High-throughput nanopore sequencing of Treponema pallidum tandem repeat genes arp and tp0470 reveals clade-specific patterns and recapitulates global whole genome phylogeny

Abstract

Sequencing of most Treponema pallidum genomes excludes repeat regions in tp0470 and the tp0433 gene, encoding the acidic repeat protein (arp). As a first step to understanding the evolution and function of these genes and the proteins they encode, we developed a protocol to nanopore sequence tp0470 and arp genes from 212 clinical samples collected from ten countries on six continents. Both tp0470 and arp repeat structures recapitulate the whole genome phylogeny, with subclade-specific patterns emerging. The number of tp0470 repeats is on average appears to be higher in Nichols-like clade strains than in SS14-like clade strains. Consistent with previous studies, we found that 14-repeat arp sequences predominate across both major clades, but the combination and order of repeat type varies among subclades, with many arp sequence variants limited to a single subclade. Although strains that were closely related by whole genome sequencing frequently had the same arp repeat length, this was not always the case. Structural modeling of TP0470 suggested that the eight residue repeats form an extended α-helix, predicted to be periplasmic. Modeling of the ARP revealed a C-terminal sporulation-related repeat (SPOR) domain, predicted to bind denuded peptidoglycan, with repeat regions possibly incorporated into a highly charged β-sheet. Outside of the repeats, all TP0470 and ARP amino acid sequences were identical. Together, our data, along with functional considerations, suggests that both TP0470 and ARP proteins may be involved in T. pallidum cell envelope remodeling and homeostasis, with their highly plastic repeat regions playing as-yet-undetermined roles.

Clinical and genomic diversity ofTreponema pallidum subsp. pallidum:A global, multi-center study of early syphilis to inform vaccine research

“…Second, we excluded several recombinant loci demonstrated to be under positive and purifying selection from our analysis; 29 novel methods for resolving these loci are greatly needed. 30 Finally, our TPA genomic analysis focused on lineageinformative variants and does not include OMP mutations subject to recombination, which are relevant to vaccine design (e.g., TPA repeat proteins). Additional investigations should be conducted to explore the full array of TPA protein variants and their impacts on vaccine efficacy and TPA biology.…”

In Vitro Isolation of Treponema pallidum subsp. pallidum From Fresh and Frozen Needle Aspirates of Primary Experimental Syphilis Lesions

“…The UW211B strain was therefore selected to circumvent the possibility that the outcome of our experiments could be due to using a strain that is considered "adapted" to the rabbit milieu. Further development of the in vitro isolation protocol, however, along with newly available protocols to perform whole genome sequencing directly from patient samples 17,19 will allow simultaneous sequencing of the strain from the original patient sample, as well as the possibility to evaluate whether in vitro isolation selected for adaptive mutations. Overall, however, we believe that treponemes aspirated from a patient lesion will easily adjust and proliferate in the in vitro environment well enough to attain isolates, and that the successful outcome of the isolation procedure will mainly depend on the initial cell inoculum size, rather than other factors, including the transfer from the human host milieu to the in vitro one containing rabbit cells.…”

“…The genome of the “parent” strain sequenced directly from the patient sample is not available to be compared with the one obtained after rabbit isolation. However, given that (a) the UW211B has been propagated in rabbits for a very limited time compared with other historical laboratory strains of T. pallidum , that (b) this pathogen is genetically monomorphic 17 and has a very slow mutation rate (ie, 1.78 × 10 −7 substitutions/site/year), 16,18 it is safe to assume that the relatively short exposure of this strain to the rabbit host milieu might not have changed its genetic background to make it more “rabbit adapted.” Considering these observations, the choice of the UW211B strain was preferred to that of strains (eg, Nichols), virtually propagated in rabbits since 1912 without solution of continuity, which could have led to such adaptive mutations. The UW211B strain was therefore selected to circumvent the possibility that the outcome of our experiments could be due to using a strain that is considered “adapted” to the rabbit milieu.…”

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

“…The gene TP0433 encodes the Acidic Repeat Protein (Arp), which contains a region of 60 nt direct repeats that translate to 20 amino acid sequences [39]. In the NC_021490.2 reference sequence, there are 14 such repeats, along with a 9 nt partial repeat.…”