Selective Whole-Genome Amplification as a Tool to Enrich Specimens with Low Treponema pallidum Genomic DNA Copies for Whole-Genome Sequencing

Thurlow, Charles M.; Joseph, Sandeep J.; Ganova-Raeva, Lilia; Katz, Samantha S.; Pereira, Lara; Chen, Cheng; Vilfort, Kendra; Workowski, Kimberly A.; Cohen, Stéphanie; Reno, Hilary; Sun, Yongcheng; Burroughs, Mark; Sheth, Mili; Chi, Kai-Hua; Danavall, Damien; Philip, Susan; Cao, Weiping; Kersh, Ellen N.; Pillay, Allan

doi:10.1128/msphere.00009-22

Cited by 19 publications

(13 citation statements)

References 44 publications

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“…Modern approaches that combine T. pallidum DNA enrichment with pathogen-specific probes (19,22), specific genome amplification before high-throughput sequencing and technologies capable of sequencing Kb-long DNA molecules will ensure the availability of complete high-quality genomes for comparative genomics analyses (23)(24)(25)(26)(27)(28). Deposition of reads and assembled genomes in public data repositories will enable more researchers to participate in vaccine development.…”

Section: Genetic Diversity and Vaccine Developmentmentioning

confidence: 99%

Notes on syphilis vaccine development

2022

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The quest for a syphilis vaccine to provide protection from infection or disease began not long after the isolation of the first Treponema pallidum subspecies pallidum (T. pallidum) strain in 1912. Yet, a practical and effective vaccine formulation continues to elude scientists. Over the last few years, however, efforts toward developing a syphilis vaccine have increased thanks to an improved understanding of the repertoire of T. pallidum outer membrane proteins (OMPs), which are the most likely syphilis vaccine candidates. More has been also learned about the molecular mechanisms behind pathogen persistence and immune evasion. Published vaccine formulations based on a subset of the pathogen’s OMPs have conferred only partial protection upon challenge of immunized laboratory animals, primarily rabbits. Nonetheless, those experiments have improved our approach to the choice of immunization regimens, adjuvants, and vaccine target selection, although significant knowledge gaps remain. Herein, we provide a brief overview on current technologies and approaches employed in syphilis vaccinology, and possible future directions to develop a vaccine that could be pivotal to future syphilis control and elimination initiatives.

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Section: Genetic Diversity and Vaccine Developmentmentioning

confidence: 99%

Notes on syphilis vaccine development

2022

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“…In recent years, efforts to catalog genomic diversity and phylodynamics of the syphilis spirochete, Treponema pallidum subsp. pallidum , have resulted in a rapid increase in the amount of sequencing data and number of near-complete genome assemblies available in public databases ( Pinto et al, 2016 ; Arora et al, 2017 ; Beale et al, 2019 , 2021 ; Grillová et al, 2019 ; Chen et al, 2021 ; Lieberman et al, 2021 ; Taouk et al, 2022 ; Thurlow et al, 2022 ). Insights gained from these efforts, including the spread of azithromycin resistance ( Beale et al, 2019 ) and high-resolution information on antigenic diversity ( Lieberman et al, 2021 ), have aided our understanding of T. pallidum evolution and are invaluable to vaccine design.…”

Section: Introductionmentioning

confidence: 99%

“…Insights gained from these efforts, including the spread of azithromycin resistance ( Beale et al, 2019 ) and high-resolution information on antigenic diversity ( Lieberman et al, 2021 ), have aided our understanding of T. pallidum evolution and are invaluable to vaccine design. Although whole genome sequencing of low abundance T. pallidum DNA directly from clinical specimens is technically challenging due to the necessity of enrichment protocols such as hybrid capture with RNA or DNA baits ( Pinto et al, 2016 ; Arora et al, 2017 ; Beale et al, 2019 , 2021 ; Lieberman et al, 2021 ; Taouk et al, 2022 ), Dpn1 enrichment ( Grillová et al, 2019 ), whole genome amplification ( Chen et al, 2021 ; Thurlow et al, 2022 ), and/or the traditional technique of passage of clinical strains through rabbits, sufficient progress has been made in development of these techniques that sequencing throughput of samples on a scale appropriate for monitoring of vaccine trials is feasible.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2022

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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.

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“…In recent years, efforts to catalog genomic diversity and phylodynamics of the syphilis spirochete, Treponema pallidum subsp. pallidum (T. pallidum), have resulted in a rapid increase in the amount of sequencing data and number of near-complete genome assemblies available in public databases (Arora et al, 2017;Beale et al, 2019Beale et al, , 2021Chen et al, 2021;Grillová et al, 2019;Lieberman et al, 2021;Pinto et al, 2016;Taouk et al, 2022;Thurlow et al, 2022). Insights gained from these efforts, including the spread of azithromycin resistance (Beale et al, 2019) and high-resolution information on antigenic diversity (Lieberman et al, 2021), have aided our understanding of T. pallidum evolution and are invaluable to vaccine design.…”

Section: Introductionmentioning

confidence: 99%

“…Insights gained from these efforts, including the spread of azithromycin resistance (Beale et al, 2019) and high-resolution information on antigenic diversity (Lieberman et al, 2021), have aided our understanding of T. pallidum evolution and are invaluable to vaccine design. Although whole genome sequencing of low abundance T. pallidum DNA directly from clinical specimens is technically challenging due to the necessity of enrichment protocols such as hybrid capture with RNA or DNA baits (Pinto et al, 2016;Arora et al, 2017;Beale et al, 2019Beale et al, , 2021Lieberman et al, 2021;Taouk et al, 2022), Dpn1 enrichment (Grillová et al, 2019), whole genome amplification (Chen et al, 2021;Thurlow et al, 2022), and/or the traditional technique of passage of clinical strains through rabbits, sufficient progress has been made in development of these techniques that sequencing throughput of samples on a scale appropriate for monitoring of vaccine trials is feasible. Most genomic analyses of T. pallidum have excluded portions of the genome difficult to resolve by short-read sequencing, including the T. pallidum repeat (tpr) family of paralogous genes, the number of 60 bp tandem near-perfect repeats in the gene encoding the acidic repeat protein (arp; tp0433), and the number of 24 bp tandem repeats in the gene encoding the tetratricopeptide repeat protein TP0470 (tp0470).…”

Section: Introductionmentioning

confidence: 99%

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

Lieberman

Armstrong

Chung

et al. 2022

Preprint

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Sequencing of most Treponema pallidum (T. 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 modelling 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.

show abstract

Selective Whole-Genome Amplification as a Tool to Enrich Specimens with Low Treponema pallidum Genomic DNA Copies for Whole-Genome Sequencing

Cited by 19 publications

References 44 publications

Notes on syphilis vaccine development

Notes on syphilis vaccine development

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

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

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