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Online Journal of Bioinformatics ©
Volume 10 (2): 241-258, 2009.
Effect of iron deprivation on expression of sphingomyelase in pathogenic serovar Lai.
Sridhar Velineni1, Sanam Ramadevi2, Swapna Asuthkar1, Manjula Sritharan1*
1Department of Animal Sciences, University of Hyderabad, Hyderabad, India; 2Informatics Division, GVK Biosciences Pvt Ltd, Hyderabad, India.
Velineni S, Ramadevi S, Asuthkar S, Sritharan M., Effect of iron deprivation on expression of sphingomyelase in pathogenic serovar Lai, Onl J Bioinform, 10 (2): 241-258, 2009. Hemolysins are one of the contributing virulence factors in pathogenic Leptospira spp. The genome of Leptospira interrogans serovar Lai contains ten hemolysin genes, encoding molecules with sphingomyelinase and phospholipase activities. The sphingomyelinase genes are absent in the non-pathogenic Leptospira biflexa serovar Patoc that, however harbors the genes for the hemolysins with phospholipase activity. In general, bacterial hemolysins are secreted into the immediate environment of the host and lyse the host cells with the release of the intracellular nutrients, including iron. Iron limitation in bacteria induces not only the iron acquisition machinery but also the expression of bacterial virulence determinants and toxins. Our earlier observations on the iron-regulated hemin-binding protein HbpA in serovar Lai was the first report on the direct acquisition of iron by this pathogen. In the present study, the iron-regulated expression of sphingomyelinase in outer membrane vesicles (OMVs) is demonstrated. The OMVs from low iron cultures showed not only sphingomyelinase but also the iron-regulated hemin-binding protein HbpA, both of which were absent in the corresponding high iron samples. LipL32 (hemolysis-associated protein, hap-1) was present in both high and low iron OMVs. None of the above three proteins were expressed by the non-pathogenic L. biflexa serovar Andamana. The release of the OMVs from the surface of the pathogen was demonstrated by transmission electron microscopy. Immunofluorescence studies using confocal microscopy showed the surface association of sphingomyelinase in low iron organisms. We also identified a 63 kDa outer membrane protein by immunoprecipitation with anti-sphingomyelinase antibodies. The protein was identified as the outer membrane efflux protein TolC (LA0957, Swiss Prot Q8F718) by sequence analysis using tandem mass spectrometry. The possible role of this protein in the transport of sphingomyelinase is discussed based on the similarity of protein folding to TolC of E. coli and the detection of hlyB and hlyD in the genome of serovar Lai.
Key words: Iron, hemolysin, Leptospira, sphingomyelinase, outer membrane efflux protein, TolC.