Mutagenesis of Treponema pallidum strain Madras with DAL-1 sequences reveals single-nucleotide mutations associated with accelerated competitive outgrowth

Although Treponema pallidum subsp. pallidum ( T. pallidum ), the causative agent of syphilis, is considered a monomorphic bacterium, in vitro growth differences have been found between individual strains, such as DAL-1 and Madras, which genomes differ by only 43 genetic variants (28 single nucleotide variants and 15 indels). In this study, we compared the in vivo growth characteristics of these two strains during serial co-infections of experimental rabbits. Over the 74-day experiment, DAL-1 multiplied faster than the Madras strain, with a median relative advantage of 1.045 generations per week. Subsequently, 30 sequence variants were selected for in vitro mutagenesis of the Madras strain. Two recombinants carrying DAL-1-like alleles in TP0233 (G13D; G242467A) and TP0720 (S123N; C789720T) were obtained; both outcompeted the parental Madras strain and became fixed in the in vitro cultures. In vitro quantification showed a similar acceleration level (approx. 0.5 generations per week) and relative growth coefficients ( r = 0.070 and 0.079, respectively) of both recombinant strains. This study supports a genetic contribution to strain-specific growth-rate differences in T. pallidum , which may underlie the clinical heterogeneity observed in syphilis patients.