We propose to explore an innovative mode of adherence to host tissue mediated by the fungal virulence factor BAD-1 (Blastomyces adhesin-1) of B. dermatitidis. Our data point to an unusual mechanism exploiting synergy between mechanochemical stress and fungal or host redox co-factors to trigger powerful catch-bonds to host cell surface glycosaminoglycans (GAGs). BAD-1 contains 3 domains: the chief one, 41 copies of a 25 aa tandem repeat. Deleting the repeats curtails fungal virulence. In solving the 3D structure of the repeats, we found parallels to the type 1 repeat of throbmospondin-1 (TSP-1) and malaria TRAP protein. Like them, BAD-1 binds heparan sulfate. BAD-1 binding of heparan-modified CD47 on T cells impairs activation and effector function, promoting immune evasion. We hypothesize that high avidity binding of BAD-1 to heparin hinges on its multiple repeats. Yet, we find that repeats do not bind heparin unless they are first reduced and re-oxidized in the presence of heparin. The BAD-1 N-terminus harbors a Cardin-Weintraub (C-W) heparin-binding motif that we posit initiates binding. We also posit that mechanochemical stress and reducing agent(s) (i.e., thioredoxins) together trigger reorganization of BAD-1 repeats, re-ordering disulfide bonds. The exposure of GAGs to serial binding sites in the multiple repeats would impact BAD-1 avidity for cell surface and matrix.
We aim to: 1) Define how BAD-1 initiates binding to heparin and reconfigures itself structurally upon attachment. The domain and aa sequence of BAD-1 that mediate the initial binding of heparin will be defined. Peptides from the BAD- 1 N-terminal C-W domain, with and without aa substitutions and adjacent repeat sequences, will be studied for binding heparin. Changes in BAD-1's shape upon association with heparin will be tested with small angle X-ray scattering, and variation in its disulfide bond order upon complexation with heparin will be defined via Mass Spec. Blastomyces lacking a BAD-1 C-W domain will be tested for virulence in vivo and heparin binding. 2) Delineate how stretching BAD-1 mechanically together with thioredoxin catalysts impacts heparin binding. Atomic force microscopy (AFM) with BAD-1 on the probe-tip will be used to measure variations in binding-affinity & avidity and elevations in tear-away force from heparin. AFM also will be used to compare heparin binding of intact BAD-1 and derivatives that lack N-terminal C-W or repeat regions, as well as the impact of endogenous thioreductases. AFM studies will guide engineering of B. dermatitidis RNAi strains with diminished endogenous thioreductase to test their impact on virulence in vivo and BAD-1 mediated interactions with heparin. This work tackles a fundamental pathogenic mechanism: adhesion to host tissue. It applies under-appreciated biophysical concepts to develop new mechanistic insight into catch-bonds: how they form, evolve & function.
Systemic fungal infections represent a significant and growing public health problem. In the U.S., invasive fungal infections are now one of the 10 leading causes of death (7th) ahead of mortality due to tuberculosis. Our work tackles significant unmet needs of developing better ways to treat and prevent these infections by investigating a basic mechanism of pathogenicity - binding host tissue - in a human fungal pathogen. The new basic knowledge gleaned here can be harnessed for better fungal disease prevention and treatment.
| Sterkel, Alana K; Lorenzini, Jenna L; Fites, J Scott et al. (2016) Fungal Mimicry of a Mammalian Aminopeptidase Disables Innate Immunity and Promotes Pathogenicity. Cell Host Microbe 19:361-74 |
| Beaussart, Audrey; Brandhorst, Tristan; Dufrêne, Yves F et al. (2015) Blastomyces Virulence Adhesin-1 Protein Binding to Glycosaminoglycans Is Enhanced by Protein Disulfide Isomerase. MBio 6:e01403-15 |
