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The performance of commonly used assays for diagnosis of syphilis varies considerably depending on stage of infection and sample type. In response to the need for improved syphilis diagnostics, we develop assays that pair PCR pre-amplification of the tpp47 gene of Treponema pallidum subsp. pallidum with CRISPR-LwCas13a. The PCR-LwCas13a assay achieves an order of magnitude better analytical sensitivity than real-time PCR with equivalent specificity. When applied to a panel of 216 biological specimens, including 135 clinically confirmed primary and secondary syphilis samples, the PCR-LwCas13a assay demonstrates 93.3% clinical sensitivity and 100% specificity, outperforming tpp47 real-time PCR and rabbit-infectivity testing. We further adapt this approach to distinguish Treponema pallidum subsp. pallidum lineages and identify genetic markers of macrolide resistance. Our study demonstrates the potential of CRISPR-based approaches to improve diagnosis and epidemiological surveillance of syphilis.

Background Macrophages play prominent roles in bacteria recognition and clearance, including Borrelia burgdorferi (Bb), the Lyme disease spirochete. To elucidate mechanisms by which MyD88/TLR signaling enhances clearance of Bb by macrophages, we studied wildtype (WT) and MyD88−/−Bb-stimulated bone marrow-derived macrophages (BMDMs). Results MyD88−/− BMDMs exhibit impaired uptake of spirochetes but comparable maturation of phagosomes following internalization of spirochetes. RNA-sequencing of infected WT and MyD88−/− BMDMs identified a large cohort of differentially expressed MyD88-dependent genes associated with re-organization of actin and cytoskeleton during phagocytosis along with several MyD88-independent chemokines involved in inflammatory cell recruitment. We computationally generated networks which identified several MyD88-dependent intermediate proteins (Rhoq and Cyfip1) that are known to mediate inflammation and phagocytosis respectively. Conclusion Our findings show that MyD88 signaling enhances, but is not required, for bacterial uptake or phagosomal maturation and provide mechanistic insights into how MyD88-mediated phagosomal signaling enhances Bb uptake and clearance.

The past two decades have seen a worldwide resurgence in infections caused by Treponema pallidum subsp. pallidum, the syphilis spirochete. The well-recognized capacity of the syphilis spirochete for early dissemination and immune evasion has earned it the designation 'the stealth pathogen'. Despite the many hurdles to studying syphilis pathogenesis, most notably the inability to culture and to genetically manipulate T. pallidum, in recent years, considerable progress has been made in elucidating the structural, physiological, and regulatory facets of T. pallidum pathogenicity. In this Review, we integrate this eclectic body of information to garner fresh insights into the highly successful parasitic lifestyles of the syphilis spirochete and related pathogenic treponemes.

In little more than 30 years, Lyme disease, which is caused by the spirochaete Borrelia burgdorferi, has risen from relative obscurity to become a global public health problem and a prototype of an emerging infection. During this period, there has been an extraordinary accumulation of knowledge on the phylogenetic diversity, molecular biology, genetics and host interactions of B. burgdorferi. In this Review, we integrate this large body of information into a cohesive picture of the molecular and cellular events that transpire as Lyme disease spirochaetes transit between their arthropod and vertebrate hosts during the enzootic cycle.

The crystal structure of recombinant TroA, a zinc-binding protein component of an ATP-binding cassette transport system in Treponema pallidum, was determined at a resolution of 1.8 A. The organization of the protein is largely similar to other periplasmic ligand-binding proteins (PLBP), in that two independent globular domains interact with each other to create a zinc-binding cleft between them. The structure has one bound zinc pentavalently coordinated to residues from both domains. Unlike previous PLBP structures that have an interdomain hinge composed of β-strands, the N- and C-domains of TroA are linked by a single long backbone helix. This unique backbone helical conformation was possibly adopted to limit the hinge motion associated with ligand exchange.

Current thinking emphasizes the primacy of CD14 in facilitating TLR recognition of microbes to initiate proinflammatory signaling events and the importance of p38-MAPK in augmenting such responses. Herein, this paradigm is challenged by demonstrating that recognition of Borrelia burgdorferi not only triggers an inflammatory response in the absence of CD14, but one that is uncontrolled as a consequence of impaired PI3K/AKT/p38-MAPK signaling and negative regulation of TLR2. CD14 deficiency results in hyperphosphorylation of AKT and reduced activation of p38. Such aberrant signaling leads to decreased negative regulation by SOCS1, SOCS3, and CIS thereby engendering a more severe and persistent inflammatory response to B. burgdorferi. Perturbation of this CD14/p38-MAPK-dependent mechanism of immune regulation may underlie development of infectious chronic inflammatory syndromes.