Research Article: Blocking two-component signalling enhances Candida albicans virulence and reveals adaptive mechanisms that counteract sustained SAPK activation

Date Published: January 30, 2017

Publisher: Public Library of Science

Author(s): Alison M. Day, Deborah A. Smith, Mélanie A. C. Ikeh, Mohammed Haider, Carmen M. Herrero-de-Dios, Alistair J. P. Brown, Brian A. Morgan, Lars P. Erwig, Donna M. MacCallum, Janet Quinn, Xiaorong Lin.


The Ypd1 phosphorelay protein is a central constituent of fungal two-component signal transduction pathways. Inhibition of Ypd1 in Saccharomyces cerevisiae and Cryptococcus neoformans is lethal due to the sustained activation of the ‘p38-related’ Hog1 stress-activated protein kinase (SAPK). As two-component signalling proteins are not found in animals, Ypd1 is considered to be a prime antifungal target. However, a major fungal pathogen of humans, Candida albicans, can survive the concomitant sustained activation of Hog1 that occurs in cells lacking YPD1. Here we show that the sustained activation of Hog1 upon Ypd1 loss is mediated through the Ssk1 response regulator. Moreover, we present evidence that C. albicans survives SAPK activation in the short-term, following Ypd1 loss, by triggering the induction of protein tyrosine phosphatase-encoding genes which prevent the accumulation of lethal levels of phosphorylated Hog1. In addition, our studies reveal an unpredicted, reversible, mechanism that acts to substantially reduce the levels of phosphorylated Hog1 in ypd1Δ cells following long-term sustained SAPK activation. Indeed, over time, ypd1Δ cells become phenotypically indistinguishable from wild-type cells. Importantly, we also find that drug-induced down-regulation of YPD1 expression actually enhances the virulence of C. albicans in two distinct animal infection models. Investigating the underlying causes of this increased virulence, revealed that drug-mediated repression of YPD1 expression promotes hyphal growth both within murine kidneys, and following phagocytosis, thus increasing the efficacy by which C. albicans kills macrophages. Taken together, these findings challenge the targeting of Ypd1 proteins as a general antifungal strategy and reveal novel cellular adaptation mechanisms to sustained SAPK activation.

Partial Text

Candida albicans is the leading cause of systemic fungal infections in humans resulting in over 400,000 deaths each year in immuno-compromised patients [1]. The ability of C. albicans to adapt to host-imposed stresses encountered during infection is an important virulence trait [2]. Central to fungal stress responses are the stress-activated protein kinases (SAPKs), which are conserved eukaryotic signalling enzymes that allow cells to adapt to environmental change [3, 4]. In C. albicans, the Hog1 SAPK is activated in response to diverse, physiologically relevant, stress conditions, and cells lacking Hog1 are acutely sensitive to such stresses [5–7]. Consistent with the vital role of the Hog1 SAPK in stress survival, C. albicans cells lacking HOG1 display significantly attenuated virulence in systemic, commensal, and phagocyte infection models [8–11].

The generation of fungal pathogen-specific drugs is hindered by the conservation of many potential drug-targets in the human host. Thus the complete absence of two-component related proteins in metazoans, but their presence in fungi, has rendered such pathways attractive drug-targets [22, 44, 45]. In S. cerevisiae, and the human fungal pathogen C. neoformans, loss of the two-component phosphorelay protein Ypd1 causes lethality due to the sustained activation of their respective SAPK pathways [20, 21]. However, as reported during the course of this work [25], a major human fungal pathogen, C. albicans, can tolerate sustained activation of the Hog1 SAPK pathway triggered by loss of Ypd1. We have significantly advanced our understanding of this observation in three main ways. Firstly we find that the constitutive activation of Hog1 in cells lacking YPD1 is mediated through two-component mediated regulation of the Ssk1 response regulator, and that the pleiotropic phenotypes associated with Ypd1 loss are dependent on Ssk1-mediated Hog1 activation. Secondly, we have provided novel insight into the mechanisms by which C. albicans survives and adapts to sustained SAPK activation. Thirdly, we show that inactivation of YPD1 promotes, rather than reduces, the virulence of C. albicans, and we provide evidence to suggest that this is mediated at least in part through effects on fungus-phagocyte interactions. A model summarising the major findings from this work is depicted in Fig 8.




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