Biology subjects

Wunder, E.

Publications and source records attributed to Wunder, E..

6 recordsLinked to original sources

bioRxiv2023

MPL36, a major plasminogen (PLG) receptor in pathogenic Leptospira, has an essential role during infection

Leptospirosis, a zoonosis with worldwide distribution, is caused by pathogenic spirochetes belonging to the genus Leptospira. Bacterial outer membrane proteins (OMPs), particularly those with surface-exposed regions, play crucial roles in pathogen dissemination and virulence mechanisms. Here we characterized the leptospiral Membrane Protein L36 (MPL36), a rare lipoprotein A (RlpA) homolog with a C-terminal Sporulation related (SPOR) domain, as an important virulence factor in pathogenic Leptospira. Our results showed that MPL36 is surface exposed and expressed during infection. Recombinant MPL36 (rMPL36) showed high plasminogen (PLG)-binding ability determined by lysine residues of the C-terminal region of the protein, with ability to convert bound-PLG to active plasmin. Using Kochs molecular postulates, we determined that a mutant of mpl36 has a reduced PLG-binding ability, leading to a decreased capacity to adhere and translocate MDCK cell monolayers. Using recombinant protein and mutant strains, we determined that the MPL36-bound plasmin (PLA) can degrade fibrinogen. Finally, our mpl36 mutant had a significant attenuated phenotype in the hamster model for acute leptospirosis. Our data indicates that MPL36 is the major PLG binding protein in pathogenic Leptospira, and crucial to the pathogens ability to attach and interact with host tissues during infection. The MPL36 characterization contributes to the expanding field of bacterial pathogens that explore PLG for their virulence, advancing the goal to close the knowledge gap regarding leptospiral pathogenesis while offering a novel potential candidate to improve diagnostic and prevention of this important zoonotic neglected disease. Author SummaryAs part of their diverse virulence machinery, bacterial pathogens bind to human plasminogen (PLG) providing them with a proteolytic platform that promotes invasiveness, dissemination, and virulence. Leptospirosis is the leading zoonotic disease in morbidity and mortality worldwide. The burden of this neglected disease will continue to raise given the effects of climate change and social inequality, important drivers of disease. Furthermore, the gap of knowledge regarding leptospiral pathogenesis has negatively impacted the development of sensitive diagnostic tools and effective prevention methods. Previous studies have shown that pathogenic Leptospira, the causative agent of leptospirosis, can interact with PLG through different protein candidates. In this work, we characterized one of those candidates, Membrane Protein L36 (MPL36), as the main leptospiral plasminogen binding protein. Using genetically modified mutants, in vivo, and in vitro assays we provided evidence that MPL36 can bound PLG, promotes adherence to host cells and subsequent translocation, and degrades fibrinogen by converting bound-PLG to PLA, thus essential to leptospiral virulence. This work contributes to the growing field of bacterial pathogens exploring PLG to increase their virulence, while highlighting important new knowledge on leptospiral pathogenesis. MPL36 is an important candidate to be explored on the continued effort to improve diagnostic and prevention of this important zoonotic disease.

microbiology↗

bioRxiv2022

Structural basis of flagellar filament asymmetry and supercoil templating by Leptospira spirochete sheath proteins

Several Leptospira species are bacterial agents of leptospirosis, a neglected tropical disease responsible for ~1 million cases and 50,000 deaths each year worldwide. Leptospira, like other members of the Spirochaeta phylum, possess specially adapted flagella that remain confined within the periplasm. These appendages drive a unique, corkscrew-like swimming style that enables efficient motility and pathogenesis. However, the composition, function, and molecular architecture of spirochetal flagellar filaments remain poorly understood. We solved single-particle cryo-EM structures of isolated Leptospira flagellar filaments, comparing the wild-type form to two mutant forms with different missing components and abrogated motilities. The structures reveal a complex proteinaceous sheath surrounding a conserved core composed of the FlaB flagellin homolog. Sheath proteins were found to fall into two distinct categories, both of which are required for motility. Filament coiling proteins, FcpA and FcpB, exert force on the filament when they bind its surface, causing the filament to stretch. In contrast, we identify sheath components FlaAP (newly discovered in this study) and FlaA2 as template factors, which have little effect on filament shape by themselves, but partition the coiling proteins to one side of the filament. In this way, the two types of Leptospira sheath factors operate collectively on the flagellar filament to bend it from a relaxed form associated with cell immobility, to a motility-competent shape that is tightly supercoiled. Our structures also indicate that core-sheath interactions are largely mediated by carbohydrate moieties from flagellin core side chain O-glycosylations. The supercoiling mechanism presented here provides a benchmark for studies with other bacteria, for which near-atomic resolution structures of flagellar filament in native supercoiled forms, are still lacking.

biophysics↗

bioRxiv2020

Escape of TLR5 Recognition by Leptospira spp: A Rationale for Atypical Endoflagella

Leptospira interrogans are invasive bacteria responsible for leptospirosis, a worldwide zoonosis. They possess two periplasmic endoflagella that allow their motility. L. interrogans are stealth pathogens that escape the innate immune responses of the NOD-like receptors NOD1/2, and the human Toll-like receptor (TLR)4, sensing peptidoglycan and lipopolysaccharide (LPS), respectively. TLR5 is another receptor of bacterial cell wall components, recognizing flagellin subunits. To study the contribution of TLR5 in the host defense against leptospires, we infected WT and TLR5 deficient mice with pathogenic L. interrogans and tracked the infection by in vivo live imaging of bioluminescent bacteria or by q-PCR. We did not identify any protective or inflammatory role of murine TLR5 to control pathogenic Leptospira. Likewise, subsequent in vitro experiments showed that infections with different live strains of L. interrogans and L. biflexa did not trigger TLR5. However, unexpectedly, heat-killed bacteria stimulated human and bovine TLR5, although barely mouse TLR5. Abolition of TLR5 recognition required extensive boiling time of the bacteria or proteinase K treatment, showing an unusual high stability of the leptospiral flagellins. Interestingly, using antimicrobial peptides to destabilize live leptospires, we detected some TLR5 activity, suggesting that TLR5 could participate in the fight against leptospires in humans or cattle. Using different Leptospira strains with mutations in flagellin proteins, we further showed that neither FlaAs nor Fcps participated in the recognition by TLR5, suggesting a role for the FlaBs. These have structural homology to Salmonella FliC, and conserved residues important for TLR5 activation, as shown by in silico analyses. Accordingly, we found that leptospires regulate the expression of FlaB mRNA according to the growth phase in vitro, and that infection with L. interrogans in hamsters and in mice downregulated the expression of the FlaBs but not the FlaAs subunits. Altogether, in contrast to different bacteria that modify their flagellin sequences to escape TLR5 recognition, our study suggests that the peculiar central localization and stability of the FlaB monomers in the periplasmic endoflagella, associated with the downregulation of FlaB subunits in hosts, constitute an efficient strategy of leptospires to escape TLR5 recognition and the immune response.

immunology↗

bioRxiv2020

A Live Attenuated Vaccine Model Confers Cross-Protective Immunity Against Different Species of Leptospira spp.

Leptospirosis is the leading zoonotic disease in terms of morbidity and mortality worldwide with a great health and economic impact in both humans and animals. Effective prevention is urgently needed as rapid urbanization, climate change and drivers of disease transmission continue to intensify. The key challenge has been developing a widely-applicable vaccine that protects against the 13 different pathogenic species and >300 serovars that can cause leptospirosis, providing a major public health benefit and opportunity to leverage One Health approaches. Live attenuated mutants that can boost immunity and induce protection are enticing vaccine candidates and poorly explored in the field. We evaluated a recently characterized motility-deficient mutant lacking the expression of a flagellar protein, FcpA. Although the fcpA- mutant has lost its ability to penetrate mucous membranes and cause disease, a transient bacteremia prior to clearance by the host immune response was observed. In two animal models, immunization with a single dose of the fcpA- mutant was sufficient to induce robust anti-protein antibodies response that promoted protection against infection with different Leptospira spp. species. Furthermore, characterization of the immune response identified a small repertoire of biologically relevant proteins that are highly conserved among pathogenic Leptospira species and potential correlates of cross-protective immunity.

microbiology↗

bioRxiv2019

An asymmetric sheath controls flagellar supercoiling and motility in the Leptospira spirochete

Spirochete bacteria, including important pathogens, exhibit a distinctive means of swimming via undulations of the entire cell. Motility is powered by the rotation of supercoiled endoflagella that wrap around the cell body, confined within the periplasmic space. To investigate the structural basis of flagellar supercoiling, which is critical for motility, we determined the structure of native flagellar filaments from the spirochete Leptospira by integrating high-resolution cryo-electron tomography and X-ray crystallography. We show that these filaments are coated by a highly asymmetric, multi-component sheath layer, contrasting with flagellin-only homopolymers previously observed in exoflagellated bacteria. Distinct sheath proteins localize to the filament inner and outer curvatures to define the supercoiling geometry, explaining a key functional attribute of the spirochete flagellum. One Sentence summaryThe corkscrew-like motility of Spirochete bacteria is enabled by a unique, asymmetrically constructed flagellum that wraps around the cell body within the periplasm.

biophysics↗

bioRxiv2019

Rainfall and other meteorological factors as drivers of urban transmission of leptospirosis

BackgroundLeptospirosis is an important public health problem affecting vulnerable urban slum populations in developing country settings. However, the complex interaction of meteorological factors driving the temporal trends of leptospirosis remain incompletely understood.\n\nMethods and findingsFrom 1996 to 2010, we investigated the association between the weekly incidence of leptospirosis and climatic variables in the city of Salvador, Brazil by using a dynamic generalized linear model that accounted for time lags, overall trend and seasonal variation. Our model showed an increase of leptospirosis cases associated with rainfall, lower temperature and higher humidity. There was a lag of one-to-two weeks between weekly values for significant meteorological variables and leptospirosis incidence. Independent of the season, a weekly cumulative rainfall of 20 mm increased the risk of the leptospirosis by 10% compared to a week without rain. Finally, over the 14 year study period the incidence of leptospirosis decreased significantly by four fold (12.8 versus 3.6 per 100,000 people), independently of variations in climate.\n\nConclusionsStrategies to control leptospirosis should focus on avoiding contact with contaminated sources of Leptospira as well as on increasing awareness in the population and health professionals within the short time window after both low-level and extreme rainfall events. Increased leptospirosis incidence was restricted to one-to-two weeks after those events suggesting that infectious Leptospira survival may be limited to short time intervals.\n\nAuthor SummaryTo determine the role of meteorological variables, seasonal variation and temporal trends in the incidence of leptospirosis, we investigated the time series of leptospirosis incidence amongst residents of Salvador, Brazil, from 1996 to 2010. Exploratory and confirmatory statistical methods detected associations between meteorological factors and disease incidence. Results showed the importance of extreme meteorological conditions, particularly rainfall, as short-term predictors of leptospirosis incidence. In addition, we found a long-term decreasing trend of in disease incidence over the observation period.

epidemiology↗