Role of sph2 Gene Regulation in Hemolytic and Sphingomyelinase Activities Produced by Leptospira interrogans.
Pathogenic members of the genus Leptospira are the causative agents of leptospirosis, a neglected disease of public and veterinary health concern. Leptospirosis is a systemic disease that in its severest forms leads to renal insufficiency, hepatic dysfunction, and pulmonary failure. Many strains of Leptospira produce hemolytic and sphingomyelinase activities, and a number of candidate leptospiral hemolysins have been identified based on sequence similarity to well-characterized bacterial hemolysins. Five of the putative hemolysins are sphingomyelinase paralogs. Although recombinant forms of the sphingomyelinase Sph2 and other hemolysins lyse erythrocytes, none have been demonstrated to contribute to the hemolytic activity secreted by leptospiral cells. In this study, we examined the regulation of sph2 and its relationship to hemolytic and sphingomyelinase activities produced by several L. interrogans strains cultivated under the osmotic conditions found in the mammalian host. The sph2 gene was poorly expressed when the Fiocruz L1-130 (serovar Copenhageni), 56601 (sv. Lai), and L495 (sv. Manilae) strains were cultivated in the standard culture medium EMJH. Raising EMJH osmolarity to physiological levels with sodium chloride enhanced Sph2 production in all three strains. In addition, the Pomona subtype kennewicki strain LC82-25 produced substantially greater amounts of Sph2 during standard EMJH growth than the other strains, and sph2 expression increased further by addition of salt. When 10% rat serum was present in EMJH along with the sodium chloride supplement, Sph2 production increased further in all strains. Osmotic regulation and differences in basal Sph2 production in the Manilae L495 and Pomona strains correlated with the levels of secreted hemolysin and sphingomyelinase activities. Finally, a transposon insertion in sph2 dramatically reduced hemolytic and sphingomyelinase activities during incubation of L. interrogans at physiologic osmolarity. Complementation of the mutation with the sph2 gene partially restored production of hemolytic and sphingomyelinase activities. These results indicate that the sph2 gene product contributes to the hemolytic and sphingomyelinase activities secreted by L. interrogans and most likely dominates those functions under the culture condition tested.
Department of Animal Biology, University of Hyderabad, Hyderabad, India.
Suneel A
SA
Narayanavari
Research Service, Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California, United States of America; Department of Medicine, David Geffen School of Medicine at University of California, Los Angeles (UCLA), Los Angeles, California, United States of America.
Kristel
K
Lourdault
Department of Animal Biology, University of Hyderabad, Hyderabad, India.
Manjula
M
Sritharan
Department of Medicine, David Geffen School of Medicine at University of California, Los Angeles (UCLA), Los Angeles, California, United States of America; Department of Urology, David Geffen School of Medicine at University of California, Los Angeles (UCLA), Los Angeles, California, United States of America; Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, California, United States of America; Division of Infectious Diseases, Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California, United States of America.
David A
DA
Haake
Research Service, Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California, United States of America; Department of Medicine, David Geffen School of Medicine at University of California, Los Angeles (UCLA), Los Angeles, California, United States of America.
James
J
Matsunaga
D000818:Animals; D004269:DNA, Bacterial / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism*; D007921:Leptospira interrogans / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D051381:Rats; D013108:Sphingomyelin Phosphodiesterase / Q000235:genetics / Q000378:metabolism*
D004269:DNA, Bacterial; D006460:Hemolysin Proteins; D013108:Sphingomyelin Phosphodiesterase