Identification of a novel Staphylococcus aureus two-component leukotoxin using cell surface proteomics.

Staphylococcus aureus is a prominent human pathogen and leading cause of bacterial infection in hospitals and the community. Community-associated methicillin-resistant S. aureus (CA-MRSA) strains such as USA300 are highly virulent and, unlike hospital strains, often cause disease in otherwise healthy individuals. The enhanced virulence of CA-MRSA is based in part on increased ability to produce high levels of secreted molecules that facilitate evasion of the innate immune response. Although progress has been made, the factors that contribute to CA-MRSA virulence are incompletely defined. We analyzed the cell surface proteome (surfome) of USA300 strain LAC to better understand extracellular factors that contribute to the enhanced virulence phenotype. A total of 113 identified proteins were associated with the surface of USA300 during the late-exponential phase of growth in vitro. Protein A was the most abundant surface molecule of USA300, as indicated by combined Mascot score following analysis of peptides by tandem mass spectrometry. Unexpectedly, we identified a previously uncharacterized two-component leukotoxin-herein named LukS-H and LukF-G (LukGH)-as two of the most abundant surface-associated proteins of USA300. Rabbit antibody specific for LukG indicated it was also freely secreted by USA300 into culture media. We used wild-type and isogenic lukGH deletion strains of USA300 in combination with human PMN pore formation and lysis assays to identify this molecule as a leukotoxin. Moreover, LukGH synergized with PVL to enhance lysis of human PMNs in vitro, and contributed to lysis of PMNs after phagocytosis. We conclude LukGH is a novel two-component leukotoxin with cytolytic activity toward neutrophils, and thus potentially contributes to S. aureus virulence.

Identification of conserved antigens for early serodiagnosis of relapsing fever Borrelia.

Borrelia hermsii is a blood-borne pathogen transmitted by the argasid tick Ornithodoros hermsi. Since spirochaete clearance in mice is associated with an IgM-mediated response, an immunoproteomic analysis was used to identify proteins reactive with IgM. We report that IgM from both mice and human patients infected with B. hermsii not only reacted with the previously identified variable membrane proteins but also identified candidate antigens including heat-shock proteins, an adhesin protein, ABC transporter proteins, flagellar proteins, housekeeping proteins, an immune evasion protein, and proteins with unknown function. Furthermore, IgM reactivity to recombinant glycerophosphodiester phosphodiesterase was detected during early spirochaete infection and prior to a detectable IgG response. Lastly, a conserved hypothetical protein was produced in Escherichia coli and tested with immune serum against B. hermsii and Borrelia recurrentis. These results identify a much larger set of immunoreactive proteins, and could help in the early serodiagnosis of this tick-borne infection.

Global analysis of community-associated methicillin-resistant Staphylococcus aureus exoproteins reveals molecules produced in vitro and during infection.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is a threat to human health worldwide. Although progress has been made, mechanisms of CA-MRSA pathogenesis are poorly understood and a comprehensive analysis of CA-MRSA exoproteins has not been conducted. To address that deficiency, we used proteomics to identify exoproteins made by MW2 (USA400) and LAC (USA300) during growth in vitro. Two hundred and fifty unique exoproteins were identified by 2-dimensional gel electrophoresis coupled with automated direct infusion-tandem mass spectrometry (ADI-MS/MS) analysis. Eleven known virulence-related exoproteins differed in abundance between the strains, including alpha-haemolysin (Hla), collagen adhesin (Cna), staphylokinase (Sak), coagulase (Coa), lipase (Lip), enterotoxin C3 (Sec3), enterotoxin Q (Seq), V8 protease (SspA) and cysteine protease (SspB). Mice infected with MW2 or LAC produced antibodies specific for known or putative virulence factors, such as autolysin (Atl), Cna, Ear, ferritin (Ftn), Lip, 1-phosphatidylinositol phosphodiesterase (Plc), Sak, Sec3 and SspB, indicating the exoproteins are made during infection in vivo. We used confocal microscopy to demonstrate aureolysin (Aur), Hla, SspA and SspB are produced following phagocytosis by human neutrophils, thereby linking exoprotein production in vitro with that during host-pathogen interaction. We conclude that the exoproteins identified herein likely account in part for the success of CA-MRSA as a human pathogen.

Carbonic anhydrase III is not required in the mouse for normal growth, development, and life span.

Carbonic anhydrase III is a cytosolic protein which is particularly abundant in skeletal muscle, adipocytes, and liver. The specific activity of this isozyme is quite low, suggesting that its physiological function is not that of hydrating carbon dioxide. To understand the cellular roles of carbonic anhydrase III, we inactivated the Car3 gene. Mice lacking carbonic anhydrase III were viable and fertile and had normal life spans. Carbonic anhydrase III has also been implicated in the response to oxidative stress. We found that mice lacking the protein had the same response to a hyperoxic challenge as did their wild-type siblings. No anatomic alterations were noted in the mice lacking carbonic anhydrase III. They had normal amounts and distribution of fat, despite the fact that carbonic anhydrase III constitutes about 30% of the soluble protein in adipocytes. We conclude that carbonic anhydrase III is dispensable for mice living under standard laboratory husbandry conditions.

Spectratype/immunoscope analysis of the expressed TCR repertoire.

Measuring the diversity of TCRs utilized by specific primary and memory T cell responses is critical to the fundamental understanding of regulation of the immune system. This unit describes the Spectratype/Immunoscope technique which permits an in depth analysis of the TCR repertoire present in a variety of biological samples from mice to humans. Spectratype analysis takes advantage of PCR technology to amplify template cDNA corresponding to rearranged transcripts with different CDR3 lengths from specific TCR variable region genes in a competitive manner. The PCR products are then resolved on polyacrylamide sequencing gels to reveal precise sizes in nucleotide base pairs. The unit also includes protocols that have been optimized to process and produce the starting materials required for spectratype analysis.