What Makes a Bacterial Species Pathogenic?:Comparative Genomic Analysis of the Genus Leptospira

Abstract

Leptospirosis, caused by spirochetes of the genus Leptospira, is a globally widespread, neglected and emerging zoonotic disease. While whole genome analysis of individual pathogenic, intermediately pathogenic and saprophytic Leptospira species has been reported, comprehensive cross-species genomic comparison of all known species of infectious and non-infectious Leptospira, with the goal of identifying genes related to pathogenesis and mammalian host adaptation, remains a key gap in the field. Infectious Leptospira, comprised of pathogenic and intermediately pathogenic Leptospira, evolutionarily diverged from non-infectious, saprophytic Leptospira, as demonstrated by the following computational biology analyses: 1) the definitive taxonomy and evolutionary relatedness among all known Leptospira species; 2) genomically-predicted metabolic reconstructions that indicate novel adaptation of infectious Leptospira to mammals, including sialic acid biosynthesis, pathogen-specific porphyrin metabolism and the first-time demonstration of cobalamin (B12) autotrophy as a bacterial virulence factor; 3) CRISPR/Cas systems demonstrated only to be present in pathogenic Leptospira, suggesting a potential mechanism for this clade’s refractoriness to gene targeting; 4) finding Leptospira pathogen-specific specialized protein secretion systems; 5) novel virulence-related genes/gene families such as the Virulence Modifying (VM) (PF07598 paralogs) proteins and pathogen-specific adhesins; 6) discovery of novel, pathogen-specific protein modification and secretion mechanisms including unique lipoprotein signal peptide motifs, Sec-independent twin arginine protein secretion motifs, and the absence of certain canonical signal recognition particle proteins from all Leptospira; and 7) and demonstration of infectious Leptospira-specific signal-responsive gene expression, motility and chemotaxis systems. By identifying large scale changes in infectious (pathogenic and intermediately pathogenic) vs. non-infectious Leptospira, this work provides new insights into the evolution of a genus of bacterial pathogens. This work will be a comprehensive roadmap for understanding leptospirosis pathogenesis. More generally, it provides new insights into mechanisms by which bacterial pathogens adapt to mammalian hosts.

Author Summary

Leptospirosis is an emerging and re-emerging globally important zoonotic infectious disease caused by spirochetes of the genus Leptospira. This genus is complex, with members that cause lethal human disease, yet mechanisms that underlie pathogenesis remain obscure. Leptospira species are divided into those that are infectious for mammals, and those that are non-infectious environmental saprophytes. Based on biological characteristics and molecular phylogeny, infectious Leptospira are further divided into pathogenic and intermediately pathogenic members. The pan-genus genomic analysis of 20 Leptospira species reported here shows the evolutionary relationship of the different Leptospira clades, and various genetic factors related to virulence and pathogenesis. Infectious Leptospira show key adaptations to mammals, for example sialic acid biosynthesis, pathogen-specific porphyrin metabolism, and the observation that pathogenic Leptospira are vitamin B12 autotrophs, able to synthesize it from a simple amino acid precursor, L-glutamine. A large novel protein family of unknown function—the Virulence Modifying proteins—is found uniquely in pathogenic Leptospira. Similarly, the CRISPR/Cas system was only found in pathogenic Leptospira. A comparative genomic analysis of a complex bacterial genus allowed us to identify large-scale changes that provides new insights into general processes by which bacteria evolve to become pathogenic.