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.

Fig 1. Phylogenetic analysis of Leptospira species.

Consensus maximum-likelihood trees are depicted using multiple alignments of 16S rRNA (A), secY (B), MLST (C) and 39 concatenated protein data sets (D). The numbers along the branches denote percent occurrence of nodes among 100 bootstrap replicates. A pan-genome tree was generated based on the mean of the BLASTP score ratio of core 1135 proteins (E). The scale bar represents the number of nucleotide (A-C), amino acid (D & E) substitutions.

Fig 2. Pan-genomic comparisons of 20 Leptospira species.

Panel A: Orthologous protein clusters were binned, counted and placed into a Venn diagram by whether clusters contained proteins from genomes in each of the three Leptospira groups: pathogenic (A), intermediate (B), saprophytic (C) and the Leptonema outgroup (D). Clusters were counted if there was a majority (50%), all-but-one, or all protein members from a particular group or groups (separated by colons). Singleton clusters, representing species-specific or strain-specific genes are noted in circles surrounding the Venn diagram. Clusters not matching any of these criteria or containing at least one protein from another group were considered as ambiguous groupings. The Venn diagram is not to scale. Panel B: Protein clusters unique to pathogenic, intermediate, and saprophytic groups or shared only between pathogenic and intermediate groups were counted by main functional role categories. See key for group colors.

Fig 3. TAT signal sequence in Leptospira sp.

The X-axis shows position in an ungapped alignment. The Y-axis shows information content, measured in bits.

Fig 4. Core and pan-metabolic capabilities of the Leptospira genus.

The core and pan metabolic content was determined for genome-scale metabolic models of 4 different Leptospira species. A) Core content, illustrated by the intersection of the Venn diagram, shared with all species. The pan content consists of all content in any model and includes the core content. The Venn diagram is not to scale. B) Classification of reactions in the core and pan reactomes by metabolic subsystem.

Fig 5. Structure of Leptospira rfb locus gene clusters.

The rfb region and beginning and ending CDSs (blue) 9 of pathogenic (A), 5 intermediate (B), and 6 saprophytic (C) representative Leptospira species were compared. rfb region CDSs are labeled by locus identifier and colored by functional role categories as noted in the boxed key. Gene symbols, when present, are noted above their respective genes. BLASTP matches between CDSs are colored by protein percent identity (see key).

Fig 6. CRISPR Spacer Sequences that Recognize Leptospira Predicted Prophages.

The CRISPR sequences are shown, which correspond to specific prophage accession numbers as listed in Table 4.

Fig 7. Phylogenetic Relationship of PF07598 Paralogous Family in Leptospira.

(A) Unrooted bootstrapped phylogenetic tree; (*) Gene duplication event; (**) gene duplication event; (***) gene deletion. (B) Principal components analysis was used to arrange PF07598 family members. Color legend indicates the PF07598 family members from specific serovars depicted as diamonds. Arrowheads indicate L. noguchii-specific orthologs. Only PF07598 family members longer than 200 amino acids are included in the analysis. Clusters (A, B and C) were defined by K-means clustering with Kendall rank correlation.