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Syphilis is a sexually transmitted infection caused by the highly invasive and immunoevasive spirochetal pathogen Treponema pallidum subsp. pallidum ( TPA ). Untreated syphilis can lead to infection of multiple organ systems, including the central nervous system. The alarming increase in syphilis cases globally underscores the importance of developing novel strategies to understand the complexities of syphilis pathogenesis. In this study, we took advantage of recent advances in in vitro cultivation and genetic manipulation of syphilis spirochetes to engineer a TPA strain that constitutively expresses green fluorescent protein (GFP). GFP + TPA grew identically to the Nichols parent strain in vitro and exhibited wild-type infectivity in the rabbit model. We then used the GFP + strain to visualize TPA interactions with host cells during co-cultivation in vitro , within infected rabbit testes, and following opsonophagocytosis by murine bone marrow-derived macrophages. Development of fluorescent strain also enabled us to develop a flow cytometric-based assay to assess antibody-mediated damage to the spirochete's fragile outer membrane (OM), demonstrating dose-dependent growth inhibition and OM disruption in vitro . Notably, we observed greater OM disruption of GFP + TPA with sera from immune rabbits infected with the TPA Nichols strain compared to sera generated against the genetically distinct SS14 strain. These latter findings highlight the importance of OM protein-specific antibody responses for clearance of TPA during syphilitic infection. The availability of fluorescent TPA strains paves the way for future studies investigating spirochete-host interactions as well as functional characterization of antibodies directed treponemal OM proteins, the presumptive targets for protective immunity., Competing Interests: J.B.P. reports research support from Gilead Sciences and non-financial support from Abbott Laboratories.

Background: The increase in syphilis rates worldwide necessitates development of a vaccine with global efficacy. We aimed to explore Treponema pallidum subspecies pallidum (TPA) molecular epidemiology essential for vaccine research by analysing clinical data and specimens from early syphilis patients using whole-genome sequencing (WGS) and publicly available WGS data., Methods: In this multicentre, cross-sectional, molecular epidemiology study, we enrolled patients with primary, secondary, or early latent syphilis from clinics in China, Colombia, Malawi, and the USA between Nov 28, 2019, and May 27, 2022. Participants aged 18 years or older with laboratory confirmation of syphilis by direct detection methods or serological testing, or both, were included. Patients were excluded from enrolment if they were unwilling or unable to give informed consent, did not understand the study purpose or nature of their participation, or received antibiotics active against syphilis in the past 30 days. TPA detection and WGS were conducted on lesion swabs, skin biopsies, skin scrapings, whole blood, or rabbit-passaged isolates. We compared our WGS data to publicly available genomes and analysed TPA populations to identify mutations associated with lineage and geography., Findings: We screened 2802 patients and enrolled 233 participants, of whom 77 (33%) had primary syphilis, 154 (66%) had secondary syphilis, and two (1%) had early latent syphilis. The median age of participants was 28 years (IQR 22-35); 154 (66%) participants were cisgender men, 77 (33%) were cisgender women, and two (1%) were transgender women. Of the cisgender men, 66 (43%) identified as gay, bisexual, or other sexuality. Among all participants, 56 (24%) had HIV co-infection. WGS data from 113 participants showed a predominance of SS14-lineage strains with geographical clustering. Phylogenomic analyses confirmed that Nichols-lineage strains were more genetically diverse than SS14-lineage strains and clustered into more distinct subclades. Differences in single nucleotide variants (SNVs) were evident by TPA lineage and geography. Mapping of highly differentiated SNVs to three-dimensional protein models showed population-specific substitutions, some in outer membrane proteins (OMPs) of interest., Interpretation: Our study substantiates the global diversity of TPA strains. Additional analyses to explore TPA OMP variability within strains is vital for vaccine development and understanding syphilis pathogenesis on a population level., Funding: US National Institutes of Health National Institute for Allergy and Infectious Disease, the Bill & Melinda Gates Foundation, Connecticut Children's, and the Czech Republic National Institute of Virology and Bacteriology., Competing Interests: Declaration of interests ACS reports royalties from UptoDate; honoraria from the University of Alabama at Birmingham; and support for meetings or travel from the American STD Association as a member of the Executive Board outside the scope of the current work. JAG-L reports honoraria from the Universidad de Antioquia; support for meetings or travel from Carnott Laboratories, Cantabria Labs, Epidermique, Pharmaderm, and Janssen; receipt of writing materials from Epidermique, Cantabria labs, Isdin, Pharmaderm, Skindrugs, Loreal, Galderma, Cetaphil, Cerave, Isispharma, Carnott, Janssen, Pharmalab, Novartis, Pfizer, and Lilly outside of the scope of work. JJJ reports membership in the Worldwide Antimalarial Resistance Network. KLH reports honoraria from the Eastern Virginia Medical School and the Lawrence Livermore National Laboratory. JDR receives royalties from Biokit, Chembio, and Span Diagnostics for syphilis serodiagnostic reagents; and support for meetings or travel from Indiana University outside the scope of the current work. JBP reports research support from Gilead Sciences; non-financial support from Abbott Diagnostics; and consulting for Zymeron Corporation, all outside the scope of the current work., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The Omp85 family of outer membrane proteins are ubiquitously distributed among diderm bacteria and play essential roles in outer membrane (OM) biogenesis. The majority of Omp85 orthologs are bipartite and consist of a conserved OM-embedded 16-stranded beta-barrel and variable periplasmic functional domains. Here, we demonstrate that Leptospira interrogans encodes four distinct Omp85 proteins. The presumptive leptospiral BamA, LIC11623, contains a noncanonical POTRA4 periplasmic domain that is conserved across Leptospiraceae. The remaining three leptospiral Omp85 proteins, LIC12252, LIC12254 and LIC12258, contain conserved beta-barrels but lack periplasmic domains. Two of the three 'noNterm' Omp85-like proteins were upregulated by leptospires in urine from infected mice compared to in vitro and/or following cultivation within rat peritoneal cavities. Mice infected with a L. interrogans lic11254 transposon mutant shed tenfold fewer leptospires in their urine compared to mice infected with the wild-type parent. Analyses of pathogenic and saprophytic Leptospira spp. identified five groups of noNterm Omp85 paralogs, including one pathogen- and two saprophyte-specific groups. Expanding our analysis beyond Leptospira spp., we identified additional noNterm Omp85 orthologs in bacteria isolated from diverse environments, suggesting a potential role for these previously unrecognized noNterm Omp85 proteins in physiological adaptation to harsh conditions., (© 2024. The Author(s).)

The global resurgence of syphilis has created a potent stimulus for vaccine development. To identify potentially protective antibodies (Abs) against Treponema pallidum ( TPA ), we used Pyrococcus furiosus thioredoxin ( Pf Trx) to display extracellular loops (ECLs) from three TPA outer membrane protein families (outer membrane factors for efflux pumps, eight-stranded β-barrels, and FadLs) to assess their reactivity with immune rabbit serum (IRS). Five ECLs from the FadL orthologs TP0856, TP0858 and TP0865 were immunodominant. Rabbits and mice immunized with these five Pf Trx constructs produced ECL-specific Abs that promoted opsonophagocytosis of TPA by rabbit peritoneal and murine bone marrow-derived macrophages at levels comparable to IRS and mouse syphilitic serum. ECL-specific rabbit and mouse Abs also impaired viability, motility, and cellular attachment of spirochetes during in vitro cultivation. The results support the use of ECL-based vaccines and suggest that ECL-specific Abs promote spirochete clearance via Fc receptor-independent as well as Fc receptor-dependent mechanisms., Competing Interests: Competing Interests: All authors have no relevant financial or non-financial competing interests to report.

Vector-borne diseases are a leading cause of death worldwide and pose a substantial unmet medical need. Pathogens binding to host extracellular proteins (the "exoproteome") represents a crucial interface in the etiology of vector-borne disease. Here, we used bacterial selection to elucidate host-microbe interactions in high throughput (BASEHIT)-a technique enabling interrogation of microbial interactions with 3,324 human exoproteins-to profile the interactomes of 82 human-pathogen samples, including 30 strains of arthropod-borne pathogens and 8 strains of related non-vector-borne pathogens. The resulting atlas revealed 1,303 putative interactions, including hundreds of pairings with potential roles in pathogenesis, including cell invasion, tissue colonization, immune evasion, and host sensing. Subsequent functional investigations uncovered that Lyme disease spirochetes recognize epidermal growth factor as an environmental cue of transcriptional regulation and that conserved interactions between intracellular pathogens and thioredoxins facilitate cell invasion. In summary, this interactome atlas provides molecular-level insights into microbial pathogenesis and reveals potential host-directed targets for next-generation therapeutics., Competing Interests: Declaration of interests N.W.P. and A.M.R. are inventors of a patent describing the BASEHIT technique., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Venereal syphilis, caused by the spirochete Treponema pallidum subsp. pallidum (TPA), is surging worldwide, underscoring the need for a vaccine with global efficacy. Vaccine development requires an understanding of syphilis epidemiology and clinical presentation as well as genomic characterization of TPA strains circulating within at-risk populations. The aim of this study was to describe the clinical, demographic, and molecular features of early syphilis cases in Cali, Colombia., Methods and Findings: We conducted a cross-sectional study to identify individuals with early syphilis (ES) in Cali, Colombia through a city-wide network of public health centers, private sector HIV clinics and laboratory databases from public health institutions. Whole blood (WB), skin biopsies (SB), and genital and oral lesion swabs were obtained for measurement of treponemal burdens by polA quantitative polymerase chain reaction (qPCR) and for whole-genome sequencing (WGS). Among 1,966 individuals screened, 128 participants met enrollment criteria: 112 (87%) with secondary (SS), 15 (12%) with primary (PS) and one with early latent syphilis; 66/128 (52%) self-reported as heterosexual, while 48 (38%) were men who have sex with men (MSM). Genital ulcer swabs had the highest polA copy numbers (67 copies/μl) by qPCR with a positivity rate (PR) of 73%, while SS lesions had 42 polA copies/μl with PR of 62%. WB polA positivity was more frequent in SS than PS (42% vs 7%, respectively; p = 0.009). Isolation of TPA from WB by rabbit infectivity testing (RIT) was achieved in 5 (56%) of 9 ES WB samples tested. WGS from 33 Cali patient samples, along with 10 other genomic sequences from South America (9 from Peru, 1 from Argentina) used as comparators, confirmed that SS14 was the predominant clade, and that half of all samples had mutations associated with macrolide (i.e., azithromycin) resistance. Variability in the outer membrane protein (OMP) and vaccine candidate BamA (TP0326) was mapped onto the protein's predicted structure from AlphaFold. Despite the presence of mutations in several extracellular loops (ECLs), ECL4, an immunodominant loop and proven opsonic target, was highly conserved in this group of Colombian and South American TPA isolates., Conclusions: This study offers new insights into the sociodemographic and clinical features of venereal syphilis in a highly endemic area of Colombia and illustrates how genomic sequencing of regionally prevalent TPA strains can inform vaccine development., Competing Interests: Outside the submitted work, ACS reports royalties from UptoDate Inc, ELM reports research grants from Sanofi Pasteur, Janssen, Moderna and GSK, grants and consulting fees from Takeda and MSD, and honoraria from Pfizer, JAGL reports support for attending meetings/travel from Janssen, MAM reports membership in an advisory board for GSK, JDR receives royalties from Biokit SA, Chembio, and Span Diagnostics for syphilis serodiagnostic reagents outside the scope of the current work, JBP reports research support from Gilead Sciences, non-financial support from Abbott Diagnostics, and consulting for Zymeron Corporation. All other authors report no competing interests. The commercial funders indicated above provided support in the form of royalties for stated authors but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript., (Copyright: © 2024 Salazar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: The global resurgence of syphilis necessitates vaccine development., Methods: We collected ulcer exudates and blood from 17 primary syphilis (PS) participants and skin biopsies and blood from 51 secondary syphilis (SS) participants in Guangzhou, China for Treponema pallidum subsp. pallidum (TPA) qPCR, whole genome sequencing (WGS), and isolation of TPA in rabbits., Results: TPA DNA was detected in 15 of 17 ulcer exudates and 3 of 17 blood PS specimens. TPA DNA was detected in 50 of 51 SS skin biopsies and 27 of 51 blood specimens. TPA was isolated from 47 rabbits with success rates of 71% (12/17) and 69% (35/51), respectively, from ulcer exudates and SS bloods. We obtained paired genomic sequences from 24 clinical samples and corresponding rabbit isolates. Six SS14- and two Nichols-clade genome pairs contained rare discordances. Forty-one of the 51 unique TPA genomes clustered within SS14 subgroups largely from East Asia, while 10 fell into Nichols C and E subgroups., Conclusions: Our TPA detection rate was high from PS ulcer exudates and SS skin biopsies and over 50% from SS blood, with TPA isolation in over two-thirds of samples. Our results support the use of WGS from rabbit isolates to inform vaccine development., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Importance: Lyme disease is a major tick-borne infection caused by a bacterial pathogen called Borrelia burgdorferi , which is transmitted by ticks and affects hundreds of thousands of people every year. These bacterial pathogens are distinct from other genera of microbes because of their distinct features and ability to transmit a multi-system infection to a range of vertebrates, including humans. Progress in understanding the infection biology of Lyme disease, and thus advancements towards its prevention, are hindered by an incomplete understanding of the microbiology of B. burgdorferi , partly due to the occurrence of many unique borrelial proteins that are structurally unrelated to proteins of known functions yet are indispensable for pathogen survival. We herein report the use of diverse technologies to examine the structure and function of a unique B. burgdorferi protein, annotated as BB0238-an essential virulence determinant. We show that the protein is structurally organized into two distinct domains, is involved in multiplex protein-protein interactions, and facilitates tick-to-mouse pathogen transmission by aiding microbial evasion of early host cellular immunity. We believe that our findings will further enrich our understanding of the microbiology of B. burgdorferi, potentially impacting the future development of novel prevention strategies against a widespread tick-transmitted infection., Competing Interests: The authors declare no conflict of interest.

Introduction: Syphilis, a sexually transmitted infection caused by the spirochete Treponema pallidum ( Tp ), is resurging globally. Tp 's repertoire of outer membrane proteins (OMPs) includes BamA (β-barrel assembly machinery subunit A/TP0326), a bipartite protein consisting of a 16-stranded β-barrel with nine extracellular loops (ECLs) and five periplasmic POTRA (polypeptide transport-associated) domains. BamA ECL4 antisera promotes internalization of Tp by rabbit peritoneal macrophages., Methods: Three overlapping BamA ECL4 peptides and a two-stage, phage display strategy, termed "Epivolve" (for epitope evolution) were employed to generate single-chain variable fragments (scFvs). Additionally, antisera generated by immunizing mice and rabbits with BamA ECL4 displayed by a Pyrococcus furiosus thioredoxin scaffold ( Pf Trx BamA/ECL4 ). MAbs and antisera reactivities were evaluated by immunoblotting and ELISA. A comparison of murine and rabbit opsonophagocytosis assays was conducted to evaluate the functional ability of the Abs ( e.g. , opsonization) and validate the mouse assay. Sera from Tp -infected mice (MSS) and rabbits (IRS) were evaluated for ECL4-specific Abs using Pf Trx BamA/ECL4 and overlapping ECL4 peptides in immunoblotting and ELISA assays., Results: Each of the five mAbs demonstrated reactivity by immunoblotting and ELISA to nanogram amounts of Pf Trx BamA/ECL4 . One mAb, containing a unique amino acid sequence in both the light and heavy chains, showed activity in the murine opsonophagocytosis assay. Mice and rabbits hyperimmunized with Pf Trx BamA/ECL4 produced opsonic antisera that strongly recognized the ECL presented in a heterologous scaffold and overlapping ECL4 peptides, including S2. In contrast, Abs generated during Tp infection of mice and rabbits poorly recognized the peptides, indicating that S2 contains a subdominant epitope., Discussion: Epivolve produced mAbs target subdominant opsonic epitopes in BamA ECL4, a top syphilis vaccine candidate. The murine opsonophagocytosis assay can serve as an alternative model to investigate the opsonic potential of vaccinogens. Detailed characterization of BamA ECL4-specific Abs provided a means to dissect Ab responses elicited by Tp infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ferguson, Delgado, McBride, Orbe, La Vake, Caimano, Mendez, Moraes, Schryvers, Moody, Radolf, Weiner and Hawley.)

Intestinal colonization with Klebsiella has been linked to necrotizing enterocolitis (NEC), but methods of analysis usually failed to discriminate Klebsiella species or strains. A novel ~ 2500-base amplicon (StrainID) that spans the 16S and 23S rRNA genes was used to generate amplicon sequence variant (ASV) fingerprints for Klebsiella oxytoca and Klebsiella pneumoniae species complexes (KoSC and KpSC, respectively) and co-occurring fecal bacterial strains from 10 preterm infants with NEC and 20 matched controls. Complementary approaches were used to identify cytotoxin-producing isolates of KoSC. Klebsiella species colonized most preterm infants, were more prevalent in NEC subjects versus controls, and replaced Escherichia in NEC subjects. Single KoSC or KpSC ASV fingerprinted strains dominated the gut microbiota, suggesting exclusionary Klebsiella competition for luminal resources. Enterococcus faecalis was co-dominant with KoSC but present infrequently with KpSC. Cytotoxin-producing KoSC members were identified in most NEC subjects and were less frequent in controls. Few Klebsiella strains were shared between subjects. We conclude that inter-species Klebsiella competition, within an environment of KoSC and E. faecalis cooperation, appears to be an important factor for the development of NEC. Preterm infants seem to acquire Klebsiella primarily through routes other than patient-to-patient transmission., (© 2023. The Author(s).)

The RNA polymerase alternative σ factor RpoS in Borrelia burgdorferi (Bb), the Lyme disease pathogen, is responsible for programmatic-positive and -negative gene regulation essential for the spirochete's dual-host enzootic cycle. RpoS is expressed during tick-to-mammal transmission and throughout mammalian infection. Although the mammalian-phase RpoS regulon is well described, its counterpart during the transmission blood meal is unknown. Here, we used Bb-specific transcript enrichment by tick-borne disease capture sequencing (TBDCapSeq) to compare the transcriptomes of WT and ΔrpoS Bb in engorged nymphs and following mammalian host-adaptation within dialysis membrane chambers. TBDCapSeq revealed dramatic changes in the contours of the RpoS regulon within ticks and mammals and further confirmed that RpoS-mediated repression is specific to the mammalian-phase of Bb's enzootic cycle. We also provide evidence that RpoS-dependent gene regulation, including repression of tick-phase genes, is required for persistence in mice. Comparative transcriptomics of engineered Bb strains revealed that the Borrelia oxidative stress response regulator (BosR), a noncanonical Fur family member, and the cyclic diguanosine monophosphate (c-di-GMP) effector PlzA reciprocally regulate the function of RNA polymerase complexed with RpoS. BosR is required for RpoS-mediated transcription activation and repression in addition to its well-defined role promoting transcription of rpoS by the RNA polymerase alternative σ factor RpoN. During transmission, ligand-bound PlzA antagonizes RpoS-mediated repression, presumably acting through BosR.

The resurgence of syphilis in the new millennium has called attention to the importance of a vaccine for global containment strategies. Studies with immune rabbit serum (IRS) indicate that a syphilis vaccine should elicit antibodies (Abs) that promote opsonophagocytosis of treponemes by activated macrophages. The availability of three-dimensional models for Treponema pallidum's ( Tp ) repertoire of outer membrane proteins (OMPs) provides an architectural framework for identification of candidate vaccinogens with extracellular loops (ECLs) as the targets for protective Abs. Herein, we used Pyrococcus furiosus thioredoxin ( Pf Trx) as a scaffold to display Tp OMP ECLs to interrogate sera and peripheral blood mononuclear cells (PBMCs) from immune rabbits for ECL-specific Abs and B cells. We validated this approach using a Pf Trx scaffold presenting ECL4 from BamA, a known opsonic target. Using scaffolds displaying ECLs of the FadL orthologs TP0856 and TP0858, we determined that ECL2 and ECL4 of both proteins are strongly antigenic. Comparison of ELISA and immunoblot results suggested that the Pf Trx scaffolds present conformational and linear epitopes. We then used the FadL ECL2 and ECL4 Pf Trx constructs as "hooks" to confirm the presence of ECL-specific B cells in PBMCs from immune rabbits. Our results pinpoint immunogenic ECLs of two newly discovered OMPs, while advancing the utility of the rabbit model for circumventing bottlenecks in vaccine development associated with large-scale production of folded OMPs. They also lay the groundwork for production of rabbit monoclonal Abs (MAbs) to characterize potentially protective ECL epitopes at the atomic level. IMPORTANCE Recent identification and structural modeling of Treponema pallidum's ( Tp ) repertoire of outer membrane proteins (OMPs) represent a critical breakthrough in the decades long quest for a syphilis vaccine. However, little is known about the antigenic nature of these β-barrel-forming OMPs and, more specifically, their surface exposed regions, the extracellular loops (ECLs). In this study, using Pyrococcus furiosus thioredoxin ( Pf Trx) as a scaffold to display Tp OMP ECLs, we interrogated immune rabbit sera and peripheral blood mononuclear cells for the presence of antibodies (Abs) and circulating rare antigen-specific B cells. Our results pinpoint immunogenic ECLs of two newly discovered OMPs, while advancing the utility of the rabbit model for surveying the entire Tp OMPeome for promising OMP vaccinogens. This work represents a major advancement toward characterizing potentially protective OMP ECLs and future vaccine studies. Additionally, this strategy could be applied to OMPs of nonspirochetal bacterial pathogens.

Leptospira interrogans, the causative agent of most cases of human leptospirosis, must respond to myriad environmental signals during its free-living and pathogenic lifestyles. Previously, we compared L. interrogans cultivated in vitro and in vivo using a dialysis membrane chamber (DMC) peritoneal implant model. From these studies emerged the importance of genes encoding the Peroxide responsive regulators PerRA and PerRB. First described in in Bacillus subtilis, PerRs are widespread in Gram-negative and -positive bacteria, where regulate the expression of gene products involved in detoxification of reactive oxygen species and virulence. Using perRA and perRB single and double mutants, we establish that L. interrogans requires at least one functional PerR for infectivity and renal colonization in a reservoir host. Our finding that the perRA/B double mutant survives at wild-type levels in DMCs is noteworthy as it demonstrates that the loss of virulence is not due to a metabolic lesion (i.e., metal starvation) but instead reflects dysregulation of virulence-related gene products. Comparative RNA-Seq analyses of perRA, perRB and perRA/B mutants cultivated within DMCs identified 106 genes that are dysregulated in the double mutant, including ligA, ligB and lvrA/B sensory histidine kinases. Decreased expression of LigA and LigB in the perRA/B mutant was not due to loss of LvrAB signaling. The majority of genes in the perRA and perRB single and double mutant DMC regulons were differentially expressed only in vivo, highlighting the importance of host signals for regulating gene expression in L. interrogans. Importantly, the PerRA, PerRB and PerRA/B DMC regulons each contain multiple genes related to environmental sensing and/or transcriptional regulation. Collectively, our data suggest that PerRA and PerRB are part of a complex regulatory network that promotes host adaptation by L. interrogans within mammals., Competing Interests: The authors have declared that no competing interests exist.