Osmotic regulation of expression of two extracellular matrix-binding proteins and a haemolysin of Leptospira interrogans: differential effects on LigA and Sph2 extracellular release

Abstract

The life cycle of the pathogen Leptospira interrogans involves stages outside and inside the host. Entry of L. interrogans from moist environments into the host is likely to be accompanied by the induction of genes encoding virulence determinants and the concomitant repression of genes encoding products required for survival outside of the host. The expression of the adhesin LigA, the haemolysin Sph2 (Lk73.5) and the outer-membrane lipoprotein LipL36 of pathogenic Leptospira species have been reported to be regulated by mammalian host signals. A previous study demonstrated that raising the osmolarity of the leptospiral growth medium to physiological levels encountered in the host by addition of various salts enhanced the levels of cell-associated LigA and LigB and extracellular LigA. In this study, we systematically examined the effects of osmotic upshift with ionic and non-ionic solutes on expression of the known mammalian host-regulated leptospiral genes. The levels of cell-associated LigA, LigB and Sph2 increased at physiological osmolarity, whereas LipL36 levels decreased, corresponding to changes in specific transcript levels. These changes in expression occurred irrespective of whether sodium chloride or sucrose was used as the solute. The increase of cellular LigA, LigB and Sph2 protein levels occurred within hours of adding sodium chloride. Extracellular Sph2 levels increased when either sodium chloride or sucrose was added to achieve physiological osmolarity. In contrast, enhanced levels of extracellular LigA were observed only with an increase in ionic strength. These results indicate that the mechanisms for release of LigA and Sph2 differ during host infection. Thus, osmolarity not only affects leptospiral gene expression by affecting transcript levels of putative virulence determinants but also affects the release of such proteins into the surroundings. INTRODUCTIONSpirochaetes of the genus Leptospira are a major cause of morbidity and mortality in developing nations (McBride et al., 2005). Leptospira interrogans is one of 14 genomospecies of pathogenic Leptospira and is the cause of endemic disease and large outbreaks of leptospirosis worldwide (Brenner et al., 1999;Levett et al., 2006). Outbreaks involving L. interrogans have been associated with flooding in rat-infested urban slums (Ko et al., 1999). L. interrogans resides chronically in the lumen of rat kidney tubules and is released within urine into the environment, where it can survive for weeks (Crawford et al., 1971;Gordon Smith & Turner, 1961;Ryu & Liu, 1966). Humans exposed to Leptospira in the environment can be infected through breaks in skin or mucous membranes. After breaching the epithelium or mucosa, leptospires migrate to the bloodstream and disseminate to numerous organs. Flulike symptoms including fever and myalgia characterize early acute-phase infection. Severe symptoms including Haake et al., 1998;Nally et al., 2001b). Recent microarray and proteomic experiments have revealed changes in the levels of numerous transcripts and s...

A Putative Regulatory Genetic Locus Modulates Virulence in the Pathogen Leptospira interrogans

“…Physiological osmolarity serves as a cue for Leptospira to both initiate expression of genes (8) and release the LigB and Sph2 proteins into the extracellular space (37). Interestingly, previous studies have also demonstrated LigB-and Sph2-mediated binding of host homeostatic proteins (38,39), suggesting a role for these proteins in leptospiral attachment to host cells.…”

Pathogenic Leptospira interrogans Exoproteins Are Primarily Involved in Heterotrophic Processes

“…Using reasoning similar to that described above, WCPs and CSPs were subjected to immunoblot analysis using antiserum cross-reactive with L. interrogans Ig-like repeat domain protein 1 (LigA with a molecular mass of 128 kDa) (Fig. 1D) to assess the bacterial response to an osmotic shift (45,46). The LigA protein was detected in CSP samples, with pronounced reactivity being observed in CSP samples from L. interrogans exposed to 120 mM NaCl (Fig.…”

Role for cis -Acting RNA Sequences in the Temperature-Dependent Expression of the Multiadhesive Lig Proteins in Leptospira interrogans

“…ligB mRNA is upregulated in the bloodstream of infected rodent models, and Lig protein is detected in the kidneys of infected hamsters (24,25). L. interrogans and the closely related species L. kirschneri are typically cultivated under low-osmolarity conditions; exposure of the organisms to higher levels of osmolarity, such as those found in the host, causes the expression level of ligA and ligB to increase markedly (26,27).…”