Surface stress induces a conserved cell wall stress response in the pathogenic fungus Candida albicans.

Surface Stress Induces a Conserved Cell Wall Stress Response in the Pathogenic Fungus Candida albicans Clemens J. Heilmann,a,b Alice G. Sorgo,a,b Sepehr Mohammadi,a,b Grazyna J. Sosinska,c Chris G. de Koster,b Stanley Brul,a Leo J. de Koning,b Frans M. Klisa Department of Molecular Biology and Microbial Food Safety,a Department of Mass Spectrometry of Biomacromolecules,b Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands; DSM Biotechnology Center, Delft, The Netherlandsc The human fungal pathogen Candida albicans can grow at temperatures of up to 45°C. Here, we show that at 42°C substantially less biomass was formed than at 37°C. The cells also became more sensitive to wall-perturbing compounds, and the wall chitin levels increased, changes that are indicative of wall stress. Quantitative mass spectrometry of the wall proteome using 15N metabolically labeled wall proteins as internal standards revealed that at 42°C the levels of the ␤-glucan transglycosylases Phr1 and Phr2, the predicted chitin transglycosylases Crh11 and Utr2, and the wall maintenance protein Ecm33 increased. Consistent with our previous results for fluconazole stress, this suggests that a wall-remodeling response is mounted to relieve wall stress. Thermal stress as well as different wall and membrane stressors led to an increased phosphorylation of the mitogen-activated protein (MAP) kinase Mkc1, suggesting activation of the cell wall integrity (CWI) pathway. Furthermore, all wall and membrane stresses tested resulted in diminished cell separation. This was accompanied by decreased secretion of the major chitinase Cht3 and the endoglucanase Eng1 into the medium. Consistent with this, cht3 cells showed a similar phenotype. When treated with exogenous chitinase, cell clusters both from stressed cells and mutant strains were dispersed, underlining the importance of Cht3 for cell separation. We propose that surface stresses lead to a conserved cell wall remodeling response that is mainly governed by Mkc1 and is characterized by chitin reinforcement of the wall and the expression of remedial wall remodeling enzymes. C andida albicans is an opportunistic fungal pathogen of humans and other warm-blooded animals. It is one of the leading causes of fungal infections among immunocompromised patients, which are often fatal if not diagnosed in time (1, 2). During infection, C. albicans encounters stresses from host defenses (e.g., fever and oxidative and nitrosative stress), environmental niches (e.g., hypoxia in the gut and antimicrobial peptides in saliva and in epithelial layers), and antifungal intervention (e.g., azoles and echinocandins). Many of these stresses directly affect the cell surface, which has distinct fungal features compared to mammalian cells. The most important difference is the presence of a cell wall. The cell wall is the initial site of host-pathogen interaction and is composed of a skeletal layer of carbohydrates, mainly ␤-glucans and chitin, which is covered with an external layer of covalently anchored mannoproteins. These mannoproteins have been shown to serve a variety of functions, from immune evasion (3, 4) and nutrient acquisition (5, 6) to adhesion, biofilm formation (7, 8), and tissue degradation (9). Many cell wall proteins also directly modulate the wall composition and architecture as carbohydrateactive enzymes (10). Reinforcement of the cell wall in response to antifungal stresses is well described, especially with respect to an increase in chitin content as a result of increased chitin synthesis (11, 12). The cell wall proteome itself is highly dynamic (13) and adaptable in response to external conditions (14, 15) as well as morphological changes (16). This dynamic surface is crucial for an opportunistic pathogen, enabling it to colonize different niches in a variety of hosts. Sites of infection differ dramatically in, for example, oxygen levels, pH, and available nutrients. Another environmental factor that has a major impact on the fungal surface and growth is temperature, which can vary considerably depending on the host species. While most fungi are not able to grow above 40°C (17), C. albicans causes infections in many animals (18), among them birds (e.g., pen- 254 ec.asm.org Eukaryotic Cell guins and pigeons), whose body temperatures are in this range (19, 20). As a consequence of prolonged thermal stress, both Saccharomyces cerevisiae and C. albicans cells have been shown to accumulate trehalose, which facilitates proper protein folding under stress conditions (21, 22). In S. cerevisiae thermal stress also leads to the activation of the cell wall integrity (CWI) pathway which, in turn, affects the composition of the wall and its proteins (reviewed in reference 23). In addition, Mkc1, the C. albicans ortholog of Slt2 in S. cerevisiae and a key signal transducer in the CWI pathway of C. albicans, has been suggested to be required for growth at elevated temperatures (24). Another member of the CWI pathway, Pkc1, was shown to be involved in the response to fluconazole (25), a widely used antimycotic agent that leads to the depletion of ergosterol in the fungal membrane (26), resulting in increased membrane fluidity (27, 28). We recently established that fluconazole does not only elicit membrane stress but also leads to a stressed cell wall. Since membrane fluidity is correlated with temperature (29), we hypothesized that a similar effect as seen for fluconazole could be achieved by growth at elevated temperatures. In this study, we examined the effect of thermal stress on the wall composition, the secretome, and the cell wall proteome as well as underlying regulatory pro- p. 254 –264 Received 3 October 2012 Accepted 7 December 2012 Published ahead of print 14 December 2012 Address correspondence to Frans M. Klis, . CJ.H. and A.G.S. contributed equally to this article. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /EC.00278-12 Copyright © 2013, American Society for Microbiology. All Rights Reserved. doi:10.1128/EC.00278-12 February 2013 Volume 12 Number 2 Surface Stress Induces a Conserved Wall Stress Response TABLE 1 Candida albicans strains used in this study Name SC5314 DSY1768 SPY24 DSY1741 CNC63 MK106 CAMM-292-4 a Parent a NA CAF-2 CAF-2 CAF-2 CAF-2 SC5314 CAI4 Relevant genotype Abbreviation Reference Wild type cht2⌬::hisG-URA3-hisG/cht2⌬::hisG cht3⌬::hisG-URA3-hisG/cht3⌬::hisG cht2⌬::hisG-URA3-hisG/cht2⌬::hisG cht3⌬::hisG/cht3⌬::hisG eng1⌬::hisG-URA3-hisG/eng1::hisG ace2⌬::FRT/ace2⌬::FRT ura3/ura3 cbk1⌬::hisG/cbk1⌬::hisG-URA3-hisG Wt ⌬/⌬cht2 ⌬/⌬cht3 ⌬/⌬cht2/3 ⌬/⌬eng1 ⌬/⌬ace2 ⌬/⌬cbk1 31 32 32 32 33 34 35 Not applicable. teins. Relative quantification revealed a set of wall-remodeling proteins that was induced during thermal stress. Intriguingly, the same set of proteins was shown previously to be involved in the response to other surface stressors (14, 30), suggesting (...truncated)