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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.

In this study, we examined the relationship between c-di-GMP and its only known effector protein, PlzA, in Borrelia burgdorferi during the arthropod and mammalian phases of the enzootic cycle. Using a B. burgdorferi strain expressing a plzA point mutant (plzA-R145D) unable to bind c-di-GMP, we confirmed that the protective function of PlzA in ticks is c-di-GMP-dependent. Unlike ΔplzA spirochetes, which are severely attenuated in mice, the plzA-R145D strain was fully infectious, firmly establishing that PlzA serves a c-di-GMP-independent function in mammals. Contrary to prior reports, loss of PlzA did not affect expression of RpoS or RpoS-dependent genes, which are essential for transmission, mammalian host-adaptation and murine infection. To ascertain the nature of PlzA's c-di-GMP-independent function(s), we employed infection models using (i) host-adapted mutant spirochetes for needle inoculation of immunocompetent mice and (ii) infection of scid mice with in vitro-grown organisms. Both approaches substantially restored ΔplzA infectivity, suggesting that PlzA enables B. burgdorferi to overcome an early bottleneck to infection. Furthermore, using a Borrelia strain expressing a heterologous, constitutively active diguanylate cyclase, we demonstrate that 'ectopic' production of c-di-GMP in mammals abrogates spirochete virulence and interferes with RpoS function at the post-translational level in a PlzA-dependent manner. Structural modeling and SAXS analysis of liganded- and unliganded-PlzA revealed marked conformational changes that underlie its biphasic functionality. This structural plasticity likely enables PlzA to serve as a c-di-GMP biosensor that in its respective liganded and unliganded states promote vector- and host-adaptation by the Lyme disease spirochete., Competing Interests: The authors have declared that no competing interests exist.

Treponema pallidum, an obligate human pathogen, has an outer membrane (OM) whose physical properties, ultrastructure, and composition differ markedly from those of phylogenetically distant Gram-negative bacteria. We developed structural models for the outer membrane protein (OMP) repertoire (OMPeome) of T. pallidum Nichols using solved Gram-negative structures, computational tools, and small-angle X-ray scattering (SAXS) of selected recombinant periplasmic domains. The T. pallidum "OMPeome" harbors two "stand-alone" proteins (BamA and LptD) involved in OM biogenesis and four paralogous families involved in the influx/efflux of small molecules: 8-stranded β-barrels, long-chain-fatty-acid transporters (FadLs), OM factors (OMFs) for efflux pumps, and T. pallidum repeat proteins (Tprs). BamA (TP0326), the central component of a β-barrel assembly machine (BAM)/translocation and assembly module (TAM) hybrid, possesses a highly flexible polypeptide-transport-associated (POTRA) 1-5 arm predicted to interact with TamB (TP0325). TP0515, an LptD ortholog, contains a novel, unstructured C-terminal domain that models inside the β-barrel. T. pallidum has four 8-stranded β-barrels, each containing positively charged extracellular loops that could contribute to pathogenesis. Three of five FadL-like orthologs have a novel α-helical, presumptively periplasmic C-terminal extension. SAXS and structural modeling further supported the bipartite membrane topology and tridomain architecture of full-length members of the Tpr family. T. pallidum's two efflux pumps presumably extrude noxious small molecules via four coexpressed OMFs with variably charged tunnels. For BamA, LptD, and OMFs, we modeled the molecular machines that deliver their substrates into the OM or external milieu. The spirochete's extended families of OM transporters collectively confer a broad capacity for nutrient uptake. The models also furnish a structural road map for vaccine development. IMPORTANCE The unusual outer membrane (OM) of T. pallidum, the syphilis spirochete, is the ultrastructural basis for its well-recognized capacity for invasiveness, immune evasion, and persistence. In recent years, we have made considerable progress in identifying T. pallidum's repertoire of OMPs. Here, we developed three-dimensional (3D) models for the T. pallidum Nichols OMPeome using structural modeling, bioinformatics, and solution scattering. The OM contains three families of OMP transporters, an OMP family involved in the extrusion of noxious molecules, and two "stand-alone" proteins involved in OM biogenesis. This work represents a major advance toward elucidating host-pathogen interactions during syphilis; understanding how T. pallidum, an extreme auxotroph, obtains a wide array of biomolecules from its obligate human host; and developing a vaccine with global efficacy.

Spores of firmicute species contain 100s of mRNAs, whose major function in Bacillus subtilis is to provide ribonucleotides for new RNA synthesis when spores germinate. To determine if this is a general phenomenon, RNA was isolated from spores of multiple firmicute species and relative mRNA levels determined by transcriptome sequencing (RNA-seq). Determination of RNA levels in single spores allowed calculation of RNA nucleotides/spore, and assuming mRNA is 3% of spore RNA indicated that only ∼6% of spore mRNAs were present at >1/spore. Bacillus subtilis, Bacillus atrophaeus, and Clostridioides difficile spores had 49, 42, and 51 mRNAs at >1/spore, and numbers of mRNAs at ≥1/spore were ∼10 to 50% higher in Geobacillus stearothermophilus and Bacillus thuringiensis Al Hakam spores and ∼4-fold higher in Bacillus megaterium spores. In all species, some to many abundant spore mRNAs (i) were transcribed by RNA polymerase with forespore-specific σ factors, (ii) encoded proteins that were homologs of those encoded by abundant B. subtilis spore mRNAs and are proteins in dormant spores, and (iii) were likely transcribed in the mother cell compartment of the sporulating cell. Analysis of the coverage of RNA-seq reads on mRNAs from all species suggested that abundant spore mRNAs were fragmented, as was confirmed by reverse transcriptase quantitative PCR (RT-qPCR) analysis of abundant B. subtilis and C. difficile spore mRNAs. These data add to evidence indicating that the function of at least the great majority of mRNAs in all firmicute spores is to be degraded to generate ribonucleotides for new RNA synthesis when spores germinate. IMPORTANCE Only ∼6% of mRNAs in spores of six firmicute species are at ≥1 molecule/spore, many abundant spore mRNAs encode proteins similar to B. subtilis spore proteins, and some abundant B. subtilis and C. difficile spore mRNAs were fragmented. Most of the abundant B. subtilis and other Bacillales spore mRNAs are transcribed under the control of the forespore-specific RNA polymerase σ factors, F or G, and these results may stimulate transcription analyses in developing spores of species other than B. subtilis. These findings, plus the absence of key nucleotide biosynthetic enzymes in spores, suggest that firmicute spores' abundant mRNAs are not translated when spores germinate but instead are degraded to generate ribonucleotides for new RNA synthesis by the germinated spore.

Background: Whole-genome sequencing (WGS) of Treponema pallidum subspecies pallidum (TPA) has been constrained by the lack of in vitro cultivation methods for isolating spirochetes from patient samples., Methods: We built upon recently developed enrichment methods to sequence TPA directly from primary syphilis chancre swabs collected in Guangzhou, China., Results: By combining parallel, pooled whole-genome amplification with hybrid selection, we generated high-quality genomes from 4 of 8 chancre-swab samples and 2 of 2 rabbit-passaged isolates, all subjected to challenging storage conditions., Conclusions: This approach enabled the first WGS of Chinese samples without rabbit passage and provided insights into TPA genetic diversity in China., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Identifying and tracking microbial strains as microbiomes evolve are major challenges in the field of microbiome research. We utilized a new sequencing kit that combines DNA extraction with PCR amplification of a large region of the rRNA operon and downstream bioinformatic data analysis. Longitudinal microbiome samples of coadmitted twins from two different neonatal intensive care units (NICUs) were analyzed using an ∼2,500-base amplicon that spans the 16S and 23S rRNA genes and mapped to a new, custom 16S-23S rRNA database. Amplicon sequence variants (ASVs) inferred using DADA2 provided sufficient resolution for the differentiation of rRNA variants from closely related but not previously sequenced Klebsiella , Escherichia coli , and Enterobacter strains, among the first bacteria colonizing the gut of these infants after admission to the NICU. Distinct ASV groups (fingerprints) were monitored between coadmitted twins over time, demonstrating the potential to track the source and spread of both commensals and pathogens. The high-resolution taxonomy obtained from long amplicon sequencing enables the tracking of strains temporally and spatially as microbiomes are established in infants in the hospital environment. IMPORTANCE Achieving strain-level resolution is a major obstacle for source tracking and temporal studies of microbiomes. In this study, we describe a novel deep-sequencing approach that provides species- and strain-level resolution of the neonatal microbiome. Using Klebsiella , E. coli , and Enterobacter as examples, we could monitor their temporal dynamics after antibiotic treatment and in pairs of twins. The strain-level resolution, combined with the greater sequencing depth and decreased cost per read of PacBio Sequel 2, enables this advantageous source- and strain-tracking analysis method to be implemented widely across more complex microbiomes., (Copyright © 2021 Graf et al.)

Borrelia ( Borreliella ) burgdorferi , along with closely related species, is the etiologic agent of Lyme disease. The spirochete subsists in an enzootic cycle that encompasses acquisition from a vertebrate host to a tick vector and transmission from a tick vector to a vertebrate host. To adapt to its environment and persist in each phase of its enzootic cycle, B. burgdorferi wields three systems to regulate the expression of genes: the RpoN-RpoS alternative sigma factor cascade, the Hk1/Rrp1 two-component system and its product c-di-GMP, and the stringent response mediated by Rel Bbu and DksA. These regulatory systems respond to enzootic phase-specific signals and are controlled or fine- tuned by transcription factors, including BosR and BadR, as well as small RNAs, including DsrABb and Bb6S RNA. In addition, several other DNA-binding and RNA-binding proteins have been identified, although their functions have not all been defined. Global changes in gene expression revealed by high-throughput transcriptomic studies have elucidated various regulons, albeit technical obstacles have mostly limited this experimental approach to cultivated spirochetes. Regardless, we know that the spirochete, which carries a relatively small genome, regulates the expression of a considerable number of genes required for the transitions between the tick vector and the vertebrate host as well as the adaptation to each.

During the natural enzootic life cycle of Borrelia burgdorferi (also known as Borreliella burgdorferi ), the bacteria must sense conditions within the vertebrate and arthropod and appropriately regulate expression of genes necessary to persist within these distinct environments. bb0345 of B. burgdorferi encodes a hypothetical protein of unknown function that is predicted to contain an N-terminal helix-turn-helix (HTH) domain. Because HTH domains can mediate protein-DNA interactions, we hypothesized that BB0345 might represent a previously unidentified borrelial transcriptional regulator with the ability to regulate events critical for the B. burgdorferi enzootic cycle. To study the role of BB0345 within mammals, we generated a bb0345 mutant and assessed its virulence potential in immunocompetent mice. The bb0345 mutant was able to initiate localized infection and disseminate to distal tissues but was cleared from all sites by 14 days postinfection. In vitro growth curve analyses revealed that the bb0345 mutant grew similar to wild-type bacteria in standard Barbour-Stoenner-Kelley II (BSK-II) medium; however, the mutant was not able to grow in dilute BSK-II medium or dialysis membrane chambers (DMCs) implanted in rats. Proteinase K accessibility assays and whole-cell partitioning indicated that BB0345 was intracellular and partially membrane associated. Comparison of protein production profiles between the wild-type parent and the bb0345 mutant revealed no major differences, suggesting BB0345 may not be a global transcriptional regulator. Taken together, these data show that BB0345 is essential for B. burgdorferi survival in the mammalian host, potentially by aiding the spirochete with a physiological function that is required by the bacterium during infection., (Copyright © 2020 American Society for Microbiology.)

Deconvolution of syphilis pathogenesis and selection of candidate syphilis vaccinogens requires detailed knowledge of the molecular architecture of the Treponema pallidum outer membrane (OM). The T. pallidum OM contains a low density of integral OM proteins, while the spirochete's many lipoprotein immunogens are periplasmic. TP0751, a lipoprotein with a lipocalin fold, is reportedly a surface-exposed protease/adhesin and protective antigen. The rapid expansion of calycin/lipocalin structures in the RCSB PDB database prompted a comprehensive reassessment of TP0751. Small angle X-ray scattering analysis of full-length protein revealed a bipartite topology consisting of an N-terminal, intrinsically disordered region (IDR) and the previously characterized C-terminal lipocalin domain. A DALI server query using the lipocalin domain yielded 97 hits, 52 belonging to the calycin superfamily, including 15 bacterial lipocalins, but no Gram-negative surface proteins. Surprisingly, Tpp17 (TP0435) was identified as a structural ortholog of TP0751. In silico docking predicted that TP0751 can bind diverse ligands along the rim of its eight-stranded β-barrel; high affinity binding of one predicted ligand, heme, to the lipocalin domain was demonstrated. qRT-PCR and immunoblotting revealed very low expression of TP0751 compared to other T. pallidum lipoproteins. Immunoblot analysis of immune rabbit serum failed to detect TP0751 antibodies, while only one of five patients with secondary syphilis mounted a discernible TP0751-specific antibody response. In opsonophagocytosis assays, neither TP0751 nor Tpp17 antibodies promoted uptake of T. pallidum by rabbit peritoneal macrophages. Rabbits immunized with intact, full-length TP0751 showed no protection against local or disseminated infection following intradermal challenge with T. pallidum. Our data argue that, like other lipoprotein lipocalins in dual-membrane bacteria, TP0751 is periplasmic and binds small molecules, and we propose that its IDR facilitates ligand binding by and offloading from the lipocalin domain. The inability of TP0751 to elicit opsonic or protective antibodies is consistent with a subsurface location., Competing Interests: The authors have declared that no competing interests exist.

Spores of Gram-positive bacteria contain 10s-1000s of different mRNAs. However, Bacillus subtilis spores contain only ∼ 50 mRNAs at > 1 molecule/spore, almost all transcribed only in the developing spore and encoding spore proteins. However, some spore mRNAs could be stabilized to ensure they are intact in dormant spores, perhaps to direct synthesis of proteins essential for spores' conversion to a growing cell in germinated spore outgrowth. Recent work shows that some growing B. subtilis cell mRNAs contain a 5'-NAD cap. Since this cap may stabilize mRNA in vivo, its presence on spore mRNAs would suggest that maintaining some intact spore mRNAs is important, perhaps because they have a translational role in outgrowth. However, significant levels of only a few abundant spore mRNAs had a 5'-NAD cap, and these were not the most stable spore mRNAs and had likely been fragmented. Even higher levels of 5'-NAD-capping were found on a few low abundance spore mRNAs, but these mRNAs were present in only small percentages of spores, and had again been fragmented. The new data are thus consistent with spore mRNAs serving only as a reservoir of ribonucleotides in outgrowth., (© FEMS 2020.)

Streptococcal glucosyltransferases (Gtf) synthesize α-glucan exopolymers which contribute to biofilm matrix. Streptococcus oralis interacts with the opportunistic pathogen Candida albicans to form hypervirulent biofilms. S. oralis 34 has a single gtf gene (gtfR). However, the role of gtfR in single and mixed species biofilms with C. albicans has never been examined. A gtfR deletion mutant, purified GtfR, and recombinant GtfR glucan-binding domain were tested in single and mixed biofilms on different substrata in vitro. A mouse oral infection model was also used. We found that in single species biofilms growing with sucrose on abiotic surfaces S. oralis gtfR increased biofilm matrix, but not bacterial biomass. In biofilms with C. albicans, S. oralis encoding gtfR showed increased bacterial biomass on all surfaces. C. albicans had a positive effect on α-glucan synthesis, and α-glucans increased C. albicans accretion on abiotic surfaces. In single and mixed infection of mice receiving sucrose S. oralis gtfR enhanced mucosal burdens. However, sucrose had a negative impact on C. albicans burdens and reduced S. oralis burdens in co-infected mice. Our data provide new insights on the GtfR-mediated interactions between the two organisms and the influence of biofilm substratum and the mucosal environment on these interactions.

Objectives: To define gut microbial patterns in preterm infants with and without necrotizing enterocolitis (NEC) and to characterize clinical factors related to the composition of the preterm intestinal microbiome. Methods: Fecal samples were collected at one-week intervals from infants with gestational ages <30 weeks at a single level IV neonatal intensive care unit. Using 16S rRNA gene sequencing, the composition and diversity of microbiota were determined in samples collected from five NEC infants and five matched controls. Hierarchical linear regression was used to identify clinical factors related to microbial diversity and specific bacterial signatures. Results: Low levels of diversity were demonstrated in samples obtained from all preterm infants and antibiotic exposure further decreased diversity among both NEC cases and controls. Fecal microbial composition differed between NEC cases and controls, with a greater abundance of Proteobacteria and bacteria belonging to the class Gammaproteobacteria among NEC infants. Control infants demonstrated a greater abundance of bacteria belonging to the phylum Firmicutes . Conclusion: These findings indicate that an association exists between intestinal Proteobacteria and NEC, and strengthens the notion that an overly exuberant response to Gram-negative products, particularly lipopolysaccharide, in the preterm intestine is involved in NEC pathogenesis. Cumulative exposure to antibiotics corresponded to a reduction in microbial diversity in both NEC cases and controls.

In order to sustain its zoonotic lifecycle, leptospires must adapt to growth within the host milieu. Signals encountered within the mammal also trigger regulatory programs required by Leptospira for the expression of virulence-related gene products. The complex transcriptional, antigenic, and physiological changes leptospires undergo within the mammal are collectively referred to as "host adaptation." In this chapter, we describe the procedures for the generation of host-adapted Leptospira spp. by cultivation within dialysis membrane chambers (DMCs) implanted in rat peritoneal cavities. In this model, Leptospira spp. diluted in EMJH medium are sequestered within sterile dialysis membrane tubing closed at both ends. The chamber then is surgically implanted within the peritoneal cavity of a rat and incubated for 7-10 days. During this period, leptospires are exposed to many, if not all, of the physiological and nutritional cues required for host adaptation while at the same time protected from clearance by host innate and adaptive immune defenses.

Maintenance of Borrelia burgdorferi within its enzootic cycle requires a complex regulatory pathway involving the alternative σ factors RpoN and RpoS and two ancillary trans -acting factors, BosR and Rrp2. Activation of this pathway occurs within ticks during the nymphal blood meal when RpoS, the effector σ factor, transcribes genes required for tick transmission and mammalian infection. RpoS also exerts a 'gatekeeper' function by repressing σ 70 -dependent tick phase genes (e.g., ospA , lp6.6 ). Herein, we undertook a broad examination of RpoS functionality throughout the enzootic cycle, beginning with modeling to confirm that this alternative σ factor is a 'genuine' RpoS homolog. Using a novel dual color reporter system, we established at the single spirochete level that ospA is expressed in nymphal midguts throughout transmission and is not downregulated until spirochetes have been transmitted to a naïve host. Although it is well established that rpoS /RpoS is expressed throughout infection, its requirement for persistent infection has not been demonstrated. Plasmid retention studies using a trans -complemented Δ rpoS mutant demonstrated that (i) RpoS is required for maximal fitness throughout the mammalian phase and (ii) RpoS represses tick phase genes until spirochetes are acquired by a naïve vector. By transposon mutant screening, we established that bba34/oppA5 , the only OppA oligopeptide-binding protein controlled by RpoS, is a bona fide persistence gene. Lastly, comparison of the strain 297 and B31 RpoS DMC regulons identified two cohorts of RpoS-regulated genes. The first consists of highly conserved syntenic genes that are similarly regulated by RpoS in both strains and likely required for maintenance of B. burgdorferi sensu stricto strains in the wild. The second includes RpoS-regulated plasmid-encoded variable surface lipoproteins ospC , dbpA and members of the ospE/ospF/elp , mlp , revA , and Pfam54 paralogous gene families, all of which have evolved via inter- and intra-strain recombination. Thus, while the RpoN/RpoS pathway regulates a 'core' group of orthologous genes, diversity within RpoS regulons of different strains could be an important determinant of reservoir host range as well as spirochete virulence.

Introduction: Premature infants often require parenteral nutrition (PN) until they reach enteral autonomy which puts them at risk of developing PN-associated cholestasis (PNAC). We sought to compare longitudinal changes in fecal microbiomes of premature infants who developed PNAC versus those who did not despite being on similar PN doses., Methods: Stool samples from premature infants (gestational age <30 weeks) who developed direct bilirubin ≥1.5 mg/dL while receiving PN were classified as precholestasis, cholestasis, or postcholestasis based on bilirubin levels at the time of sample acquisition and were compared to matched control groups 1, 2, and 3, respectively., Results: A total of 102 fecal samples from 8 cases and 10 controls were analyzed. Precholestasis samples were more abundant in phylum Firmicutes and genus Staphylococcus, whereas control 1 was more abundant in phylum Proteobacteria and genus Escherichia-Shigella. Nonmetric multidimensional scaling ordination plots based on the taxonomic composition of early fecal samples revealed significant separation between cases and controls. On indicator species analysis, genus Bacilli was more prevalent in samples from the precholestasis group, whereas genus Escherichia-Shigella was more prevalent in control 1. With feeding advances, weaning of PN and resolution of PNAC, most differences in microbiota resolved with the exception of control 3 group being more diverse compared to the postcholestasis group., Conclusions: Premature neonates who develop PNAC, compared to those who do not, show significantly different fecal microbiomes preceding the biochemical detection of cholestasis.

Bacillus spores incubated on plates for 2 to 98 days at 37°C had identical Ca-dipicolinic acid contents, exhibited identical viability on rich- or poor-medium plates, germinated identically in liquid with all germinants tested, identically returned to vegetative growth in rich or minimal medium, and exhibited essentially identical resistance to dry heat and similar resistance to UV radiation. However, the oldest spores had a lower core water content and significantly higher wet heat and NaOCl resistance. In addition, 47- and 98-day spores had lost >98% of intact 16S and 23S rRNA and 97 to 99% of almost all mRNAs, although minimal amounts of mononucleotides were generated in 91 days. Levels of 3-phosphoglyceric acid (3PGA) also fell 30 to 60% in the oldest spores, but how the 3PGA was lost is not clear. These results indicate that (i) translation of dormant spore mRNA is not essential for completion of spore germination, nor is protein synthesis from any mRNA; (ii) in sporulation for up to 91 days at 37°C, the RNA broken down generates minimal levels of mononucleotides; and (iii) the lengths of time that spores are incubated in sporulation medium should be considered when determining conditions for spore inactivation by wet heat, in particular, in using spores to test for the efficacy of sterilization regimens. IMPORTANCE We show that spores incubated at 37°C on sporulation plates for up to 98 days have lost almost all mRNAs and rRNAs, yet the aged spores germinated and outgrew as well as 2-day spores, and all these spores had identical viability. Thus, it is unlikely that spore mRNA, rRNA, or protein synthesis is important in spore germination. Spores incubated for 47 to 98 days also had much higher wet heat resistance than 2-day spores, suggesting that spore "age" should be considered in generating spores for tests of sterilization assurance. These data are the first to show complete survival of hydrated spores for ∼100 days, complementing published data showing dry-spore survival for years., (Copyright © 2019 American Society for Microbiology.)

Large-scale shotgun sequencing (RNA-seq) analysis of mRNAs in dormant Bacillus subtilis spores prepared on plates or in liquid generally found the same ∼46 abundant mRNA species, with >250 mRNAs detected at much lower abundances. Knowledge of the amount of phosphate in a single B. subtilis spore allowed calculation of the amount of mRNA in an individual spore as ∼10 6  nucleotides (nt). Given the levels of abundant spore mRNAs compared to those of other mRNAs, it was calculated that the great majority of low-abundance mRNAs are present in only small fractions of spores in populations. Almost all of the most abundant spore mRNAs are encoded by genes expressed late in sporulation in the developing spore under the control of the forespore-specific RNA polymerase sigma factor, σ G , and most of the encoded proteins are in spores. Levels of the most abundant spore mRNAs were also relatively stable for a week at 4°C after spore harvest. RNA-seq analysis of mRNAs in highly purified and less-well-purified spores made in liquid, as well as from spores that were chemically decoated to remove possible contaminating mRNA, indicated that low-abundance mRNAs in spores were not contaminants in purified spore preparations, and several sources of low-abundance mRNAs in spores are suggested. The function of at least the great majority of spore mRNAs seems most likely to be the generation of ribonucleotides for new RNA synthesis by their degradation early in spore revival. IMPORTANCE Previous work indicates that dormant Bacillus subtilis spores have many hundreds of mRNAs, some of which are suggested to play roles in spores' "return to life" or revival. The present work finds only ∼46 mRNAs at ≥1 molecule spore, with others in only fractions of spores in populations, often very small fractions. Less-abundant spore mRNAs are not contaminants in spore preparations, but how spores accumulate them is not clear. Almost all abundant spore mRNAs are synthesized in the developing spore late in its development, most encode proteins in spores, and abundant mRNAs in spores are relatively stable at 4°C. These findings will have a major impact on thinking about the roles that spore mRNAs may play in spore revival., (Copyright © 2019 American Society for Microbiology.)

In recent years, considerable progress has been made in topologically and functionally characterizing integral outer membrane proteins (OMPs) of Treponema pallidum subspecies pallidum , the syphilis spirochete, and identifying its surface-exposed β-barrel domains. Extracellular loops in OMPs of Gram-negative bacteria are known to be highly variable. We examined the sequence diversity of β-barrel-encoding regions of tprC , tprD , and bamA in 31 specimens from Cali, Colombia; San Francisco, California; and the Czech Republic and compared them to allelic variants in the 41 reference genomes in the NCBI database. To establish a phylogenetic framework, we used T. pallidum 0548 ( tp0548 ) genotyping and tp0558 sequences to assign strains to the Nichols or SS14 clades. We found that (i) β-barrels in clinical strains could be grouped according to allelic variants in T. pallidum subsp. pallidum reference genomes; (ii) for all three OMP loci, clinical strains within the Nichols or SS14 clades often harbored β-barrel variants that differed from the Nichols and SS14 reference strains; and (iii) OMP variable regions often reside in predicted extracellular loops containing B-cell epitopes. On the basis of structural models, nonconservative amino acid substitutions in predicted transmembrane β-strands of T. pallidum repeat C (TprC) and TprD2 could give rise to functional differences in their porin channels. OMP profiles of some clinical strains were mosaics of different reference strains and did not correlate with results from enhanced molecular typing. Our observations suggest that human host selection pressures drive T. pallidum subsp. pallidum OMP diversity and that genetic exchange contributes to the evolutionary biology of T. pallidum subsp. pallidum They also set the stage for topology-based analysis of antibody responses to OMPs and help frame strategies for syphilis vaccine development. IMPORTANCE Despite recent progress characterizing outer membrane proteins (OMPs) of Treponema pallidum , little is known about how their surface-exposed, β-barrel-forming domains vary among strains circulating within high-risk populations. In this study, sequences for the β-barrel-encoding regions of three OMP loci, tprC , tprD , and bamA , in T. pallidum subsp. pallidum isolates from a large number of patient specimens from geographically disparate sites were examined. Structural models predict that sequence variation within β-barrel domains occurs predominantly within predicted extracellular loops. Amino acid substitutions in predicted transmembrane strands that could potentially affect porin channel function were also noted. Our findings suggest that selection pressures exerted within human populations drive T. pallidum subsp. pallidum OMP diversity and that recombination at OMP loci contributes to the evolutionary biology of syphilis spirochetes. These results also set the stage for topology-based analysis of antibody responses that promote clearance of T. pallidum subsp. pallidum and frame strategies for vaccine development based upon conserved OMP extracellular loops., (Copyright © 2018 Kumar et al.)

The transmission, survival, and virulence of Borrelia burgdorferi depend upon the spirochete's ability to modulate its transcriptome as it cycles between its arthropod vector and reservoir host. This complex adaptive process is collectively referred to as "host-adaptation." The paucibacillary nature of borrelial infections precludes the detailed analysis of host adaptation within infected mammalian tissues. To circumvent this limitation, we (J Clin Invest 101:2240-2250, 1998) developed a model system whereby spirochetes are cultivated within dialysis membrane chambers (DMCs) surgically implanted within the peritoneal cavity of a rat. Spirochetes within DMCs are exposed to many, if not all, of the environmental signals and physiological cues required for mammalian host adaptation but are protected from clearance by the host's immune system.

Borrelia burgdorferi is an extreme amino acid (AA) auxotroph whose genome encodes few free AA transporters and an elaborate oligopeptide transport system ( B. burgdorferi Opp [ Bb Opp]). Bb Opp consists of five oligopeptide-binding proteins (OBPs), two heterodimeric permeases, and a heterodimeric nucleotide-binding domain (NBD). Homology modeling based on the crystal structure of liganded Bb OppA4 revealed that each OBP likely binds a distinct range of peptides. Transcriptional analyses demonstrated that the OBPs are differentially and independently regulated whereas the permeases and NBDs are constitutively expressed. A conditional NBD mutant failed to divide in the absence of inducer and replicated in an IPTG (isopropyl-β-d-thiogalactopyranoside) concentration-dependent manner. NBD mutants grown without IPTG exhibited an elongated morphotype lacking division septa, often with flattening at the cell center due to the absence of flagellar filaments. Following cultivation in dialysis membrane chambers, NBD mutants recovered from rats not receiving IPTG also displayed an elongated morphotype. The NBD mutant was avirulent by needle inoculation, but infectivity was partially restored by oral administration of IPTG to infected mice. We conclude that peptides are a major source of AAs for B. burgdorferi both in vitro and in vivo and that peptide uptake is essential for regulation of morphogenesis, cell division, and virulence. IMPORTANCE Borrelia burgdorferi , the causative agent of Lyme disease, is an extreme amino acid (AA) auxotroph with a limited repertoire of annotated single-AA transporters. A major issue is how the spirochete meets its AA requirements as it transits between its arthropod vector and mammalian reservoir. While previous studies have confirmed that the B. burgdorferi oligopeptide transport ( opp ) system is capable of importing peptides, the importance of the system for viability and pathogenesis has not been established. Here, we evaluated the opp system structurally and transcriptionally to elucidate its ability to import a wide range of peptides during the spirochete's enzootic cycle. Additionally, using a novel mutagenesis strategy to abrogate opp transporter function, we demonstrated that peptide uptake is essential for bacterial viability, morphogenesis, and infectivity. Our studies revealed a novel link between borrelial physiology and virulence and suggest that peptide uptake serves an intracellular signaling function regulating morphogenesis and division., (Copyright © 2017 Groshong et al.)

The major outer sheath protein (MOSP) is a prominent constituent of the cell envelope of Treponema denticola (TDE) and one of its principal virulence determinants. Bioinformatics predicts that MOSP consists of N- and C-terminal domains, MOSP N and MOSP C . Biophysical analysis of constructs refolded in vitro demonstrated that MOSP C , previously shown to possess porin activity, forms amphiphilic trimers, while MOSP N forms an extended hydrophilic monomer. In TDE and E. coli expressing MOSP with a PelB signal sequence (PelB-MOSP), MOSP C is OM-embedded and surface-exposed, while MOSP N resides in the periplasm. Immunofluorescence assay, surface proteolysis, and novel cell fractionation schemes revealed that MOSP in TDE exists as outer membrane (OM) and periplasmic trimeric conformers; PelB-MOSP, in contrast, formed only OM-MOSP trimers. Although both conformers form hetero-oligomeric complexes in TDE, only OM-MOSP associates with dentilisin. Mass spectrometry (MS) indicated that OM-MOSP interacts with proteins in addition to dentilisin, most notably, oligopeptide-binding proteins (OBPs) and the β-barrel of BamA. MS also identified candidate partners for periplasmic MOSP, including TDE1658, a spirochete-specific SurA/PrsA ortholog. Collectively, our data suggest that MOSP destined for the TDE OM follows the canonical BAM pathway, while formation of a stable periplasmic conformer involves an export-related, folding pathway not present in E. coli.

Syphilis is a multi-stage, sexually transmitted disease caused by the spirochete Treponema pallidum ( Tp ). Considered broadly, syphilis can be conceptualized as a dualistic process in which spirochete-driven inflammation, the cause of clinical manifestations, coexists to varying extents with bacterial persistence. Inflammation is elicited in the tissues, along with the persistence of spirochetes to keep driving a robust immune response while evading host defenses; this duality is best exemplified during the florid, disseminated stage called secondary syphilis (SS). SS lesions typically contain copious amounts of spirochetes along with a mixed cellular infiltrate consisting of CD4 + T cells, CD8 + T cells, NK cells, plasma cells, and macrophages. In the rabbit model, Tp are cleared by macrophages via antibody-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. Herein, we used monocyte-derived macrophages to study Tp -macrophage interactions ex vivo . In the absence of HSS, monocyte-derived macrophages internalized low numbers of Tp and secreted little cytokine (e.g., TNF). By contrast, these same macrophages internalized large numbers of unopsonized Borrelia burgdorferi and secreted robust levels of cytokines. Maturation of macrophages with M-CSF and IFNγ resulted in a macrophage phenotype with increased expression of HLA-DR, CD14, inducible nitric oxide synthase, TLR2, TLR8, and the Fcγ receptors (FcγR) CD64 and CD16, even in the absence of LPS. Importantly, IFNγ-polarized macrophages resulted in a statistically significant increase in opsonophagocytosis of Tp accompanied by enhanced production of cytokines, macrophage activation markers (CD40, CD80), TLRs (TLR2, TLR7, TLR8), chemokines (CCL19, CXCL10, CXCL11), and T H 1-promoting cytokines (IL-12, IL-15). Finally, the blockade of FcγRs, primarily CD64, significantly diminished spirochetal uptake and proinflammatory cytokine secretion by IFNγ-stimulated macrophages. Our ex vivo studies demonstrate the importance of CD64-potentiated uptake of opsonized Tp and suggest that IFNγ-activated macrophages have an important role in the context of early syphilis. Our study results also provide an ex vivo surrogate system for use in future syphilis vaccine studies.

The alternative sigma factor RpoS plays a key role modulating gene expression in Borrelia burgdorferi , the Lyme disease spirochete, by transcribing mammalian host-phase genes and repressing σ 70 -dependent genes required within the arthropod vector. To identify cis regulatory elements involved in RpoS-dependent repression, we analyzed green fluorescent protein (GFP) transcriptional reporters containing portions of the upstream regions of the prototypical tick-phase genes ospAB , the glp operon, and bba74 As RpoS-mediated repression occurs only following mammalian host adaptation, strains containing the reporters were grown in dialysis membrane chambers (DMCs) implanted into the peritoneal cavities of rats. Wild-type spirochetes harboring ospAB - and glp-gfp constructs containing only the minimal (-35/-10) σ 70 promoter elements had significantly lower expression in DMCs relative to growth in vitro at 37°C; no reduction in expression occurred in a DMC-cultivated RpoS mutant harboring these constructs. In contrast, RpoS-mediated repression of bba74 required a stretch of DNA located between -165 and -82 relative to its transcriptional start site. Electrophoretic mobility shift assays employing extracts of DMC-cultivated B. burgdorferi produced a gel shift, whereas extracts from RpoS mutant spirochetes did not. Collectively, these data demonstrate that RpoS-mediated repression of tick-phase borrelial genes occurs by at least two distinct mechanisms. One (e.g., ospAB and the glp operon) involves primarily sequence elements near the core promoter, while the other (e.g., bba74 ) involves an RpoS-induced transacting repressor. Our results provide a genetic framework for further dissection of the essential "gatekeeper" role of RpoS throughout the B. burgdorferi enzootic cycle. IMPORTANCE Borrelia burgdorferi , the Lyme disease spirochete, modulates gene expression to adapt to the distinctive environments of its mammalian host and arthropod vector during its enzootic cycle. The alternative sigma factor RpoS has been referred to as a "gatekeeper" due to its central role in regulating the reciprocal expression of mammalian host- and tick-phase genes. While RpoS-dependent transcription has been studied extensively, little is known regarding the mechanism(s) of RpoS-mediated repression. We employed a combination of green fluorescent protein transcriptional reporters along with an in vivo model to define cis regulatory sequences responsible for RpoS-mediated repression of prototypical tick-phase genes. Repression of ospAB and the glp operon requires only sequences near their core promoters, whereas modulation of bba74 expression involves a putative RpoS-dependent repressor that binds upstream of the core promoter. Thus, Lyme disease spirochetes employ at least two different RpoS-dependent mechanisms to repress tick-phase genes within the mammal., (Copyright © 2017 Grove et al.)

Pathogenic species of Leptospira cause leptospirosis, a bacterial zoonotic disease with a global distribution affecting over one million people annually. Reservoir hosts of leptospirosis, including rodents, dogs, and cattle, exhibit little to no signs of disease but shed large numbers of organisms in their urine. Transmission occurs when mucosal surfaces or abraded skin come into contact with infected urine or urine-contaminated water or soil. Whilst little is known about how Leptospira adapt to and persist within a reservoir host, in vitro studies suggest that leptospires alter their transcriptomic and proteomic profiles in response to environmental signals encountered during mammalian infection. We applied the dialysis membrane chamber (DMC) peritoneal implant model to compare the whole cell proteome of in vivo derived leptospires with that of leptospires cultivated in vitro at 30°C and 37°C by 2-dimensional difference in-gel electrophoresis (2-D DIGE). Of 1,735 protein spots aligned across 9 2-D DIGE gels, 202 protein spots were differentially expressed ( p < 0.05, fold change >1.25 or < -1.25) across all three conditions. Differentially expressed proteins were excised for identification by mass spectrometry. Data are available via ProteomeXchange with identifier PXD006995. The greatest differences were detected when DMC-cultivated leptospires were compared with IV30- or IV37-cultivated leptospires, including the increased expression of multiple isoforms of Loa22, a known virulence factor. Unexpectedly, 20 protein isoforms of LipL32 and 7 isoforms of LipL41 were uniformly identified by DIGE as differentially expressed, suggesting that unique post-translational modifications (PTMs) are operative in response to mammalian host conditions. To test this hypothesis, a rat model of persistent renal colonization was used to isolate leptospires directly from the urine of experimentally infected rats. Comparison of urinary derived leptospires to IV30 leptospires by 2-D immunoblotting confirmed that modification of proteins with trimethyllysine and acetyllysine occurs to a different degree in response to mammalian host signals encountered during persistent renal colonization. These results provide novel insights into differential protein and PTMs present in response to mammalian host signals which can be used to further define the unique equilibrium that exists between pathogenic leptospires and their reservoir host of infection.

Human milk-associated microbes are among the first to colonize the infant gut and may help to shape both short- and long-term infant health outcomes. We performed a systematic review to characterize the microbiota of human milk. Relevant primary studies were identified through a comprehensive search of PubMed (January 1, 1964, to June 31, 2015). Included studies were conducted among healthy mothers, were written in English, identified bacteria in human milk, used culture-independent methods, and reported primary results at the genus level. Twelve studies satisfied inclusion criteria. All varied in geographic location and human milk collection/storage/analytic methods. Streptococcus was identified in human milk samples in 11 studies (91.6%) and Staphylococcus in 10 (83.3%); both were predominant genera in 6 (50%). Eight of the 12 studies used conventional ribosomal RNA (rRNA) polymerase chain reaction (PCR), of which 7 (87.5%) identified Streptococcus and 6 (80%) identified Staphylococcus as present. Of these 8 studies, 2 (25%) identified Streptococcus and Staphylococcus as predominant genera. Four of the 12 studies used next-generation sequencing (NGS), all of which identified Streptococcus and Staphylococcus as present and predominant genera. Relative to conventional rRNA PCR, NGS is a more sensitive method to identify/quantify bacterial genera in human milk, suggesting the predominance of Streptococcus and Staphylococcus may be underestimated in studies using older methods. These genera, Streptococcus and Staphylococcus, may be universally predominant in human milk, regardless of differences in geographic location or analytic methods. Primary studies designed to evaluate the effect of these 2 genera on short- and long-term infant outcomes are warranted.

Borrelia burgdorferi, the causative agent of Lyme disease (along with closely related genospecies), is in the deeply branching spirochete phylum. The bacterium is maintained in nature in an enzootic cycle that involves transmission from a tick vector to a vertebrate host and acquisition from a vertebrate host to a tick vector. During its arthropod sojourn, B. burgdorferi faces a variety of stresses, including nutrient deprivation. Here, we review some of the spirochetal factors that promote persistence, maintenance and dissemination of B. burgdorferi in the tick, and then focus on the utilization of available carbohydrates as well as the exquisite regulatory systems invoked to adapt to the austere environment between blood meals and to signal species transitions as the bacteria traverse their enzootic cycle. The spirochetes shift their source of carbon and energy from glucose in the vertebrate to glycerol in the tick. Regulation of survival under limiting nutrients requires the classic stringent response in which RelBbu controls the levels of the alarmones guanosine tetraphosphate and guanosine pentaphosphate (collectively termed (p)ppGpp), while regulation at the tick-vertebrate interface as well as regulation of protective responses to the blood meal require the two-component system Hk1/Rrp1 to activate production of the second messenger cyclic-dimeric-GMP (c-di-GMP)., (© 2016 John Wiley & Sons Ltd.)

The Treponema denticola FhbB protein contributes to immune evasion by binding factor H (FH). Cleavage of FH by the T. denticola protease, dentilisin, may contribute to the local immune dysregulation that is characteristic of periodontal disease (PD). Although three FhbB phyletic types have been defined (FhbB1, FhbB2, and FhbB3), the in vivo expression patterns and antigenic heterogeneity of FhbB have not been assessed. Here, we demonstrate that FhbB is a dominant early antigen that elicits FhbB type-specific antibody (Ab) responses. Using the murine skin abscess model, we demonstrate that the presence or absence of FhbB or dentilisin significantly influences Ab responses to infection and skin abscess formation. Competitive binding analyses revealed that α-FhbB Ab can compete with FH for binding to T. denticola and block dentilisin-mediated FH cleavage. Lastly, we demonstrate that dentilisin cleavage sites reside within critical functional domains of FH, including the complement regulatory domain formed by CCPs 1 to 4. Analysis of the FH cleavage products revealed that they lack cofactor activity. The data presented here provide insight into the in vivo significance of dentilisin, FhbB and its antigenic diversity, and the potential impact of FH cleavage on the regulation of complement activation., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retro-transposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing ∼57% of the genome, reveals 20,486 protein-coding genes and expansions of gene families associated with tick-host interactions. We report insights from genome analyses into parasitic processes unique to ticks, including host 'questing', prolonged feeding, cuticle synthesis, blood meal concentration, novel methods of haemoglobin digestion, haem detoxification, vitellogenesis and prolonged off-host survival. We identify proteins associated with the agent of human granulocytic anaplasmosis, an emerging disease, and the encephalitis-causing Langat virus, and a population structure correlated to life-history traits and transmission of the Lyme disease agent.

Two-component signal transduction systems are the primary mechanisms by which bacteria perceive and respond to changes in their environment. The Hk1/Rrp1 two-component system (TCS) in Borrelia burgdorferi consists of a hybrid histidine kinase and a response regulator with diguanylate cyclase activity, respectively. Phosphorylated Rrp1 catalyzes the synthesis of c-di-GMP, a second messenger associated with bacterial life-style control networks. Spirochetes lacking either Hk1 or Rrp1 are virulent in mice but destroyed within feeding ticks. Activation of Hk1 by exogenous stimuli represents the seminal event for c-di-GMP signaling. We reasoned that structural characterization of Hk1's sensor would provide insights into the mechanism underlying signal transduction and aid in the identification of activating ligands. The Hk1 sensor is composed of three ligand-binding domains (D1-3), each with homology to periplasmic solute-binding proteins (PBPs) typically associated with ABC transporters. Herein, we determined the structure for D1, the most N-terminal PBP domain. As expected, D1 displays a bilobed Venus Fly Trap-fold. Similar to the prototypical sensor PBPs HK29S from Geobacter sulfurreducens and VFT2 from Bordetella pertussis, apo-D1 adopts a closed conformation. Using complementary approaches, including SAXS, we established that D1 forms a dimer in solution. The D1 structure enabled us to model the D2 and D3 domains. Differences in the ligand-binding pockets suggest that each PBP recognizes a different ligand. The ability of Hk1 to recognize multiple stimuli provides spirochetes with a means of distinguishing between the acquisition and transmission blood meals and generate a graded output response that is reflective of the perceived environmental threats., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Borrelia burgdorferi, the Lyme disease spirochete, couples environmental sensing and gene regulation primarily via the Hk1/Rrp1 two-component system (TCS) and Rrp2/RpoN/RpoS pathways. Beginning with acquisition, we reevaluated the contribution of these pathways to spirochete survival and gene regulation throughout the enzootic cycle. Live imaging of B. burgdorferi caught in the act of being acquired revealed that the absence of RpoS and the consequent derepression of tick-phase genes impart a Stay signal required for midgut colonization. In addition to the behavioral changes brought on by the RpoS-off state, acquisition requires activation of cyclic di-GMP (c-di-GMP) synthesis by the Hk1/Rrp1 TCS; B. burgdorferi lacking either component is destroyed during the blood meal. Prior studies attributed this dramatic phenotype to a metabolic lesion stemming from reduced glycerol uptake and utilization. In a head-to-head comparison, however, the B. burgdorferi Δglp mutant had a markedly greater capacity to survive tick feeding than B. burgdorferi Δhk1 or Δrrp1 mutants, establishing unequivocally that glycerol metabolism is only one component of the protection afforded by c-di-GMP. Data presented herein suggest that the protective response mediated by c-di-GMP is multifactorial, involving chemotactic responses, utilization of alternate substrates for energy generation and intermediary metabolism, and remodeling of the cell envelope as a means of defending spirochetes against threats engendered during the blood meal. Expression profiling of c-di-GMP-regulated genes through the enzootic cycle supports our contention that the Hk1/Rrp1 TCS functions primarily, if not exclusively, in ticks. These data also raise the possibility that c-di-GMP enhances the expression of a subset of RpoS-dependent genes during nymphal transmission., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Leptospira interrogans can infect a myriad of mammalian hosts, including humans (Bharti et al. , 2003; Ko et al. , 2009). Following acquisition by a suitable host, leptospires disseminate via the bloodstream to multiple tissues, including the kidneys, where they adhere to and colonize the proximal convoluted renal tubules (Athanazio et al. , 2008). Infected hosts shed large number of spirochetes in their urine and the leptospires can survive in different environmental conditions before transmission to another host. Differential gene expression by Leptospira spp. permits adaption to these new conditions. Here we describe a protocol for the cultivation of Leptospira interrogans within Dialysis Membrane Chambers (DMCs) implanted into the peritoneal cavities of Sprague-Dawley rats (Caimano et al. , 2014). This technique was originally developed to study mammalian adaption by the Lyme disease spirochete, Borrelia burgdorferi (Akins et al. , 1998; Caimano, 2005). The small pore size (8,000 MWCO) of the dialysis membrane tubing used for this procedure permits access to host nutrients but excludes host antibodies and immune effector cells. Given the physiological and environmental similarities between DMCs and the proximal convoluted renal tubule, we reasoned that the DMC model would be suitable for studying in vivo gene expression by L. interrogans . In a 20 to 30 min procedure, DMCs containing virulent leptospires are surgically-implanted into the rat peritoneal cavity. Nine to 11 days post-implantation, DMCs are explanted and organisms recovered. Typically, a single DMC yields ~10 9 mammalian host-adapted leptospires (Caimano et al. , 2014). In addition to providing a facile system for studying the transcriptional and physiologic changes pathogenic L. interrogans undergo within the mammal, the DMC model also provides a rationale basis for selecting new targets for mutagenesis and the identification of novel virulence determinants. Caution: Leptospira interrogans is a BSL-2 level pathogen and known to be excreted in the urine of infected animals. Animals should be handled and disposed of using recommended Animal Biosafety Levels (ABSL) for infectious agents using vertebrate animal guidelines. Note: All protocols using live animals must conform to governmental regulations regarding the care and use of laboratory animals. The success of this protocol is dependent on the proper use of aseptic techniques during all stages of both dialysis membrane chamber preparation and animal surgery.

Unlabelled: We recently demonstrated that TP&#95;0326 is a bona fide rare outer membrane protein (OMP) in Treponema pallidum and that it possesses characteristic BamA bipartite topology. Herein, we used immunofluorescence analysis (IFA) to show that only the β-barrel domain of TP&#95;0326 contains surface-exposed epitopes in intact T. pallidum. Using the solved structure of Neisseria gonorrhoeae BamA, we generated a homology model of full-length TP&#95;0326. Although the model predicts a typical BamA fold, the β-barrel harbors features not described in other BamAs. Structural modeling predicted that a dome comprised of three large extracellular loops, loop 4 (L4), L6, and L7, covers the barrel's extracellular opening. L4, the dome's major surface-accessible loop, contains mainly charged residues, while L7 is largely neutral and contains a polyserine tract in a two-tiered conformation. L6 projects into the β-barrel but lacks the VRGF/Y motif that anchors L6 within other BamAs. IFA and opsonophagocytosis assay revealed that L4 is surface exposed and an opsonic target. Consistent with B cell epitope predictions, immunoblotting and enzyme-linked immunosorbent assay (ELISA) confirmed that L4 is an immunodominant loop in T. pallidum-infected rabbits and humans with secondary syphilis. Antibody capture experiments using Escherichia coli expressing OM-localized TP&#95;0326 as a T. pallidum surrogate further established the surface accessibility of L4. Lastly, we found that a naturally occurring substitution (Leu(593) → Gln(593)) in the L4 sequences of T. pallidum strains affects antibody binding in sera from syphilitic patients. Ours is the first study to employ a "structure-to-pathogenesis" approach to map the surface topology of a T. pallidum OMP within the context of syphilitic infection., Importance: Previously, we reported that TP&#95;0326 is a bona fide rare outer membrane protein (OMP) in Treponema pallidum and that it possesses the bipartite topology characteristic of a BamA ortholog. Using a homology model as a guide, we found that TP&#95;0326 displays unique features which presumably relate to its function(s) in the biogenesis of T. pallidum's unorthodox OM. The model also enabled us to identify an immunodominant epitope in a large extracellular loop that is both an opsonic target and subject to immune pressure in a human population. Ours is the first study to follow a structure-to-pathogenesis approach to map the surface topology of a T. pallidum rare OMP within the context of syphilitic infection., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Borrelia burgdorferi, the agent of Lyme disease, is maintained in nature within an enzootic cycle involving a mammalian reservoir and an Ixodes sp. tick vector. The transmission, survival and pathogenic potential of B. burgdorferi depend on the bacterium's ability to modulate its transcriptome as it transits between vector and reservoir host. Herein, we employed an amplification-microarray approach to define the B. burgdorferi transcriptomes in fed larvae, fed nymphs and in mammalian host-adapted organisms cultivated in dialysis membrane chambers. The results show clearly that spirochetes exhibit unique expression profiles during each tick stage and during cultivation within the mammal; importantly, none of these profiles resembles that exhibited by in vitro grown organisms. Profound shifts in transcript levels were observed for genes encoding known or predicted lipoproteins as well as proteins involved in nutrient uptake, carbon utilization and lipid synthesis. Stage-specific expression patterns of chemotaxis-associated genes also were noted, suggesting that the composition and interactivities of the chemotaxis machinery components vary considerably in the feeding tick and mammal. The results as a whole make clear that environmental sensing by B. burgdorferi directly or indirectly drives an extensive and tightly integrated modulation of cell envelope constituents, chemotaxis/motility machinery, intermediary metabolism and cellular physiology. These findings provide the necessary transcriptional framework for delineating B. burgdorferi regulatory pathways throughout the enzootic cycle as well as defining the contribution(s) of individual genes to spirochete survival in nature and virulence in humans., (© 2014 John Wiley & Sons Ltd.)

The spirochete periplasmic flagellum has many unique attributes. One unusual characteristic is the flagellar hook. This structure serves as a universal joint coupling rotation of the membrane-bound motor to the flagellar filament. The hook is comprised of about 120 FlgE monomers, and in most bacteria these structures readily dissociate to monomers (∼ 50 kDa) when treated with heat and detergent. However, in spirochetes the FlgE monomers form a large mass of over 250 kDa [referred to as a high molecular weight complex (HMWC)] that is stable to these and other denaturing conditions. In this communication, we examined specific aspects with respect to the formation and structure of this complex. We found that the Lyme disease spirochete Borrelia burgdorferi synthesized the HMWC throughout the in vitro growth cycle, and also in vivo when implanted in dialysis membrane chambers in rats. The HMWC was stable to formic acid, which supports the concept that the stability of the HMWC is dependent on covalent cross-linking of individual FlgE subunits. Mass spectrometry analysis of the HMWC from both wild type periplasmic flagella and polyhooks from a newly constructed ΔfliK mutant indicated that other proteins besides FlgE were not covalently joined to the complex, and that FlgE was the sole component of the complex. In addition, mass spectrometry analysis also indicated that the HMWC was composed of a polymer of the FlgE protein with both the N- and C-terminal regions remaining intact. These initial studies set the stage for a detailed characterization of the HMWC. Covalent cross-linking of FlgE with the accompanying formation of the HMWC we propose strengthens the hook structure for optimal spirochete motility.

The Borrelia burgdorferi bba64 gene product is a surface-localized lipoprotein synthesized within mammalian and tick hosts and is involved in vector transmission of disease. These properties suggest that BBA64 may be a vaccine candidate against Lyme borreliosis. In this study, protective immunity against B. burgdorferi challenge was assessed in mice immunized with the BBA64 protein. Mice developed a high-titer antibody response following immunization with soluble recombinant BBA64 but were not protected when challenged by needle inoculation of culture-grown spirochetes. Likewise, mice passively immunized with an anti-BBA64 monoclonal antibody were not protected against needle-inoculated organisms. BBA64-immunized mice were subjected to B. burgdorferi challenge by the natural route of a tick bite, but these trials did not demonstrate significant protective immunity in either outbred or inbred strains of mice. Lipidated recombinant BBA64 produced in Escherichia coli was assessed for possible improved elicitation of a protective immune response. Although inoculation with this antigen produced a high-titer antibody response, the lipidated BBA64 also was unsuccessful in protecting mice from B. burgdorferi challenge by tick bites. Anti-BBA64 antibodies raised in rats eradicated the organisms, as evidenced by in vitro borreliacidal assays, thus demonstrating the potential for BBA64 to be effective as a protective immunogen. However, passive immunization with the same monospecific rat anti-BBA64 polyclonal serum failed to provide protection against tick bite-administered challenge. These results reveal the challenges faced in not only identifying B. burgdorferi proteins with potential protective capability but also in producing recombinant antigens conducive to preventive therapies against Lyme borreliosis.

Leptospirosis, an emerging zoonotic disease with worldwide distribution, is caused by spirochetes belonging to the genus Leptospira. More than 500,000 cases of severe leptospirosis are reported annually, with >10% of these being fatal. Leptospires can survive for weeks in suitably moist conditions before encountering a new host. Reservoir hosts, typically rodents, exhibit little to no signs of disease but shed large numbers of organisms in their urine. Transmission occurs when mucosal surfaces or abraded skin come into contact with infected urine or urine-contaminated water or soil. In humans, leptospires can cause a variety of clinical manifestations, ranging from asymptomatic or mild fever to severe icteric (Weil's) disease and pulmonary haemorrhage. Currently, little is known about how Leptospira persist within a reservoir host. Prior in vitro studies have suggested that leptospires alter their transcriptomic and proteomic profiles in response to environmental signals encountered during mammalian infection. However, no study has examined gene expression by leptospires within a mammalian host-adapted state. To obtain a more faithful representation of how leptospires respond to host-derived signals, we used RNA-Seq to compare the transcriptome of L. interrogans cultivated within dialysis membrane chambers (DMCs) implanted into the peritoneal cavities of rats with that of organisms grown in vitro. In addition to determining the relative expression levels of "core" housekeeping genes under both growth conditions, we identified 166 genes that are differentially-expressed by L. interrogans in vivo. Our analyses highlight physiological aspects of host adaptation by leptospires relating to heme uptake and utilization. We also identified 11 novel non-coding transcripts that are candidate small regulatory RNAs. The DMC model provides a facile system for studying the transcriptional and antigenic changes associated with mammalian host-adaptation, selection of targets for mutagenesis, and the identification of previously unrecognized virulence determinants.