The lack of genetic tools has hampered molecular analyses of putative virulence factors in pathogenic Leptospira spp. In this work, we showed for the first time that pathogen-specific proteins can be expressed in a saprophytic Leptospira and that expression of such proteins are accompanied by an in vitro virulence associated phenotype. The approach used in this study demonstrates that the fast-growing non pathogenic species L. biflexa serves a model for examining pathogenetic mechanisms of L. interrogans. In contrast to L. biflexa, data obtained when E. coli was used as a surrogate host revealed that most of the spirochetal promoters functioned poorly in this genetic background. Even when leptospiral proteins are expressed in E. coli, many are found to be insoluble. An additional consideration is that a number of leptospiral proteins undergo post-translational modifications that may not occur in Gram negative bacteria .
In this study, the L. interrogans LigA and LigB lipoproteins were expressed and exposed on the surface of L. biflexa cells. However, the ligB-transformed L. biflexa produced almost no full length LigB protein. This suggests that L. biflexa is an appropriate surrogate host for expression of at least some L. interrogans outer membrane proteins . These experimental results confirm genome sequence analyses indicating that most of the known protein export and processing systems of L. interrogans and L. biflexa are highly conserved . Surface localization of Ligs in the model bacterium L. biflexa presents a unique opportunity to study the translocation of lipoproteins through leptospiral membranes. Further study could, for instance, include the analysis of the leptospiral lipobox which is distinct from the motifs of E. coli and other gram-negative bacteria. For example, the leptospiral surface lipoprotein, LipL41 was not efficiently expressed in E. coli until its lipobox was altered to mimic that of murein lipoprotein . Analysis of leptospiral lipobox sequences indicates that most leptospiral lipoproteins would be anticipated to not be processed correctly in E. coli .
Bacterial adhesion is a crucial step in the infectious process.
Among members of the superfamily of bacterial immunoglobulin (Ig)-like (Big) proteins, previous studies have demonstrated that in comparison to the wild type strain, an intimin-deficient enteropathogenic E. coli strain is defective in adherence to cultured cells and in intestinal colonization . In Y. enterocolitica, an invasin mutant was impaired in its ability to translocate the intestinal epithelium . By contrast, we found that a L. interrogans ligB
mutant retained its virulence and ability to adhere to MDCK cells . This may be due to functional redundancy of other Lig proteins such as LigA. To determine the function of lig genes in pathogens, it may therefore be necessary to knock-out multiple genes, which would not be feasible in pathogenic Leptospira strains.
This study is a complete description of our approach for heterologous expression of pathogen-specific proteins in the saprophyte, L. biflexa serovar Patoc, resulting in the acquisition of virulence-associated phenotype. We demonstrate that Patoc ligA is able to adhere to epithelial cells in a time-dependent fashion, comparable to the pathogen L. interrogans. In addition, levels of binding of Patoc ligA and Patoc ligB to fibronectin and laminin were significantly higher in comparison to Patoc wt. However, lig transformants did not appear to bind collagens (type I and IV) or elastin better than wild-type cells. Analysis of Patoc ligA and Patoc ligB suggests that the Lig proteins are not sufficient for the efficient translocation of the bacteria across the cell monolayers, a characteristic that distinguishes leptospiral pathogens from saprophytes . This result suggests that invasion is a more complex process than adherence and may require additional properties unique to leptospiral pathogens. In other words, invasion of cellular monolayers may require a stepwise adherence process involving interactions with a series of host ligands. Recently, we described enhanced fibrinogen binding of L. biflexa expressing LigA and LigB using the same plasmid constructs described here as part of a general examination of Lig-fibrinogen interactions , validating the relevance of our heterologous expression system.
Studies involving recombinant proteins, including LigA and LigB, have revealed a number of proteins that bind to extracellular matrix proteins [37–43]. Whether the functions of these putative adhesins are overlapping or synergistic in the interactions of leptospiral cells with eukaryotic cells or monolayers is unknown. LigA and LigB proteins contain related yet distinct Big domains that may share redundant function [13–15]. For example Choy et al demonstrated that portions of both LigA and LigB proteins bind fibronectin in vitro . Thus the function of LigB can be substituted to varying extents by other lipoproteins, including LigA, which may play a role in host-cell interactions. The use of L. biflexa as a surrogate host enables functional studies of virulence factors in isolation without interference from activities of competing or redundant outer membrane proteins. Further studies expressing distinct regions of LigA and LigB in L. biflexa are required to understand the precise role of each domain in the binding of components of the extracellular matrix.
L. interrogans is an invasive pathogen that can adhere and translocate through host cells [30, 44]. In contrast to the increased adherence of the ligA-transformed L. biflexa strain to MDCK renal cells, the ligB transformants did not exhibit enhanced attachment to the eukaryotic cells following four hours of incubation. This may be due to the partial degradation of LigB observed in these transformants by Western blots (Figure 1B). In contrast, we found that both ligA- and ligB-transformed L. biflexa bound fibronectin in significantly greater numbers than wild-type L. biflexa in a solid-phase assay format (Figure 5A). The large remaining LigB fragment appears slightly larger than intact LigA, suggesting that the degraded LigB may comprise the immunoglobulin-like repeats containing the fibronectin-binding domain . These findings suggest that lig-mediated host cell adhesion may involve receptors in addition to fibronectin. The expression and localization of fibronectin and its binding integrins as well as other components of extracellular matrix in MDCK cells are dependent on culture conditions [45, 46]. It is possible that the large proteolytic fragment of LigB remaining with the ligB transformants retains the fibronectin-binding region but has lost sequences mediating the interaction of LigB with a different and distinct renal cell receptor. Further studies with lig transformants could include analyzing lig-mediated host cell adhesion by using additional cell lines representing different species and cell types.