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

Predicting Impacts of Climate Change on Fasciola hepatica Risk

Fasciola hepatica (liver fluke) is a physically and economically devastating parasitic trematode whose rise in recent years has been attributed to climate change. Climate has an impact on the free-living stages of the parasite and its intermediate host Lymnaea truncatula, with the interactions between rainfall and temperature having the greatest influence on transmission efficacy. There have been a number of short term climate driven forecasts developed to predict the following season's infection risk, with the Ollerenshaw index being the most widely used. Through the synthesis of a modified Ollerenshaw index with the UKCP09 fine scale climate projection data we have developed long term seasonal risk forecasts up to 2070 at a 25 km square resolution. Additionally UKCIP gridded datasets at 5 km square resolution from 1970-2006 were used to highlight the climate-driven increase to date. The maps show unprecedented levels of future fasciolosis risk in parts of the UK, with risk of serious epidemics in Wales by 2050. The seasonal risk maps demonstrate the possible change in the timing of disease outbreaks due to increased risk from overwintering larvae. Despite an overall long term increase in all regions of the UK, spatio-temporal variation in risk levels is expected. Infection risk will reduce in some areas and fluctuate greatly in others with a predicted decrease in summer infection for parts of the UK due to restricted water availability. This forecast is the first approximation of the potential impacts of climate change on fasciolosis risk in the UK. It can be used as a basis for indicating where active disease surveillance should be targeted and where the development of improved mitigation or adaptation measures is likely to bring the greatest benefits

A Mycobacterium tuberculosis-Derived Lipid Inhibits Membrane Fusion by Modulating Lipid Membrane Domains

Tuberculosis is an infectious and potentially fatal disease caused by the acid-fast bacillus Mycobacterium tuberculosis (MTB). One hallmark of a tuberculosis infection is the ability of the bacterium to subvert the normal macrophage defense mechanism of the host immune response. Lipoarabinomannan (LAM), an integral component of the MTB cell wall, is released when MTBs are taken into phagosomes and has been reported to be involved in the inhibition of phago-lysosomal (P-L) fusion. However, the physical chemistry of the effects of LAM on lipid membrane structure relative to P-L fusion has not been studied. We produced membranes in vitro composed of dioleoylphosphatidylcholine, sphingomyelin, and cholesterol to simulate phagosomal lipid membranes and quantified the effects of the addition of LAM to these membranes, using fluorescence resonance energy transfer assays and atomic force microscopy. We found that LAM inhibits vesicle fusion and markedly alters lipid membrane domain morphology and sphingomyelin-chollesterol/dioleoylphosphatidylcholine ratios. These data demonstrate that LAM induces a dramatic reorganization of lipid membranes in vitro and clarifies the role of LAM in the inhibition of P-L fusion and the survival of the MTB within the macrophage

Altered membrane structure and surface potential in homozygous hemoglobin C erythrocytes.

BACKGROUND:Hemoglobin C differs from normal hemoglobin A by a glutamate-to-lysine substitution at position 6 of beta globin and is oxidatively unstable. Compared to homozygous AA erythrocytes, homozygous CC erythrocytes contain higher levels of membrane-associated hemichromes and more extensively clustered band 3 proteins. These findings suggest that CC erythrocytes have a different membrane matrix than AA erythrocytes. METHODOLOGY AND FINDINGS:We found that AA and CC erythrocytes differ in their membrane lipid composition, and that a subset of CC erythrocytes expresses increased levels of externalized phosphatidylserine. Detergent membrane analyses for raft marker proteins indicated that CC erythrocyte membranes are more resistant to detergent solubilization. These data suggest that membrane raft organization is modified in CC erythrocytes. In addition, the average zeta potential (a measure of surface electrochemical potential) of CC erythrocytes was approximately 2 mV lower than that of AA erythrocytes, indicating that substantial rearrangements occur in the membrane matrix of CC erythrocytes. We were able to recapitulate this low zeta potential phenotype in AA erythrocytes by treating them with NaNO(2) to oxidize hemoglobin A molecules and increase levels of membrane-associated hemichromes. CONCLUSION:Our data support the possibility that increased hemichrome deposition and altered lipid composition induce molecular rearrangements in CC erythrocyte membranes, resulting in a unique membrane structure

Zeta potential (ZP) analyses.

<p>(A) Diagram of ZP principle. ZP is defined as the electrochemical potential at the shear plane. Outside the shear plane, ions are not closely associated with the internal ion cloud. (B) ZP measurements from AA and CC erythrocyte populations. Peak values were estimated by Gaussian fitting the histogram. (C) Levels of membrane-associated hemichromes (mean±SD) in control and NaNO₂-treated AA erythrocytes. For reference, native CC erythrocytes show 1.8-fold greater hemichrome levels than native AA erythrocytes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005828#pone.0005828-Fairhurst1" target="_blank">[31]</a>. (D) ZP measurements from control and NaNO₂-treated AA erythrocyte populations.</p