Site-directed homologous recombination has not been successful in pathogenic Leptospira and there exist only a few examples of mutants obtained by targeted mutagenesis. We have generated a library of random mutants in the pathogen L. interrogans by Himar1 transposon mutagenesis. A library of ≈1,400 random mutants of L. interrogans serovar Manilae strain L495 is now available and we are pursuing to generate random transposon mutants to enrich our library. We selected a set of mutants with transposon insertion in genes encoding putative virulence factors. The phenotype of the mutants were then characterized in vitro and in animal models.
We demonstrated that the genes encodingthe bacterial homolog of the molecular chaperone Hsp90, and ClpB are essential for virulence in the animal models of acute leptospirosis. The clpB mutant exhibited in vitro growth defects and showed increased susceptibility to oxidative stress but, surprisingly, the hsp90 mutant did not exhibit increased sensitivity to heat, oxidative, or osmotic stress. We also found reduced virulence and survival to oxidative stress when the gene encoding the L. interrogans catalase (KatE) was inactivated, further demonstrating the requirement for a functional ROS (reactive oxygen species) resistance mechanism for full leptospiral pathogenesis. A transposon mutant in a gene encoding for a putative HtpX-like metalloprotease related to the M48 family of metalloproteases, which is usually associated with the degradation of misfolded proteins, was also studied. Under high iron conditions, the mutant produces an iron precipitate and form of outer membrane vesicles. The lipL41 gene, which encodes the third most abundant OM lipoprotein, is located immediately upstream of a smaller gene, with which it is co-transcribed. The expression of this second gene, called “lep” for lipL41 expression partner, appeared to be required for stable expression of LipL41, perhaps by acting as a chaperone. Neither a lipL41 nor a lep mutant was attenuated for virulence in hamsters. Similar results have been reported for the major lipoproteins LipL32 and LigB , which are also not required for virulence in animals, further suggesting a high degree of functional redundancy in leptospiral virulence genes. We identified and characterized a novel ABC ATPase in Leptospira whose inactivation led to the decreased growth of the pathogen L. interrogans when Fe2+ was replaced by Mn2+. The structure of this ATPase was solved from a crystal containing two monomers in the asymmetric unit. The two monomers were arranged in a head-to-tail orientation, forming a V-shaped particle with all the conserved ABC motifs at the dimer interface, similar to functional ABC ATPases. Characterization of an avirulent mutant with a transposon insertion in a putative regulatory locus containing genes encoding a sensor protein with a phosphatase domain and an anti-sigma factor antagonist prompted us to further characterize the transcriptome of the insertion mutant. Whole-transcriptome analyses revealed a bias toward the downregulation of genes involved in motility/chemotaxis and signal transduction, and genes encoding predicted extracellular proteins . Leptospira encode two genes annotated as UDP-3-O-[3-hydroxymyristoyl] glucosamine N-acyltransferases (lpxD1 and lpxD2), that in other bacteria are involved in the early steps of lipid A biosynthesis, the membrane lipid anchor of lipopolysaccharide. Inactivation of only one of these genes, lpxD1, imparted sensitivity to host physiological temperature and rendered the bacteria avirulent in an animal infection model. Polymyxin B sensitivity assays revealed compromised outer membrane integrity in the lpxD1 mutant, but structural analysis of lipid A in this mutant revealed only minor changes in the lipid A moiety compared to that found in the wild type strain. In accord, an in-trans complementation restored phenotypes to a level comparable to the wild type strain. These results suggest that the gene annotated as lpxD1 in L. interrogans plays an important role in temperature adaptation and virulence in the animal infection model.
This library of random mutants provides an opportunity to investigate genes that contribute to pathogenesis and will continue to provide a better understanding of the biology of L. interrogans.