Genome-scale analysis of the non-cultivable Treponema pallidum reveals extensive within-patient genetic variation

Abstract

Insights into the genomic adaptive traits of Treponema pallidum, the causative bacterium of syphilis, have long been hampered due to the absence of in vitro culture models and the constraints associated with its propagation in rabbits. Here, we have bypassed the culture bottleneck by means of a targeted strategy never applied to uncultivable bacterial human pathogens to directly capture whole-genome T. pallidum data in the context of human infection. This strategy has unveiled a scenario of discreet T. pallidum interstrain single-nucleotide-polymorphism-based microevolution, contrasting with a rampant within-patient genetic heterogeneity mainly targeting multiple phase-variable loci and a major antigen-coding gene (tprK). TprK demonstrated remarkable variability and redundancy, intra- and interpatient, suggesting ongoing parallel adaptive diversification during human infection. Some bacterial functions (for example, flagella- and chemotaxis-associated) were systematically targeted by both inter- and intrastrain single nucleotide polymorphisms, as well as by ongoing within-patient phase variation events. Finally, patient-derived genomes possess mutations targeting a penicillin-binding protein coding gene (mrcA) that had never been reported, unveiling it as a candidate target to investigate the impact on the susceptibility to penicillin. Our findings decode the major genetic mechanisms by which T. pallidum promotes immune evasion and survival, and demonstrate the exceptional power of characterizing evolving pathogen subpopulations during human infection.

Syphilis

“…However, research on virulence determinants of T. pallidum , and our understanding of protective immunity against it, have been hindered by our inability to culture the bacteria in vitro. To overcome this limitation, genome sequencing of T. pallidum directly from clinical samples is now possible 92,238 This advance should enable understanding of strain variation on a global scale, and help to identify outer membrane proteins and other surface antigens as possible vaccine candidates 81 . A recent study showed that immunization of rabbits with the lipoprotein TP071 prevented dissemination of T. pallidum and, hence, has become a promising vaccine candidate 81 .…”

“…Of these, TprK (TP0897) has received the greatest attention because of its presumed role in immune evasion by the spirochete 87,88 ; it has been shown to undergo antigenic variation in seven regions believed to be extracellular loops harbouring B-cell epitopes 89–92 . DNA sequence cassettes that correspond to V-region sequences in an area of the T. pallidum chromosome located away from the tprK gene have been proposed to serve as unidirectional donor sites for the generation of variable regions by nonreciprocal gene conversion 89 .…”

“…The picture emerging from our evolving concepts of the spirochete’s molecular architecture is multi-factorial and likely involves copious production of antibodies against subsurface lipoprotein ‘decoys’ 57,110 ; poor target availability owing to low copy numbers of outer membrane proteins and surface-exposed lipoproteins 67,77,82,84,93 ; in the case of bipartite outer membrane proteins, limited production of antibodies against surface-exposed epitopes along with skewed production of antibodies against periplasmic domain 84,93 ; organism-to-organism variation in the levels of expression of outer membrane proteins and outer surface lipoproteins through a variety of mechanisms, including phase variation 82,92,115,116 ; and, in the case of TprK, antigenic variation as a result of intra-genomic recombination 89,92,117 . Additionally, the ability of motile spirochetes to ‘outrun’ infiltrating phagocytes and reach sequestered locations, including the epidermis, could be an under-appreciated aspect of immune evasion 10,102 .…”

Human Treponema pallidum 11q/j isolate belongs to subsp. endemicum but contains two loci with a sequence in TP0548 and TP0488 similar to subsp. pertenue and subsp. pallidum, respectively

“…The small amount of treponemal DNA within the only available sample of the 11q/j isolate (copy number less than 10 molecules of treponemal DNA per 1 μl) with an excessive amount of contaminating human DNA, which exceeded the treponemal DNA by at least 100,000 times, precluded the use of other techniques that have been recently reported to be effective in sequencing treponemal DNA directly from clinical samples [34, 35]. Efficient enrichment of treponemal DNA requires the number of treponemal copies > 1x10 4 per 1 μl [34].…”

“…Efficient enrichment of treponemal DNA requires the number of treponemal copies > 1x10 4 per 1 μl [34]. In fact, enrichment of the TEN Iraq B DNA sample, containing 10 4 copies per 1 μl, revealed genome coverage less than 12.4% [35].…”