Stress-induced nuclear accumulation is dispensable for Hog1-dependent gene expression and virulence in a fungal pathogen

The authors thank E. Veal for intellectual input. This work was funded by the UK Biotechnology and Biological Research Council [J.Q. BB/K016393/1; A.J.P.B. BB/K017365/1], the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) [D.M.M. NC/N002482/1] and the Wellcome Trust Strategic Award in Medical Mycology and Fungal Immunology [097377]). D.M.M. and A.J.P.B. are also supported by the MRC Centre for Medical Mycology at the University of Aberdeen (MR/N006364/1).Peer reviewedPublisher PD

Sfp1 and Rtg3 reciprocally modulate carbon source-conditional stress adaptation in the pathogenic yeast Candida albicans

Acknowledgements We thank Aaron Mitchell, Dominique Sanglard and Suzanne Noble for their generosity in providing mutant collections, and Linghuo Jiang for generously providing strains. We also thank Susan Budge for her support and excellent technical assistance. We also thank the qPCR Facility in the Institute of Medical Sciences, and particularly Fiona Saunders for her great advice and help. SLK was supported by a PhD scholarship from the University of Aberdeen. AJPB was supported by the UK Biotechnology and Biological Research Council (BB/F00513X/1; BB/K017365/1), by the European Research Council (STRIFE Advanced Grant; ERC-2009-AdG-249793), and by the UK Medical Research Council (MR/M026663/1). AJPB and CAM were also supported by the Wellcome Trust (088858; 097377), and by the MRC Centre for Medical Mycology and the University of Aberdeen (MR/N006364/1).Peer reviewedPublisher PD

Redox Regulation, Rather than Stress-Induced Phosphorylation, of a Hog1 Mitogen-Activated Protein Kinase Modulates Its Nitrosative-Stress-Specific Outputs

Data availability. The RNA sequencing dataset is available at EBI (www.ebi.ac.uk/arrayexpress/) under accession number E-MTAB-5990. Other data that support the findings of this study are available from the corresponding author upon reasonable request. ACKNOWLEDGMENTS We thank Debbie Smith for constructing the strains JC41 and JC310, Arnab Pradhan for help with DHE control experiments, and our colleagues in the Aberdeen Fungal Group and Newcastle Yeast Group for insightful discussions. We are also grateful to Mike Gustin for his advice. We are grateful to the Centre for Genome Enabled Biology and Medicine, Aberdeen Proteomics, the Iain Fraser Cytometry Centre, the Microscopy and Histology Facility, and the qPCR facility at the University of Aberdeen for their help, advice, and support. This work was funded by the UK Biotechnology and Biological Research Council (http://www.bbsrc.ac.uk) (grants BB/K017365/1 and BB/F00513X/1 to A.J.P.B. and grant BB/K016393/1 to J.Q.). This work was also supported by the European Research Council (http://erc.europa.eu/) (STRIFE advanced grant C-2009-AdG-249793 to A.J.P.B.), the UK Medical Research Council (http://www.mrc.ac.uk) (grant MR/M026663/1 to A.J.P.B. and grant MR/M000923/1 to P.S.S.), the Wellcome Trust (https://wellcome.ac.uk) (grant 097377 to A.J.P.B. and J.Q.), the MRC Centre for Medical Mycology and the University of Aberdeen (grant MR/M026663/1 to A.J.P.B.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

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

This work was funded by the UK Biotechnology and Biological Research Council [www.bbsrc.ac.uk] JQ (BB/K016393/1); AJPB (BB/K017365/1). The work was also supported by the Wellcome Trust [www.wellcome.ac.uk], JQ (086048, 097377); AJPB (097377)); LPE (097377). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Integrative Model of Oxidative Stress Adaptation in the Fungal Pathogen Candida albicans

Acknowledgments We are grateful to the Ian Fraser Cytometry Centre and our Mass Spetrometry and qPCR Facilities for help with the flow cytometry, glutathione and qRT-PCR assays, respectively. We also thank our many colleagues in the CRISP Consortium and in the medical mycology and systems biology communities for insightful discussions. Funding: This work was supported by the CRISP project (Combinatorial Responses In Stress Pathways), which was funded by the UK Biotechnology and Biological Research Council (www.bbsrc.ac.uk): AJPB, KH, CG, ADM, NARG, MT, MCR. (Research Grants; BB/F00513X/1, BB/F005210/1-2). AJPB and JQ received additional support from the BBSRC (Research Grants; BB/K016393/1; BB/K017365/1). NARG and AJPB were also supported by the Wellcome Trust (www.wellcome.ac.uk), (Grants: 080088; 097377). AJPB was also supported by the European Research Council (http://erc.europa.eu/), (STRIFE Advanced Grant; ERC-2009-AdG-249793). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Pho4 mediates phosphate acquisition in Candida albicans and is vital for stress resistance and metal homeostasis

ACKNOWLEDGMENTS We thank Karl Kuchler for the C. albicans superoxide dismutase mutants used in this study. This work was funded by a Medical Research Council Doctoral Training Program studentship to M.A.C.I.; Wellcome Trust Grants 089930 to J.Q., 080088 to A.J.P.B., and 097377 to J.Q., A.J.P.B., and L.P.E.; Biotechnology and Biological Sciences Research Council Grants BB/K016393/1 to J.Q. and BB/F00513X/1 and BB/K017365/1 to A.J.P.B.; European Research Council STRIFE Advanced Grant ERC-2009-AdG-249793 to A.J.P.B.; and Wellcome Trust and Royal Society Sir Henry Dale Fellowship 098375/Z/12/Z to K.J.W. and E.T. The funders had no role in study design, data collection, or interpretation or the decision to submit the work for publication.Peer reviewedPublisher PD

Elevated catalase expression in a fungal pathogen is a double-edged sword of iron

We thank our colleagues in the Aberdeen Fungal Group, Lloyd Peck (British Antarctic Survey) and John Helmann (Cornell University) for insightful discussions. We thank Christophe d’Enfert and Melanie Legrand (Institut Pasteur) for help with the design of barcodes and provision of the CIp10-PTET-GTw overexpression vector and CEC2908 strain. We are grateful to the following Core Facilities at the University of Aberdeen for their excellent technical assistance, advice and support: the Medical Research Facility; the Centre for Genome Enabled Biology and Medicine; the Iain Fraser Cytometry Centre; the Microscopy and Histology Facility; Aberdeen Proteomics; and the qPCR Facility.Peer reviewedPublisher PD

Phenotypes associated with loss of Ypd1 are dependent on Hog1 and Ssk1.

<p>(A) Repression or deletion of <i>YPD1</i> triggers flocculation and a swollen pseudohyphal filamentous phenotype. Micrographs of <i>Wt</i>, <i>ypd1Δ</i>, and <i>tetO-YPD1</i> cells plus or minus doxycycline (DOX) grown overnight in rich media. Images of culture tubes demonstrate the rapid sedimentation rate of cells lacking <i>YPD1</i>. (B) Repression or deletion of <i>YPD1</i> results in pleiotropic stress phenotypes. 10⁴ cells, and 10-fold dilutions thereof, of exponentially growing <i>tetO-YPD1</i> cells, or wild-type (<i>Wt</i>), <i>ypd1Δ</i> and <i>ypd1Δ+YPD1</i> cells, were spotted onto rich media plates (plus or minus DOX for <i>tetO-YPD1</i> cells) containing NaCl (1.0 M), calcofluor white (CFW, 30 μg/ml), NaAsO₂ (1.5 mM) and <i>t</i>-BOOH (2 mM), and incubated at 30°C for 24h. (C) The morphological defects exhibited by <i>ypd1Δ</i> cells are dependent on Hog1 and Ssk1. Micrographs of wild-type (<i>Wt</i>), <i>ypd1Δ</i>, <i>hog1Δ</i> (JC50), <i>ssk1Δ</i> (JC1552), <i>hog1Δ ypd1Δ</i> (JC1475) <i>hog1Δypd1Δ</i>+<i>HOG1</i> (JC1478), <i>ssk1Δ ypd1Δ</i> (JC1683), and <i>ssk1Δ ypd1Δ</i>+<i>SSK1</i> (JC1704) cells. (D) The high glycerol levels in <i>ypd1Δ</i> cells are dependent on Hog1. The mean ± SD is shown for 3 biological replicates. (E) The stress phenotypes exhibited by <i>ypd1Δ</i> cells are dependent on Hog1 and Ssk1. Exponentially growing strains were spotted onto rich media plates containing the additives detailed in B above, and incubated at 30°C for 24h. (F) The sustained Hog1 activation in <i>ypd1Δ</i> cells is dependent on Ssk1. Western blots depicting basal levels of Hog1 phosphorylation in the indicated strains. Blots were probed for phosphorylated Hog1 (Hog1-P), stripped and reprobed for total Hog1 (Hog1).</p

Ypd1 inactivation in <i>C</i>. <i>albicans</i> triggers Hog1 hyperactivation, increased virulence, and in the long term a reduction in Hog1 activity.

<p>Model depicting outcomes following <i>YPD1</i> loss in <i>C</i>. <i>albicans</i>. Loss of Ypd1 results in the accumulation of the unphosphorylated Ssk1 response regulator, which drives the activation of Hog1 under non-stressed conditions. The levels of Hog1 phosphorylation are modulated by the induction of the negative regulators Ptp2 and Ptp3 which allows cells to adapt and survive Ypd1 loss. Loss of Ypd1 function during infection increases the virulence of <i>C</i>. <i>albicans</i>, by possibly enhancing Hog1 activity promoting stress resistance and/or filamentation. Furthermore, <i>C</i>. <i>albicans</i> adapts to long-term activation of Hog1 by reducing the levels of the phosphorylated Hog1 kinase. This adaptation process prevents phenotypes associated with sustained SAPK activation and <i>ypd1Δ</i> cells now phenotypically resemble wild-type cells. Notably, however, this adaptation mechanism to circumvent Hog1 phosphorylation can be over-ridden following transient stress exposure and thus sustained Hog1 activation is restored.</p

Repression of <i>YPD1</i> expression during infection potentiates <i>C</i>. <i>albicans</i> virulence.

<p>(A) <i>C</i>. <i>elegans model of infection</i>. Nematodes were infected with the conditional <i>tetO-YPD1</i> strain (JC1586) and transferred to liquid medium either with (+DOX) or without (-DOX) doxycycline. Doxycycline treatment consistently increased the rate of killing of the nematodes infected with the <i>tetO-YPD1</i> strain (<i>P</i><0.001). These data are from a single experiment representative of three independent biological replicates. (B) <i>Mouse model of infection</i>. Kidney fungal burden measurements, percentage weight loss, and outcome score measurements of mice infected with <i>tetO-YPD1</i> cells and administered doxycycline (+DOX) or not (-DOX). Comparison of +DOX and -DOX treated groups by Kruskal-Wallis statistical analysis demonstrates significant differences for all three parameters with doxycycline treated mice giving a significantly greater outcome score (*<i>P</i><0.05, ** <i>P</i><0.01). (C) Increased virulence of cells with repressed <i>YPD1</i> expression is associated with increased numbers of fungi and inflammation in the infected kidneys. Representative images from kidneys of mice infected with <i>tetO-YPD1</i> cells and treated with (+DOX; i-iii) or without (-DOX; iv-vi) doxycycline. Sections (5 μm) were Periodic Acid-Schiff stained and post-stained with Hematoxylin. Low magnification images in (i) and (iv) (scale bar = 200 μm) show the difference in lesion number and extent of inflammatory response. Higher magnification (scale bar = 50μm) shows the presence of clusters of filamentous fungal cells (white arrows) in the lesions of doxycycline-treated mouse kidneys (ii & iii) and isolated single pseudohyphal fungal cells (white arrows) in placebo-treated mouse kidneys (v & vi). (D) Macrophage model of infection. <i>C</i>. <i>albicans</i> mediated killing of macrophages was determined by detecting the number of ruptured macrophages following co-culture with or without <i>tetO-YPD1</i> cells in the presence (+DOX) or absence (-DOX) of doxycycline. ANOVA was used to determine statistical significance (** <i>P</i> ≤ 0.01). (E) Assessment of hyphal growth following phagocytosis by <i>tetO-YPD1</i> cells in the presence (+DOX) or absence (-DOX) of doxycycline. The left panel shows that +DOX treatment resulted in a faster rate of hyphal growth. ANOVA was used to determine statistical significance (** <i>P</i> ≤ 0.01). The right panel shows images taken 61 mins post engulfment of yeast cells. The scale bar is 9 μm, and the white arrows indicate hyphal cells within the macrophage.</p