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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, Online J Bioinformatics, 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.