טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentBar-Yosef Hagit
SubjectSuppression of Filamentation in Candida albicans
DepartmentDepartment of Medicine
Supervisor Professor Daniel Kornitzer


Abstract

Candida albicans is a human commensal microorganism that typically inhabits the oral cavity and/or the gastrointestinal (GI) tract.  In immunocompromised individuals, (HIV patients, cancer patients, etc.) C. albicans can cause candidemia - a bloodstream infection - with a crude mortality rate of 40%.

A significant virulence trait of C. albicans is its ability to undergo transition between yeast and hyphal (filamentous) morphologies. The yeast-to-hyphae morphogenetic switch is activated by well-established, kinase-based signal transduction pathways that are induced by extracellular stimuli. The hyphal switch allows effective invasion of host tissues, and confers the ability to form biofilms and to block an adaptive immune response. Thus the suppression of hyphal morphogenesis is of both fundamental and therapeutic importance.

Both genetic research and pharmacological reagents can be used to study pathways of inhibition of hyphal morphogenesis. In a screen using libraries of compounds that either serve as drugs or have undergone clinical trials (“repurposing screen”), a collaborating laboratory identified several molecules out of over 3000 screened that suppress hyphal morphogenesis at concentrations that do not affect yeast growth. One of these molecules, Trifluoperazine, suppresses hyphal morphogenesis and hyphal-specific gene expression. Trifluoperazine exposure also resulted in a reduction of biofilm formation. Using different C. albicans and S. cerevisiae genomic library screens, we identified candidate genes for the sites of action of this hyphal morphogenesis-suppressing drug. Many of these genes are involved in endocytosis. Indeed, using Lucifer Yellow as a marker, we found that Trifluoperazine suppresses fluid phase endocytosis.

In an additional attempt to identify possible inhibitory pathways of the yeast-to-hyphae transition, a collection of C. albicans protein kinases and phosphatases ectopically expressed under the regulation of the TET-on promoter had been interrogated in our laboratory. AKL1 was identified, whose homolog in baker’s yeast is involved in the regulation of endocytosis. We found that C. albicans AKL1 overexpression suppresses hyphal morphogenesis, whereas deletion of this gene leads to a more filamentous morphology. CaAkl1 specifically affected hyphal elongation: deletion of CaAKL1 resulted in an acceleration in the rate of hyphal elongation. CaAkl1 overexpression suppressed fluid phase endocytosis while its knockout strain exhibited higher levels of endocytosis. In a Caume6-/- strain, which is not filamentous even under hyphal inducing conditions, CaAkl1 still suppressed Lucifer Yellow uptake, indicating that it affects endocytosis directly rather than via an effect on morphogenesis, and suggesting that it affects hyphal morphogenesis via its effect on endocytosis.

Akl1 was suggested to inhibit endocytosis in S. cerevisiae via phosphorylation of endocytic protein Pan1. C. albicans Pan1 was identified as a relevant substrate of CaAkl1 as well: strains overexpressing CaPAN1 showed increased filamentous morphology and sedimentation compared to strains overexpressing only CaAKL1. Western blotting analysis revealed accelerated migration of CaPan1 in the absence of CaAkl1, consistent with a reduced phosphorylation. We also found that CaAkl1 affects CaPan1 localization in the growing hyphal cells.

Our results highlight the importance of the little-studied role of endocytosis in hyphal morphogenesis and indicate that under standard conditions, endocytosis is rate-limiting for hyphal elongation.