Development of a Multilocus Sequence Typing (MLST) scheme for Treponema pallidum subsp. pertenue: Application to yaws in Lihir Island, Papua New Guinea

Abstract

Background: Yaws is a neglected tropical disease, caused by Treponema pallidum subsp. pertenue. The disease causes chronic lesions, primarily in young children living in remote villages in tropical climates. As part of a global yaws eradication campaign initiated by the World Health Organization, we sought to develop and evaluate a molecular typing method to distinguish different strains of T. pallidum subsp. pertenue for disease control and epidemiological purposes.

Methods and principal findings: Published genome sequences of strains of T. pallidum subsp. pertenue and pallidum were compared to identify polymorphic genetic loci among the strains. DNA from a number of existing historical Treponema isolates, as well as a subset of samples from yaws patients collected in Lihir Island, Papua New Guinea, were analyzed using these targets. From these data, three genes (tp0548, tp0136 and tp0326) were ultimately selected to give a high discriminating capability among the T. pallidum subsp. pertenue samples tested. Intragenic regions of these three target genes were then selected to enhance the discriminating capability of the typing scheme using short readily amplifiable loci. This 3-gene multilocus sequence typing (MLST) method was applied to existing historical human yaws strains, the Fribourg-Blanc simian isolate, and DNA from 194 lesion swabs from yaws patients on Lihir Island, Papua New Guinea. Among all samples tested, fourteen molecular types were identified, seven of which were found in patient samples and seven among historical isolates or DNA. Three types (JG8, TD6, and SE7) were predominant on Lihir Island.

Conclusions: This MLST approach allows molecular typing and differentiation of yaws strains. This method could be a useful tool to complement epidemiological studies in regions where T. pallidum subsp. pertenue is prevalent with the overall goals of improving our understanding of yaws transmission dynamics and helping the yaws eradication campaign to succeed.

Fig 1. Sequence alignment of Tp0548 types A through X.

Sequence alignment is for different strains of T. pallidum subsp. pallidum and T. pallidum subsp. pertenue. The coordinates of nucleotides 130–212 shown above the alignment are based on the Nichols strain genome (AE 000520.1) as indicated by **. Published reference sequences for each tp0548 type are as follows: Types A-I:[11]; Type J: [36]; Type K: [17]; Type L: [37]; Type M-N:[38]; Type O:[33]; Type P: [34] Type Q: [35]; Type R,V,W: [5]; Type S-T and X: this work; Type U: [7] Type Q (indicated by *) was originally incorrectly published as Type O; it was renamed in this manuscript.

Fig 2. Phylogenetic relationships of the tp0548 types.

tp0548 types are shown for T.p. pallidum, T.p. pertenue, and Fribourg Blanc isolates/strains and for PNG samples, as shown in Fig 1. Sequences were first aligned using the Muscle algorithm, using default parameters. The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree is shown, with branch lengths equivalent to the evolutionary distance as indicated by the scale. Evolutionary distance was measured using the number of differences per sequence, with pairwise deletion of gaps. The percentage of replicate trees in which the associated molecular types clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Analyses were conducted in MEGA version 7.0 [32].

Fig 3. Sequence alignment of Tp0136 types A through G from T. pallidum subsp. pertenue isolates and Papua New Guinea samples.

The coordinates in the alignment between nucleotides 223 and 675 in T. pallidum subsp. pertenue strains and Papua Guinea samples are in reference to strain CDC2 in GenBank (Accession No. CP002375.1) as indicated by **. A: Fribourg-Blanc, CDC2; B: Samoa D, Samoa F; C: Gauthier, F: CDC1, Ghana051; D, E, and G: Papua New Guinea samples.

Fig 4. Sequence alignment of tp0136 type G with Treponema paraluiscuniculi A.

The very unusual sequence (Type G) found in tp0136 from the majority of PNG samples was more closely aligned with the sequence from T. paraluiscuniculi than with the other T.p. pertenue strains (types A-F in Fig 3). The coordinates in the alignment are in reference to T. paraluiscuniculi A strain (Accession No. CP002103) as indicated by **.

Fig 5. Phylogenetic relationships of the tp0136 typing region.

tp0136 types are shown for T.p. pertenue and Fribourg Blanc isolates and PNG samples; typing designations are as described in Fig 3. Sequences were first aligned using the Muscle algorithm, using default parameters. The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree is shown, with branch lengths equivalent to the evolutionary distance as indicated by the scale. Evolutionary distance was measured using the number of differences per sequence, with pairwise deletion of gaps. The percentage of replicate trees in which the associated molecular types clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Analyses were conducted in MEGA version 7.0 [32].

Fig 6. Sequence alignment of tp0326 typing region.

Sequence alignment of tp0326 types from T. pallidum subsp. pertenue isolates and Papua New Guinea samples. Nucleotide numbering between 2031and 2342 in the alignment refers to coordinates in strain CDC2 in GenBank (Accession No. CP002375.1) as indicated by **. The sequences between coordinates 2117 and 2297 are conserved in all strains examined. 1: Samoa D, Samoa F; 2: Gauthier; 3: Ghana051, CDC1; 4: CDC2; 5: Fribourg-Blanc; 6–8: Papua New Guinea samples.

Fig 7. Phylogenetic relationships of the tp0326 types.

tp0326 types are shown for T.p. pertenue and Fribourg Blanc isolates and PNG samples; typing designations are as described in Fig 6. Sequences were first aligned using the Muscle algorithm, using default parameters. The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree is shown, with branch lengths equivalent to the evolutionary distance as indicated by the scale. Evolutionary distance was measured using the number of differences per sequence, with pairwise deletion of gaps. The percentage of replicate trees in which the associated molecular types clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Analyses were conducted in MEGA version 7.0 [32].

Fig 8. T. pallidum subsp. pertenue types collected on Lihir Island, Papua New Guinea, from May 2013 and October 2016.

Data for the 194 fully typeable samples are included here, and proportions for each type are shown.

Fig 9. Phylogenetic analysis of Tp multilocus sequence types.

Multilocus Sequence Types (MLSTs) were defined by sequencing regions of three genes: tp0548, tp0136 and tp0326. Concatenated sequences were first aligned using the Muscle algorithm, using default parameters. The evolutionary history of the MLSTs was inferred using the Neighbor-Joining method. The optimal tree is shown, with branch lengths equivalent to the evolutionary distance as indicated by the scale. Evolutionary distance was measured using the number of differences per sequence, with pairwise deletion of gaps. The percentage of replicate trees in which the associated molecular types clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Analyses were conducted in MEGA version 7.0 [32].