Poster abstracts
Poster number 26 submitted by Dale Lingo
Functional characterization of Histoplasma capsulatum chitinases
Dale E Lingo (Molecular, Cellular, and Developmental Biology Graduate Program), Dr. Kristie Goughenour (Division of Pulmonary and Critical Care Medicine, University of Michigan ), Dr. Chad Rappleye (Dept. of Microbiology, The Ohio State University)
Abstract:
Histoplasma capsulatum is a dimorphic fungal pathogen which can cause severe lung infection in both immunocompetent and immunocompromised people. Histoplasma yeast survive and proliferate in the phagocytic compartment of macrophages. Chitin, a core constituent of the fungal cell wall, is crucial for cell structure and protection from extracellular stressors. However, structural changes during morphological transitions as well as yeast cell division requires the remodeling of cell wall components, including chitin. To better understand how chitin impacts Histoplasma cell structure and cell wall dynamics, we are characterizing the eight chitinases (chitin hydrolyzing enzymes; Cts1-Cts8) encoded within the Histoplasma genome. For functional tests, we generated strains depleted of each chitinase via RNA-interference (RNAi), as well as CRISPR/Cas9-mediated genome editing. Individual chitinase depletion did not alter the overall level of cell wall chitin as assessed by staining with the fluorescent chitin stain Uvitex. However, depletion of Cts2 (CTS2-RNAi) caused increased yeast cell aggregation. The aggregation of Cts2-deficient cells requires cell growth and division and in mixed cultures with wild type, only the Cts2-deficient cells aggregated. These data suggest Cts2-deficient yeasts have defects in separation following division. Either supernatant from wild-type yeasts or purified Cts2 can prevent aggregation of Cts2-deficient cells indicating Cts2 acts in trans to facilitate cell separation. Electron microscopy of Cts2-deficient yeasts indicate altered cell surface. Infections of mice with Cts2-deficient yeasts compared to wild-type yeasts showed similar fungal burdens in the lungs, but reduced colonization of the spleen, indicating a defect in dissemination. As Histoplasma is an intracellular pathogen, we hypothesize that Cts2 acts to prevent aggregation of yeasts following division thereby allowing macrophages to engulf isolated yeasts cells and subsequently traffic them to extrapulmonary organs.
Keywords: Histoplasma, Cell wall, Chitinase