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

Gram-negative spirochetes of the genus Leptospira cause the disease Leptospirosis, which is one of the most important zoonotic infectious conditions worldwide, including in Thailand [1, 2]. Pathogenic Leptospira strains can infect most mammals and produce a wide range of clinical manifestations varying from acute life-threatening disease to chronic asymptomatic forms [3]. In humans, leptospirosis is estimated to have a worldwide annual average morbidity of 1.03 million cases with a mortality of 58,900 cases, and these figures are poised to increase due to climate change [4]. Asymptomatic animals that are carrying leptospires in the convoluted tubules of the kidney play an important role in disease maintenance and in transmission via infected urine [1].

Based on DNA-DNA hybridization and 16S rRNA phylogeny, Leptospira can be classified into more than 22 species located within three groups: the pathogenic, intermediate and non-pathogenic groups. In addition, more than 300 serovars have been described based on antigenic diversity in the lipopolysaccharide (LPS) structure on the cell surface. These different serovars can be identified using the cross-agglutination absorption test (CAAT) [3].

Pathogenic L. interrogans serovar Lai strain 56601 was the first leptospire to be subjected to genome sequencing, and the data obtained revealed the unique physiological characteristics and virulence factors of this strain [5]. Subsequently, the genomes of a number of other both pathogenic and non-pathogenic Leptospira strains have been subjected to next-generation sequencing (NGS) to investigate the genetic basis of pathogenesis [6]. Using comparative genomic analysis, pathogenic markers were defined based on gene gain and loss events, and genome rearrangements were identified [6–8]. An aspect that has not been explored for Leptospira is measurement of selective pressure, inferred by non-synonymous to synonymous substitution rate in genes in conserved genome regions, signaling that the genes are under positive selection: in other species these adaptations have been shown to have an important role in species diversification and adaptation in different ecological niches and hosts [9].

In pathogenic bacteria, positive selection is an evolutionary driving force arising by mutations in protein-coding genes that are associated with bacterial adaptation to changes in their environment or host species [10]. Advantageous point mutations enhance bacterial physiological fitness or virulence factor profiles appropriate to survival under various conditions [11]. Genome-wide analyses of positive selection associated with increased rates of point mutations in specific genes has been reported for several bacteria, including Bacteroides fragilis [12], Escherichia coli [13, 14], Mycobacterium tuberculosis [15], Salmonella serotypes [16] and Pasteurella multocida [17]. To date reports of positive selection in Leptospira remain scarce, and there is still a lack of genomic information for Leptospira isolates recovered from asymptomatic animals.

In the present study, we determined the serotypes and then sequenced the genomes of two L. interrogans isolates that were recently recovered from the urine of asymptomatic Thai dogs. We then undertook comparative analysis with these genomes and 22 other representatives of validly published Leptospira genomes obtained from the NCBI database. The Thai L. interrogans strains were subjected to genomic characterization and phylogenetic analysis to examine evolutionary relationships. Within-species, comparative genome analysis was performed with 97 strains of L. interrogans to obtain insight into the pan-genome structure. Strain-specific genes of the two Thai strains that were associated with specific functions including virulence also were identified. Furthermore, a genome-wide analysis for positive selection was undertaken on the core genes of L. interrogans to determine genes involved in adaptive evolution. The results provide insight into the Leptospira genome and contribute to a more comprehensive understanding of factors involved in molecular pathogenesis and adaptive evolution in pathogenic Leptospira.

The present study reports the first genomic characterization of L. interrogans strains isolated from dogs in Thailand, and presents a comparative genome analysis of these with the published genomes of another 12 Leptospira species. Moreover, a genome-wide scan of genes under positive selection, focusing on potential virulence factors of L. interrogans, was undertaken to obtain insights into genetic diversity, molecular pathogenesis and molecular adaptation of the Leptospira strains.

L. interrogans serovar Paidjan strain CUDO5 and L. interrogans serovar Dadas strain CUDO8 were isolated from the urine of asymptomatic dogs in an area of Thailand with a high frequency of human leptospirosis, and a 9.92% prevalence in animals. Evidence from previous rrs phylogenetic analysis has revealed close genetic links between isolates from animals and humans in this area [19]. Therefore, theses strains have epidemiological importance and potentially may pose a risk to public health in the region. The availability of genome sequences from both Thai Leptospira serovars provides useful new information relating to disease diagnosis and prevention.

Analysis of the genomes of the two Thai strains demonstrated that they had similar numbers of CDSs and sub-system categories to other analyzed L. interrogans strains. Most of the genes encoded proteins responsible for basic biological processes such as transcription, translation, cell wall synthesis and motility, and the findings confirm the high conservation of CDSs among L. interrogans strains.

Phages are viruses that infect specific bacterial hosts, and are abundant in the environment [20]. Prophage-related genes were found in the Leptospira genomes, presenting as incomplete phages without any obvious association with pathogenicity islands or antimicrobial resistant genes, but with diverse prophage types (Additional file 1: Table S3). In this study, Sphingomonas phage (PHAGE_Sphing_PAU_NC_019521) was found in 11 of the 13 representative L. interrogans strains, and this phage potentially could be exchanged between L. interrogans and Sphingonomas paucimobilis in shared environments such as in water sources [21].

The CRISPR-Cas system is defined as part of the bacterial adaptive immune system. This system can remember and destroy phages and plasmids invading the host cell by producing specific crRNA against these elements [18]. Our results are in agreement with a previous study where the CRISPR-Cas system was found only in intermediate and pathogenic group strains, which harbored at least one subtype of CRISPR1, whereas non-pathogenic Leptospira species did not have this system [6]. The CRISPR-Cas system has been thought to be a necessary functional system in pathogenic Leptospira [6]. The strains from dogs in this study possessed two CRISPR1 loci (subtypes I-B and I-C). CRISPR1 subtype I-B targets foreign DNA invasion, while the function of subtype I-C is still uncharacterized. These two loci of the CRISPR-Cas system also are found in Pyrococcus furiosus, suggesting its ability to use crRNA to defend against a wide range of foreign genetic material, including both DNA and RNA [22]. Various crRNAs targeting either plasmids or phages in various species were identified in L. interrogans serovar Dadas strain CUDO8 (n = 40) and to a lesser extent in L. interrogans serovar Paidjan strain CUDO5 (n = 31) (Additional file 1: Table S4). The difference suggests that CUDO8 may have been subjected to infection with more phages and plasmids than CUDO5. The fact that the plasmid of L. interrogans serovar Linhai strain 56609 was found in CUDO5 and CUDO8 might reflect their shared evolutionary history, or transfer of the plasmid during their occupation of shared microenvironmental niches before their dissemination to different hosts. On the other hand, it should be noted that crRNAs targets predicted by bioinformatics programs quite commonly have non-specific or coincidental hits (both plasmid and phage), especially when querying with short sequences. Therefore, a CRISPR interference assay is required to validate the crRNAs target results relating to interference with foreign genetic material in Leptospira [23].

The presence of virulence genes associated with the pathogenesis of leptospirosis have been used to divide Leptospira species and strains into three groups: pathogenic, intermediate and non-pathogenic [24]. In this study, we identified virulence factors present in strains CUDO5 and CUDO8 genomes using the VFDB database. However, VFDB searching has some limitations since virulence genes/proteins with confirmed roles in other bacteria might not reflect their function in strains CUDO5 and CUDO8. Therefore, a combination search with orthologous virulence genes with established roles in acute and chronic leptospirosis also was undertaken. All pathogenic Leptospira strains contained genes encoding catalase, collagenase, TonB-hemin binding receptor, immunoglobulin-like protein B and sphingomyelinases, which all are confirmed as virulence markers during the early stage of infection [24]. On the other hand, intermediate and non-pathogenic Leptospira lack many of these virulence genes: their acquisition may reflect an evolutionary process favoring host infection and disease [6], whilst loss of these genes may result in attenuation and reduction in virulence. Although strains CUDO5 and CUDO8 have genes with confirmed roles as virulence factors, they were isolated from the urine of asymptomatic dogs. Their ability to establish persistent infections with long-term kidney colonization is likely to reflect a balance of gene content and expression that favors an advantageous carrier state - maximizing the opportunity for further transmission via urine.

Comparative genome analysis can be used to understand genomic diversity and provide insights into biological functions and evolution [25]. Using the power law exponents of Heap’s law, our data indicated that L. interrogans strains have an open pan-genome [6, 7]. This finding suggests that the gene pool of the spirochetes can expand, and the observed genomic plasticity probably results from multiple ways of incorporating new genetic material into their genomes such as through horizontal gene transfer and/or replication of advantageous genes via gene duplication [7]. This phenomenon is beneficial in relation to adaptations resulting in increased infectivity and survival in a broad range of mammalian hosts and the environment [1].

The Leptospira species that were analyzed were separated into three groups using DNA-DNA hybridization as well as by virulence gene profiles [6]. The 13 representative strains of L. interrogans had ANI values of over 98%, suggesting that nucleotide sequences of L. interrogans strains are mostly conserved, even though the strains were of different serovars and were from different origins and epidemiological units. The ML-based core-SNPs phylogenetic analysis identified both distinct and clustered Leptospira serovars in the same branch of the phylogenetic tree. This finding reflected the diversity of the Leptospira strains, even amongst those coming from the same geographical origins. It also inferred different accumulations of SNPs in the core genome in each strain. Moreover, there were instances of connectivity between human and animal Leptospira isolates, providing genetic evidence supporting the occurrence of disease maintenance in animals and cross-species transmission. Notably, the phylogenetic tree confirmed that the two Thai canine strains were closely related to each other. However, the serotyping results revealed that they belonged to different serogroups and serovars (serogroup Bataviae serovar Paidjan for strain CUDO5 and serogroup Grippotyphosa serovar Dadas for strain CUDO8). Consistent with the rfb locus analysis, serovars Paidjan and Dadas had gene variations in these regions. Nevertheless, genes association with rhamnose biosynthesis (rfb ABCD) and rhamnose containing glycan (rfbB and galE) were distributed in both serovars. Previously, the LPS of pathogenic Leptospira has been shown to contain more rhamnose than intermediate Leptospira, and moreover specific sugars were found related to particular serovars [26]. This finding was in agreement with our results in the case of serovar Dadas that contained three additional genes associated with capsular heptose biosynthesis. Furthermore, the gene encoding aminotransferase PglE that was found in both serovars might play a crucial role in creating novel sugar composition during the glycosylation process of outer membrane proteins [27]. The three aminotransferases belonging to the DegT family in serovar Paidjan might be associated with LPS O-antigen side chain biosynthesis, leading to differences between the serological characteristics of the serovars [28]. These findings provided possible explanations for the difference in LPS composition and outer membrane protein post-translational modifications between serovars Paidjan and Dadas. However, more experiments are required to compare the relationship of the genes in the rfb locus, sugar composition, sugar ramification and structural determination of the LPS, since there are large variations and complexities in this material [1]. A previous investigation of the LPS of pathogenic and intermediately pathogenic Leptospira species using multiple methods including LPS extraction, gas chromatography-mass spectrometry (GC-MS), and electron microscopy revealed significant differences in terms of carbohydrate and fatty acid composition, including in Leptospira isolated from common reservoir hosts [26]. The presence of large quantities of a specific sugar in a particular serovar is believed to play an important role for survival and colonization in certain environments [26]. Therefore, a combination of genomic detail and biochemical and structural analysis will allow a better understanding of LPS biology in the adaptation strategies in these two pathogenic Leptospira.

In bacteria, strain-specific genes are associated with specific functions and niche adaptations [25]. Strains CUDO5 and CUDO8 harbored strain-specific genes compared to the other strains in this study (n = 97), but these were mostly predicted as hypothetical proteins with unknown functions. Nevertheless, strain-specific genes of known function were found in these strains, consisting of; a type III restriction endonuclease and phage terminase large subunit. The function of these genes may be related to the pathogenesis of Leptospira. Notably, type III restriction endonucleases are functionally associated with defense mechanisms against invading foreign DNA. Therefore, this gene possibly functions to protect L. interrogans from phage infection, and could modulate the genetic exchange barrier [29]. As no complete phage was reported in this study, the cryptic prophage including phage terminase large subunit might indicate the existence of a prior phage infection that was probably degraded by the CRISPR-Cas system and/or a restriction modification system, and was subsequently trapped in the Leptospira chromosome [30]. Although the role of phage terminase large subunit in the Leptospira strains was not established, cryptic prophages in E. coli contribute to survival in adverse environment conditions [31]. Therefore, this cryptic prophage might help Leptospira to cope with stress in new hosts and environments.

Beneficial mutations in bacterial genes under positive selection can assist in molding bacterial-host adaptation [11], whilst recombination helps positive selection to become fixed in the population [32]. Our investigation of positive selection among the genomes of 13 representative L. interrogans strains focused on genes encoding virulence-associated proteins. Genes under significant positive selection included 101 predicted virulence-associated genes and 352 genes with various functions, including those involved in cell wall/envelope biosynthesis; amino acid transport and coenzyme transport and metabolism; and signal transduction and cell translation: these function in binding and transport of essential nutrients in the early stages of infection and bacterial replication. Using the VFDB database, the genes under positive selection were grouped by virulence functions consisting of adhesion, anti-phagocytosis and iron uptake systems. In Leptospira infection an initial step is adhesion at the site of abrasion or on mucous membranes. In general, several adhesion factors are responsible for attachment to a variety of cell surfaces, such as the extracellular matrix (ECM), complement factors, macrophages, fibroblasts, endothelium cells and kidney cells [6, 33]. Therefore, positive selection could enhance adhesion ability and assist Leptospira in spreading throughout the body during the initial stages of infection. Moreover, positive selection was found on binding sites involved in immune recognition. Protein conformational changes may be part of a strategy for anti-phagocytosis by using surface adhesion-related proteins to trap complement factors and in turn avoid opsonization and IgG deposition on the leptospiral surface membrane [34]. In this study, four genes encoding experimentally confirmed virulence factors in pathogenic Leptospira were selected to construct 3D-structure models with positive selection site mapping to investigate possible functional implications of changes to the proteins (genes tlyA, hbpA, fliN2 and lpxD1). Specific amino acids in each molecule were identified as being under positive selection, but the significance of these in relation to their structure and function was not investigated further. Genes related to iron uptake systems were under positive selection, including the tlyA gene encoding hemolysin A and hbpA encoding the TonB-hemin binding receptor for which the structures were analyzed. These genes may provide a synergistic effect for iron acquisition from red blood cells during Leptospira infection [35]. Previously, endoflagella and protein-related LPS biosynthesis were confirmed as true virulence factors in pathogenic Leptospira [36]. In the present study, we found positive selection on genes associated with endoflagella, including fliN2 encoding for flagella motor switch protein, flgK encoding for flagella hook-associated protein, and flhB encoding for polar flagella protein. This positive selection is likely to be important for motility of Leptospira, allowing translocation, penetration and dissemination in host tissues [37, 38]. The gene lpxD1 encoding for UDP-3-O-(3-hydroxymyristoryl) glucosamine N-acyltransferase that is involved with lipid A biosynthesis as part of the LPS structure also was subjected to positive selection. Studies have shown that alterations to the gene lpxD contribute to modification in lipid A, and subsequently influence outer membrane integrity when bacteria are subjected to changing temperatures [36, 39]. Therefore, the positive selection on lpxD1 may be involved in assisting Leptospira to adapt to temperature differences in mammalian hosts. Additionally, mutations in lpxD1 have been considered to be involved with kidney and liver colonization by Leptospira in an asymptomatic animal model. It also has been suggested that pathogenic Leptospira have lipid A modification strategies to evade immune response in the early stage of infection, leading to development of chronic leptospirosis in mammalian hosts [40]. The gene colA encoding for collagenase (ColA) showed evidence of both positive selection and recombination in this study. ColA has been experimentally confirmed as a virulence factor in Leptospira [41]. Positive selection in colA might promote Leptospira invasiveness in the host body, whilst recombination of this gene might facilitate spread of advantageous mutations to other Leptospira [32, 41].

Pathogenic Leptospira have the ability to cause chronic infection by evading the host immune response [42]. Several different strategies have been described for immune evasion including biofilm formation, an increase in LPS O-antigen, and down-regulation of outer membrane proteins [43–45]. Moreover, during prolonged infections immune pressures acting on such pathogens are likely to select for advantageous genetic variation within the genome, leading to accumulation of mutations that increase survival fitness or that cause loss of function in virulence factors [46]. The balance of these genetic alterations may contribute to the strain’s adaptation and establishment of a reservoir/carrier state in an animal. Our study has improved understanding of genetic adaptation in this important zoonotic pathogen and provided promising gene targets for further research. This should lead to development of better control strategies, such as improved vaccines and more accurate diagnostic tests for use in the region.

This study provided genomic data and characterization of Thai L. interrogans serovar Paidjan stain CUDO5 and L. interrogans serovar Dadas strain CUDO8 isolated from the urine of asymptomatic dogs; furthermore, comparative genomic analysis with the genomes of 97 L. interrogans strains were undertaken and revealed an open pan-genome for the strains. Genes with established roles in acute and chronic leptospirosis were found in both Thai canine strains. Core-SNPs phylogeny confirmed their close genetic relatedness, but the rfb locus analysis identified genetic variations between them that were consistent with the different serotyping results. Genes under positive selection were identified, including genes encoding factors involved in motility, temperature adaptation, and iron acquisition: these may be involved in bacterial dissemination, thermal adaptation and nutrient uptake. Further investigations into the functions and expression of these genes, including in vivo experiments are required to expand these findings.