Characterization of the novel clinical isolate X-4 containing a new tp0548 sequence-type

Background A novel tp0548 sequence-type was identified in one clinical isolate (X-4) from a patient diagnosed with primary syphilis in Xiamen, China. To precisely define and characterize this new clinical isolate, we performed further genome-scale molecular analysis. Methodology/Principal findings The alignment of all published tp0548 genotypes revealed that this new genotype had a unique nucleotide substitution G->T at position 167, and the letter “ao” was assigned to the genotype. Phylogenetic analysis showed that the “ao” genotype belonged to the SS14-like clade of Treponema pallidum (TPA) strains. The genome of the X-4 isolate was then sequenced and analyzed, and the result of a multi-locus sequence analysis using a set of nine chromosomal loci showed that the X-4 isolate was clustered with a monophyletic group of TPA strains, which clearly identified the isolate as a TPA strain. Whole-genome phylogenetic analysis was subsequently conducted to corroborate the TPA strain classification of the X-4 isolate. And the isolate was genetically related to the SS14 strain, with 42 single nucleotide variations and 12 insertions/deletions. In addition, high intrastrain heterogeneity in the length of the poly G/C tracts was found in the TPAChi_0347 locus, which indicated that this gene was most likely involved in phase variation events. The first investigation of the length heterogeneity of the poly A/T tracts showed the variability of the ploy A/T was lower, and all the observed intrastrain variations fell within coding regions. Conclusions/Significance The study demonstrated the X-4 isolate was a TPA isolate containing a novel tp0548 sequence-type. The identification of intrastrain genetic heterogeneity at poly G/C tracts and poly A/T tracts of the isolate could provide a snapshot of the genes that potentially involved in genotype-phenotype variations. These findings provide an unequivocal characterization for better understanding the molecular variation of this emerging isolate. Author summary Three subspecies of Treponema pallidum (pallidum, pertenue, and endemicum) are increasingly showing overlap in terms of transmission and clinical manifestations. We recently identified a novel tp0548 genotype in the X-4 isolate, which was obtained from an adult male with genital lesions. The novel genotype contained a unique nucleotide substitution G->T at position 167 and belonged to the SS14-like clade of TPA strains, as determined by phylogenetic analysis. We conducted an in-depth exploration of the genome of the X-4 isolate using the pooled segment genome sequencing method followed by Illumina sequencing. Multi-locus sequence analysis of nine chromosomal loci demonstrated that the X-4 isolate was clustered within a monophyletic group of TPA strains, which identified the isolate as a TPA strain. Whole-genome phylogenetic analysis subsequently corroborated the TPA strain classification of the X-4 isolate and revealed that the isolate was very closely related to the SS14 strain, with 42 single nucleotide variations and 12 insertions/deletions. In addition, characterization of the intrastrain heterogeneity in the lengths of homopolymeric tracts in the X-4 isolate showed that the heterogeneity of the poly G/C tracts was greater than that of the poly A/T tracts, and high poly G/C tract diversity was observed in the TPAChi_0347 locus.


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The oft-stated belief was that these three subspecies could be distinguished based on 23 their mode of transmission, clinical manifestations and host specificity, but this 24 probably appears to not be the case. In a previous study, one Paris isolate 11q/j was 25 obtained from a syphilis-like primary genital lesion and was first reported as a syphilis 26 case containing a novel tp0548 sequence-type "j" by enhanced CDC (ECDC) typing 27 system [4]. However, the novel tp0548 genotype "j" was further found to be similar to 28 those of TPE strains, and the "q" RFLP pattern of the tpr subfamily II genes was also 29 consistent with that of most TPE strains. Hence, this 11q/j isolate was defined as an 30 imported case of yaws [5]. After extensive molecular locus analysis, the isolate was 31 finally clearly characterized as a TEN isolate that contained two recombination events 32 in the tp0548 and tp0488 loci, resulting in sequences similar to those of TPE and TPA  Whole-genome sequencing and de novo assembly of the X-4 genome 72 The genome of the X-4 isolate was determined using the pooled segment genome 73 sequencing (PSGS) method as reported previously [15,16]. Library construction and 74 sequencing were performed by Beijing Novogene Bioinformatics Technology Co., Ltd., 75 with an Illumina HiSeq 1500 platform in the paired-end mode. Prior to genome 76 assembly, quality control evaluation on the raw sequencing data using the Trimmomatic 77 tool was performed. The Illumina sequencing reads corresponding to individual pools 78 were handled separately and de novo assembled using SOAP de novo. The resulting 79 contigs from the X-4 isolate were aligned to sequences from the T. pallidum subsp.      Table. 140

Evaluation of intrastrain heterogeneous G/C and A/T regions in DNA
We separately aligned the resulting contigs to the Nichols genome, and 13 genome gaps  published: two were defined as type "r" and "s" by Grillová el al. [11], and one was 153 defined as type "y" by Kumar et al. [23]. For consistency with the already extensive list 154 of tp0548 genotype sequences (a-ak), we redefined types "r", "s" and "y" as "al", "am" 155 and "an" and added the new tp0548 sequence type of the X-4 isolate to the updated list 156 as type "ao" (Fig. 1). Phylogenetic analysis of all tp0548 genotypes divided the 157 treponemas into three clades (S2 Fig) and the novel genotype "ao" was found to belong 158 to the SS14-like clade of TPA strains.

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Because of the novel tp0548 genotype, the ECDC subtype (13d/ao) of the isolate was  Analysis of the X-4 isolate at the whole-genome scale A whole-genome phylogenetic 176 analysis of the X-4 isolate and six TPA reference strains available at GenBank was 177 performed and the results showed that the seven TPA strains were grouped into three 178 clades, and among these clades, the X-4 isolate was segregated in a separate branch and 179 closely related to the SS14 strain (Fig. 3). 180 We subsequently analyzed the genome of the X-4 isolate by referring to the SS14  Table).  sequences from the assembled contigs of X-4 and conducted a phylogenetic analysis.

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The results were consistent with the classification of the X-4 isolate as a TPA strain.

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Moreover, whole-genome-based phylogenetic analysis indicated that the X-4 isolate 239 and SS14 strain might have originated from the same ancestor, which further confirmed 240 that the X-4 isolate was a TPA strain.

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Comparative genomic results showed that the X-4 isolate had a 9-nt-long repetitive sequence pattern to that of the X-4 isolate (S3 Table).    The new genotype "ao" is shown in gray.