Cell biology of Candida albicans-host interactions

Acknowledgements The authors are supported by the Wellcome Trust via a Senior Investigator Award to NG, an ISST award and a Wellcome Trust Strategic Award in Medical Mycology and Fungal Immunology. The authors are also part of the MRC Centre for Medical Mycology at Aberdeen.Peer reviewedPublisher PD

The Rewiring of Ubiquitination Targets in a Pathogenic Yeast Promotes Metabolic Flexibility, Host Colonization and Virulence

Funding: This work was funded by the European Research Council [http://erc.europa.eu/], AJPB (STRIFE Advanced Grant; C-2009-AdG-249793). The work was also supported by: the Wellcome Trust [www.wellcome.ac.uk], AJPB (080088, 097377); the UK Biotechnology and Biological Research Council [www.bbsrc.ac.uk], AJPB (BB/F00513X/1, BB/K017365/1); the CNPq-Brazil [http://cnpq.br], GMA (Science without Borders fellowship 202976/2014-9); and the National Centre for the Replacement, Refinement and Reduction of Animals in Research [www.nc3rs.org.uk], DMM (NC/K000306/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Acknowledgments We thank Dr. Elizabeth Johnson (Mycology Reference Laboratory, Bristol) for providing strains, and the Aberdeen Proteomics facility for the biotyping of S. cerevisiae clinical isolates, and to Euroscarf for providing S. cerevisiae strains and plasmids. We are grateful to our Microscopy Facility in the Institute of Medical Sciences for their expert help with the electron microscopy, and to our friends in the Aberdeen Fungal Group for insightful discussions.Peer reviewedPublisher PD

Metabolic flexibility in <i>C</i>. <i>albicans</i> promotes resistance to macrophage killing.

<p>Survival of <i>C</i>. <i>albicans</i> strains following co-incubation with J774.1 macrophages for 48 h: WT, SC5314; <i>icl1Δ/Δ</i>, DCY65; <i>ICL1</i>, DCY75 (<i>ICL1-Myc</i>_<i>3</i><i>-NAT1</i>); <i>ICL1-Ubi</i>, DCY82 (<i>ICL1-Ubi-Myc</i>_<i>3</i><i>-NAT1</i>) (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005566#ppat.1005566.s007" target="_blank">S2 Table</a> in the supplementary information). Data represent three independent biological experiments performed in technical triplicate (mean values plus standard error of the mean (SEM)). The data were analysed using one-way ANOVA with Tukey’s post-hoc test: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.</p

Many <i>S</i>. <i>cerevisiae</i> clinical isolates are Crabtree negative.

<p><i>S</i>. <i>cerevisiae</i> clinical isolates were pre-grown in YNB-lactate and spotted on SC medium with glucose or lactate in the presence or absence of 200 μg/mL 2-deoxyglucose. Nine clinical isolates are presented here, with data for a further 12 isolates presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005566#ppat.1005566.s004" target="_blank">S4 Fig</a> in the supplementary information.</p

The lack of catabolite inactivation in <i>C</i>. <i>albicans</i> permits simultaneous assimilation of alternative carbon sources and glucose.

<p>In <i>S</i>. <i>cerevisiae</i>, glucose triggers the rapid ubiquitination and degradation via the GID complex of enzymes involved in the assimilation of alternative carbon sources (catabolite inactivation). Consequently, <i>S</i>. <i>cerevisiae</i> displays sequential assimilation of these carbon sources, only utilizing alternative carbon sources once glucose has been exhausted. In contrast, <i>C</i>. <i>albicans</i> enzymes involved in the utilization of alternative carbon sources lack ubiquitination sites and hence are not subject to catabolite inactivation. Consequently, these pathways remain active in <i>C</i>. <i>albicans</i> following glucose exposure and this yeast displays simultaneous assimilation of alternative carbon sources and glucose [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005566#ppat.1005566.ref018" target="_blank">18</a>].</p

Crabtree negative <i>S</i>. <i>cerevisiae</i> strains are more resistant to macrophage killing.

<p>(A) <i>S</i>. <i>cerevisiae</i> wild-type (black bars, S288c) and <i>gid8Δ</i> cells (red, DCY122) were pre-grown in YNB-glucose or YNB-lactate, and then co-incubated with J774.1 macrophages. The viability of the <i>gid8Δ</i> cells after 48 hours is expressed relative to the corresponding wild type control. (B) Using the same approach, the resistance of three Crabtree positive <i>S</i>. <i>cerevisiae</i> clinical isolates (black) and three Crabtree negative clinical isolates (red) to macrophage killing was compared. (C) Isocitrate lyase activities were measured in the same <i>S</i>. <i>cerevisiae</i> strains after pre-growth overnight in YNB-lactate followed by growth in YNB-glucose or YNB-lactate for 2 h. The fold-reduction in Icl1 activity in cells exposed to glucose is expressed relative to the control cells grown in lactate. Statistical significance was calculated relative to the fold-reduction observed in the wild type control (S288c). (D) The phagocytic uptake of the <i>S</i>. <i>cerevisiae</i> strains was determined after pre-growth in YNB-lactate, and then co-incubation with J774.1 macrophages for 2 h. No significant differences (ns) were observed relative to the wild type control. The data represent two independent biological experiments performed in technical triplicate ± SEM. They were analysed relative to the glucose grown controls by two-way ANOVA with multiple comparisons test: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.</p

A Crabtree negative <i>S</i>. <i>cerevisiae</i> strain is more virulent in immunocompromised mice.

<p>(A) Inactivation of <i>GID8</i> in <i>S</i>. <i>cerevisiae</i> clinical isolate NCPF8313 renders it 2-deoxyglucose resistant. <i>S</i>. <i>cerevisiae</i> strains were pre-grown in YNB-lactate and spotted on SC medium with glucose (Glu) or lactate (Lac) in the presence or absence of 2-deoxyglucose (2DG): WT, DCY145; <i>gid8Δ</i>, DCY150; <i>gid8Δ</i>+GID8, DCY148 (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005566#ppat.1005566.s007" target="_blank">S2 Table</a> in the supplementary information). (B) Immunodeficient DBA/2 mice were injected via lateral tail vein with the same <i>S</i>. <i>cerevisiae</i> strains and renal fungal burdens were measured after 72 h (n = 6 per group). Points represent the CFUs for each animal and the bar denotes the mean. (C) The infection outcome scores were then determined by combining the renal fungal burdens with the percentage weight change for the mice ± SEM [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005566#ppat.1005566.ref039" target="_blank">39</a>]. The data were analysed using the Mann Whitney test: *, P ≤ 0.05; **, P ≤ 0.01.</p

Evolutionary rewiring of metabolic ubiquitination targets across yeast species.

<p>The phylogenetic tree of the <i>Candida</i> and <i>Saccharomyces</i> species analysed was generated by aligning ITS sequences using ClustalW. The <i>in silico</i> prediction of ubiquitination sites was performed using Ubpred (<a href="http://www.ubpred.org" target="_blank">www.ubpred.org</a>) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005566#ppat.1005566.ref032" target="_blank">32</a>] on the metabolic enzymes listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005566#ppat.1005566.s006" target="_blank">S1 Table</a> in the supplementary information: grey, the presence of at least one high confidence ubiquitination site; red, human pathogen; green, not a human pathogen; blue, Crabtree positive yeast; yellow, Crabtree negative yeast.</p