Genetic Complexity of Fusidic Acid-Resistant Small Colony Variants (SCV) in Staphylococcus aureus

FusE mutants are fusidic acid-resistant small colony variants (SCVs) of Staphylococcus aureus that can be selected with aminoglycosides. All FusE SCVs have mutations in rplF, encoding ribosomal protein L6. However, individual FusE mutants including some with the same mutation in rplF display auxotrophy for either hemin or menadione, suggesting that additional mutations are involved. Here we show that FusE SCVs can be divided into three genetic sub-groups and that some carry an additional mutation, in one of the genes required for hemin biosynthesis, or in one of the genes required for menadione biosynthesis. Reversion analysis and genome sequencing support the hypothesis that these combinations of mutations in the rplF, hem, and/or men genes can account for the SCV and auxotrophic phenotypes of FusE mutants

Potentially virulence-related extracellular proteins of Streptococcus equi

Equine strangles, a disease of the upper respiratory tract caused by the bacterium Streptococcus equi subspecies equi, is one of the most commonly diagnosed and serious diseases in horses. However, the molecular basis of S. equi subsp. equi infection is poorly understood and there are no safe and effective vaccines on the market. The main objective of this study was to identify and characterise extracellular proteins used by S. equi subsp. equi to initiate infection and cause disease. Extracellular proteins, which can be secreted or cell surface-located, play an important role in the initiation of infection and in continued bacterial survival inside the host. Adhesins are a specific class of virulence-related proteins that are used by the bacteria to attach to host tissues. This study focused on a number of cell-surface anchored proteins that specifically adhere to collagen and fibronectin, two major extracellular matrix proteins of vertebrates. The binding characteristics of the fibronectin-binding protein FNEB were compared to two previously studied fibronectin-binding proteins, FNE and SFS. A follow-up investigation showed that FNE and FNEB are part of a family of six similar proteins encoded by S. equi subsp. equi. One of the four novel proteins identified (FNEE) was shown to bind to fibronectin and all four to collagen. In another study, a secreted immunoglobulin-specific protease was characterised. This enzyme could be used by the bacteria to interfere with the immune response of the infected horse. A useful tool in this research was the public S. equi subsp. equi genome database, which can be used to identify homologues to virulence-related proteins of other pathogens. An applied objective of the present study was to identify potential components for a future vaccine against strangles. One of the collagen-binding proteins characterised (CNE) is currently being used in vaccination trials as a component of a protein subunit vaccine for horses

Genetic Determinants of Resistance to Fusidic Acid among Clinical Bacteremia Isolates of Staphylococcus aureus▿

Resistance to fusidic acid in Staphylococcus aureus is caused by mutation of the elongation factor G (EF-G) drug target (FusA class) or by expression of a protein that protects the drug target (FusB and FusC classes). Recently, two novel genetic classes of small-colony variants (SCVs) were identified among fusidic acid-resistant mutants selected in vitro (FusA-SCV and FusE classes). We analyzed a phylogenetically diverse collection of fusidic acid-resistant bacteremia isolates to determine which resistance classes were prevalent and whether these were associated with particular phylogenetic lineages. Each isolate was shown by DNA sequencing and plasmid curing to carry only one determinant of fusidic acid resistance, with approximately equal frequencies of the FusA, FusB, and FusC genetic classes. The FusA class (mutations in fusA) were distributed among different phylogenetic types. Two distinct variants of the FusC class (chromosomal fusC gene) were identified, and FusC was also distributed among different phylogenetic types. In contrast, the FusB class (carrying fusB on a plasmid) was found in closely related types. No FusE-class mutants (carrying mutations in rplF) were found. However, one FusA-class isolate had multiple mutations in the fusA gene, including one altering a codon associated with the FusA-SCV class. SCVs are frequently unstable and may undergo compensatory evolution to a normal growth phenotype after their initial occurrence. Accordingly, this normal-growth isolate might have evolved from a fusidic acid-resistant SCV. We conclude that at least three different resistance classes are prevalent among fusidic acid-resistant bacteremia isolates of S. aureus

Genetic and Phenotypic Identification of Fusidic Acid-Resistant Mutants with the Small-Colony-Variant Phenotype in Staphylococcus aureus▿

Small-colony variants (SCVs) of Staphylococcus aureus are a slow-growing subpopulation whose phenotypes can include resistance to aminoglycosides, defects in electron transport, and enhanced persistence in mammalian cells. Here we show that a subset of mutants selected as SCVs by reduced susceptibility to aminoglycosides are resistant to the antibiotic fusidic acid (FA) and conversely that a subset of mutants selected for resistance to FA are SCVs. Mutation analysis reveals different genetic classes of FA-resistant SCVs. One class, FusA-SCVs, have amino acid substitution mutations in the ribosomal translocase EF-G different from those found in classic FusA mutants. Most of these mutations are located in structural domain V of EF-G, but some are in domain I or III. FusA-SCVs are auxotrophic for hemin. A second class of FA-resistant SCVs carry mutations in rplF, coding for ribosomal protein L6, and are designated as FusE mutants. FusE mutants fall into two phenotypic groups: one auxotrophic for hemin and the other auxotrophic for menadione. Accordingly, we have identified new genetic and phenotypic classes of FA-resistant mutants and clarified the genetic basis of a subset of S. aureus SCV mutants. A clinical implication of these data is that FA resistance could be selected by antimicrobial agents other than FA

Studies of Fibronectin-Binding Proteins of Streptococcus equi

Streptococcus equi subsp. equi is the causative agent of strangles, a disease of the upper respiratory tract in horses. The initiation of S. equi subsp. equi infection is likely to involve cell surface-anchored molecules mediating bacterial adhesion to the epithelium of the host. The present study describes the cloning and characterization of FNEB, a fibronectin-binding protein with cell wall-anchoring motifs. FNEB can thus be predicted as cell surface located, contrary to the two previously characterized fibronectin-binding proteins in S. equi subsp. equi, FNE and SFS. Assays of antibody titers in horses and in experimentally infected mice indicate that the protein is immunogenic and expressed in vivo during S. equi subsp. equi infection. Using Western ligand blotting, it was shown that FNEB binds to the N-terminal 29-kDa fragment of fibronectin, while SFS and FNE both bind to the adjacent 40-kDa fragment. S. equi subsp. equi is known to bind fibronectin to a much lower degree than the closely related S. equi subsp. zooepidemicus, but the binding is primarily directed to the 29-kDa fragment. Inhibition studies using S. equi subsp. equi cells indicate that FNEB mediates cellular binding to fibronectin in this species

Two novel IgG endopeptidases of Streptococcus equi

Streptococcus equi ssp. equi causes strangles, a highly contagious and serious disease in the upper respiratory tract of horses. Streptococcus equi ssp. zooepidemicus, another subspecies of this genus, is regarded as an opportunistic commensal in horses. The present study describes the characterization of two novel immunoglobulin G (IgG) endopeptidases of these subspecies, IdeE2 and IdeZ2. Both enzymes display sequence similarities with two previously characterized IgG endopeptidases, IdeE of S. equi ssp. equi and IdeZ of S. equi ssp. zooepidemicus. IdeE2 and IdeZ2 display high substrate-specificity in comparison with IdeE and IdeZ, as they both completely cleave horse IgG, while the activity against IgG from mouse, rabbit, cat, cow, sheep and goat is low or absent. The potential use of IdeE and IdeE2 as vaccine components was studied in a mouse infection model. In this vaccination and challenge study, both enzymes induced protection against S. equi ssp. equi infection

The Hypervariable Region of Streptococcus pyogenes M Protein Escapes Antibody Attack by Antigenic Variation and Weak Immunogenicity.

Sequence variation of antigenic proteins allows pathogens to evade antibody attack. The variable protein commonly includes a hypervariable region (HVR), which represents a key target for antibodies and is therefore predicted to be immunodominant. To understand the mechanism(s) of antibody evasion, we analyzed the clinically important HVR-containing M proteins of the human pathogen Streptococcus pyogenes. Antibodies elicited by M proteins were directed almost exclusively against the C-terminal part and not against the N-terminal HVR. Similar results were obtained for mice and humans with invasive S. pyogenes infection. Nevertheless, only anti-HVR antibodies protected efficiently against infection, as shown by passive immunizations. The HVR fused to an unrelated protein elicited no antibodies, implying that it is inherently weakly immunogenic. These data indicate that the M protein HVR evades antibody attack not only through antigenic variation but also by weak immunogenicity, a paradoxical observation that may apply to other HVR-containing proteins

Sequence variability is correlated with weak immunogenicity in Streptococcus pyogenes M protein.

The M protein of Streptococcus pyogenes, a major bacterial virulence factor, has an amino-terminal hypervariable region (HVR) that is a target for type-specific protective antibodies. Intriguingly, the HVR elicits a weak antibody response, indicating that it escapes host immunity by two mechanisms, sequence variability and weak immunogenicity. However, the properties influencing the immunogenicity of regions in an M protein remain poorly understood. Here, we studied the antibody response to different regions of the classical M1 and M5 proteins, in which not only the HVR but also the adjacent fibrinogen-binding B repeat region exhibits extensive sequence divergence. Analysis of antisera from S. pyogenes-infected patients, infected mice, and immunized mice showed that both the HVR and the B repeat region elicited weak antibody responses, while the conserved carboxy-terminal part was immunodominant. Thus, we identified a correlation between sequence variability and weak immunogenicity for M protein regions. A potential explanation for the weak immunogenicity was provided by the demonstration that protease digestion selectively eliminated the HVR-B part from whole M protein-expressing bacteria. These data support a coherent model, in which the entire variable HVR-B part evades antibody attack, not only by sequence variability but also by weak immunogenicity resulting from protease attack

Differential binding of Y402 and H402 FH to different M proteins: enrichment of the Y402 protein on an M6 derived construct.

<p>(<b>A</b>) Binding of pure Y402 and H402 FH to <i>S. pyogenes</i> M5, M6 and M18 bacteria. The pure FH proteins were added at the concentrations indicated. Bound FH was detected with sheep anti-FH and radiolabeled protein G. The binding to M-negative control strains was low (not shown). (<b>B</b>) Differential binding of the Y402 and H402 proteins to pure M5, M6 and M18 immobilized in microtiter wells. The FH proteins were added at the concentrations indicated. Bound FH was detected with sheep anti-FH and radiolabeled protein G. The data in A and B represent mean values with SD from three experiments. Two-way ANOVA analysis was performed on the binding values corresponding to the highest concentrations of FH corrected for the difference between individual experiments (not set to 100 % as in the graph) (n=3). The differences in binding to Y402 and H402 FH were found to be significant with P<0.001 for M5, P<0.001 for M6 and P=0.004 for M18. (<b>C</b>) Schematic representation of the 6161 construct. See text. (<b>D</b>) Affinity purification of FH on the 6161 and 5151 constructs. Serum containing Y402 and H402 FH (from a heterozygote) was applied to columns with immobilized 6161 or 5151. Bound protein was eluted and analyzed by SDS-PAGE (left panel) and western blot (middle and right panels). The blots were probed with mAbs specific for Y402 or H402 FH, as indicated. Pure Y402 and H402 FH were included as controls.</p