Clonal isolates of Treponema pallidum subsp. pallidum Nichols provide evidence for the occurrence of microevolution during experimental rabbit infection and in vitro culture

Abstract

The recent development of a system for long-term in vitro culture of the syphilis spirochete, Treponema pallidum subsp. pallidum, has introduced the possibility of detailed genetic analysis of this bacterium. In this study, the in vitro culture system was used to isolate and characterize clonal populations of T. pallidum subsp. pallidum Nichols, the most widely studied strain. In limiting dilutions experiments, it was possible to establish cultures with inocula as low as 0.5 T. pallidum per well despite the long generation time (~35 to 40 hours) of this organism. Six Nichols strain clones isolated by limiting dilution were characterized in detail. All clones exhibited indistinguishable morphology and motility, highly similar in vitro multiplication rates, and comparable infectivity in the rabbit model (ID50 ≤ 100 bacteria). Genomic sequencing revealed sequence heterogeneity in the form of insertions or deletions at 5 sites, single nucleotide variations at 20 sites, and polynucleotide (polyG/C) tract length differences at 22 locations. Genomic sequences of the uncloned Nichols strain preparations propagated in rabbits or in vitro cultures exhibited substantial heterogeneity at these locations, indicating coexistence of many varied ‘clonotypes’ within these populations. Nearly all genetic variations were specific for the Nichols strain and were not detected in the >280 T. pallidum genomic sequences that are currently available. We hypothesize that these Nichols strain-specific sequence variations arose independently either during human infection or within the 110 years since the strain’s initial isolation, and thus represent examples of microevolution and divergence.

In-Depth Proteome Coverage of In Vitro-Cultured Treponema pallidum and Quantitative Comparison Analyses with In Vivo-Grown Treponemes

“…pallidum Nichols strain revealed that in vivo-grown and in vitro-cultured T. pallidum genome sequences are highly similar and any observed sequence differences are unlikely to account for the 11 novel genome errors identified in the present in vitro study. Another limitation is that the Nichols strain used in our previous and present study was originally isolated in 1912 from the cerebrospinal fluid (CSF) of a secondary syphilis patient .…”

“…A limitation of these findings is the possibility that the remaining nine potential proteome errors identified in this study but not detected from in vivo-grown treponemes 25 are due to potential “artificial” conditions introduced during in vitro treponeme culture and/or possible post-translational modifications such as N-terminal proteolytic processing. However, genome sequencing of an in vitro-cultured T. pallidum Nichols strain revealed that in vivo-grown and in vitro-cultured T. pallidum genome sequences are highly similar 78 and any observed sequence differences are unlikely to account for the 11 novel genome errors identified in the present in vitro study. Another limitation is that the Nichols strain used in our previous 25 and present study was originally isolated in 1912 from the cerebrospinal fluid (CSF) of a secondary syphilis patient.…”

Treponema pallidum subsp. pallidum strains DAL-1 and Philadelphia 1 differ in generation times in vitro as well as during experimental rabbit infection

“…Genetic adaptation of TPA strains to rabbit infection during long-term propagations in rabbits is widely speculated, but there is no clear genetic evidence of this process. While Giacani et al [20] suggested the presence of such mutations by genetic comparisons of the Nichols strain to other Nicholslike strains and Edmondson et al [21] demonstrated the microevolution of the Nichols strain, Grillova ´et al [22] tested the DAL-1 strain for genome stability and adaptation in continual rabbit cultivation representing more than 100 TPA generations (i.e., 142 days) during which the allelic profile of DAL-1 remained stable. Moreover, the genetic adaptation to rabbits appears to be a relatively long process corresponding to the relatively low estimated mutation rates of pathogenic T. pallidum, where it took over a decade to fix a single mutation [23,24].…”

A Novel Treponema pallidum Subspecies pallidum Strain Associated With a Painful Oral Lesion Is a Member of a Potentially Emerging Nichols-Related Subgroup

“…Assemblies were then produced using MiniMap2 14 and the NYMC01 genomic sequence as template, followed by iterative refinements using refined templates to resolve discrepancies. 15 Sequence ambiguities with <80% nucleotide agreement at a given position were designated as N. The numbers of N's in the NYCM01, P-22-20198_RA, and R-22-10139_RA assemblies were 120, 180, and 213, respectively, with most of these being in the highly variable tprK gene.…”