Diversification of Orientia tsutsugamushi genotypes by intragenic recombination and their potential expansion in endemic areas

Abstract

Background: Scrub typhus is a mite-borne febrile disease caused by O. tsutsugamushi infection. Recently, emergence of scrub typhus has attracted considerable attention in several endemic countries in Asia and the western Pacific. In addition, the antigenic diversity of the intracellular pathogen has been a serious obstacle for developing effective diagnostics and vaccine.

Methodology/principal findings: To understand the evolutionary pathway of genotypic diversification of O. tsutsugamushi and the environmental factors associated with the epidemiological features of scrub typhus, we analyzed sequence data, including spatiotemporal information, of the tsa56 gene encoding a major outer membrane protein responsible for antigenic variation. A total of 324 tsa56 sequences covering more than 85% of its open reading frame were analyzed and classified into 17 genotypes based on phylogenetic relationship. Extensive sequence analysis of tsa56 genes using diverse informatics tools revealed multiple intragenic recombination events, as well as a substantially higher mutation rate than other house-keeping genes. This suggests that genetic diversification occurred via frequent point mutations and subsequent genetic recombination. Interestingly, more diverse bacterial genotypes and dominant vector species prevail in Taiwan compared to other endemic regions. Furthermore, the co-presence of identical and sub-identical clones of tsa56 gene in geographically distant areas implies potential spread of O. tsutsugamushi genotypes.

Conclusions/significance: Fluctuation and diversification of vector species harboring O. tsutsugamushi in local endemic areas may facilitate genetic recombination among diverse genotypes. Therefore, careful monitoring of dominant vector species, as well as the prevalence of O. tsutsugamushi genotypes may be advisable to enable proper anticipation of epidemiological changes of scrub typhus.

Fig 1. Epidemiological trends of scrub typhus incidence in several endemic countries from 2000 to 2014.

Regional map shows the distribution of scrub typhus (gray area) and the annual incidence of several endemic countries during 2000 ~ 2014 are presented. The graphs are based on the data summarized in S1 Table. Red line: reported cases, pink area: incidence rate/10⁶.

Fig 2. Phylogenetic analysis of 206 tsa56 genes and their classification into genotypes and genogroups.

Phylogenetic relationships of 206 complete or nearly complete tsa56 genes (listed in S2 Table) covering more than 85% of coding sequences are presented. 17 genotypes were defined based on the branching supporting values (SH-like value ≥ 0.90) and the relative branch length from a node. They were further classified into 5 genogroups (Karp, Gilliam, TA763, Kato, and Shimokoshi) based on phylogenetic distances.

Fig 3. Similarity and identity matrix for amino acid sequences of 206 TSA56 proteins.

Pairwise identity (upper triangle) and similarity (lower triangle) matrix of TSA56 sequences were constructed using the MatGAT2.1 program. The order of TSA56 sequences is the same as in Fig 2. Raw data values are presented in S5 Table.

Fig 4. Detection of intragenic recombination in tsa56 genes using 17 proto-genotype sequences.

A. Recombination breakpoints within tsa56 sequences were detected using the GARD program. Support probabilities for inferred recombination break-points are shown on the left side of the breakpoint plots. B. Intragenic recombination events in tsa56 sequences found using the RDP suite. The schematic diagrams of indicated genotypes present the putative major and minor parent sequences of each genotype (by color codes) and the location of predicted breakpoints. Detailed information of the recombination events predicted by the RDP suite is summarized in S6 Table. The genotypes without significant recombination are marked with (*). C. Representative BOOTSCAN evidence for recombination origin on the basis of pairwise distance, modeled with a window size 200 nt, step size 20 nt, and 100 Bootstrap replicates. The threshold of significance for the analysis was set as 70% bootstrapping value (dash line).

Fig 5. Hierarchical relationship of 17 proto-genotype sequences.

A. Based on the recombination events and the parental origins of the tsa56 sequences shown in Fig 4, the hierarchical relationship of 17 genotypes was estimated and is presented as sequential generations. B. Recombination events in Gilliam genotype sequence, the last generation in A, were analyzed by similarity plot on the basis of pairwise comparison with Boryong, Karp_B, and JG_C genotypes as parental sequences (scanned with a window size 200 and step size 20 nt).

Fig 6. Estimation of recombination and mutation rates of O. tsutsugamushi genes.

Recombination and mutation rate per site of 53 genes were calculated by LDhat installed in RDP program. Sequences of 53 gene sets were extracted from nine O. tsutsugamushi genomes available in NCBI database. Average recombination rate per base pair (ρ/bp) of 53 gene sets is 0.083 and average mutation rate per base pair (θ/bp) is 0.020 (blue lines). Genes encoding known membrane proteins are indicated as red dots. Detailed information of the gene sets is presented in S7 Table.

Fig 7. Geographical distribution of O. tsutsugamushi genotypes in endemic countries.

A. The relative proportion of each genotype reported in the indicated endemic country is presented and the pie size is proportional to the number of sequences reported. The number of tsa56 sequences used for each country is as follows: Taiwan (123), Korea (69), Japan (52), Thailand (29), Cambodia (28), China (9), Malaysia (2), Myanmar (1), and Papua New Guinea (1). B. Relative proportion of each genotype reported from Taiwan, northern endemic area (Korea, Japan, and China), and southern countries (Thailand, Cambodia, Vietnam, Malaysia, Myanmar, and Papua New Guinea) is presented. The number of sequences from each group is indicated below the bar graphs.

Fig 8. Geographical distribution of nine Leptotrombidium species, the major vectors of scrub typhus.

Geographical distributions of nine representative Leptotrombidium species mediating scrub typhus are presented. If the collection sites of vector identification were specified with coordinates, they are indicated as red dots, otherwise the collection sites were indicated at province or county level as colored area. Blue: collected before 1974, red: collected after 1974, green: collected before and after 1974. Detailed information is available in S3 Table.

Fig 9. Potential expansion of O. tsutsugamushi clones throughout the endemic region.

A. Linkage map indicates the presence of identical (solid line) or sub-identical (one or two base difference, dotted line) tsa56 genes in geographically distant endemic countries. B. The East Asia/Australasia major flyway of migratory birds is presented as blue lines. The primary habitats of 21 key bird species in the flyway are overlaid on the regional map (Data available at http://www.eaaflyway.net/).