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In Vitro Transformation and Selection of Treponema pallidum subsp. pallidum.

ABSTRACT: Although the isolation of Treponema pallidum subsp. pallidum (T. pallidum) from a syphilis patient dates to 1912, for the duration of the 20^th century, this pathogen has remained an exceedingly difficult organism to study due to the lack of a system to support its viability in vitro. This limitation, in turn, has precluded the application of genetic engineering techniques via transformation and subsequent selection of T. pallidum transformants. A recently described method for in vitro cultivation of T. pallidum, however, has made it possible for us to experiment with transformation and selection methods. Here we describe the approach that we adopted to successfully transform T. pallidum with foreign DNA and select the resulting recombinant strain using kanamycin. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Transformation of T. pallidum Support Protocol 1: Quantification of T. pallidum in suspensions using dark-field microscopy Support Protocol 2: Counting cells using a hemacytometer Basic Protocol 2: Selection, initial passaging, and expansion of transformed cultures Basic Protocol 3: Isolation of a clonal strain through limiting dilution.

SUBMITTER: Phan A

PROVIDER: S-EPMC9389596 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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In Vitro Transformation and Selection of Treponema pallidum subsp. pallidum.

Phan Amber A Romeis Emily E Tantalo Lauren L Giacani Lorenzo L

Current protocols 20220801 8

Although the isolation of Treponema pallidum subsp. pallidum (T. pallidum) from a syphilis patient dates to 1912, for the duration of the 20<sup>th</sup> century, this pathogen has remained an exceedingly difficult organism to study due to the lack of a system to support its viability in vitro. This limitation, in turn, has precluded the application of genetic engineering techniques via transformation and subsequent selection of T. pallidum transformants. A recently described method for in vitro ...[more]

PMID: 35976045

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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.

Project description:In the rabbit model of syphilis, infection phenotypes associated with the Nichols and Chicago strains of Treponema pallidum (T. pallidum), though similar, are not identical. Between these strains, significant differences are found in expression of, and antibody responses to some candidate virulence factors, suggesting the existence of functional genetic differences between isolates. The Chicago strain genome was therefore sequenced and compared to the Nichols genome, available since 1998. Initial comparative analysis suggested the presence of 44 single nucleotide polymorphisms (SNPs), 103 small (?3 nucleotides) indels, and 1 large (1204 bp) insertion in the Chicago genome with respect to the Nichols genome. To confirm the above findings, Sanger sequencing was performed on most loci carrying differences using DNA from Chicago and the Nichols strain used in the original T. pallidum genome project. A majority of the previously identified differences were found to be due to errors in the published Nichols genome, while the accuracy of the Chicago genome was confirmed. However, 20 SNPs were confirmed between the two genomes, and 16 (80.0%) were found in coding regions, with all being of non-synonymous nature, strongly indicating action of positive selection. Sequencing of 16 genomic loci harboring SNPs in 12 additional T. pallidum strains, (SS14, Bal 3, Bal 7, Bal 9, Sea 81-3, Sea 81-8, Sea 86-1, Sea 87-1, Mexico A, UW231B, UW236B, and UW249C), was used to identify "Chicago-" or "Nichols -specific" differences. All but one of the 16 SNPs were "Nichols-specific", with Chicago having identical sequences at these positions to almost all of the additional strains examined. These mutations could reflect differential adaptation of the Nichols strain to the rabbit host or pathoadaptive mutations acquired during human infection. Our findings indicate that SNPs among T. pallidum strains emerge under positive selection and, therefore, are likely to be functional in nature.

Project description:Immune evasion and disease progression of Treponema pallidum subsp. pallidum are associated with sequence diversity in the hypervariable outer membrane protein TprK. Previous attempts to study variation within TprK have sequenced at depths insufficient to fully appreciate the hypervariable nature of the protein, failed to establish linkage between the protein's seven variable regions, or were conducted on isolates passed through rabbits. As a consequence, a complete profile of tprK during infection in the human host is still lacking. Furthermore, prior studies examining how T. pallidum subsp. pallidum uses its repertoire of genomic donor sites to generate diversity within the variable regions of the tprK have yielded a partial understanding of this process due to the limited number of tprK alleles examined. In this study, we used short- and long-read deep sequencing to directly characterize full-length tprK alleles from T. pallidum subsp. pallidum collected from early lesions of patients attending two sexually transmitted infection clinics in Italy. We demonstrate that strains collected from cases of secondary syphilis contain significantly more unique variable region sequences and full-length TprK sequences than those from cases of primary syphilis. Our data, combined with recent data available on Chinese T. pallidum subsp. pallidum specimens, show the near-complete absence of overlap in TprK sequences among the 41 specimens profiled to date. We further estimate that the potential antigenic variability carried by TprK rivals that of current estimates of the human adaptive immune system. These data underscore the immunoevasive ability of TprK that allows T. pallidum subsp. pallidum to establish lifelong infection.IMPORTANCE Syphilis continues to be a significant public health issue in both low- and high-income countries, including the United States where the rate of syphilis infection has increased over the past 5 years. Treponema pallidum subsp. pallidum, the causative agent of syphilis, carries the outer membrane protein TprK that undergoes segmental gene conversion to constantly create new sequences. We performed full-length deep sequencing of TprK to examine TprK diversity in clinical T. pallidum subsp. pallidum strains. We then combined our results with data from all samples for which TprK deep sequencing results were available. We found almost no overlap in TprK sequences between different patients. Moreover, our data allowed us to estimate the total number of TprK variants that T. pallidum subsp. pallidum can potentially generate. Our results support how the T. pallidum subsp. pallidum TprK antigenic variation system is an equal adversary of the human immune system leading to pathogen persistence in the host.

Dataset Information

In Vitro Transformation and Selection of Treponema pallidum subsp. pallidum.

Publications

In Vitro Transformation and Selection of Treponema pallidum subsp. pallidum.

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