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OJBTM
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.