Vaccine candidates are typically identified through characterization of microbial surface antigens expressed under laboratory conditions. Here, we studied the major human pathogen Streptococcus pyogenes and showed that Slr, a lipoprotein encoded by all strains, is not detected on the bacterial surface during growth in broth but nevertheless is targeted by protective antibodies in vivo, as demonstrated by passive and active immunizations in a mouse model of invasive infection. The expression of Slr is governed by the zinc-controlled regulator AdcR, indicating that zinc depletion triggers surface expression of Slr in vivo. During infection in humans and mice, the antibody response to Slr is comparable to that elicited by the classical M protein and is, intriguingly, directed almost exclusively against a region with histidine triad (HT) motifs, an outcome that may represent a mechanism of immune escape. For vaccine development, these data focus interest on Slr and other microbial surface proteins selectively expressed in vivo.

Maternal vaccination is a promising strategy for preventing neonatal disease caused by group B Streptococcus. The safety and immunogenicity of the prototype vaccine GBS-NN, a fusion protein consisting of the N-terminal domains of the alpha-like proteins (Alp) αC and Rib, were recently evaluated favorably in healthy adult women in a phase 1 trial. Here we demonstrate robust immunoglobulin G (IgG) and immunoglobulin A (IgA) responses against αC and Rib, as well as against the heterotypic Alp family members Alp1–Alp3. IgA and heterotypic IgG responses are more variable between subjects and correlate with pre-existing immunity. Vaccine-induced IgG mediates opsonophagocytic killing and prevents bacterial invasion of epithelial cells. Like the vaccine-induced response, naturally acquired IgG against the vaccine domains is dominated by IgG1. Consistent with the high IgG1 cross-placental transfer rate, naturally acquired IgG against both domains reaches higher concentrations in neonatal than maternal blood, as assessed in a separate group of non-vaccinated pregnant women and their babies.

Maternal vaccination, resulting in transfer of protective IgG across the placenta, represents a promising strategy to prevent neonatal disease caused by group B Streptococcus (GBS). The prototype vaccine GBS-NN, a fusion protein consisting of the N-terminal domains of the alpha-like protein (Alp) family members AlphaC and Rib, has been shown to display a good safety profile and to elicit a strong antibody response against the intact GBS-NN protein in a randomized placebo-controlled double-blind phase 1 trial in healthy adult women. Here we show that the vaccinees achieve robust IgG and IgA responses against both AlphaC and Rib and that vaccination additionally gives rise to antibodies against the heterotypic Alp family members Alp1-3. Responses against the heterotypic N-domains were more variable between subjects and correlated strongly with levels of pre-existing antibodies. Postvaccination sera mediated opsonophagocytic killing that correlated strongly with IgG but not IgM responses elicited by the vaccine. These sera also consistently prevented bacterial invasion of human cervical epithelial cells. Like the vaccine-induced response, naturally acquired IgG against the N-domains of AlphaC and Rib was dominated by the IgG1 subclass. Consistent with the enhanced ability of IgG1 to cross the placenta, naturally acquired IgG against both domains accumulated in neonatal relative to maternal blood, as assessed in a separate group of non-vaccinated pregnant women and their babies.

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.

Many pathogens express a surface protein that binds the human complement regulator factor H (FH), as first described for Streptococcus pyogenes and the antiphagocytic M6 protein. It is commonly assumed that FH recruited to an M protein enhances virulence by protecting the bacteria against complement deposition and phagocytosis, but the role of FH-binding in S. pyogenes pathogenesis has remained unclear and controversial. Here, we studied seven purified M proteins for ability to bind FH and found that FH binds to the M5, M6 and M18 proteins but not the M1, M3, M4 and M22 proteins. Extensive immunochemical analysis indicated that FH binds solely to the hypervariable region (HVR) of an M protein, suggesting that selection has favored the ability of certain HVRs to bind FH. These FH-binding HVRs could be studied as isolated polypeptides that retain ability to bind FH, implying that an FH-binding HVR represents a distinct ligand-binding domain. The isolated HVRs specifically interacted with FH among all human serum proteins, interacted with the same region in FH and showed species specificity, but exhibited little or no antigenic cross-reactivity. Although these findings suggested that FH recruited to an M protein promotes virulence, studies in transgenic mice did not demonstrate a role for bound FH during acute infection. Moreover, phagocytosis tests indicated that ability to bind FH is neither sufficient nor necessary for S. pyogenes to resist killing in whole human blood. While these data shed new light on the HVR of M proteins, they suggest that FH-binding may affect S. pyogenes virulence by mechanisms not assessed in currently used model systems.

Recent studies indicate that defective activity of complement factor H (FH) is associated with several human diseases, suggesting that pure FH may be used for therapy. Here, we describe a simple method to isolate human FH, based on the specific interaction between FH and the hypervariable region (HVR) of certain Streptococcus pyogenes M proteins. Special interest was focused on the FH polymorphism Y402H, which is associated with the common eye disease age-related macular degeneration (AMD) and has also been implicated in the binding to M protein. Using a fusion protein containing two copies of the M5-HVR, we found that the Y402 and H402 variants of FH could be efficiently purified by single-step affinity chromatography from human serum containing the corresponding protein. Different M proteins vary in their binding properties, and the M6 and M5 proteins, but not the M18 protein, showed selective binding of the FH Y402 variant. Accordingly, chromatography on a fusion protein derived from the M6-HVR allowed enrichment of the Y402 protein from serum containing both variants. Thus, the exquisite binding specificity of a bacterial protein can be exploited to develop a simple and robust procedure to purify FH and to enrich for the FH variant that protects against AMD.

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.

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.

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.

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.

ABSTRACT Small-colony variants (SCVs) of Staphylococcus aureus are associated with persistent infections and may be selectively enriched during antibiotic therapy. Three pairs of clonally related S. aureus isolates were recovered from patients receiving systemic antibiotic therapy. Each pair consisted of an isolate with a normal phenotype and an isolate with an SCV phenotype. These SCVs were characterized by reduced susceptibility to gentamicin, reduced hemolytic activity, slow growth, and menadione auxotrophy. Sequencing of the genes involved in menadione biosynthesis revealed mutations in menB , the gene encoding naphthoate synthase, in all three strains with the SCV phenotype. The menB mutations were (i) a 9-bp deletion from nucleotides 55 to 63, (ii) a frameshift mutation that resulted in a premature stop codon at position 230, and (iii) a point mutation that caused the amino acid substitution Gly to Val at codon 233. Fluctuation tests showed that growth-compensated mutants arose in the SCV population of one strain, strain OM1b, at a rate of 1.8 × 10 −8 per cell per generation. Sequence analyses of 23 independently isolated growth-compensated mutants of this strain revealed alterations in the menB sequence in every case. These alterations included reversions to the wild-type sequence and intragenic second-site mutations. Each of the growth-compensated mutants showed a restoration of normal growth and a loss of menadione auxotrophy, increased susceptibility to gentamicin, and restored hemolytic activity. These data show that mutations in menB cause the SCV phenotype in these clinical isolates. This is the first report on the genetic basis of menadione-auxotrophic SCVs determined in clinical S. aureus isolates.

Strangles is an upper respiratory tract infection in horses, which is highly contagious and one of the more costly diseases of the horse. Three recombinant antigens were used to vaccinate horses, which were then experimentally challenged with Streptococcus equi, the causative agent for strangles. The vaccinated horses showed significantly reduced bacterial growth (p=0.02) and nasal discharge (p=0.0004), a typical symptom of strangles. Other clinical signs of strangles were also reduced and at post mortem examination, lower rate of empyaema or scarring of the guttural pouches was found in the vaccinated group (p=0.01). The antigens used were EAG (α2-macroglobulin, albumin, and IgG-binding protein), CNE (a collagen-binding protein), and SclC (a collagen-like protein). The adjuvant used was Abisco, a saponin derived matrix. No adverse effects were observed following vaccination with the antigens and adjuvant.

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.

Fibroblast-mediated collagen gel contraction depends on collagen-binding β1 integrins. Perturbation of these integrins reveals an alternative contraction process that is integrin αVβ3-dependent and platelet-derived growth factor (PDGF) BB-stimulated. Connective tissue cells actively control interstitial fluid pressure (IFP), and inflammation-induced lowering of IFP provides a driving force for edema formation. PDGF-BB normalizes a lowered IFP by an αVβ3-dependent process. A potential modulation of IFP by extracellular matrix-binding bacterial proteins has previously not been addressed. The fibronectin (FN)-binding protein FNE is specifically secreted by the highly virulent Streptococcus equi subspecies equi. FNE bound FN and native collagen type I with Kd values of ∼20 and ∼50 nm determined by solid-phase binding assays. Rotary shadowing revealed a single FNE binding site located at on average 122 nm from the C terminus of procollagen type I. FNE induced αVβ3-mediated contraction by C2C12 cells in a concentration-dependent manner having a maximal effect at ∼100 nm. This activity of FNE required cellular FN, and FNE acted synergistically to added plasma FN or PDGF-BB. FNE enhanced binding of soluble FN to immobilized collagen, and conversely the binding of collagen to immobilized FN. Marked bell-shaped concentration dependences for these interactions suggest that FNE forms a bridge between FN and collagen. Finally, FNE normalized dermal IFP lowered by anaphylaxis. Our data suggest that secreted FNE normalized lowering of IFP by stimulating connective tissue cell contraction.

Streptococcus equi ssp. equi is the causative agent of strangles, a highly contagious and serious disease in the upper respiratory tract of horses. The present study describes the characterization of IdeE, a homolog of the secreted IgG-specific protease IdeS/Mac of Streptococcus pyogenes. The activity of IdeE is compared with the activity of IdeZ, the corresponding enzyme of the closely related S. equi ssp. zooepidemicus. A study of the proteolytic activity of recombinant IdeE and IdeZ on IgG from a selection of mammals shows that only antibodies containing the substrate site of IdeS/Mac are cleaved, indicating that the specificities of these enzymes are similar. Interestingly, IgG from horse is less effectively cleaved than IgG from e.g. dog or humans, as the dominating IgG isotype in horse sera (IgG4) lacks a distinct substrate site for IdeE/IdeZ. IgG-degradation is observed when S. equi ssp. equi is grown in the presence of horse serum, but not when grown with purified IgG. As the fraction of degraded IgG contains IgG4, the observed activity might be due to the expression of an unknown enzyme rather than IdeE. In a similar assay, no proteolysis of IgG was detected in the growth media of S. equi ssp. zooepidemicus.

This thesis aimed at studying mechanisms involved in control of tissue fluid homeostasis during inflammation.The interstitial fluid pressure (PIF) is of importance for control of tissue fluid balance. A lowering of PIF in vivo will result in a transport of fluid from the circulation into the tissue, leading to edema. Loose connective tissues that surround blood vessels have an intrinsic ability to take up fluid and swell. The connective tissue cells exert a tension on the fibrous network of the tissues, thereby preventing the tissues from swelling. Under normal homeostasis, the interactions between the cells and the fibrous network are mediated by β1 integrins. Connective tissue cells are in this way actively controlling PIF.Here we show a previously unrecognized function for the integrin αVβ3, namely in the control of PIF. During inflammation the β1 integrin function is disturbed and the connective tissue cells release their tension on the fibrous network resulting in a lowering of PIF. Such a lowering can be restored by platelet-derived growth factor (PDGF) -BB. We demonstrated that PDGF-BB restored PIF through a mechanism that was dependent on integrin αVβ3. This was shown by the inability of PDGF-BB to restore a lowered PIF in the presence of anti-integrin β3 IgG or a peptide inhibitor of integrin αVβ3. PDGF-BB was in addition unable to normalize a lowered PIF in β3 null mice. Furthermore, we demonstrated that extracellular proteins from Streptococcus equi modulated αVβ3-mediated collagen gel contraction. Because of the established concordance between collagen gel contraction in vitro and control of PIF in vivo, a potential role for these proteins in control of tissue fluid homeostasis during inflammation could be assumed. Sepsis and septic shock are severe, and sometimes lethal, conditions. Knowledge of how bacterial components influence PIF and the mechanisms for tissue fluid control during inflammatory reactions is likely to be of clinical importance in treating sepsis and septic shock.

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.

A mouse model resembling Streptococcus equi subspecies equi infection in the horse, strangles, was used to assess the protective effect of vaccination with selected recombinant proteins from S. equi subsp. equi. After challenge the infection was monitored by weight loss and by nasal colonisation with S. equi subsp. equi. Vaccination with a collagen-binding protein (CNE) and a collagen-like protein (SclC) resulted in protective antibodies, whereas a novel fibronectin-binding protein (FNEB) did not. Co-administration of CNE with EAG, a poorly immunogenic alpha2-macroglobulin-, albumin- and immunoglobulin G-binding protein, resulted in a significant synergistic effect and enhanced the protective immune response against EAG.

The N-terminal fragment (FNZN) of the fibronectin-binding protein FNZ from Streptococcus equi subspecies zooepidemicus was investigated as to effects on murine cell interactions with extracellular matrix proteins. FNZN bound to immobilized fibronectin (FN) and native, but not denatured, collagen type I. FNZN had no effect on primary adhesion of cells from the murine myoblastic C2C12 cell line to immobilized fibronectin. C2C12 cells adhered to immobilized FNZN, a process that was not inhibited by anti-human FN IgG or by an inhibitor of integrin alphaVbeta3. C2C12 cells lack collagen-binding beta1 integrins and neither adhere to native collagen nor mediate contraction of three-dimensional collagen gels. FNZN stimulated collagen gel contraction by C2C12 cells but not adhesion of C2C12 cells to collagen. Experiments with an alphaVbeta3-inhibitor suggested that FNZN promoted contraction by a process requiring alphaVbeta3. Our data suggest that FNZN by binding to cells, collagen, and FN modulate complex adhesive processes mediated by the alphaVbeta3 integrin. Since alphaVbeta3-mediated contractile events function to counteract edema formation during inflammation, it is possible that FNZN and its secreted homologue FNE modulate edema responses in infected tissues.