Claudia F. Deobald

Staphylococcal fibronectin binding protein interacts with heat shock protein 60 and integrins : Role in internalization by epithelial cells

ABSTRACT We reported previously that internalization of Staphylococcus aureus by nonprofessional phagocytes involves an interaction between fibronectin (Fn) binding protein (FnBP) and the host cell, resulting in signal transduction, tyrosine kinase activity, and cytoskeletal rearrangement (K. Dziewanowska, J. M. Patti, C. F. Deobald, K. W. Bayles, W. R. Trumble, and G. A. Bohach, Infect. Immun. 67:4673–4678, 1999). The goal of the present study was to identify the host molecules responsible for uptake of the organism through an interaction with FnBP. First, Fn was required for internalization. Addition of small amounts of exogenous Fn stimulated the uptake of S. aureus by HEp-2 cells, which are deficient in Fn synthesis. Fn antibodies blocked internalization of the organism by MAC-T cell monolayers, a bovine epithelial cell line which expresses Fn. Second, a monoclonal antibody (MAb) specific for β1integrins dramatically reduced S. aureus invasion, suggesting that the formation of a Fn bridge linking the host cell β1 integrin and FnBP precedes internalization. However, ligand blotting of cell membrane proteins with a functional fragment of FnBP consistently identified an additional ∼55-kDa receptor on both human and bovine epithelial cells. This protein was purified and identified by N-terminal microsequencing as heat shock protein 60 (Hsp60). The interaction between FnBP and Hsp60 also occurred when the whole cells were used. Cell membrane localization of Hsp60 was confirmed by biotinylation with an agent nonpermeable to the cell membrane. Pretreatment of epithelial cells with a MAb specific for eukaryotic Hsp60 significantly reduced internalization of S. aureus. Combined, these results suggest that the FnBP binds directly to both Hsp60 and Fn and is linked to β1integrins through a Fn bridge. The simultaneous involvement of Fn and two host cell ligands, β1 integrins and Hsp60, suggests that FnBP is a multifunctional adhesin that mediates internalization in a manner similar to that proposed for OpaA, the Neisseria gonorrhoeae FnBP homolog (J. P. M. van Putten, T. D. Duensing, and R. L. Cole, Mol. Microbiol. 29:369–379, 1998).

An enterotoxin-bearing pathogenicity island in Staphylococcus epidermidis

Cocolonization of human mucosal surfaces causes frequent encounters between various staphylococcal species, creating opportunities for the horizontal acquisition of mobile genetic elements. The majority of Staphylococcus aureus toxins and virulence factors are encoded on S. aureus pathogenicity islands (SaPIs). Horizontal movement of SaPIs between S. aureus strains plays a role in the evolution of virulent clinical isolates. Although there have been reports of the production of toxic shock syndrome toxin 1 (TSST-1), enterotoxin, and other superantigens by coagulase-negative staphylococci, no associated pathogenicity islands have been found in the genome of Staphylococcus epidermidis, a generally less virulent relative of S. aureus. We show here the first evidence of a composite S. epidermidis pathogenicity island (SePI), the product of multiple insertions in the genome of a clinical isolate. The taxonomic placement of S. epidermidis strain FRI909 was confirmed by a number of biochemical tests and multilocus sequence typing. The genome sequence of this strain was analyzed for other unique gene clusters and their locations. This pathogenicity island encodes and expresses staphylococcal enterotoxin C3 (SEC3) and staphylococcal enterotoxin-like toxin L (SElL), as confirmed by quantitative reverse transcription-PCR (qRT-PCR) and immunoblotting. We present here an initial characterization of this novel pathogenicity island, and we establish that it is stable, expresses enterotoxins, and is not obviously transmissible by phage transduction. We also describe the genome sequence, excision, replication, and packaging of a novel bacteriophage in S. epidermidis FRI909, as well as attempts to mobilize the SePI element by this phage.

Characterization of Staphylococcal Bovine Mastitis Isolates Using the Polymerase Chain Reaction

A polymerase chain reaction (PCR) assay was adapted to detect toxin genes of staphylococcal isolates from cases of bovine mastitis. Samples were obtained from three geographical areas: Korea and Idaho and Washington in the northwest United States. Samples from Korea and Washington were randomly chosen. Idaho samples were from a prospective study of mastitis etiology. Forty-one milk samples from 25 commercial farms in south-central Idaho were collected from cows with symptoms of mastitis. Although Staphylococcus aureus constituted 37.5% of mastitis isolates, these isolates lacked genes for staphylococcal enterotoxins (SEs), toxic shock syndrome toxin, and exfoliative toxins. In contrast, 4 of 13 isolates from Washington and 6 of 20 isolates from South Korea expressed SEs. These results suggest that PCR may be an effective means of screening bovine isolates for toxins. They also emphasize the potential for significant geographic differences in mastitis etiology.

Induction of innate immunity by lipid A mimetics increases survival from pneumonic plague

This study analysed the effect of priming the innate immune system using synthetic lipid A mimetics in a Yersinia pestis murine pulmonary infection model. Two aminoalkyl glucosaminide 4-phosphate (AGP) Toll-like receptor 4 (TLR4) ligands, delivered intranasally, extended time to death or protected against a lethal Y. pestis CO92 challenge. The level of protection was dependent upon the challenge dose of Y. pestis and the timing of AGP therapy. Protection correlated with cytokine induction and a decreased bacterial burden in lung tissue. AGP protection was TLR4-dependent and was not evidenced in transgenic TLR4-deficient mice. AGP therapy augmented with subtherapeutic doses of gentamicin produced dramatically enhanced survival. Combined, these results indicated that AGPs may be useful in protection of immunologically naive individuals against plague and potentially other infectious agents, and that AGP therapy may be used synergistically with other therapies.

Lipid A Mimetics are Potent Adjuvants for an Intranasal Pneumonic Plague Vaccine

An effective intranasal (i.n.) vaccine against pneumonic plague was developed. The formulation employed two synthetic lipid A mimetics as adjuvant combined with Yersinia pestis-derived V- and F1-protective antigens. The two nontoxic lipid A mimetics, classed as amino-alkyl glucosaminide 4-phosphates (AGPs) are potent ligands for the Toll-like receptor (TLR) 4. Using a murine (BALB/c) pneumonic plague model, we showed a single i.n. application of the vaccine provided 63% protection within 21 days against a Y. pestis CO92 100 LD50 challenge. Protection reached 100% by 150 days. Using a homologous i.n. 1 degrees /2 degrees dose regimen, with the boost administered at varying times, 63% protection was achieved within 7 days and 100% protection was achieved by 21 days after the first immunization. Little or no protection was observed in animals that received antigens alone, and no protection was observed when the vaccine was administered to BALB/c TLR4 mutant mice. Vaccine-induced serum IgG titers to F1 and V-antigen were reflected in high titers for IgG1 and IgG2a, the latter reflecting a bias for a cell-mediated (TH1) immune response. This intranasal vaccine showed 90% protection in Sprague-Dawley rats challenged with 1000 LD50. We conclude that lipid A mimetics are highly effective adjuvants for an i.n. plague vaccine.

Brucella sp. vertebral osteomyelitis with intercurrent fatal Staphylococcus aureus toxigenic enteritis in a bottlenose dolphin (Tursiops truncatus).

A previously beach-stranded, juvenile, male, bottlenose dolphin (Tursiops truncatus) was diagnosed with vertebral osteomyelitis of unknown etiology. Antemortem serological testing suggested past or current Brucella sp. infection; however, this could not be confirmed prior to death despite multiple isolation attempts from aspirates, blood, and biopsies. Systemic antibiotics were administered for over a year to control the suspected infection; however, the animal succumbed peracutely to infection by a highly pathogenic, enterotoxin-secreting Staphylococcus sp. Gross necropsy findings included a fistulous tract leading to locally extensive osteomyelitis of a coccygeal vertebra with sequestra and osteophytes from which a Brucella species was isolated. Histopathological examination of intestine revealed pseudomembranous enteritis with a uniform population of intraluminal Gram-positive cocci. Staphylococcus aureus was isolated in pure culture from the intestine and tested positive for the staphylococcal enterotoxin A gene by polymerase chain reaction analysis. Serum taken shortly before death had endotoxin and elevated antibody titers to staphylococcal enterotoxin A when compared to samples collected during a period of apparent good health 18 months earlier. The isolation of a pyrogenic toxin superantigen-producing staphylococcal isolate, clinical signs, and diagnostic findings in this animal resembled some of those noted in human toxic shock syndrome. The present case highlights the clinical challenges of treating chronic illnesses, complications of long-term antibiotic use, and promotion of pathogenic strains in cases of prolonged rehabilitation of marine mammals.

Role of a New Intimin/Invasin-Like Protein in Yersinia pestis Virulence

ABSTRACT A comprehensive TnphoA mutant library was constructed in Yersinia pestis KIM6 to identify surface proteins involved in Y. pestis host cell invasion and bacterial virulence. Insertion site analysis of the library repeatedly identified a 9,042-bp chromosomal gene (YPO3944), intimin/invasin-like protein (Ilp), similar to the Gram-negative intimin/invasin family of surface proteins. Deletion mutants of ilp were generated in Y. pestis strains KIM5(pCD1+) Pgm− (pigmentation negative)/, KIM6(pCD1−) Pgm+, and CO92. Comparative analyses were done with the deletions and the parental wild type for bacterial adhesion to and internalization by HEp-2 cells in vitro, infectivity and maintenance in the flea vector, and lethality in murine models of systemic and pneumonic plague. Deletion of ilp had no effect on bacterial blockage of flea blood feeding or colonization. The Y. pestis KIM5 Δilp strain had reduced adhesion to and internalization by HEp-2 cells compared to the parental wild-type strain (P < 0.05). Following intravenous challenge with Y. pestis KIM5 Δilp, mice had a delayed time to death and reduced dissemination to the lungs, livers, and kidneys as monitored by in vivo imaging using a lux reporter system (in vivo imaging system [IVIS]) and bacterial counts. Intranasal challenge in mice with Y. pestis CO92 Δilp had a 55-fold increase in the 50% lethal dose ([LD50] 1.64 × 104 CFU) compared to the parental wild-type strain LD50 (2.98 × 102 CFU). These findings identified Ilp as a novel virulence factor of Y. pestis.

Conjugates of Group A and W135 Capsular Polysaccharides of Neisseria meningitidis Bound to Recombinant Staphylococcus aureus Enterotoxin C1: Preparation, Physicochemical Characterization, and Immunological Properties in Mice

ABSTRACT Neisseria meningitidis groups A (GAM) and W135 capsular polysaccharides (CPs) were bound to recombinant Staphylococcus aureus enterotoxin C1 (rSEC). The CPs were activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate and then bound to adipic acid dihydrazide derivatives of rSEC. Syntheses were conducted with native GAM CP (GAMP), W135 CP (W135P), and ultrasonicated or hydrazine-treated W135P at various concentrations of reactants, pHs, and ionic strengths. The conjugates were characterized by compositional and serologic analyses, high-performance size-exclusion chromatography with multi-angle laser light scattering detection, and immunogenicity in 5- to 6-week-old mice. Conjugates injected subcutaneously in phosphate-buffered saline elicited immunoglobulin G (IgG) responses against their respective CPs and rSEC, whereas GAMP and W135P alone did not induce detectable CP antibodies. The O-acetyl content of W135P was low, and its removal had no adverse effect upon the conjugates immunogenicity. Reduction of the molecular size of W135P by treatment with hydrazine improved the immunogenicity of W135P-rSEC. IgG anti-CP elicited by the conjugates showed complement-dependent bactericidal activity against their respective organisms, and IgG anti-rSEC neutralized the T-cell proliferative activity of native SEC. A bivalent formulation of GAMP-rSEC and W135P-rSEC elicited IgG anti-CP at comparable levels to those induced by the conjugates administered separately.