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Multi-locus sequence typing of Treponema pallidum subsp. pallidum present in clinical samples from France: Infecting treponemes are genetically diverse and belong to 18 allelic profiles.

ABSTRACT: Treponema pallidum subsp. pallidum, the causative agent of sexually transmitted syphilis, detected in clinical samples from France, was subjected to molecular typing using the recently developed Multilocus Sequence Typing system. The samples (n = 133) used in this study were collected from 2010-2016 from patients with diagnosed primary or secondary syphilis attending outpatient centers or hospitals in several locations in France. Altogether, 18 different allelic profiles were found among the fully typed samples (n = 112). There were five allelic variants identified for TP0136, 12 for TP0548, and eight for TP0705. Out of the identified alleles, one, seven, and three novel alleles were identified in TP0136, TP0548, and TP0705, respectively. Partial allelic profiles were obtained from 6 samples. The majority of samples (n = 110) belonged to the SS14-like cluster of TPA isolates while 7 clustered with Nichols-like isolates. Patients infected with Nichols-like samples were more often older (p = 0.041) and more often diagnosed with secondary syphilis (p = 0.033) compared to patients infected with SS14-like samples. In addition, macrolide resistance caused by the A2058G mutation was found to be associated with allelic profile 1.3.1 or with strains belonging to the 1.3.1 lineage (p<0.001). The genetic diversity among TPA strains infecting the European population was surprisingly high, which suggests that additional studies are needed to reveal the full genetic diversity of TPA pathogens infecting humans.

SUBMITTER: Pospisilova P

PROVIDER: S-EPMC6053231 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Multi-locus sequence typing of Treponema pallidum subsp. pallidum present in clinical samples from France: Infecting treponemes are genetically diverse and belong to 18 allelic profiles.

Pospíšilová Petra P Grange Philippe Alain PA Grillová Linda L Mikalová Lenka L Martinet Pervenche P Janier Michel M Vermersch Annie A Benhaddou Nadjet N Del Giudice Pascal P Alcaraz Isabelle I Truchetet François F Dupin Nicolas N Šmajs David D

PloS one 20180719 7

Treponema pallidum subsp. pallidum, the causative agent of sexually transmitted syphilis, detected in clinical samples from France, was subjected to molecular typing using the recently developed Multilocus Sequence Typing system. The samples (n = 133) used in this study were collected from 2010-2016 from patients with diagnosed primary or secondary syphilis attending outpatient centers or hospitals in several locations in France. Altogether, 18 different allelic profiles were found among the ful ...[more]

PMID: 30024965

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Project description:Many non-human primate species in sub-Saharan Africa are infected with Treponema pallidum subsp. pertenue, the bacterium causing yaws in humans. In humans, yaws is often characterized by lesions of the extremities and face, while T. pallidum subsp. pallidum causes venereal syphilis and is typically characterized by primary lesions on the genital, anal or oral mucosae. It remains unclear whether other T. pallidum subspecies found in humans also occur in non-human primates and how the genomic diversity of non-human primate T. pallidum subsp. pertenue lineages is distributed across hosts and space. We observed orofacial and genital lesions in sooty mangabeys (Cercocebus atys) in Taï National Park, Côte d'Ivoire and collected swabs and biopsies from symptomatic animals. We also collected non-human primate bones from 8 species in Taï National Park and 16 species from 11 other sites across sub-Saharan Africa. Samples were screened for T. pallidum DNA using polymerase chain reactions (PCRs) and we used in-solution hybridization capture to sequence T. pallidum genomes. We generated three nearly complete T. pallidum genomes from biopsies and swabs and detected treponemal DNA in bones of six non-human primate species in five countries, allowing us to reconstruct three partial genomes. Phylogenomic analyses revealed that both orofacial and genital lesions in sooty mangabeys from Taï National Park were caused by T. pallidum subsp. pertenue. We showed that T. pallidum subsp. pertenue has infected non-human primates in Taï National Park for at least 28 years and has been present in two non-human primate species that had not been described as T. pallidum subsp. pertenue hosts in this ecosystem, western chimpanzees (Pan troglodytes verus) and western red colobus (Piliocolobus badius), complementing clinical evidence that started accumulating in Taï National Park in 2014. More broadly, simian T. pallidum subsp. pertenue strains did not form monophyletic clades based on host species or the symptoms caused, but rather clustered based on geography. Geographical clustering of T. pallidum subsp. pertenue genomes might be compatible with cross-species transmission of T. pallidum subsp. pertenue within ecosystems or environmental exposure, leading to the acquisition of closely related strains. Finally, we found no evidence for mutations that confer antimicrobial resistance.

Project description:Despite more than a century of research, genetic manipulation of Treponema pallidum subsp. pallidum (T. pallidum), the causative agent of syphilis, has not been successful. The lack of genetic engineering tools has severely limited understanding of the mechanisms behind T. pallidum success as a pathogen. A recently described method for in vitro cultivation of T. pallidum, however, has made it possible to experiment with transformation and selection protocols in this pathogen. Here, we describe an approach that successfully replaced the tprA (tp0009) pseudogene in the SS14 T. pallidum strain with a kanamycin resistance (kanR) cassette. A suicide vector was constructed using the pUC57 plasmid backbone. In the vector, the kanR gene was cloned downstream of the tp0574 gene promoter. The tp0574prom-kanR cassette was then placed between two 1-kbp homology arms identical to the sequences upstream and downstream of the tprA pseudogene. To induce homologous recombination and integration of the kanR cassette into the T. pallidum chromosome, in vitro-cultured SS14 strain spirochetes were exposed to the engineered vector in a CaCl2-based transformation buffer and let recover for 24 hours before adding kanamycin-containing selective media. Integration of the kanR cassette was demonstrated by qualitative PCR, droplet digital PCR (ddPCR), and whole-genome sequencing (WGS) of transformed treponemes propagated in vitro and/or in vivo. ddPCR analysis of RNA and mass spectrometry confirmed expression of the kanR message and protein in treponemes propagated in vitro. Moreover, tprA knockout (tprAko-SS14) treponemes grew in kanamycin concentrations that were 64 times higher than the MIC for the wild-type SS14 (wt-SS14) strain and in infected rabbits treated with kanamycin. We demonstrated that genetic manipulation of T. pallidum is attainable. This discovery will allow the application of functional genetics techniques to study syphilis pathogenesis and improve syphilis vaccine development.

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Dataset Information

Multi-locus sequence typing of Treponema pallidum subsp. pallidum present in clinical samples from France: Infecting treponemes are genetically diverse and belong to 18 allelic profiles.

Publications

Multi-locus sequence typing of Treponema pallidum subsp. pallidum present in clinical samples from France: Infecting treponemes are genetically diverse and belong to 18 allelic profiles.

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