Treponema pallidum subsp. pallidum with an Artificially impaired TprK antigenic variation system is attenuated in the Rabbit model of syphilis

Abstract

Background The TprK protein of the syphilis agent, Treponema pallidum subsp. pallidum (T. pallidum), undergoes antigenic variation in seven discrete variable (V) regions via non-reciprocal segmental gene conversion. These recombination events transfer information from a repertoire of 53 silent chromosomal donor cassettes (DCs) into the single tprK expression site to continually generate TprK variants. Several lines of research developed over the last two decades support the theory that this mechanism is central to T. pallidum’s ability for immune avoidance and persistence in the host. Structural and modeling data, for example, identify TprK as an integral outer membrane porin with the V regions exposed on the pathogen’s surface. Furthermore, infection-induced antibodies preferentially target the V regions rather than the predicted β-barrel scaffolding, and sequence variation abrogates the binding of antibodies elicited by antigenically different V regions. Here, we engineered a T. pallidum strain to impair its ability to vary TprK and assessed its virulence in the rabbit model of syphilis. Principal findings A suicide vector was transformed into the wild-type (WT) SS14 T. pallidum isolate to eliminate 96% of its tprK DCs. The resulting SS14-DCKO strain exhibited an in vitro growth rate identical to the untransformed strain, supporting that the elimination of the DCs did not affect strain viability in absence of immune pressure. In rabbits injected intradermally with the SS14-DCKO strain, generation of new TprK sequences was impaired, and the animals developed attenuated lesions with a significantly reduced treponemal burden compared to control animals. During infection, clearance of V region variants originally in the inoculum mirrored the generation of antibodies to these variants, although no new variants were generated in the SS14-DCKO strain to overcome immune pressure. Naïve rabbits that received lymph node extracts from animals infected with the SS14-DCKO strain remained uninfected. Conclusion These data further support the critical role of TprK in T. pallidum virulence and persistence during infection.

Genomic Epidemiology of Treponema pallidum and Circulation of Strains With Diminished tprK Antigen Variation Capability in Seattle, 2021–2022

“…Owing to the potential confounding effects of lineage and stage on the analysis, we compared the total V region diversity (number of unique V regions recovered) from deletion strains only to the diversity in other Nichols secondary syphilis samples. Strains harboring the tprK donor site deletion had significantly reduced diversity ( P < .05; Welch's t test) ( Figure 5 A ), consistent with the phenotype seen in rabbits infected with T. pallidum with t prK donor sites knocked out [ 11 ].…”

“…There were no differences in the T. pallidum genome sequence between the 3 samples, strongly suggestive of transmission of a single deletion-bearing strain rather than independent deletion events. Although experimental elimination of 96% of donor sites results in reduced tprK diversity, severe attenuation of early lesions, and diminished ability to persist in the rabbit host [ 11 ], ablation of only 9 donor sites did not appear to prevent lesion appearance or prevent transmission in humans as these 3 patients had clinically evident secondary syphilis.…”

“…The V1 variable region is enriched in variant sequences comprising a complete donor site sequence, and our best interpretation is that this V1 sequence was likely present when the tprK donor site locus deletion occurred and had been recombined in only 1 of the 4 specimens [ 9 ]. Certainly, more work is required to understand the mechanism of variable region creation both in vitro and in vivo [ 10 , 11 , 33 ].…”

“…An additional, and likely more important, immune evasion strategy used by T. pallidum involves continuous antigenic variation of 7 predicted surface-exposed variable (V) loop regions of the putative TprK outer membrane porin by nonreciprocal recombination of DNA sequences from donor sites flanking the tprD ( tp0131 ) locus [ 6–10 ]. Recent studies in rabbits using a T. pallidum strain genetically modified to ablate 96% of donor sites demonstrated that the inability to vary the antigen composition of TprK leads to reduced V region diversity, attenuated disease manifestations, and decreased ability of T. pallidum to persist in the host, highlighting the importance of this virulence factor in pathogen persistence [ 11 ].…”

Neurosyphilis: insights into its pathogenesis, susceptibility, diagnosis, treatment, and prevention

“…The latter view seems more reliable due to the normal or only slightly increased quotient (Q alb ) level in neurosyphilis patients in clinical practice ( 40 ). In addition to interacting with macrophages and endothelial cells, the biochemical system, antigenic variation system, and matrix metalloproteinases (MMPs)/tissue inhibitors of metalloproteinases (TIMPs) imbalance are involved in the pathogenesis of T. pallidum ( 41 – 43 ).…”