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Recent studies have revealed that bacterial protein methylation is a widespread post-translational modification that is required for virulence in selected pathogenic bacteria. In particular, altered methylation of outer-membrane proteins has been shown to modulate the effectiveness of the host immune response. In this study, 2D gel electrophoresis combined with MALDI-TOF MS identified a Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 protein, corresponding to ORF LIC11848, which undergoes extensive and differential methylation of glutamic acid residues. Immunofluorescence microscopy implicated LIC11848 as a surface-exposed outer-membrane protein, prompting the designation OmpL32. Indirect immunofluorescence microscopy of golden Syrian hamster liver and kidney sections revealed expression of OmpL32 during colonization of these organs. Identification of methylated surface-exposed outer-membrane proteins, such as OmpL32, provides a foundation for delineating the role of this post-translational modification in leptospiral virulence.

Leptospirosis is a widespread zoonosis caused by invasive spirochaetes belonging to the genus Leptospira. Pathogenic leptospires disseminate via the bloodstream to colonize the renal tubules of reservoir hosts. Little is known about leptospiral outer-membrane proteins expressed during the dissemination stage of infection. In this study, a novel surface-exposed lipoprotein is described; it has been designated LipL46 to distinguish it from a previously described 31 kDa peripheral membrane protein, P31(LipL45), which is exported as a 45 kDa probable lipoprotein. The lipL46 gene encodes a 412 aa polypeptide with a 21 aa signal peptide. Lipid modification of cysteine at the lipoprotein signal peptidase cleavage site FSISC is supported by the finding that Leptospira interrogans intrinsically labels LipL46 during incubation in medium containing [(14)C]palmitate. LipL46 appears to be exported to the leptospiral outer membrane as a 46 kDa lipoprotein, based on Triton X-114 solubilization and phase partitioning studies, which included the outer and inner membrane controls LipL32 and LipL31, respectively. Surface immunoprecipitation and whole-cell ELISA experiments indicate that LipL46 is exposed on the leptospiral surface. Immunohistochemistry studies demonstrated expression of LipL46 by leptospires found in the bloodstream of acutely infected hamsters. Leptospires expressing LipL46 were also found in the intercellular spaces of the liver, within splenic phagocytes, and invading the glomerular hilum of the kidney. Infection-associated expression is supported by the finding that LipL46 is a major antigen recognized by sera from infected hamsters. These findings indicate that LipL46 may be important in leptospiral dissemination, and that it may serve as a useful serodiagnostic antigen.

The search for novel antigens suitable for improved vaccines and diagnostic reagents against leptospirosis led to the identification of LigA and LigB. LigA and LigB expression were not detectable at the translation level but were detectable at the transcription level in leptospires grown in vitro. Lig genes were present in pathogenic serovars of Leptospira, but not in non-pathogenic Leptospira biflexa. The conserved and variable regions of LigA and LigB (Con, VarA and VarB) were cloned, expressed and purified as GST-fusion proteins. Purified recombinant LigA and LigB were evaluated for their diagnostic potential in a kinetic ELISA (KELA) using sera from vaccinated and microscopic agglutination test (MAT)-positive dogs. Sera from vaccinated dogs showed reactivity to whole-cell antigens of leptospires but did not show reactivity in the KELA assay with recombinant antigens, suggesting a lack of antibodies to Lig proteins in the vaccinated animals. The diagnostic potential of recombinant Lig antigens in the KELA assay was evaluated by using 67 serum samples with MAT > or =1600, which showed reactivity of 76, 41 and 35% to rConA, rVarA and rVarB, respectively. These findings suggest that recombinant antigen to the conserved region of LigA and LigB can differentiate between vaccinated and naturally infected animals.

The intestinal spirochaete Brachyspira pilosicoli causes colitis in a wide variety of host species. Little is known about the structure or protein constituents of the B. pilosicoli outer membrane (OM). To identify surface-exposed proteins in this species, membrane vesicles were isolated from B. pilosicoli strain 95-1000 cells by osmotic lysis in dH(2)O followed by isopycnic centrifugation in sucrose density gradients. The membrane vesicles were separated into a high-density fraction (HDMV; rho=1.18 g cm(-3)) and a low-density fraction (LDMV; rho=1.12 g cm(-3)). Both fractions were free of flagella and soluble protein contamination. LDMV contained predominantly OM markers (lipo-oligosaccharide and a 29 kDa B. pilosicoli OM protein) and was used as a source of antigens to produce mAbs. Five B. pilosicoli-specific mAbs reacting with proteins with molecular masses of 23, 24, 35, 61 and 79 kDa were characterized. The 23 kDa protein was only partially soluble in Triton X-114, whereas the 24 and 35 kDa proteins were enriched in the detergent phase, implying that they were integral membrane proteins or lipoproteins. All three proteins were localized to the B. pilosicoli OM by immunogold labelling using specific mAbs. The gene encoding the abundant, surface-exposed 23 kDa protein was identified by screening a B. pilosicoli 95-1000 genome library with the mAb and was expressed in Escherichia coli. Sequence analysis showed that it encoded a unique lipoprotein, designated BmpC. Recombinant BmpC partitioned predominantly in the OM fraction of E. coli strain SOLR. The mAb to BmpC was used to screen a collection of 13 genetically heterogeneous strains of B. pilosicoli isolated from five different host species. Interestingly, only strain 95-1000 was reactive with the mAb, indicating that either the surface-exposed epitope on BmpC is variable between strains or that the protein is restricted in its distribution within B. pilosicoli.