Matthias, M. A. | bioRxiv Science

bioRxiv2022

Culture-Independent Detection and Identification of Leptospira Serovars

Pathogenic Leptospira, the causative agents of leptospirosis, comprise >200 serotypes (called serovars). Most have a restricted reservoir-host range, and some, e.g., serovar Copenhageni, are cosmopolitan and of public health importance owing to their propensity to produce severe, fatal disease in humans. Available serotyping approaches--such as multi-locus sequence typing, core genome sequence typing, pulsed-field gel electrophoresis, and the cross-agglutination absorption test--are tedious and expensive, and require isolation of the organisms in culture media--a protracted and incredibly inefficient process-- precluding their use in prospective studies or outbreak investigations. The unavailability of culture-independent assays capable of distinguishing Leptospira serotypes remains a crucial gap in the field. Here, we have developed a simple yet specific real-time qPCR assay--targeting a Leptospira-unique gene encoding a putative polysaccharide flippase--that provides intra-species, serotype-defining (i.e., epidemiologically useful) information, and improves upon the sensitivity of preferred lipL32-based qPCR-based diagnostic tests. The assay, dubbed RAgI ("rage one"), is rapid and affordable, and reliably and specifically detects group I pathogenic Leptospira in culture, serum and urine, with no detectable off-target amplification--even of the genetically related but low virulence group II pathogenic (formerly "intermediate") or non-pathogenic Leptospira. It retained 100% diagnostic specificity when tested against difficult sample types, including field-collected dog urine-samples and environmental samples containing varied and complex microbial species-consortia. And holds considerable promise in the clinical setting, and for routine epidemiological and environmental surveillance studies.

microbiology↗

bioRxiv2020

Novel R-type Lectin Domain-Containing Cytotoxins Comprise a Family of Virulence-Modifying Proteins in Pathogenic Leptospira

Leptospirosis is a globally important neglected zoonotic disease subject to both small scale outbreaks and weather-driven, large-scale epidemics. Due to gaps in our understanding of Leptospira biology, pathogenetic mechanisms of leptospirosis remain largely unknown. Previous data suggest that a gene family, PF07598, unique amongst most known bacterial pathogens and encoding so-called "Virulence-Modifying (VM)" proteins, are important virulence determinants. Here, we show that VM proteins are potent cytotoxins, sharing a distinct domain organization while exhibiting varied mechanisms of cellular toxicity. Structural homology searches using Phyre2 suggest that VM proteins are novel R-type lectins containing an N-terminal ricin B chain-like domain. As is known for native ricin B-chain, recombinant full-length rLA3490 (most highly up-regulated in vivo) and an N-terminal fragment, t3490, containing a partial ricin B-domain, bound to asialofetuin and directly competed for asialofetuin binding with recombinant ricin B chain. While t3490 bound to the HeLa cell surface but was neither internalized nor cytotoxic, rLA3490 bound to the HeLa cell surface, was rapidly internalized, translocated to the nucleus inducing chromosomal fragmentation, and was rapidly cytolethal, providing strong evidence that Leptospira VM proteins are bona fide cytotoxins. Because monoclonal antibodies impeding cell entry or intracellular trafficking of ricin holotoxin clearly mitigate its toxicity, that VM proteins share binding and intracellular trafficking mechanisms suggests that anti-VM-protein antibody-based (anti-toxin) therapeutics could ameliorate severe complications of leptospirosis thereby improving prognosis. As most VM proteins are restricted to high-virulence Leptospira species with some, e.g., LA3490, being exceptionally potent, their level in serum might be a potentially useful indicator of a poor prognosis, thus identifying high risk patients. Author SummaryThe PF07598 gene family encoding Virulence-Modifying (VM) proteins in pathogenic Leptospira species is associated with severe manifestations of leptospirosis. Structural homology searches indicate that VM proteins contain an N-terminal ricin B chain-like domain, biochemically confirmed in asialofetuin binding and competitive-binding assays suggesting that VM proteins bind to terminal galactosyl residues of this model ricin B domain binding protein. The leptospiral N-terminal ricin B chain-like domain mediated VM protein binding to HeLa cells. Full-length recombinant protein rapidly led to cell death. Amino acid conservation among PF07598 family members at the N-terminal ricin B chain-like domain suggests that VM protein levels in serum might be a useful biomarker for quickly identifying at-risk patients, and that novel "anti-toxin"-based therapeutics could ameliorate severe complications of leptospirosis, both of which remain to be explored.

microbiology↗