Molecular strain typing of the yaws pathogen, Treponema pallidum subspecies pertenue

Abstract

Yaws is a neglected tropical disease caused by the bacterium Treponema pallidum subspecies pertenue. The disease primarily affects children under 15 years of age living in low socioeconomic conditions in tropical areas. As a result of a renewed focus on the disease owing to a recent eradication effort initiated by the World Health Organization, we have evaluated a typing method, adapted from and based on the enhanced Centers for Disease Control and Prevention typing method for T. pallidum subsp. pallidum, for possible use in epidemiological studies. Thirty DNA samples from yaws cases in Vanuatu and Ghana, 11 DNA samples extracted from laboratory strains, and 3 published genomic sequences were fully typed by PCR/RFLP analysis of the tpr E, G, and J genes and by determining the number of 60-bp repeats within the arp gene. Subtyping was performed by sequencing a homonucleotide "G" tandem repeat immediately upstream of the rpsA gene and an 84-bp region of tp0548. A total of 22 complete strain types were identified; two strain types in clinical samples from Vanuatu (5q11/ak and 5q12/ak), nine strain types in clinical samples from Ghana (3q12/ah, 4r12/ah, 4q10/j, 4q11/ah, 4q12/ah, 4q12/v, 4q13/ah, 6q10/aj, and 9q10/ai), and twelve strain types in laboratory strains and published genomes (2q11/ae, 3r12/ad, 4q11/ad, 4q12/ad, 4q12/ag, 4q12/v, 5r12/ad, 6r12/x, 6q11/af, 10q9/r, 10q12/r, and 12r12/w). The tpr RFLP patterns and arp repeat sizes were subsequently verified by sequencing analysis of the respective PCR amplicons. This study demonstrates that the typing method for subsp. pallidum can be applied to subsp. pertenue strains and should prove useful for molecular epidemiological studies on yaws.

Fig 1. RFLP patterns of T. pallidum subsp. pertenue.

(A) The tpr E, G, and J genes were amplified in a nested PCR to produce a mixed amplicon approximately 1830–1848 bp in length. Following PCR, amplicons were digested with the enzyme Mse I and analyzed on an Agilent Bioanalyzer. Two RFLP patterns (q and r) were observed among the clinical specimens and laboratory strains analyzed, including those for strains only partially typed (data not shown). The sizes of each fragment were confirmed through sequencing and are indicated in red. The Nichols TPA strain was used as a control for RFLP. The violet and green bands visible in each lane depict the Bioanalyzer kit’s internal control upper and lower bands, respectively. (B) Each of the tpr E, G, and J genes was amplified directly from the genome and sequenced. Regions of the products between the IP6 and IP7 primer annealing sequences were analyzed for Mse I restriction sites for both patterns. Approximate positions of each site (indicated as “M”) are shown. Sizes between each restriction site are listed in red.

Fig 2. Analysis of the arp 60-bp repeat region.

(A) A portion of the arp gene was amplified through PCR and run on an Agilent Bioanalyzer for each sample. Seven patterns containing 2, 3, 4, 5, 6, 9, and 10 repeats were identified among the samples tested in the lab, and an additional pattern (12 repeats) was seen following in silico analysis of one of the published genomes (not shown). The Nichols TPA strain, which contains 14 repeats, was used as a reference. The violet and green bands visible in each lane depict the Bioanalyzer kit’s internal control upper and lower bands, respectively. (B) Distribution of repeat sizes in clinical specimens from Vanuatu, Ghana clinical specimens, and among laboratory strains.

Fig 3. Alignment of the sequences using the variable region within tp0548.

Amplicons containing a region of the tp0548 locus were sequenced and analyzed for their genetic composition. The alignment compares the subtypes identified in this study to the Nichols TPA strain (pattern a). Thirteen subtypes, designated following nomenclature used for TPA strains and previously applied to TPE strains[12], were identified among the clinical specimens and laboratory strains, including those only partially strain typed.

Fig 4. Distribution of TPE strains.

Twenty-two strain TPE strain types were identified among the 44 specimens and genomic sequences analyzed. The overall distribution between each of the geographic locations is shown.