Stephen B. Olmsted

Contact with epithelial cells induces the formation of surface appendages on Salmonella typhimurium

The enteric bacteria Salmonella typhimurium has the ability to invade (enter) nonphagocytic cells. The internalization process occurs as a result of an intimate interaction between the bacteria and the host cell, in which S. typhimurium triggers a cascade of host cell-signaling events leading to the formation of host cell membrane ruffles and bacterial uptake. Using high resolution scanning electron microscopy, we have observed that contact with cultured epithelial cells results in the formation of appendages on the surface of S. typhimurium. The formation of such appendages did not require de novo protein synthesis, and it was transient, since these surface structures were no longer present on bacteria that had initiated the internalization event. Salmonella mutants defective in the transient formation of these surface organelles were unable to enter into cultured epithelial cells, indicating that such structures are required for bacterial internalization.

Proteomic Analysis and Identification of Streptococcus pyogenes Surface-Associated Proteins

Streptococcus pyogenes is a gram-positive human pathogen that causes a wide spectrum of disease, placing a significant burden on public health. Bacterial surface-associated proteins play crucial roles in host-pathogen interactions and pathogenesis and are important targets for the immune system. The identification of these proteins for vaccine development is an important goal of bacterial proteomics. Here we describe a method of proteolytic digestion of surface-exposed proteins to identify surface antigens of S. pyogenes. Peptides generated by trypsin digestion were analyzed by multidimensional tandem mass spectrometry. This approach allowed the identification of 79 proteins on the bacterial surface, including 14 proteins containing cell wall-anchoring motifs, 12 lipoproteins, 9 secreted proteins, 22 membrane-associated proteins, 1 bacteriophage-associated protein, and 21 proteins commonly identified as cytoplasmic. Thirty-three of these proteins have not been previously identified as cell surface associated in S. pyogenes. Several proteins were expressed in Escherichia coli, and the purified proteins were used to generate specific mouse antisera for use in a whole-cell enzyme-linked immunosorbent assay. The immunoreactivity of specific antisera to some of these antigens confirmed their surface localization. The data reported here will provide guidance in the development of a novel vaccine to prevent infections caused by S. pyogenes.

Antibody against Surface-Bound C5a Peptidase Is Opsonic and Initiates Macrophage Killing of Group B Streptococci

ABSTRACT The capsular polysaccharides of group B streptococci (GBS) are a primary focus of vaccine development. Immunogenicity and long-lasting protection are best achieved by conjugating polysaccharides to a T-cell-dependent protein antigen. Streptococcal C5a peptidase (SCPB) is a conserved surface protein that is expressed by all streptococcal serotypes tested to date, and it is a possible carrier protein that could itself induce a protective immune response. Clearance of GBS from lungs, mucosal surfaces, or blood probably depends on the opsonophagocytic response of tissue-specific macrophages and polymorphonuclear leukocytes (PMNs). In this study, we examined the potential of antibody directed against SCPB from a serotype II strain to enhance the capacity of mouse bone marrow macrophages (from primary cultures) and human PMNs in whole blood to kill GBS in vitro. Our experiments demonstrated that Streptococcus serotypes Ia, Ib, II, III, and V, preopsonized with anti-SCPB antibody, were killed more rapidly by cultured macrophages and PMNs in whole blood than were nonopsonized GBS. The increased rate of killing was accompanied by an increased macrophage oxidative burst. Furthermore, opsonization was serotype transparent. Immunization with SCPB conjugated to capsular polysaccharide type III produced polysaccharide-specific antibodies. It is interesting that this antiserum promoted serotype-independent killing of streptococci. These data support the use of SCPB in a GBS polysaccharide conjugate vaccine. SCPB not only enhanced the immunogenicity of polysaccharide components of the vaccine, but it might also induce additional serotype-independent protective antibodies.

Toxoids of streptococcal pyrogenic exotoxin A are protective in rabbit models of streptococcal toxic shock syndrome

ABSTRACT Streptococcal pyrogenic exotoxins (SPEs) are superantigens that have been implicated in causing streptococcal toxic shock syndrome (STSS). Most notably, SPE serotype A is made by nearly all M-protein serotype 1 and 3 streptococci, the M types most associated with the illness (these strains contain one or more other SPEs, and those proteins are likely also to contribute to disease). We have prepared double-, triple-, and hexa-amino-acid mutants of SPE A by PCR and other mutagenesis procedures. The sites chosen for mutation were solvent-exposed residues thought to be important for T-cell receptor (TCR) or major histocompatibility complex (MHC) class II interaction. These mutants were nonsuperantigenic for human peripheral blood mononuclear cells and rabbit and mouse splenocytes and were nonlethal in two rabbit models of STSS. In addition, these mutants stimulated protective antibody responses. Interestingly, mutants that altered toxin binding to MHC class II were more immunogenic than mutants altering TCR binding. Collectively, these studies indicate that multiple-site mutants of SPE A are toxoids that may have use in protecting against the toxins effects in STSS.

Development of Streptococcal Pyrogenic Exotoxin C Vaccine Toxoids That Are Protective in the Rabbit Model of Toxic Shock Syndrome

Streptococcal pyrogenic exotoxin C (SPE C) is a superantigen produced by many strains of Streptococcus pyogenes that (along with streptococcal pyrogenic exotoxin A) is highly associated with streptococcal toxic shock syndrome (STSS) and other invasive streptococcal diseases. Based on the three-dimensional structure of SPE C, solvent-exposed residues predicted to be important for binding to the TCR or the MHC class II molecule, or important for dimerization, were generated. Based on decreased mitogenic activity of various single-site mutants, the double-site mutant Y15A/N38D and the triple-site mutant Y15A/H35A/N38D were constructed and analyzed for superantigenicity, toxicity (lethality), immunogenicity, and the ability to protect against wild-type SPE C-induced STSS. The Y15A/N38D and Y15A/H35A/N38D mutants were nonmitogenic for rabbit splenocytes and human PBMCs and nonlethal in two rabbit models of STSS, yet both mutants were highly immunogenic. Animals vaccinated with the Y15A/N38D or Y15A/H35A/N38D toxoids were protected from challenge with wild-type SPE C. Collectively, these data indicate that the Y15A/N38D and Y15A/H35A/N38D mutants may be useful as toxoid vaccine candidates.

Immunization with C5a peptidase or peptidase-type III polysaccharide conjugate vaccines enhances clearance of group B Streptococci from lungs of infected mice.

ABSTRACT Group B streptococci (GBS) are among the most common causes of life-threatening neonatal infections. Vaccine development since the late 1970s has focused on the capsular polysaccharides, but a safe, effective product is still not available. Our quest for a vaccine turned to the streptococcal C5a peptidase (SCPB). This surface protein is antigenically conserved across most if not all serotypes. A murine model was used to assess the impact of SCPB on clearance of GBS from the lungs of intranasally infected animals. Mutational inactivation of SCPB resulted in more-rapid clearance of streptococci from the lung. Immunization with recombinant SCPB alone or SCPB conjugated to type III capsular polysaccharide produced serotype-independent protection, which was evidenced by more-rapid clearance of the serotype VI strain from the lungs. Immunization of mice with tetanus toxoid-type III polysaccharide conjugate did not produce protection, confirming that protection induced by SCPB conjugates was independent of type III polysaccharide antigen. Histological evaluation of lungs from infected mice revealed that pathology in animals immunized with SCPB or SCPB conjugates was significantly less than that in animals immunized with a tetanus toxoid-polysaccharide conjugate. These experiments suggest that inclusion of C5a peptidase in a vaccine will both add another level to and broaden the spectrum of the protection of a polysaccharide vaccine.

Bacterial translocation in cultured enterocytes: magnitude, specificity, and electron microscopic observations of endocytosis.

Previous in vivo evidence has shown that bacterial phagocytosis by enterocytes may be an initial step in bacterial translocation across the intestinal epithelium. This study analyzed the interactions of cultured enterocytes, namely Caco-2 cells, with nine strains of enteric bacteria, tested in pure culture and in mixed culture. These nine strains had a spectrum of invasive potential and included Salmonella typhimurium, Listeria monocytogenes (three strains), Escherichia coli (three strains), Proteus mirabilis, and Enterococcus faecalis. Numbers of viable intracellular bacteria recovered from Caco-2 cells were: L. monocytogenes>S. typhimurium>P. mirabilis>E. coli>E. faecalis. Uptake of a given microbe by enterocytes was strain-specific and was not influenced by the presence of another strain, regardless of the invasive ability of the coinfecting strain. Electron microscopic visualization of bacterial adherence and uptake by Caco-2 cells indicated that the epithelial interactions of normal enteric bacteria were similar to these observed with invasive strains of salmonella and listeria.

The early interferon response of nasal-associated lymphoid tissue to Streptococcus pyogenes infection

Streptococcus pyogenes is a major causative agent of tonsillitis or pharyngitis in children. Streptococcus pyogenes can persist in tonsils, and one-third of children treated with antibiotics continue to shed streptococci and have recurrent infections. Mouse nasal-associated lymphoid tissue (NALT) is functionally analogous to human oropharyngeal lymphoid tissues, and serves as a model for characterization of the mucosal innate immune response to S. pyogenes. Wild-type S. pyogenes induces transcription of both type I and interferon-gamma (IFN-gamma)-responsive genes, proinflammatory genes and acute-phase response proteins 24 h after intranasal infection. Invasion of NALT and the induction of the interferon response were not dependent on expression of antiphagocytic M protein. Intranasal infection induces a substantial influx of neutrophils into NALT at 24 h, which declines by 48 h after infection. Infection of IFN-gamma(-/-) [IFN-gamma knock-out mouse (GKO)] C57BL/6 mice with wild-type S. pyogenes resulted in local dissemination of bacteria to draining lymph nodes (LN), but did not lead to systemic infection by 48 h after infection. Infected GKO mice had an increased influx of neutrophils into NALT compared with immunocompetent mice. Thus, IFN-gamma-induced responses are required to prevent local dissemination of streptococci to the draining LN.