scholarly journals Comparative Genomics and Full-Length TprK Profiling of Treponema pallidum subsp. pallidum Reinfection

2019 ◽  
Author(s):  
Amin Addetia ◽  
Lauren C. Tantalo ◽  
Michelle J. Lin ◽  
Hong Xie ◽  
Meei-Li Huang ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Causative Agent ◽  
Genetic Basis ◽  
Antigenic Variation ◽  
Treponema Pallidum ◽  
Variable Region ◽  
Full Length ◽  
Comparative Genomic ◽  
Definition Of

AbstractDeveloping a vaccine against Treponema pallidum subspecies pallidum, the causative agent of syphilis, remains a public health priority. Syphilis vaccine design efforts have been complicated by lack of an in vitro T. pallidum culture system, prolific antigenic variation in outer membrane protein TprK, and lack of functional annotation for nearly half of the genes. Understanding the genetic basis of T. pallidum reinfection can provide insights into variation among strains that escape cross-protective immunity. Here, we present comparative genomic sequencing and deep, full-length tprK profiling of two T. pallidum isolates from blood from the same patient that were collected six years apart. Notably, this patient was diagnosed with syphilis four times, with two of these episodes meeting the definition of neurosyphilis, during this interval. Outside of the highly variable tprK gene, we identified 14 coding changes in 13 genes. Nine of these genes putatively localized to the periplasmic or outer membrane spaces, consistent with a potential role in serological immunoevasion. Using a newly developed full-length tprK deep sequencing protocol, we profiled the diversity of this gene that far outpaces the rest of the genome. Intriguingly, we found that the reinfecting isolate demonstrated less diversity across each tprK variable region compared to the isolate from the first infection. Notably, the two isolates did not share any full-length TprK sequences. Our results are consistent with an immunodominant-evasion model in which the diversity of TprK explains the ability of T. pallidum to successfully reinfect individuals, even when they have been infected with the organism multiple times.Author SummaryThe causative agent of syphilis, Treponema pallidum subspecies pallidum, is capable of repeat infections in people, suggesting that the human immune response does not develop sufficiently broad or long-lasting immunity to cover treponemal diversity. Here, we examined the genomes from two blood-derived isolates of T. pallidum derived 6 years apart from a patient who had syphilis four times during the same period to understand the genetic basis of reinfection. We found a paucity of coding changes across the genome outside of the highly variable tprK gene. Using deep profiling of the full-length tprK gene, we found surprisingly that the two isolates did not share any full-length TprK sequences.

scholarly journals Prospective Characterization of Full-Length Hepatitis C Virus NS5A Quasispecies during Induction and Combination Antiviral Therapy

2000 ◽  
Vol 74 (19) ◽  
pp. 9028-9038 ◽  
Author(s):  
J.-B. Nousbaum ◽  
S. J. Polyak ◽  
S. C. Ray ◽  
D. G. Sullivan ◽  
A. M. Larson ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Antiviral Therapy ◽  
Variable Region ◽  
Response To Therapy ◽  
Full Length ◽  
Variable Regions ◽  
C Terminus

ABSTRACT The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein has been controversially implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy in clinical studies. In this study, the relationship between NS5A mutations and selection pressures before and during antiviral therapy and virologic response to therapy were investigated. Full-length NS5A clones were sequenced from 20 HCV genotype 1-infected patients in a prospective, randomized clinical trial of IFN induction (daily) therapy and IFN plus ribavirin combination therapy. Pretreatment NS5A nucleotide and amino acid phylogenies did not correlate with clinical IFN responses and domains involved in NS5A functions in vitro were all well conserved before and during treatment. A consensus IFN sensitivity-determining region (ISDR237–276) sequence associated with IFN resistance was not found, although the presence of Ala245 within the ISDR was associated with nonresponse to treatment in genotype 1a-infected patients (P < 0.01). There were more mutations in the 26 amino acids downstream of the ISDR required for PKR binding in pretreatment isolates from responders versus nonresponders in both HCV-1a- and HCV-1b-infected patients (P < 0.05). In HCV-1a patients, more amino acid changes were observed in isolates from IFN-sensitive patients (P < 0.001), and the mutations appeared to be concentrated in two variable regions in the C terminus of NS5A, that corresponded to the previously described V3 region and a new variable region, 310 to 330. Selection of pretreatment minor V3 quasispecies was observed within the first 2 to 6 weeks of therapy in responders but not nonresponders, whereas the ISDR and PKR binding domains did not change in either patient response group. These data suggest that host-mediated selective pressures act primarily on the C terminus of NS5A and that NS5A can perturb or evade the IFN-induced antiviral response using sequences outside of the putative ISDR. Mechanistic studies are needed to address the role of the C terminus of NS5A in HCV replication and antiviral resistance.

scholarly journals Definition of a minimal munc18c domain that interacts with syntaxin 4

2000 ◽  
Vol 350 (3) ◽  
pp. 741-746 ◽  
Author(s):  
Julian GRUSOVIN ◽  
Violet STOICHEVSKA ◽  
Keith H. GOUGH ◽  
Katrina NUNAN ◽  
Colin W. WARD ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Full Length ◽  
Yeast Two Hybrid ◽  
Protein Protein Interaction ◽  
Interaction Studies ◽  
Syntaxin 4 ◽  
Definition Of ◽  
Two Hybrid

munc18c is a critical protein involved in trafficking events associated with syntaxin 4 and which also mediates inhibitory effects on vesicle docking and/or fusion. To investigate the domains of munc18c responsible for its interaction with syntaxin 4, fragments of munc18c were generated and their interaction with syntaxin 4 examined in vivo by the yeast two-hybrid assay. In vitro protein–protein interaction studies were then used to confirm that the interaction between the proteins was direct. Full-length munc18c1–592, munc18c1–139 and munc18c1–225, but not munc18c226–592, munc18c1–100, munc18c43–139 or munc18c66–139, interacted with the cytoplasmic portion of syntaxin 4, Stx42–273, as assessed by yeast two-hybrid assay of growth on nutritionally deficient media and by β-galactosidase reporter induction. The N-terminal predicted helix-a-helix-b-helix-c region of syntaxin 4, Stx429–157, failed to interact with full-length munc18c1–592, indicating that a larger portion of syntaxin 4 is necessary for the interaction. The yeast two-hybrid results were confirmed by protein–protein interaction studies between Stx42–273 and glutathione S-transferase fusion proteins of munc18c. Full-length munc18c1–592, munc18c1–139 and munc18c1–225 interacted with Stx42–273 whereas munc18c1–100 did not, consistent with the yeast two-hybrid data. These data thus identify a region of munc18c between residues 1 and 139 as a minimal domain for its interaction with syntaxin 4.

scholarly journals Estimation of Full-Length TprK Diversity in Treponema pallidum subsp. pallidum

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Amin Addetia ◽  
Michelle J. Lin ◽  
Quynh Phung ◽  
Hong Xie ◽  
Meei-Li Huang ◽  
...  
Keyword(s):  
Immune System ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Deep Sequencing ◽  
Outer Membrane Protein ◽  
Treponema Pallidum ◽  
The United States ◽  
Full Length ◽  
Variable Regions ◽  
Content Type

ABSTRACT Immune evasion and disease progression of Treponema pallidum subsp. pallidum are associated with sequence diversity in the hypervariable outer membrane protein TprK. Previous attempts to study variation within TprK have sequenced at depths insufficient to fully appreciate the hypervariable nature of the protein, failed to establish linkage between the protein’s seven variable regions, or were conducted on isolates passed through rabbits. As a consequence, a complete profile of tprK during infection in the human host is still lacking. Furthermore, prior studies examining how T. pallidum subsp. pallidum uses its repertoire of genomic donor sites to generate diversity within the variable regions of the tprK have yielded a partial understanding of this process due to the limited number of tprK alleles examined. In this study, we used short- and long-read deep sequencing to directly characterize full-length tprK alleles from T. pallidum subsp. pallidum collected from early lesions of patients attending two sexually transmitted infection clinics in Italy. We demonstrate that strains collected from cases of secondary syphilis contain significantly more unique variable region sequences and full-length TprK sequences than those from cases of primary syphilis. Our data, combined with recent data available on Chinese T. pallidum subsp. pallidum specimens, show the near-complete absence of overlap in TprK sequences among the 41 specimens profiled to date. We further estimate that the potential antigenic variability carried by TprK rivals that of current estimates of the human adaptive immune system. These data underscore the immunoevasive ability of TprK that allows T. pallidum subsp. pallidum to establish lifelong infection. IMPORTANCE Syphilis continues to be a significant public health issue in both low- and high-income countries, including the United States where the rate of syphilis infection has increased over the past 5 years. Treponema pallidum subsp. pallidum, the causative agent of syphilis, carries the outer membrane protein TprK that undergoes segmental gene conversion to constantly create new sequences. We performed full-length deep sequencing of TprK to examine TprK diversity in clinical T. pallidum subsp. pallidum strains. We then combined our results with data from all samples for which TprK deep sequencing results were available. We found almost no overlap in TprK sequences between different patients. Moreover, our data allowed us to estimate the total number of TprK variants that T. pallidum subsp. pallidum can potentially generate. Our results support how the T. pallidum subsp. pallidum TprK antigenic variation system is an equal adversary of the human immune system leading to pathogen persistence in the host.

scholarly journals Persistence, Immune Response, and Antigenic Variation of Mycoplasma genitalium in an Experimentally Infected Pig-Tailed Macaque (Macaca nemestrina)

2013 ◽  
Vol 81 (8) ◽  
pp. 2938-2951 ◽  
Author(s):  
Gwendolyn E. Wood ◽  
Stefanie L. Iverson-Cabral ◽  
Dorothy L. Patton ◽  
Peter K. Cummings ◽  
Yvonne T. Cosgrove Sweeney ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Antigenic Variation ◽  
Immune Escape ◽  
Variable Region ◽  
Mycoplasma Genitalium ◽  
Macaca Nemestrina ◽  
Suitable Model ◽  
Content Type

ABSTRACTMycoplasma genitaliumis a sexually transmitted pathogen associated with several acute and chronic reproductive tract disease syndromes in men and women. To evaluate the suitability of a pig-tailed macaque model ofM. genitaliuminfection, we inoculated a pilot animal withM. genitaliumstrain G37 in the uterine cervix and in salpingeal pockets generated by transplanting autologous Fallopian tube tissue subcutaneously. Viable organisms were recovered throughout the 8-week experiment in cervicovaginal specimens and up to 2 weeks postinfection in salpingeal pockets. Humoral and cervicovaginal antibodies reacting to MgpB were induced postinoculation and persisted throughout the infection. The immunodominance of the MgpB adhesin and the accumulation ofmgpBsequence diversity previously observed in persistent human infections prompted us to evaluate sequence variation in this animal model. We found that after 8 weeks of infection, sequences withinmgpBvariable region B were replaced by novel sequences generated by reciprocal recombination with an archived variant sequence located elsewhere on the chromosome. In contrast,mgpBregion B of the same inoculum propagated for 8 weeksin vitroremained unchanged. Notably, serum IgG reacted strongly with a recombinant protein spanning MgpB region B of the inoculum, while reactivity to a recombinant protein representing the week 8 variant was reduced, suggesting that antibodies were involved in the clearance of bacteria expressing the original infecting sequence. Together these results suggest that the pig-tailed macaque is a suitable model to studyM. genitaliumpathogenesis, antibody-mediated selection of antigenic variantsin vivo, and immune escape.

scholarly journals New Tools for Syphilis Research

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Sheila A. Lukehart
Keyword(s):  
Protein Function ◽  
Antigenic Variation ◽  
Treponema Pallidum ◽  
Content Type ◽  
Genetic Tools ◽  
Rapid Isolation

ABSTRACTSyphilis research has been severely limited by the necessity to propagateTreponema pallidumin vivoin rabbits. After decades of erroneous or irreproducible reports of cultivation ofT. pallidum, the recent very convincing report of its successful long-termin vitropropagation opens numerous opportunities for development of genetic tools for studying pathogenesis and protein function, antigenic variation, and surface exposure of antigens. The possibility of more rapid isolation of new strains will expand our knowledge of this organism beyond the century-old Nichols strain.

scholarly journals Genetic Basis of De Novo Appearance of Carotenoid Ornamentation in Bare Parts of Canaries

2020 ◽  
Vol 37 (5) ◽  
pp. 1317-1328 ◽  
Author(s):  
Małgorzata Anna Gazda ◽  
Matthew B Toomey ◽  
Pedro M Araújo ◽  
Ricardo J Lopes ◽  
Sandra Afonso ◽  
...  
Keyword(s):  
Genetic Basis ◽  
De Novo ◽  
Bird Species ◽  
Beta Carotene ◽  
Full Length ◽  
Nonsynonymous Mutation ◽  
Retinal Tissue ◽  
Cleavage Activity ◽  
Important Mediator

Abstract Unlike wild and domestic canaries (Serinus canaria), or any of the three dozen species of finches in genus Serinus, the domestic urucum breed of canaries exhibits bright red bills and legs. This novel trait offers a unique opportunity to understand the mechanisms of bare-part coloration in birds. To identify the mutation producing the colorful phenotype, we resequenced the genome of urucum canaries and performed a range of analyses to search for genotype-to-phenotype associations across the genome. We identified a nonsynonymous mutation in the gene BCO2 (beta-carotene oxygenase 2, also known as BCDO2), an enzyme involved in the cleavage and breakdown of full-length carotenoids into short apocarotenoids. Protein structural models and in vitro functional assays indicate that the urucum mutation abrogates the carotenoid-cleavage activity of BCO2. Consistent with the predicted loss of carotenoid-cleavage activity, urucum canaries tended to have increased levels of full-length carotenoid pigments in bill tissue and reduced levels of carotenoid-cleavage products (apocarotenoids) in retinal tissue compared with other breeds of canaries. We hypothesize that carotenoid-based bare-part coloration might be readily gained, modified, or lost through simple switches in the enzymatic activity or regulation of BCO2 and this gene may be an important mediator in the evolution of bare-part coloration among bird species.

scholarly journals Genetic Basis of De Novo Appearance of Carotenoid Ornamentation in Bare-Parts of Canaries

2019 ◽  
Author(s):  
Małgorzata Anna Gazda ◽  
Matthew B. Toomey ◽  
Pedro M. Araújo ◽  
Ricardo J. Lopes ◽  
Sandra Afonso ◽  
...  
Keyword(s):  
Genetic Basis ◽  
De Novo ◽  
Bird Species ◽  
Beta Carotene ◽  
Full Length ◽  
Nonsynonymous Mutation ◽  
Retinal Tissue ◽  
Cleavage Activity ◽  
Causative Locus

ABSTRACTUnlike wild and domestic canaries (Serinus canaria), or any of the three dozen species of finches in genus Serinus, the domestic urucum breed of canaries exhibits bright red bills and legs. This novel bare-part coloration offers a unique opportunity to understand how leg and bill coloration evolve in birds. To identify the causative locus, we resequenced the genome of urucum canaries and performed a range of analyses to search for genotype-to-phenotype associations across the genome. We identified a nonsynonymous mutation in the gene BCO2 (beta-carotene oxygenase 2, also known as BCDO2), an enzyme involved in the cleavage and breakdown of full-length carotenoids into short apocarotenoids. Protein structural models and in vitro functional assays indicate that the urucum mutation abrogates the carotenoid cleavage activity of BCO2. Consistent with the predicted loss of carotenoid cleavage activity, urucum canaries had increased levels of full-length carotenoid pigments in bill tissue and a significant reduction in levels of carotenoid cleavage products (apocarotenoids) in retinal tissue compared to other breeds of canaries. We hypothesize that carotenoid-based bare-part coloration might be readily gained, modified, or lost through simple switches in the enzymatic activity or regulation of BCO2 and this gene may be an important mediator in the evolution of bare-part coloration among bird species.

scholarly journals Longitudinal TprK profiling of in vivo and in vitro-propagated Treponema pallidum subsp. pallidum reveals accumulation of antigenic variants in absence of immune pressure

2021 ◽  
Vol 15 (9) ◽  
pp. e0009753
Author(s):  
Michelle J. Lin ◽  
Austin M. Haynes ◽  
Amin Addetia ◽  
Nicole A. P. Lieberman ◽  
Quynh Phung ◽  
...  
Keyword(s):  
Gene Conversion ◽  
Antigenic Variation ◽  
Treponema Pallidum ◽  
Sequence Diversity ◽  
Immune Selection ◽  
Syphilis Infection ◽  
Variable Regions ◽  
Immune Pressure

Immune evasion by Treponema pallidum subspecies pallidum (T. pallidum) has been attributed to antigenic variation of its putative outer-membrane protein TprK. In TprK, amino acid diversity is confined to seven variable (V) regions, and generation of sequence diversity within the V regions occurs via a non-reciprocal segmental gene conversion mechanism where donor cassettes recombine into the tprK expression site. Although previous studies have shown the significant role of immune selection in driving accumulation of TprK variants, the contribution of baseline gene conversion activity to variant diversity is less clear. Here, combining longitudinal tprK deep sequencing of near clonal Chicago C from immunocompetent and immunosuppressed rabbits along with the newly developed in vitro cultivation system for T. pallidum, we directly characterized TprK alleles in the presence and absence of immune selection. Our data confirm significantly greater sequence diversity over time within the V6 region during syphilis infection in immunocompetent rabbits compared to immunosuppressed rabbits, consistent with previous studies on the role of TprK in evasion of the host immune response. Compared to strains grown in immunocompetent rabbits, strains passaged in vitro displayed low level changes in allele frequencies of TprK variable region sequences similar to that of strains passaged in immunosuppressed rabbits. Notably, we found significantly increased rates of V6 allele generation relative to other variable regions in in vitro cultivated T, pallidum strains, illustrating that the diversity within these hypervariable regions occurs in the complete absence of immune selection. Together, our results demonstrate antigenic variation in T. pallidum can be studied in vitro and occurs even in the complete absence of immune pressure, allowing the T. pallidum population to continuously evade the immune system of the infected host.

scholarly journals Antigenic Variation of Ehrlichia chaffeensis Resulting from Differential Expression of the 28-Kilodalton Protein Gene Family

2002 ◽  
Vol 70 (4) ◽  
pp. 1824-1831 ◽  
Author(s):  
S. Wesley Long ◽  
Xiao-Feng Zhang ◽  
Hai Qi ◽  
Steven Standaert ◽  
David H. Walker ◽  
...  
Keyword(s):  
Gene Family ◽  
Outer Membrane ◽  
Differential Expression ◽  
Protein Gene ◽  
Antigenic Variation ◽  
Outer Membrane Proteins ◽  
Pcr Amplification ◽  
Ehrlichia Chaffeensis ◽  
Allele Variation

ABSTRACT The transcriptional activity and allele variation of the 28-kDa outer membrane protein gene (p28) of Ehrlichia chaffeensis were analyzed to determine the mechanism of the antigenic variation of the 28-kDa outer membrane proteins. Reverse transcriptase PCR amplification of mRNA indicated that 16 of the 22 members of the p28 multigene family were transcribed. Amino acid sequence analysis indicated that the p28-19 protein was produced in vitro in the Arkansas strain. The p28-19 gene and its promoter region were sequenced and compared in 12 clinical isolates of E. chaffeensis to determine allele variation. The variation of the p28-19 gene among the isolates is limited to three types represented by strains Arkansas, 91HE17, and Sapulpa, respectively. These results indicate that the majority of the p28 genes are active genes and that antigenic variation of the E. chaffeensis 28-kDa proteins may result from differential expression of the p28 gene family members rather than gene conversion.

scholarly journals Antigenic Variation of TprK Facilitates Development of Secondary Syphilis

2014 ◽  
Vol 82 (12) ◽  
pp. 4959-4967 ◽  
Author(s):  
Tara B. Reid ◽  
Barbara J. Molini ◽  
Mark C. Fernandez ◽  
Sheila A. Lukehart
Keyword(s):  
Immune Response ◽  
Outer Membrane ◽  
Antigenic Variation ◽  
Rabbit Model ◽  
Variable Region ◽  
Skin Lesions ◽  
Secondary Syphilis ◽  
Variable Regions ◽  
Robust Immune Response ◽  
Immune Pressure

ABSTRACTAlthough primary syphilis lesions heal spontaneously, the infection is chronic, with subsequent clinical stages. Healing of the primary chancre occurs as antibodies against outer membrane antigens facilitate opsonophagocytosis of the bacteria by activated macrophages. TprK is an outer membrane protein that undergoes antigenic variation at 7 variable regions, and variants are selected by immune pressure. We hypothesized that individual TprK variants escape immune clearance and seed new disseminated lesions to cause secondary syphilis. As in human syphilis, infected rabbits may develop disseminated secondary skin lesions. This study explores the nature of secondary syphilis, specifically, the contribution of antigenic variation to the development of secondary lesions. Our data from the rabbit model show that the odds of secondary lesions containing predominately TprK variant treponemes is 3.3 times higher than the odds of finding TprK variants in disseminated primary lesions (odds ratio [OR] = 3.3 [95% confidence interval {CI}, 0.98 to 11.0];P= 0.055) and that 96% of TprK variant secondary lesions are likely seeded by single treponemes. Analysis of antibody responses demonstrates significantly higher antibody titers totprKvariable region sequences found in the inoculum compared to reactivity totprKvariant sequences found in newly arising secondary lesions. This suggests thattprKvariants escape the initial immune response raised against the V regions expressed in the inoculum. These data further support a role for TprK in immune evasion and suggest that the ability of TprK variants to persist despite a robust immune response is instrumental in the development of later stages of syphilis.

Sign in / Sign up

Export Citation Format

Share Document