Genomic analysis of Leptospira interrogans serovar Paidjan and Dadas isolates from carrier dogs and comparative genomic analysis to detect genes under positive selection

Abstract

Background: Leptospirosis is an emerging infectious disease worldwide that can cause high morbidity and mortality rates in humans and animals. The causative spirochetes have reservoirs in mammalian hosts, but there has been limited analysis of the genomes of isolates recovered from animals. The aims of this study were to characterize genomic features of two Leptospira interrogans strains recently isolated from asymptomatic dogs in Thailand (strains CUDO5 and CDUO8), and to perform comparative genome analyses with other strains. Molecular adaptive evolution in L. interrogans as signaled by positive selection also was analyzed.

Results: Whole genome sequence analysis revealed that strains CUDO5 and CUDO8 had genome sizes of approximately 4.9 Mbp with 35.1% GC contents. Using monoclonal antibodies, strains CUDO5 and CUDO8 were identified as serovars Paidjan and Dadas, respectively. These strains harbored genes known to be associated with acute and chronic infections. Using Single Nucleotide Polymorphisms phylogeny (SNPs) with 97 L. interrogans strains, CUDO5 and CUDO8 had closest genetic relatedness with each other. Nevertheless, the serovar determinant region (rfb locus) showed variations in the genes encoding sugar biosynthesis. Amongst 13 representative L. interrogans strains examined for molecular adaptive evolution through positive selection under the site-model of Phylogenetic Analysis of Maximum Likelihood, genes responsible for iron acquisition (tlyA and hbpA), motility (fliN2, flgK, and flhB) and thermal adaptation (lpxD1) were under increased selective pressure.

Conclusions: L. interrogans serovar Paidjan strain CUDO5 and serovar Dadas strain CUDO8 had close genetic relatedness as analyzed by SNPs phylogeny. They contained genes with established roles in acute and chronic leptospirosis. The rfb locus in both serovars showed gene variation associated with sugar biosynthesis. Positive selection analysis indicated that genes encoding factors involved in motility, temperature adaptation, and iron acquisition were under strong positive selection in L. interrogans. These may be associated with adaptation in the early stages of infection.

Keywords: Dog; Genome; Leptospira interrogans; Positive selection; Serovar Dadas; Serovar Paidjan.

Fig. 1

RAST subsystem annotation in L. interrogans serovar Paidjan strain CUDO5 (pink bar) and serovar Dadas strain CUDO8 (green bar). The functional subsystem category with the largest number of annotations is RNA metabolism, following by Cofactors, vitamins, prosthetic groups, pigments, and Amino acids and derivatives, respectively

Fig. 2

CRISPR-Cas gene organization in L. interrogans serovar Paidjan strain CUDO5 and serovar Dadas strain CUDO8. Strains CUDO5 and CUDO8 contained CRISPR1 subtypes I-B and I-C. CRISPR1 subtype I-B is a complete locus flanked with a CRISPR array which harbors specific direct repeats (DRs) and spacers (panel a), with different quantities based on the time of exogenous DNA exposure (panel b)

Fig. 3

Graphical display of CRISPR RNA (crRNA) and target protospacer DNA in L. interrogans serovar Paidjan strain CUDO5 and serovar Dadas strain CUDO8. Spacer specific sequences are transcribed to crRNA to protect against phage and plasmid re-infection. The crRNA in strains CUDO5 and CUDO8 gave highest matching scores to a plasmid of L. interrogans serovar Linhai strain 56609. Colored yellow indicates the complementary matching between spacer and target protospacer sequence; red indicates the mismatching of spacer and target protospacer sequence and blue represents the flanking sequence between spacer and target protospacer

Fig. 4

The distribution and conservation of 33 representative confirmed virulence genes in pathogenic, intermediate and non-pathogenic Leptospira species. Genes encoding for outer membrane protein (loa22), flagella motor switch protein (fliY), and hemolysins B and C (tlyB and tlyC) were conserved in all 24 Leptospira strains. Genes encoding was conserved only in pathogenic Leptospira strains. Strains CUDO5 and CUDO8 contained all of the 33 virulence genes

Fig. 5

Whole genome core-single nucleotide polymorphisms (core-SNPs) phylogeny of L. interrogans based on the maximum likelihood method with 1000 bootstrap replicates. The core-SNPs phylogenomic analyses was visualized by EvolView [63]. The tree shows that L. interrogans serovar Paidjan strain CUDO5 and serovar Dadas strain CUDO8 were placed in the same branch with a bootstrap percentage of 100 as highlight in peach color. Serovar specific clades with bootstrap percentage of more than of 75 are highlight in blue color, including serovar Australis, Grippotyphosa, Hardjo, Pomona and Pyrogenes. L. kirscheneri serovar Cynopteri strain 3522 CT (CYN) was used as an outgroup in this study

Fig. 6

Pan-genome analysis of L. interrogans (n = 97) using the BPGA pipeline. The power and exponential law equations were used to create pan- and core-genome curves of L. interrogans (panel a). The COG categories distribution of core, accessory and strain-specific genes in 97 L. interrogans strains are shown in panel b, and according to Heap’s law the Leptospira genome was predicted to be open. The COG categories are denoted as follows: D (Cell cycle control, cell division, chromosome partitioning), M (Cell wall/membrane/envelope biogenesis), N (Cell motility), O (Posttranslational modification, protein turnover, chaperones), T (Signal transduction mechanisms), U (Intracellular trafficking, secretion, and vesicular transport), V (Defense mechanisms), J (Translation, ribosomal structure and biogenesis), K (Transcription), L (Replication, recombination and repair), C (Energy production and conversion), G (Carbohydrate transport and metabolism), E (Amino acid transport and metabolism), F (Nucleotide transport and metabolism), H (Coenzyme transport and metabolism), I (Lipid transport and metabolism), Q (Secondary metabolites biosynthesis, transport and catabolism), P (Inorganic ion transport and metabolism), R (General function prediction only), and S (Function unknown)

Fig. 7

Circular genome map of L. interrogans serovar Lai strain 56601 and the other 13 representative strains studied, with the location of 101 predicted virulence genes under positive selection marked. Comparative sequence analysis was performed with BRIG [76] using L. interrogans strain 56601 as a reference (thick black innermost ring) against other representative related L. interrogans strains. The second inner ring indicates GC contents (black color) followed by the GC skew (purple and green colors). Each colored ring shows the host origin in each strain of chromosomal DNA of Leptospira presented as follows: magenta for dogs, blue for pigs, cyan for cattle and light-green for humans. The outermost labels show the location of 101 predicted virulence genes under positive selection in L. interrogans. The intensity of the ring color represents the percentage of nucleotide identity from 50 to 90

Fig. 8

Three-dimensional protein models of Hemolysin A (encoded by tlyA: panel a), Ton-B hemin binding receptor (encoded by hbpA: panel b), Flagella motor switch protein (encoded by fliN2: panel c), and UDP-3-O-(3-hydroxymyristoryl) glucosamine N-acyltransferase (encoded by lpxD1: panel d) under positive selection in L. interrogans. Sites of strong positive selection are highlighted as red spheres (posterior probability > 0.95)