Treponema pallidum Periplasmic and Membrane Proteins Are Recognized by Circulating and Skin CD4+ T Cells.

BACKGROUND: Histologic and serologic studies suggest the induction of local and systemic Treponema pallidum-specific CD4+ T-cell responses to T. pallidum infection. We hypothesized that T. pallidum-specific CD4+ T cells are detectable in blood and in the skin rash of secondary syphilis and persist in both compartments after treatment. METHODS: Peripheral blood mononuclear cells collected from 67 participants were screened by interferon-γ (IFN-γ) ELISPOT response to T. pallidum sonicate. T. pallidum-reactive T-cell lines from blood and skin were probed for responses to 89 recombinant T. pallidum antigens. Peptide epitopes and HLA class II restriction were defined for selected antigens. RESULTS: We detected CD4+ T-cell responses to T. pallidum sonicate ex vivo. Using T. pallidum-reactive T-cell lines we observed recognition of 14 discrete proteins, 13 of which localize to bacterial membranes or the periplasmic space. After therapy, T. pallidum-specific T cells persisted for at least 6 months in skin and 10 years in blood. CONCLUSIONS: T. pallidum infection elicits an antigen-specific CD4+ T-cell response in blood and skin. T. pallidum-specific CD4+ T cells persist as memory in both compartments long after curative therapy. The T. pallidum antigenic targets we identified may be high-priority vaccine candidates.

In-Depth Proteome Coverage of In Vitro-Cultured Treponema pallidum and Quantitative Comparison Analyses with In Vivo-Grown Treponemes.

Previous mass spectrometry (MS)-based global proteomics studies have detected a combined total of 86% of all Treponema pallidum proteins under infection conditions (in vivo-grown T. pallidum). Recently, a method was developed for the long-term culture of T. pallidum under in vitro conditions (in vitro-cultured T. pallidum). Herein, we used our previously reported optimized MS-based proteomics approach to characterize the T. pallidum global protein expression profile under in vitro culture conditions. These analyses provided a proteome coverage of 94%, which extends the combined T. pallidum proteome coverage from the previously reported 86% to a new combined total of 95%. This study provides a more complete understanding of the protein repertoire of T. pallidum. Further, comparison of the in vitro-expressed proteome with the previously determined in vivo-expressed proteome identifies only a few proteomic changes between the two growth conditions, reinforcing the suitability of in vitro-cultured T. pallidum as an alternative to rabbit-based treponemal growth. The MS proteomics data have been deposited in the MassIVE repository with the data set identifier MSV000093603 (ProteomeXchange identifier PXD047625).

Treponema pallidum periplasmic and membrane proteins are recognized by circulating and skin CD4+ T cells.

Background: Histologic and serologic studies suggest the induction of local and systemic Treponema pallidum ( Tp )-specific CD4+ T cell responses to Tp infection. We hypothesized that Tp -specific CD4+ T cells are detectable in blood and in the skin rash of secondary syphilis and persist in both compartments after treatment. Methods: PBMC collected from 67 participants were screened by IFNγ ELISPOT response to Tp sonicate. Tp -reactive T cell lines from blood and skin were probed for responses to 88 recombinant Tp antigens. Peptide epitopes and HLA class II restriction were defined for selected antigens. Results: We detected CD4+ T cell responses to Tp sonicate ex vivo. Using Tp -reactive T cell lines we observed recognition of 14 discrete proteins, 13 of which localize to bacterial membranes or the periplasmic space. After therapy, Tp -specific T cells persisted for at least 6 months in skin and 10 years in blood. Conclusions: Tp infection elicits an antigen-specific CD4+ T cell response in blood and skin. Tp -specific CD4+ T cells persist as memory in both compartments long after curative therapy. The Tp antigenic targets we identified may be high priority vaccine candidates.

Syphilis and the host: multi-omic analysis of host cellular responses to Treponema pallidum provides novel insight into syphilis pathogenesis.

Introduction: Syphilis is a chronic, multi-stage infection caused by the extracellular bacterium Treponema pallidum ssp. pallidum. Treponema pallidum widely disseminates through the vasculature, crosses endothelial, blood-brain and placental barriers, and establishes systemic infection. Although the capacity of T. pallidum to traverse the endothelium is well-described, the response of endothelial cells to T. pallidum exposure, and the contribution of this response to treponemal traversal, is poorly understood. Methods: To address this knowledge gap, we used quantitative proteomics and cytokine profiling to characterize endothelial responses to T. pallidum. Results: Proteomic analyses detected altered host pathways controlling extracellular matrix organization, necroptosis and cell death, and innate immune signaling. Cytokine analyses of endothelial cells exposed to T. pallidum revealed increased secretion of interleukin (IL)-6, IL-8, and vascular endothelial growth factor (VEGF), and decreased secretion of monocyte chemoattractant protein-1 (MCP-1). Discussion: This study provides insight into the molecular basis of syphilis disease symptoms and the enhanced susceptibility of individuals infected with syphilis to HIV co-infection. These investigations also enhance understanding of the host response to T. pallidum exposure and the pathogenic strategies used by T. pallidum to disseminate and persist within the host. Furthermore, our findings highlight the critical need for inclusion of appropriate controls when conducting T. pallidum-host cell interactions using in vitro- and in vivo-grown T. pallidum.

Deep proteome coverage advances knowledge of Treponema pallidum protein expression profiles during infection.

Comprehensive proteome-wide analysis of the syphilis spirochete, Treponema pallidum ssp. pallidum, is technically challenging due to high sample complexity, difficulties with obtaining sufficient quantities of bacteria for analysis, and the inherent fragility of the T. pallidum cell envelope which further complicates proteomic identification of rare T. pallidum outer membrane proteins (OMPs). The main aim of the present study was to gain a deeper understanding of the T. pallidum global proteome expression profile under infection conditions. This will corroborate and extend genome annotations, identify protein modifications that are unable to be predicted at the genomic or transcriptomic levels, and provide a foundational knowledge of the T. pallidum protein expression repertoire. Here we describe the optimization of a T. pallidum-specific sample preparation workflow and mass spectrometry-based proteomics pipeline which allowed for the detection of 77% of the T. pallidum protein repertoire under infection conditions. When combined with prior studies, this brings the overall coverage of the T. pallidum proteome to almost 90%. These investigations identified 27 known/predicted OMPs, including potential vaccine candidates, and detected expression of 11 potential OMPs under infection conditions for the first time. The optimized pipeline provides a robust and reproducible workflow for investigating T. pallidum protein expression during infection. Importantly, the combined results provide the deepest coverage of the T. pallidum proteome to date.

Lipid A structural diversity among members of the genus Leptospira.

Lipid A is the hydrophobic component of bacterial lipopolysaccharide and an activator of the host immune system. Bacteria modify their lipid A structure to adapt to the surrounding environment and, in some cases, to evade recognition by host immune cells. In this study, lipid A structural diversity within the Leptospira genus was explored. The individual Leptospira species have dramatically different pathogenic potential that ranges from non-infectious to life-threatening disease (leptospirosis). Ten distinct lipid A profiles, denoted L1-L10, were discovered across 31 Leptospira reference species, laying a foundation for lipid A-based molecular typing. Tandem MS analysis revealed structural features of Leptospira membrane lipids that might alter recognition of its lipid A by the host innate immune receptors. Results of this study will aid development of strategies to improve diagnosis and surveillance of leptospirosis, as well as guide functional studies on Leptospira lipid A activity.

Immunization with a tri-antigen syphilis vaccine significantly attenuates chancre development, reduces bacterial load, and inhibits dissemination of Treponema pallidum.

Syphilis continues to be a significant public health concern worldwide. The disease is endemic in many low- and middle-income countries, and rates have risen sharply in high-income countries over the last decade. The continued prevalence of infectious and congenital syphilis worldwide highlights the need for the development of an effective syphilis vaccine to complement public health measures for syphilis control. The complex, multi-stage course of syphilis infection necessitates a holistic approach to the development of an effective vaccine, in which immunization prevents both the localized stage of infection (typified by the highly infectious chancre) and the disseminated stages of infection (typified by the secondary rash, neurosyphilis, and destructive tertiary lesions, as well as congenital syphilis). Inhibiting development of the infectious chancre would reduce transmission thus providing community- level protection, while preventing dissemination would provide individual-level protection by reducing serious sequelae and may also provide community level protection by reducing shedding during secondary syphilis. In the current study we build upon prior investigations which demonstrated that immunizations with individual, well characterized T. pallidum TprK, TprC, and Tp0751 peptides elicits partial protection against infection in the animal model. Specifically, we show here that immunization with a TprC/TprK/Tp0751 tri-antigen cocktail protects animals from progressive syphilis lesions and substantially inhibits dissemination of the infection.

Identification and Functional Characterization of Peptides With Antimicrobial Activity From the Syphilis Spirochete, Treponema pallidum.

The etiological agent of syphilis, Treponema pallidum ssp. pallidum, is a highly invasive "stealth" pathogen that can evade the host immune response and persist within the host for decades. This obligate human pathogen is adept at establishing infection and surviving at sites within the host that have a multitude of competing microbes, sometimes including pathogens. One survival strategy employed by bacteria found at polymicrobial sites is elimination of competing microorganisms by production of antimicrobial peptides (AMPs). Antimicrobial peptides are low molecular weight proteins (miniproteins) that function directly via inhibition and killing of microbes and/or indirectly via modulation of the host immune response, which can facilitate immune evasion. In the current study, we used bioinformatics to show that approximately 7% of the T. pallidum proteome is comprised of miniproteins of 150 amino acids or less with unknown functions. To investigate the possibility that AMP production is an unrecognized defense strategy used by T. pallidum during infection, we developed a bioinformatics pipeline to analyze the complement of T. pallidum miniproteins of unknown function for the identification of potential AMPs. This analysis identified 45 T. pallidum AMP candidates; of these, Tp0451a and Tp0749 were subjected to further bioinformatic analyses to identify AMP critical core regions (AMPCCRs). Four potential AMPCCRs from the two predicted AMPs were identified and peptides corresponding to these AMPCCRs were experimentally confirmed to exhibit bacteriostatic and bactericidal activity against a panel of biologically relevant Gram-positive and Gram-negative bacteria. Immunomodulation assays performed under inflammatory conditions demonstrated that one of the AMPCCRs was also capable of differentially regulating expression of two pro-inflammatory chemokines [monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8)]. These findings demonstrate proof-of-concept for our developed AMP identification pipeline and are consistent with the novel concept that T. pallidum expresses AMPs to defend against competing microbes and modulate the host immune response.

Treponema pallidum Disrupts VE-Cadherin Intercellular Junctions and Traverses Endothelial Barriers Using a Cholesterol-Dependent Mechanism.

Treponema pallidum subspecies pallidum, the causative agent of syphilis, traverses the vascular endothelium to gain access to underlying tissue sites. Herein, we investigate the mechanisms associated with T. pallidum traversal of endothelial barriers. Immunofluorescence microscopy reveals that a subpopulation of T. pallidum localizes to intercellular junctions and that viable T. pallidum, as well as a T. pallidum vascular adhesin (Tp0751), disrupts the architecture of the main endothelial junctional protein VE-cadherin. Intriguingly, in this study we show that T. pallidum traverses endothelial barriers with no disruption in barrier permeability. Furthermore, barrier traversal by T. pallidum is reduced by pretreatment of endothelial cells with filipin, an inhibitor that blocks cholesterol-mediated endocytosis. Collectively, these results suggest that T. pallidum can use a cholesterol-dependent, lipid raft-mediated endocytosis mechanism to traverse endothelial barriers. Further, treponemal localization to, and disruption of, intercellular junctions suggests that a paracellular route may also be utilized, a dual traversal strategy that has also been observed to occur for leukocytes and other invasive bacteria.

Identification of the Neuroinvasive Pathogen Host Target, LamR, as an Endothelial Receptor for the Treponema pallidum Adhesin Tp0751.

Treponema pallidum subsp. pallidum is the causative agent of syphilis, a human-specific sexually transmitted infection that causes a multistage disease with diverse clinical manifestations. Treponema pallidum undergoes rapid vascular dissemination to penetrate tissue, placental, and blood-brain barriers and gain access to distant tissue sites. The rapidity and extent of T. pallidum dissemination are well documented, but the molecular mechanisms have yet to be fully elucidated. One protein that has been shown to play a role in treponemal dissemination is Tp0751, a T. pallidum adhesin that interacts with host components found within the vasculature and mediates bacterial adherence to endothelial cells under shear flow conditions. In this study, we further explore the molecular interactions of Tp0751-mediated adhesion to the vascular endothelium. We demonstrate that recombinant Tp0751 adheres to human endothelial cells of macrovascular and microvascular origin, including a cerebral brain microvascular endothelial cell line. Adhesion assays using recombinant Tp0751 N-terminal truncations reveal that endothelial binding is localized to the lipocalin fold-containing domain of the protein. We also confirm this interaction using live T. pallidum and show that spirochete attachment to endothelial monolayers is disrupted by Tp0751-specific antiserum. Further, we identify the 67-kDa laminin receptor (LamR) as an endothelial receptor for Tp0751 using affinity chromatography, coimmunoprecipitation, and plate-based binding methodologies. Notably, LamR has been identified as a receptor for adhesion of other neurotropic invasive bacterial pathogens to brain endothelial cells, including Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae, suggesting the existence of a common mechanism for extravasation of invasive extracellular bacterial pathogens.IMPORTANCE Syphilis is a sexually transmitted infection caused by the spirochete bacterium Treponema pallidum subsp. pallidum. The continued incidence of syphilis demonstrates that screening and treatment strategies are not sufficient to curb this infectious disease, and there is currently no vaccine available. Herein we demonstrate that the T. pallidum adhesin Tp0751 interacts with endothelial cells that line the lumen of human blood vessels through the 67-kDa laminin receptor (LamR). Importantly, LamR is also a receptor for meningitis-causing neuroinvasive bacterial pathogens such as Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae Our findings enhance understanding of the Tp0751 adhesin and present the intriguing possibility that the molecular events of Tp0751-mediated treponemal dissemination may mimic the endothelial interaction strategies of other invasive pathogens.