Complete genome sequences of two strains of Treponema pallidum subsp. pertenue from Indonesia: Modular structure of several treponemal genes

Abstract

Background: Treponema pallidum subsp. pertenue (TPE) is the causative agent of yaws, a multistage disease endemic in tropical regions in Africa, Asia, Oceania, and South America. To date, seven TPE strains have been completely sequenced and analyzed including five TPE strains of human origin (CDC-2, CDC 2575, Gauthier, Ghana-051, and Samoa D) and two TPE strains isolated from the baboons (Fribourg-Blanc and LMNP-1). This study revealed the complete genome sequences of two TPE strains, Kampung Dalan K363 and Sei Geringging K403, isolated in 1990 from villages in the Pariaman region of Sumatra, Indonesia and compared these genome sequences with other known TPE genomes.

Methodology/principal findings: The genomes were determined using the pooled segment genome sequencing method combined with the Illumina sequencing platform resulting in an average coverage depth of 1,021x and 644x for the TPE Kampung Dalan K363 and TPE Sei Geringging K403 genomes, respectively. Both Indonesian TPE strains were genetically related to each other and were more distantly related to other, previously characterized TPE strains. The modular character of several genes, including TP0136 and TP0858 gene orthologs, was identified by analysis of the corresponding sequences. To systematically detect genes potentially having a modular genetic structure, we performed a whole genome analysis-of-occurrence of direct or inverted repeats of 17 or more nucleotides in length. Besides in tpr genes, a frequent presence of repeats was found in the genetic regions spanning TP0126-TP0136, TP0856-TP0858, and TP0896 genes.

Conclusions/significance: Comparisons of genome sequences of TPE Kampung Dalan K363 and Sei Geringging K403 with other TPE strains revealed a modular structure of several genomic loci including the TP0136, TP0856, and TP0858 genes. Diversification of TPE genomes appears to be facilitated by intra-strain genome recombination events.

Fig 1. A phylogenetic tree constructed from the whole genome sequence alignment of available TPE and TEN complete genome sequences (S3 Table).

The tree was constructed using the Maximum Likelihood method based on the Tamura-Nei model [19] and MEGA software [20]. The bar scale corresponds to a difference of 0.0001 nucleotides per site. Bootstrap values based on 1,000 replications are shown next to the branches. There were a total of 1,189 informative positions in the final dataset. Genome sequence of TEN strain Bosnia A was used as an outgroup. Both TPE strains of Indonesian origin were highly related to each other when compared to the genetic diversity detected among other, previously characterized TPE strains. Both intergenic spacer regions within the rrn operons, the tprK (TP0897) and tprD (TP0131) gene sequences and the repetitive sequences within the arp (TP0433) and TP0470 genes were omitted from the analysis. The TPE Kampung Dalan K363 and TPE Sei Geringging K403 strains are shown in bold.

Fig 2. The modular structure of the TP0856 and TP0858 genes among completely sequenced treponemal strains.

A. Please note the differences between sequence patterns of TPE Kampung Dalan K363 and the other TPE strains, the TPA Sea81-4 and the other TPA strains, and TPE and TPA/TEN strains in TP0858 gene. The r3 sequence (in TPA Sea81-4), r4 sequence (in TPA and TEN strains), r5 sequence (in Kampung Dalan K363 and TPA Sea81-4) and r6 sequence (in TEN Bosnia A) within TP0858 or TP0856 genes resulted probably from intra-strain recombination events. B. A list of repetitive (r3, r4, r5, r6) and non-repetitive (unique) sequences (r1, r2, r7, r8, r9).

Fig 3

A. Modular structure of the TP0136 gene of the TPE Kampung Dalan K363, TPE Sei Geringging K403 and additional TPE, TEN, and TPA strains. While the TPE Kampung Dalan K363 and TPE Sei Geringging K403 strains resemble TPA strains, other TPE strains showed a deleted version of the TP0136 gene (Samoa D and Gauthier) or a deleted version with a duplicated subregion r6 and r4 (CDC-2, CDC 2575, Ghana-051, Fribourg-Blanc, and LMNP-1). The sequence of the TP0136 gene from the Treponema paraluisleporidarum ecovar Cuniculus strain Cuniculi A was not included in this analysis since this gene locus contains a genetically different sequence, which is identical (99.7% at the DNA level) to the TP0133 gene sequence. B. A list of repetitive sequences (r1, r2, r3, r4, r5, r6).

Fig 4. Number of identified k-mers of different length (9–33 nt) derived from TPE genome sequences.

The number of unique k-mers saturated at a length of 17 nts, which was subsequently used as the cutoff length for further evaluations.

Fig 5. A schematic representation of examples of detected modular structure in several treponemal genes.

A. The modular structure of TP0126/TP0126b and TP0126c genes (coordinates 148661–148691 and 149217–149247, respectively, according to the TPE Samoa D genome [6]). The TPE Fribourg-Blanc strain has identical sequences between regions where the TP0126 and TP0126b genes overlap and in TP0126c, while other analyzed genomes have different sequences in the TP0126/TP0126b. B. The modular structure of the TP0127b, TP0128, TP0130, and TP0898 genes (coordinates 150108–150120, 150605–150619, 151643–151659, and 977776–977789, respectively, according to the TPE Samoa D genome [6]). The TEN Bosnia A strain has identical sequences in regions of TP0128, TP0130, and TP0898 genes, while other genomes have the same sequences between regions of the TP0128 and TP0130 genes and between the TP0127b and TP0898 genes. C. The modular structure of the TP0117, TP0131, TP0316, and TP0620 genes (coordinates 134610–134655, 152056–152101, 331882–331927, and 672661–672706, respectively, according to the TPE Samoa D genome [6]). The TPE LMNP-1 strain has identical sequences in all of these genes while other genomes have the same sequences between regions of the TP0117 and TP0131 genes and between the TP0316 and TP0620 genes. D. The modular structure of the TP0117, TP0131, TP0316, and TP0620 genes (coordinates 134676–134672, 152122–152138, 331948–331964, and 672727–672743, respectively, according to the TPE Samoa D genome [6]). The TPE LMNP-1 and TEN Bosnia A strains have identical sequences in all of these genes while other genomes have the same sequences between regions of the TP0117 and TP0131 genes and between the TP0316 and TP0620 genes. E. The modular structure of the TP0117, TP0131, TP0316, and TP0620 genes (coordinates 134780–134804, 152226–152250, 332052–332076, and 672831–672855, respectively, according to the TPE Samoa D genome [6]). The TPE LMNP-1 strain has identical sequences in all these genes while TPE strains Gauthier, CDC 2575, and Ghana-051 have identical sequences between regions of the TP0117 and TP0131 genes and between the TP0316 and TP0620 genes. The remaining genomes have the same sequences between the TP0316 and TP0620 genes.