Introduction Outer membrane proteins (OMPs) play critical roles in disease pathogenesis and are vaccinogens. Topologic characterisation of surface-exposed β-barrels of Treponema pallidum (Tp) Nichols rare OMPs enabled a novel strategy to assess sequence diversity and evolution of Tp in geographically diverse locations. Methods Through early 2017, sequences encoding TprC (TP0117), TprD (TP0131), and BamA (TP0326) β-barrels were amplified from secondary syphilis patients from Cali (n=16) and swabs from patients in San Francisco (SF, n=6) and Czech Republic (CZ, n=9). Strains were assigned to the Nichols or SS14 clade based on tp0548 and/or tp0558 sequences. Results 23 assignable CZ and Cali strains belonged to either the SS14 or Nichols clade (SS14 predominant), while all 6 SF strains belong to the SS14 clade. Sequence diversity at the three OMP loci was greatest in Cali, with evidence of recombination within tprC and bamA alleles, as well as between strains and clades at all 3 genetic loci. SF strains contained nearly identical sequences at all 3 genetic loci. The SS14 and Mexico A reference strains, both belonging to the SS14 clade, have identical tprDs (tprD2) but different tprC and bamA alleles. Mexico A tprCs were common at all three geographic locations, including Nichols clade strains from Cali. Mexico A bamAs were prevalent in Cali and SF, while CZ SS14 clades contained only SS14 bamAs. OMP sequences were obtained from all three loci in 7 of 8 Nichols clade strains. Of these 7, only 1 matched the Nichols reference strain, while the other 6 contained Mexico A alleles in at least 1 OMP locus. Of the 21 SS14 clade strains, 10 contained Mexico A alleles at all 3 loci; 2 contained Mexico A trpCs and Nichols bamAs; and 9 contained Mexico A tprCs and SS14 bamAs. Conclusion OMP loci are evolving independently within Tp. Recombination of OMP sequences appears to be occurring between Tp strains and clades within patients. Mexico A OMP alleles are circulating widely among Tp strains. These findings have major ramifications for syphilis vaccine development.

Introduction For treatment of Mycoplasma genitalium (Mg) infection, azithromycin is first line initial treatment but persistent NGU caused by Mg may not respond to repeat azithromycin treatment; quinolones, in particular moxifloxcin, have been shown to be effective. The molecular epidemiology of mutations in macrolide- and quinolone-resistance determining regions (RDRs) of 23S rRNA, parC and gyrA genes remains unclear in China. Methods From April 2011 to August 2015, male subjects with clinical and microscopic evidence of urethritis provided a first-voided urine specimen and the mgpA gene was partially amplified and sequenced to diagnose Mg infection. RDRs of the 23s rRNA, parC and gyrA genes were sequenced. Results Among 1816 male-patients, the overall prevalence of M. genitalium was 19.7% (358/1816). N. gonorrhoeae prevalence was 46.6% (847/1816). Among 969 NGU-cases, prevalence of: C. trachomatis was 42.3% (409/968), M. genitalium 27.9% (271/969), U. urealyticum 21.3% (182/856), T. vaginalis1.8% (17/969) and M. hominis 0.7% (6/855). Based on data available from partial (5.4%) sequencing of the MgPa gene (mgpA) an evolutionary tree was constructed that divided the 358 Mg mgpA sequences into five major clusters. Among 358 Mg positive samples, successful sequencing was accomplished for: the 23s rRNA gene (341 specimens), the parC gene (344 specimens) and the gyrA gene (339 specimens). 88.9% (303/341) had mutations in 23s rRNA, 89.5% (308/344) had sense mutations in parC, and 12.4% (42/339) had sense mutations in gyrA.The most common single base pair mutation in the 23s rRNA gene was A2059G (211/303; 69.6%) followed by A2058G (60/303; 19.8%) and A2059T (30/303; 9.9%). The most common mutation in the parC gene was G248T (289/344; 84%) and in the gyrA gene G285A (22/339, 6.5%). Conclusion The high mutation rate in 23s rRNA and parC in Mg strains in Nanjing are a harbinger of resistance to macrolides and quinolones that may follow. Molecular surveillance of indigenous strains of Mg is warranted to anticipate the development of antimicrobial resistance.

Event Abstract Back to Event Human macrophage polarization enhances opsonophagocytosis and inflammatory response of the stealth pathogen, Treponema pallidum Kelly L. Hawley1*, Adriana R. Cruz2, Jorge Cervantes1, 3, Carson Karanian4, Morgan LeDoyt4, Rodolfo Trujillo1, Lady G. Ramirez2, Justin Radolf1, 4, 5 and Juan C. Salazar1, 3, 4 1 University of Connecticut Health Center, Department of Pediatrics, United States 2 Centro Internacional de Entrenamiento e Investigaciones Médicas CIDEIM, Colombia 3 Connecticut Children’s Medical Center, Division of Infectious Disease and Immunology, United States 4 University of Connecticut Health Center, Departments of Medicine, United States 5 University of Connecticut Health Center, Genetics and Developmental Biology, United States Venereal syphilis is a multi-stage, sexually transmitted disease caused by the spirochete Treponema pallidum (Tp). Congenital syphilis, a result of transplacental migration of Tp, is a major global threat to child health. Clinical manifestations result from the treponeme's ability to elicit a robust immune response while evading host defenses; this duality is best exemplified during the florid, disseminated stage called secondary syphilis (SS). SS lesions contain copious spirochetes along with a mixed cellular infiltrate consisting of CD4 T cells, CD8 T cells, NK cells, plasma cells and macrophages (MΦ). In the rabbit model, Tp are cleared by MΦs via antibody (Ab)-mediated opsonophagocytosis. Previously, we demonstrated that human syphilitic serum (HSS) promotes efficient uptake of Tp by human monocytes and that opsonophagocytosis of Tp markedly enhances cytokine production. The purpose of this study is to establish a potential role for MΦs and opsonic Ab in clearance of treponemes and generation of tissue-based inflammation during human syphilis. We used monocyte-derived MΦs to develop an ex vivo model for studying spirochete-MΦ interactions and cellular responses (e.g., phagosomal signaling) following uptake of Tp. We also examined SS skin biopsies for evidence of internalization of Tp by dermal MΦs. In the presence or absence of HSS, untreated (i.e., no M-CSF or IFNγ) MΦs internalized low numbers of Tp and secreted little cytokine (e.g., TNF and IL-6). In contrast, untreated MΦs internalized unopsonized Lyme disease spirochetes (Borrelia burgdorferi) and secreted robust levels of cytokines. Maturation of MΦs with M-CSF and IFNγ [MΦ(IFNγ)] resulted in increased TLR signaling and enhanced expression of the Fc receptors CD64 and CD16. Importantly, IFNγ polarization of MΦs led to a statistically significant, but modest, increase in opsonophagocytosis of Tp and cytokine production. Interestingly, immunofluorescence analysis of SS skin biopsies yielded only scant evidence for internalization of Tp by dermal MΦs. Our ex vivo studies demonstrate a potential role for MΦs in clearance of Tp during human syphilis. On the other hand, the in vivo data suggest that Tp, the stealth pathogen, is adept at evading MΦ-dependent clearance and suggest the possibility of MΦ-independent clearance mechanisms. Acknowledgements We gratefully acknowledge the patients who participated in this study. This work received support from NIAID grant A1090166, CCMC Arrison and Burr Curtis Research Funds and Fogarty/NIH R03TW009172. Keywords: Macrophages, Syphilis, FcγR, phagosomal signaling, Clearance Conference: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015. Presentation Type: Oral Presentation Topic: Innate Immunity Citation: Hawley KL, Cruz AR, Cervantes J, Karanian C, LeDoyt M, Trujillo R, Ramirez LG, Radolf J and Salazar JC (2015). Human macrophage polarization enhances opsonophagocytosis and inflammatory response of the stealth pathogen, Treponema pallidum. Front. Immunol. Conference Abstract: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología. doi: 10.3389/conf.fimmu.2015.05.00306 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 29 May 2015; Published Online: 15 Sep 2015. * Correspondence: PhD. Kelly L Hawley, University of Connecticut Health Center, Department of Pediatrics, Farmington, Connecticut, United States, Hawley@uchc.edu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Kelly L Hawley Adriana R Cruz Jorge Cervantes Carson Karanian Morgan LeDoyt Rodolfo Trujillo Lady G Ramirez Justin Radolf Juan C Salazar Google Kelly L Hawley Adriana R Cruz Jorge Cervantes Carson Karanian Morgan LeDoyt Rodolfo Trujillo Lady G Ramirez Justin Radolf Juan C Salazar Google Scholar Kelly L Hawley Adriana R Cruz Jorge Cervantes Carson Karanian Morgan LeDoyt Rodolfo Trujillo Lady G Ramirez Justin Radolf Juan C Salazar PubMed Kelly L Hawley Adriana R Cruz Jorge Cervantes Carson Karanian Morgan LeDoyt Rodolfo Trujillo Lady G Ramirez Justin Radolf Juan C Salazar Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

We previously identified Treponema pallidum repeat proteins TprC/D, TprF, and TprI as candidate outer membrane proteins (OMPs) and subsequently demonstrated that TprC is not only a rare OMP but also forms trimers and has porin activity. We also reported that TprC contains N- and C-terminal domains (TprC(N) and TprC(C)) orthologous to regions in the major outer sheath protein (MOSP(N) and MOSP(C)) of Treponema denticola and that TprC(C) is solely responsible for β-barrel formation, trimerization, and porin function by the full-length protein. Herein, we show that TprI also possesses bipartite architecture, trimeric structure, and porin function and that the MOSP(C)-like domains of native TprC and TprI are surface-exposed in T. pallidum, whereas their MOSP(N)-like domains are tethered within the periplasm. TprF, which does not contain a MOSP(C)-like domain, lacks amphiphilicity and porin activity, adopts an extended inflexible structure, and, in T. pallidum, is tightly bound to the protoplasmic cylinder. By thermal denaturation, the MOSP(N) and MOSP(C)-like domains of TprC and TprI are highly thermostable, endowing the full-length proteins with impressive conformational stability. When expressed in Escherichia coli with PelB signal sequences, TprC and TprI localize to the outer membrane, adopting bipartite topologies, whereas TprF is periplasmic. We propose that the MOSP(N)-like domains enhance the structural integrity of the cell envelope by anchoring the β-barrels within the periplasm. In addition to being bona fide T. pallidum rare outer membrane proteins, TprC/D and TprI represent a new class of dual function, bipartite bacterial OMP.

Background:Treponema pallidum contains a paucity of outer membrane proteins. Results: The C-terminal domains of TprC/D and TprI are -barrels with porin function; their N-terminal domains provide periplasmic anchors for the -barrels. Conclusion: Full-length TprC subfamily proteins possess a dual domain topology. Significance: TprC/D are bona fide rare outer membrane proteins and a new class of dual function, bipartite, bacterial outer membrane protein.We previously identified Treponemapallidumrepeat proteins TprC/D, TprF, and TprI as candidate outer membrane proteins (OMPs) and subsequently demonstrated that TprC is not only a rare OMP but also forms trimers and has porin activity. We also reported that TprC contains N- and C-terminal domains (TprC(N) and TprC(C)) orthologous to regions in the major outer sheath protein (MOSPN and MOSPC) of Treponema denticola and that TprC(C) is solely responsible for -barrel formation, trimerization, and porin function by the full-length protein. Herein, we show that TprI also possesses bipartite architecture, trimeric structure, and porin function and that the MOSPC-like domains of native TprC and TprI are surface-exposed in T. pallidum, whereas their MOSPN-like domains are tethered within the periplasm. TprF, which does not contain a MOSPC-like domain, lacks amphiphilicity and porin activity, adopts an extended inflexible structure, and, in T. pallidum, is tightly bound to the protoplasmic cylinder. By thermal denaturation, the MOSPN and MOSPC-like domains of TprC and TprI are highly thermostable, endowing the full-length proteins with impressive conformational stability. When expressed in Escherichia coli with PelB signal sequences, TprC and TprI localize to the outer membrane, adopting bipartite topologies, whereas TprF is periplasmic. We propose that the MOSPN-like domains enhance the structural integrity of the cell envelope by anchoring the -barrels within the periplasm. In addition to being bona fide T. pallidum rare outer membrane proteins, TprC/D and TprI represent a new class of dual function, bipartite bacterial OMP.

Volume 194, no. 9, pages [2321–2333][1], 2012. Page 2330: The legend to Fig. 6E should read as follows. “Immunoblot of unboiled, recombinant TprCN and TprCC separated by SDS-PAGE and then probed with rabbit anti-TprCFl polyclonal antiserum (asterisk indicates TprCC trimer).” Page 2331: The

ABSTRACTIdentification ofTreponema pallidumrare outer membrane proteins (OMPs) has been a longstanding objective of syphilis researchers. We recently developed a consensus computational framework that employs a battery of cellular localization and topological prediction tools to generate ranked clusters of candidate rare OMPs (D. L. Cox et al., Infect. Immun. 78:5178–5194, 2010). TP0117/TP0131 (TprC/D), a member of theT. pallidumrepeat (Tpr) family, was a highly ranked candidate. Circular dichroism, heat modifiability by SDS-PAGE, Triton X-114 phase partitioning, and liposome incorporation confirmed that full-length, recombinant TprC (TprCFl) forms a β-barrel capable of integrating into lipid bilayers. Moreover, TprCFlincreased efflux of terbium-dipicolinic acid complex from large unilamellar vesicles and migrated as a trimer by blue-native PAGE. We found that inT. pallidum, TprC is heat modifiable, trimeric, expressed in low abundance, and, based on proteinase K accessibility and opsonophagocytosis assays, surface exposed. From these collective data, we conclude that TprC is a bona fide rare OMP as well as a functional ortholog ofEscherichia coliOmpF. We also discovered that TprC has a bipartite architecture consisting of a soluble N-terminal portion (TprCN), presumably periplasmic and bound directly or indirectly to peptidoglycan, and a C-terminal β-barrel (TprCC). Syphilitic rabbits generate antibodies exclusively against TprCC, while secondary syphilis patients fail to mount a detectable antibody response against either domain. The syphilis spirochete appears to have resolved a fundamental dilemma arising from its extracellular lifestyle, namely, how to enhance OM permeability without increasing its vulnerability to the antibody-mediated defenses of its natural human host.