LipL53, a temperature regulated protein from Leptospira interrogans that binds to extracellular matrix molecules

Abstract

The regulation of gene expression by environmental signals, such as temperature and osmolarity, has been correlated with virulence. In this study, we characterize the protein LipL53 from Leptospira interrogans, previously shown to react with serum sample of individual diagnosed with leptospirosis and to be up-regulated by shift to physiological osmolarity. The recombinant protein was expressed in Escherichia coli system, in insoluble form, recovered by urea solubilization and further refolded by decreasing the denaturing agent concentration during the purification procedure. The secondary structure content of the recombinant LipL53, as assessed by circular dichroism, showed a mixture of beta-strands and alpha-helix. The presence of LipL53 transcript at 28 degrees C was only detected within the virulent strains. However, upon shifted of attenuated cultures of pathogenic strains from 28 degrees C to 37 degrees C and to 39 degrees C, this transcript could also be observed. LipL53 binds laminin, collagen IV, cellular and plasma fibronectin in dose-dependent and saturable manner. Animal challenge studies showed that LipL53, although immunogenic, elicited only partial protection in hamsters. LipL53 is probably surface exposed as seen through immunofluorescence confocal microscopy. Our results suggest that LipL53 is a novel temperature regulated adhesin of L. interrogans that may be relevant in the leptospiral pathogenesis.

Characterization of Three Novel Adhesins of Leptospira interrogans

“…We performed a proteinase K accessibility assay by using a described assay 14,39 with some modifications. Live L. interrogans serovar Copenhageni strain M-20 was treated with 25 μg/mL of proteinase K, and aliquots of the bacterial suspensions were obtained at 0, 30, 60, 120, and 240 minutes.…”

“…[8][9][10][11][12][13][14][15][16][17][18] Several laminin-binding proteins were identified, which indicated that leptospires have a redundant repertoire of adhesion molecules that are probably part of their invasion strategies. Lsa23, Lsa26, and Lsa36 bind laminin in a dose-dependent, specific, and saturable manner, fulfilling the properties of receptor-ligand interaction.…”

“…Some of these proteins were identified as novel leptospiral adhesins that might be involved in the first steps of host-Leptospira interaction. [8][9][10][11][12][13][14][15][16][17][18] After attachment, leptospires have to overcome tissue barriers and reach target organs. Proteolytic activity by plasmin (PLA) generation at the surface of Leptospira spp.…”

“…Enzymatic digestion was performed as described by Oliveira and others 14 with some modifications. In brief, 5-mL suspensions of live leptospires (L. interrogans serovar Copenhageni strain M-20)/per each treatment (approximately 10 8 cells/mL) were harvested by centrifugation at 8,000 + g for 10 minutes at room temperature and washed once with PBS low salt containing 50 mM NaCl (PBS ls).…”

The Novel Leptospiral Surface Adhesin Lsa20 Binds Laminin and Human Plasminogen and Is Probably Expressed during Infection

“…These proteins are exposed to hosts and then are potential targets for inducing immune response during infection. In addition, it is possible that some of these membrane proteins mediate the initial adhesion to host cells, as has been reported by several researchers (5,7,13,14,22,23,26,29,37,41,50,55). After adherence, pathogens have to surmount host tissue barriers in order to reach blood circulation and organs.…”