS-C and CPS-D strains and passive protection with diheteroglycan-specific antiserum reduced bacterial counts by 1.4 – 3.4 logs in mice infected with E. faecalis strains of the CPS-C and CPS-D serotype. Diheteroglycan-specific opsonic antibodies were absorbed by whole bacterial cells of E. faecalis FA2-2 (CPS-C) but not by its isogenic acapsular cpsI-mutant and on native PAGE purified diheteroglycan co-migrated with the gene product of the cps-locus, suggesting that it is synthesized by this locus. In summary, two polysaccharide antigens, LTA and a novel diheteroglycan, are targets of opsonic antibodies against typeable E. faecalis strains. These cell-wall associated polymers are promising candidates for active and passive vaccination and add to our armamentarium to fight this important nosocomial pathogen.

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.

Deletion of the glycosyltransferase bgsB of Enterococcus faecalis leads to a complete loss of glycolipids from the cell membrane and to impaired biofilm formation

BackgroundDeletion of the glycosyltransferase bgsA in Enterococcus faecalis leads to loss of diglucosyldiacylglycerol from the cell membrane and accumulation of its precursor monoglucosyldiacylglycerol, associated with impaired biofilm formation and reduced virulence in vivo. Here we analyzed the function of a putative glucosyltransferase EF2890 designated biofilm-associated glycolipid synthesis B (bgsB) immediately downstream of bgsA.ResultsA deletion mutant was constructed by targeted mutagenesis in E. faecalis strain 12030. Analysis of cell membrane extracts revealed a complete loss of glycolipids from the cell membrane. Cell walls of 12030ΔbgsB contained approximately fourfold more LTA, and 1H-nuclear magnetic resonance (NMR) spectroscopy suggested that the higher content of cellular LTA was due to increased length of the glycerol-phosphate polymer of LTA. 12030ΔbgsB was not altered in growth, cell morphology, or autolysis. However, attachment to Caco-2 cells was reduced to 50% of wild-type levels, and biofilm formation on polystyrene was highly impaired. Despite normal resistance to cationic antimicrobial peptides, complement and antibody-mediated opsonophagocytic killing in vitro, 12030ΔbgsB was cleared more rapidly from the bloodstream of mice than wild-type bacteria. Overall, the phenotype resembles the respective deletion mutant in the bgsA gene. Our findings suggest that loss of diglucosyldiacylglycerol or the altered structure of LTA in both mutants account for phenotypic changes observed.ConclusionsIn summary, BgsB is a glucosyltransferase that synthesizes monoglucosyldiacylglycerol. Its inactivation profoundly affects cell membrane composition and has secondary effects on LTA biosynthesis. Both cell-membrane amphiphiles are critical for biofilm formation and virulence of E. faecalis.

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 SagA as a novel vaccine target for the prevention of Enterococcus faecium infections.

Infections caused by multiresistant Gram-positive bacteria represent a major health burden in the community as well as in hospitalized patients. Enterococci, especially Enterococcus faecium, are well-known pathogens of hospitalized patients and are frequently linked with resistance against multiple antibiotics, which compromises effective therapy. Rabbit immune serum raised against heat-killed E. faecium E155, a HiRECC clone, was used in an opsonophagocytic assay, an inhibition assay and a mouse bacteraemia model to identify targets of opsonic and protective antibodies. Serum against whole heat-killed bacteria was opsonic and recognized a protein of about 72 kDa that was abundantly secreted. This protein, identified as SagA by LC-ES-MS/MS, was expressed in Escherichia coli and purified. Rabbit serum raised against the purified protein showed opsonic killing activity that was inhibited by almost 100% using 100 µg purified protein ml(-1). In a mouse bacteraemia model, a statistically significant reduction of the colony counts in blood was shown with immune rabbit serum compared with preimmune serum using the homologous and a heterologous vancomycin-resistant enterococci (VRE) strain. These results indicate that SagA could be used as a promising vaccine target to treat and/or prevent VRE bacteraemia.

Light fluorous synthesis of glucosylated glycerol teichoic acids.

We here describe the synthesis of glucosylated teichoic acid (TA) fragments using two complementary fluorous scaffolds. The use of a perfluorooctylpropylsulfonylethyl (F-Pse) linker in combination with (glucosyl)glycerol phosphoramidite building blocks allows for the assembly of TA fragments with a terminal phosphate mono-ester, whereas the use of a perfluorooctylsuccinyl spacer delivers TA oligomers featuring a terminal alcohol functionality. These complementary linker systems have been developed because the nature of the TA chain terminus can play a role in the biological activity of the synthetic TAs. A novel α-glucosylated glycerolphosphoramidite building block is introduced to allow for a robust light fluorous synthetic protocol.

In vitro and in vivo activity of hyperimmune globulin preparations against multiresistant nosocomial pathogens

PurposeWe compared different immunoglobulin preparations containing IgG (Intraglobin/Intratect) or a mixture of IgG, IgA, and IgM (Pentaglobin) to assess the opsonic and protective efficacy of human immunoglobulin preparations against multiresistent nosocomial pathogens.Materials and methodsClinical isolates of E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecalis, Enterococcus faecium, and Staphylococcus aureus were tested by opsonophagocytic assay using immunologobulin preparations at dilutions usually obtained in patients. The target antigens of opsonic antibodies were characterized by opsonophagocytic inhibition assays, and the protective efficacy in vivo was tested in a mouse bacteremia model as previously described.ResultsAll strains were killed to at least 50xa0% by Pentaglobin. One P. aeruginosa strain was not efficiently killed by Intraglobin (23xa0%)xa0but the other strains were killed by Intraglobinxa0to a similar degree compared to Pentaglobin. Opsonic IgG antibodies against E. faecalis were directed against LTA, while opsonic antibodies in Pentaglobin were primarily directed against other cell wall carbohydrates. In a mouse bacteremia model, Pentaglobin was more protective than Intratectxa0against Staphylococcus aureus, while Intratect reduced colony counts better than normal rabbit serum or saline.ConclusionsAll tested human immunoglobulin preparations contain opsonic and protective antibodies against targets present on multiresistant Gram-positive and Gram-negative bacteria. Enrichment of these preparations with IgM increases the protective efficacy against some strains, probably due to antibodies directed against cell wall carbohydrates.

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.

Targeting Type IV Secretion System Proteins to Combat Multidrug-Resistant Gram-positive Pathogens

For many gram-positive pathogens, conjugative plasmid transfer is an important means of spreading antibiotic resistance . Therefore, the search for alternative treatments to fight and prevent infections caused by these bacteria has become of major interest. In the present study, we evaluated the protein TraM, from the conjugative plasmid pIP501, as a potential vaccine candidate. Anti-TraM antiserum mediated in vitro opsonophagocytic killing of the strain harboring the pIP501 plasmid and also proved to be cross-reactive against other clinically relevant enterococcal and staphylococcal strains. Specificity of antibodies toward TraM was confirmed by results of an opsonophagocytic inhibition assay and Western blot. In addition, conjugative transfer experiments proved that TraM is essential for the transfer of pIP501. Finally, immunization with either TraM or anti-TraM antiserum reduced significantly the colony counts in mice livers, demonstrating that TraM is a promising vaccine candidate against enterococci and other gram-positive pathogens.