Lvr, a Signaling System That Controls Global Gene Regulation and Virulence in Pathogenic Leptospira

Abstract

Leptospirosis is an emerging zoonotic disease with more than 1 million cases annually. Currently there is lack of evidence for signaling pathways involved during the infection process of Leptospira. In our comprehensive genomic analysis of 20 Leptospira spp. we identified seven pathogen-specific Two-Component System (TCS) proteins. Disruption of two these TCS genes in pathogenic Leptospira strain resulted in loss-of-virulence in a hamster model of leptospirosis. Corresponding genes lvrA and lvrB (leptospira virulence regulator) are juxtaposed in an operon and are predicted to encode a hybrid histidine kinase and a hybrid response regulator, respectively. Transcriptome analysis of lvr mutant strains with disruption of one (lvrB) or both genes (lvrA/B) revealed global transcriptional regulation of 850 differentially expressed genes. Phosphotransfer assays demonstrated that LvrA phosphorylates LvrB and predicted further signaling downstream to one or more DNA-binding response regulators, suggesting that it is a branched pathway. Phylogenetic analyses indicated that lvrA and lvrB evolved independently within different ecological lineages in Leptospira via gene duplication. This study uncovers a novel-signaling pathway that regulates virulence in pathogenic Leptospira (Lvr), providing a framework to understand the molecular bases of regulation in this life-threatening bacterium.

Keywords: Leptospira; branched signaling; gene duplication; hybrid histidine kinase; hybrid response regulator; pathogenic; two-component system; virulence.

Figure 1

In vivo screening of selected Leptospira TCS mutants. Golden Syrian male hamsters were challenged with wild type, lvrA/B (M1529), lvrA/BII (M1529 II), lvrB (M1419), lic13192 (M480), and lic13087 (M854) mutants of L. interrogans Manilae L495 sp via conjunctival route in doses of 5 × 10⁶, 10⁷, and 10⁸ leptospires. Animals were monitored for 21 days post-challenge with death as a primary outcome. The survival probability plot was based on a proportional hazards model. Treatment effects (mutations in L. interrogans Manilae strain) and day effects were estimated based on this model and P values were calculated. ^*P < 0.0001.

Figure 2

Gene arrangement and domain organization of Lvr dyad in pathogenic Leptospira spp. (A) This figure is a schematic representation of the Lvr loci in pathogenic Leptospira. Arrow length is proportional to the gene length. lvrA (blue): Putative hybrid histidine kinase-response regulator gene and lvrB (green): Putative hybrid response regulator gene in L. interrogans serovar Manilae strain L495. Himar1 insertion site in lvrA gene (lvrA/B mutant) and in intergenic region between lvrA and lvrB (lvrB mutant) has been indicated. (B) In vitro gene expression of lvrA and lvrB by RT-qPCR (C) Domain organization of Lvr proteins; signature segments of kinase and receiver domains were identified by multiple sequence alignment and indicated. Conserved histidine and aspartate residues as putative phosphorylation sites are denoted for each protein. REC, receiver domain; PAS, Per, Arnt, Sim domains; DhP, Dimerization histidine phosphotransfer; CA, catalytic and ATP-binding domain. (D) Phosphotransfer from LvrA to LvrB. An LvrB mutant devoid of autokinase activity (LvrB_H161A) was incubated with [Y-33P] ATP for 30 min with LvrA wild type, LvrA_H218A or LvrA_D524A (as labeled in each lane). Reaction products were separated by SDS-PAGE and visualized using autoradiography. (E) Wild-type LvrB and the point mutants D56A and H161A were incubated with [Y-33P] ATP and MgCl2 for 30 min, in presence (lanes 1 to 3) or absence (lanes 4 to 6) of LvrA_D524A (as labeled in each lane). Reaction products were separated by SDS-PAGE and visualized using autoradiography.

Figure 3

Global transcriptional changes in lvr mutants. (A) Venn diagram depicting the number of differentially expressed genes in lvr mutants, lvrA/B (M1529) and lvrB (M1419) with ± log 2-fold change cut-off and P ≤ 0.05. (B) Validation of RNA-Seq Analysis was performed by RT-qPCR and correlation coefficient has been indicated. (C) Heatmap depicting clusters of differentially expressed genes in lvr mutants when compared to L. interrogans Manilae L495 WT. Computationally we identified five arbitrary clusters that are marked in the heat map. (D) Lvr regulatory functions inferred from transcriptome analysis of lvr mutants, lvrA/B (M1529) and lvrB (M1419). Solid and dashed lines depict positive regulation and negative regulation, respectively. Inferences are based on relative abundance of COG categories (>5%) across each cluster. (E) Functional categorization of upregulated and downregulated genes in lvr mutants, lvrA/B (M1529) and lvrB (M1419) during late-log phase of growth at 30°C. Percent distribution is calculated for the total number of differentially expressed genes (according to the RNA-Seq analysis; log 2-fold change, P < 0.05) in each COG category.

Figure 4

Lvr dyad governs leptospiral virulence. (A) In vivo expression of lvr genes determined by RT-qPCR. Relative expression of the target lvr genes was studied by quantifying transcripts in sample collected from blood of hamsters (n = 2) at 3 days post-infection intraperitoneally with L. interrogans serovar Manilae WT at a dose of 10⁸ leptospires. Transcripts of in vitro cultures were obtained from a late-log phase culture of L. interrogans serovar Manilae WT incubated in EMJH at 30°C. In vivo results represent the expression levels of lvr genes in comparison to in vitro conditions and normalized to flaB gene expression. Results are the average of two independent assays and the error bars indicate ±1 SD. (B) Categorization of differentially expressed virulence-related genes (P < 0.05) in lvrA/B (M1529) and lvrB (M1419) mutants into genetically characterized (red) genes and putative (black) genes.

Figure 5

Lvr dyad regulates motility of Leptospira spp. (A) Hierarchical clustering heatmap representing the normalized expression levels of indicated motility genes in Leptospira Manilae L495 wild type, lvrA/B (M1529) and lvrB (M1419) mutants. (B) EMJH plates (0.5% agar) inoculated with 10⁵ cells of Leptospira interrogans Manilae L495 wild type strain or lvrA/B (M1529) and lvrB (M1419) mutants. Plates were incubated at 30°C and colony diameter was measured on 14th day. A representative plate from one of the three experiments is shown. Images were captured by Chemidoc XRS system (BioRad) (C) Graphical representation of colony diameter for Leptospira interrogans Manilae L495 wild type, lvrA/B (M1529) and lvrB (M1419) mutants measured after 17 days of incubation on 0.5% semisolid media. Points are plotted at the mean of three biological replicates and error bars indicate ±1 SD. ^***P ≤ 0.001; ns, not significant.

Figure 6

Evolution of LvrA and LvrB. Phylogenetic relationships of lvr genes in pathogenic Leptospira (indicated in red) with intermediate Leptospira (indicated in blue) and related two-component systems in sampled bacteria were inferred from an amino acid alignment using Bayesian approaches with models averaged parameter sets of rate matrix. The trees were rooted with sequences from two Mycobacterium species. The majority-rule consensus of 8001 MCMCMC-sampled trees with averaged branch length is present, and branches with strong support (BPP > 0.98) are in boldface. Bar indicates the substitutions per amino acid site. The tree is broken at a node for a better presentation, and a dashed line is used to link the node. Exceedingly long branches are foreshortened, as indicated with the symbol -//-.

Figure 7

Model for branched signaling pathway of Lvr hybrid two-component system: An unknown input signal modulates the autokinase activity of N-terminal HK module in LvrA. Upon switching to a kinase-on state, there would be phosphotransfer to its downstream HRR module, as well as to LvrB in a branched pathway. Alternatively, LvrB can be activated by a small molecule signal such as AcP (Acetyl Phosphate). After phosphotransfer events, Lvr proteins influence the expression of virulence and motility genes either by activation of putative downstream RRs or by protein-protein interactions.