Gursharan S. Chhatwal

alpha-Enolase of Streptococcus pneumoniae is a plasmin(ogen)-binding protein displayed on the bacterial cell surface.

Binding of human plasminogen to Streptococcus pneumoniae and its subsequent activation promotes penetration of bacteria through reconstituted basement membranes. In this study, we have characterized a novel pneumococcal surface protein with a molecular mass of 47 kDa, designated Eno, which specifically binds human plasmin(ogen), exhibits α‐enolase activity and is necessary for viability. Using enzyme assays, we have confirmed the α‐enolase activity of both pneumococcal surface‐displayed Eno and purified recombinant Eno protein. Immunoelectron microscopy indicated the presence of Eno in the cytoplasm as well as on the surface of encapsulated and unencapsulated pneumococci. Plasminogen‐binding activity was demonstrated with whole pneumococcal cells and purified Eno protein. Binding of activated plasminogen was also shown for Eno; however, the affinity for plasmin is significantly reduced compared with plasminogen. Results from competitive inhibition assays indicate that binding is mediated through the lysine binding sites in plasmin(ogen). Carboxypeptidase B treatment and amino acid substitutions of the C‐terminal lysyl residues of Eno indicated that the C‐terminal lysine is pivotal for plasmin(ogen)‐binding activity. Eno is ubiquitously distributed among pneumococcal serotypes, and binding experiments suggested the reassociation of secreted Eno to the bacterial cell surface. The reassociation was also confirmed by immunoelectron microscopy. The results suggest a mechanism of plasminogen activation for human pathogens that might contribute to their virulence potential in invasive infectious processes.

SPSA, A NOVEL PNEUMOCOCCAL SURFACE PROTEIN WITH SPECIFIC BINDING TO SECRETORY IMMUNOGLOBULIN A AND SECRETORY COMPONENT

The interaction of pathogenic bacteria with host serum and matrix proteins is a common strategy to enhance their virulence. Streptococcus pneumoniae colonizes the human upper respiratory tract in healthy individuals and is also able to cause invasive diseases. Here, we describe a novel pneumococcal surface protein, SpsA, capable of binding specifically to human secretory immunoglobulin A (SIgA). The dissociation constant of SIgA binding to SpsA was 9.3 × 10−9 M. Free secretory component (SC) also binds to S. pneumoniae, whereas serum IgA does not, suggesting that pneumococcal binding to SIgA is mediated by the SC. To our knowledge, this is the first defined interaction of SC with a prokaryotic protein. The spsA gene encodes a polypeptide of 523 amino acids with a predicted molecular mass of 59 151 Da. The SIgA‐ or SC‐binding domain is located in the N‐terminal part of SpsA and exhibits no significant homology to any other proteins. The purified SIgA‐binding domain of SpsA could completely inhibit the binding of SIgA to pneumococci. SpsA was expressed by 73% of the tested S. pneumoniae isolates and was substantially conserved between different serotypes. The interaction between S. pneumoniae and SC via SpsA represents a novel biological interaction that might increase virulence by the impairment of bacterial clearance.

DNase Sda1 provides selection pressure for a switch to invasive group A streptococcal infection.

Most invasive bacterial infections are caused by species that more commonly colonize the human host with minimal symptoms. Although phenotypic or genetic correlates underlying a bacteriums shift to enhanced virulence have been studied, the in vivo selection pressures governing such shifts are poorly understood. The globally disseminated M1T1 clone of group A Streptococcus (GAS) is linked with the rare but life-threatening syndromes of necrotizing fasciitis and toxic shock syndrome. Mutations in the GAS control of virulence regulatory sensor kinase (covRS) operon are associated with severe invasive disease, abolishing expression of a broad-spectrum cysteine protease (SpeB) and allowing the recruitment and activation of host plasminogen on the bacterial surface. Here we describe how bacteriophage-encoded GAS DNase (Sda1), which facilitates the pathogens escape from neutrophil extracellular traps, serves as a selective force for covRS mutation. The results provide a paradigm whereby natural selection exerted by the innate immune system generates hypervirulent bacterial variants with increased risk of systemic dissemination.

Housekeeping enzymes as virulence factors for pathogens

Housekeeping enzymes are ubiquitously present in almost all living beings to perform essential metabolic functions for the purpose of survival. These enzymes have been characterized in detail for many years. In recent years, there has been a number of reports indicating that some of these enzymes perform a variety of other functions. In case of many pathogens, certain enzymes play a role to enhance virulence. To perform such a function, enzymes must be located on the surface of pathogens. Although they do not have the typical signal sequence or membrane anchoring mechanisms, they do get secreted and are displayed on the surface, probably by their reassociation. Once on the surface, these enzymes interact with host components, such as fibronectin and plasminogen, or interact directly with the host cells, to trigger signal transduction and thereby enable the pathogens to colonize, persist and invade the host tissue. Therefore, certain housekeeping enzymes may act as putative virulence factors and targets for the development of new strategies to control the infection by using agents that can block their secretion and/or reassociation.

Clostridium botulinum type C produces a novel ADP-ribosyltransferase distinct from botulinum C2 toxin

The culture medium of certain strains of Clostridium botulinum type C contains two separable ADP‐ribosyltransferases. Besides the ADP‐ribosylation of actin due to botulinum C2 I toxin, a second microbial enzyme causes the mono‐ADP‐ribosylation of a eukaryotic protein with a molecular mass of about 20 kDa found in platelets, neuroblastoma × glioma hybrid cells, S49 lymphoma cells, chick embryo fibroblasts and sperm. The eukaryotic substrate is inactivated by heating and trypsin treatment. In contrast, the novel ADP‐ribosyltransferase, which can be separated by DEAE‐Sephadex chromatography, is largely resistant in the short term to trypsin digestion.

Identification of a novel plasmin(ogen)‐binding motif in surface displayed α‐enolase of Streptococcus pneumoniae

The interaction of Streptococcus pneumoniae with human plasmin(ogen) represents a mechanism to enhance bacterial virulence by capturing surface‐associated proteolytic activity in the infected host. Plasminogen binds to surface displayed pneumococcal α‐enolase (Eno) and is subsequently activated to the serine protease plasmin by host‐derived tissue plasminogen activator (tPA) or urokinase (uPA). The C‐terminal lysyl residues of Eno at position 433 and 434 were identified as a binding site for the kringle motifs of plasmin(ogen) which contain lysine binding sites. In this report we have identified a novel internal plamin(ogen)‐binding site of Eno by investigating the protein–protein interaction. Plasmin(ogen)‐binding activity of C‐terminal mutated Eno proteins used in binding assays as well as surface plasmon resonance studies suggested that an additional binding motif of Eno is involved in the Eno‐plasmin(ogen) complex formation. The analysis of spot synthesized synthetic peptides representing Eno sequences identified a peptide of nine amino acids located between amino acids 248–256 as the minimal second binding epitope mediating binding of plasminogen to Eno. Binding of radiolabelled plasminogen to viable pneumococci was competitively inhibited by a synthetic peptide FYDKERKVYD representing the novel internal plasmin(ogen)‐binding motif of Eno. In contrast, a synthetic peptide with amino acid substitutions at critical positions in the internal binding motif identified by systematic mutational analysis did not inhibit binding of plasminogen to pneumococci. Pneumococcal mutants expressing α‐enolase with amino acid substitutions in the internal binding motif showed a substantially reduced plasminogen‐binding activity. The virulence of these mutants was also attenuated in a mouse model of intranasal infection indicating the significance of the novel plasminogen‐binding motif in the pathogenesis of pneumococcal diseases.

Anchorless adhesins and invasins of Gram-positive bacteria: a new class of virulence factors

Bacterial adherence to and invasion of eukaryotic cells are important mechanisms of pathogenicity. Most Gram-positive bacteria interact with the components of the host extracellular matrix (ECM) to adhere to, colonize and invade cells and tissues. The bacterial proteins that bind to components of the ECM harbour signal sequences for their secretion and mechanisms of anchoring to the host cell surface. However, in recent years, some cell-surface adhesins and invasins of Gram-positive bacteria have been described that do not possess a signal sequence or a membrane anchor. These proteins are secreted by an as-yet-unknown mechanism and are probably localized on the bacterial surface by reassociation. These anchorless but surface-located adhesins and invasins represent a new class of virulence factors.

Characterization of a novel fibronectin‐binding surface protein in group A streptococci

Streptococcus pyogenes interacts with host fibronectin via distinct surface components. One of these components is the Sfbl protein (streptococcal fibronectin‐binding protein, now specified as class I), an adhesin that represents a protein family with characteristic features. Here we present the complete structure of a novel fibronectin‐binding protein of S. pyogenes, designated SfbII, which is distinct from the previously described Sfbl proteins. The sfbII gene originated from a λ EMBL3 library of chromosomal DNA from group A streptococcal strain A75 and coded for a 113kDa protein exhibiting features of membrane‐anchored surface proteins of Gram‐positive cocci. The expression of biologically active fusion proteins allowed the determination of the location of the fibronectin‐binding domain within the C‐terminal part of the protein. It consisted of two and a half repeats which share common motifs with fibronectin‐binding repeats of other streptococcal and staphylococcal proteins. Purified recombinant fusion protein containing this domain competitively inhibited the binding of fibronectin to the parental S. pyogenes strain. Furthermore, polyclonal antibodies against the binding domain specifically blocked the Sfbll receptor site on the streptococcal surface. No cross‐reactivity could be detected between anti‐Sfbll antibodies and the sfbl gene product, and vice versa, indicating that the two proteins do not share common immunogenic epitopes. Southern hybridization experiments performed with specific sfbll gene probes revealed the presence of the sfbll gene in more than 55% of 93 streptococcal isolates tested. The majority of the strains also harboured the sfbl gene, and 86% carried at least one of the two sfb genes.

Species-specific binding of human secretory component to SpsA protein of Streptococcus pneumoniae via a hexapeptide motif

SpsA, a pneumococcal surface protein belonging to the family of choline‐binding proteins, interacts specifically with secretory immunglobulin A (SIgA) via the secretory component (SC). SIgA and free SC from mouse, rat, rabbit and guinea‐pig failed to interact with SpsA indicating species‐specific binding to human SIgA and SC. SpsA is the only pneumococcal receptor molecule for SIgA and SC as confirmed by complete loss of SIgA and SC binding to a spsA mutant. Analysis of recombinant SpsA fusion proteins showed that the binding domain is located in the N‐terminal region of SpsA. By the use of different truncated N‐terminal SpsA fusion proteins, the minimum binding domain was shown to be composed of 112 amino acids (residues 172–283). The sequence of this 112‐amino‐acids domain was used to spot synthesize 34 overlapping peptides, consisting of 15 amino acids each, with an offset of three amino acids on a cellulose membrane. One of the peptides reacted specifically with both SIgA and SC. By using a second membrane with immobilized synthetic peptides of decreasing length containing parts of the identified 15‐amino‐acid motif a hexapeptide, YRNYPT was identified as the binding motif for SC and SIgA. SpsA proteins with a size smaller than the assay‐positive domain of 112 amino acids were able to inhibit the interaction of SIgA and pneumococci provided they contained the binding motif. The results indicated that the hexapeptide YRNYPT located in SpsA of pneumococcal strain type 1 (ATCC 33400) between amino acids 198 and 203 is involved in SIgA and SC binding. Because synthetic peptides containing only parts of the hexapeptide also assayed positive, these results further suggest that at least the amino acids YPT of the identified hexapeptide are critical for binding to SC and SIgA. Amino acid substitutions in the identified putative binding motif abolished SC‐/SIgA‐binding activity of the mutated SpsA protein, confirming the functional activity of this hexapeptide and the critical role of the amino acids YPT in SC and SIgA binding. Identification of this motif, which is highly conserved in SpsA protein among different serotypes, might contribute towards a new peptide based vaccine strategy.

Protective Immune Response against Streptococcus pyogenes in Mice after Intranasal Vaccination with the Fibronectin-Binding Protein SfbI

Despite the significant impact on human health of Streptococcus pyogenes, an efficacious vaccine has not yet been developed. Here, the potential as a vaccine candidate of a major streptococcal adhesin, the fibronectin-binding protein SfbI, was evaluated. Intranasal immunization of mice with either SfbI alone or coupled to cholera toxin B subunit (CTB) triggered efficient SfbI-specific humoral (mainly IgG) and lung mucosal (14% of total IgA) responses. CTB-immunized control mice were not protected against challenge with S. pyogenes (90%-100% lethality), whereas SfbI-vaccinated animals showed 80% and 90% protection against homologous and heterologous challenge, respectively. Multiple areas of consolidation with diffused cellular infiltrates (macrophages and neutrophils) were observed in lungs from control mice; the histologic structure was preserved in SfbI-vaccinated animals, which occasionally presented focal infiltrates confined to the perivascular, peribronchial, and subpleural areas. These results suggest that SfbI is a promising candidate for inclusion in acellular vaccines against S. pyogenes.