An extensive circuitry for cell wall regulation in Candida albicans

Abstract

Protein kinases play key roles in signaling and response to changes in the external environment. The ability of Candida albicans to quickly sense and respond to changes in its environment is key to its survival in the human host. Our guiding hypothesis was that creating and screening a set of protein kinase mutant strains would reveal signaling pathways that mediate stress response in C. albicans. A library of protein kinase mutant strains was created and screened for sensitivity to a variety of stresses. For the majority of stresses tested, stress response was largely conserved between C. albicans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe. However, we identified eight protein kinases whose roles in cell wall regulation (CWR) were not expected from functions of their orthologs in the model fungi Saccharomyces cerevisiae and Schizosaccharomyces pombe. Analysis of the conserved roles of these protein kinases indicates that establishment of cell polarity is critical for CWR. In addition, we found that septins, crucial to budding, are both important for surviving and are mislocalized by cell wall stress. Our study shows an expanded role for protein kinase signaling in C. albicans cell wall integrity. Our studies suggest that in some cases, this expansion represents a greater importance for certain pathways in cell wall biogenesis. In other cases, it appears that signaling pathways have been rewired for a cell wall integrity response.

Figure 1. PKs play conserved and novel roles in CWR.

A wild type marker-matched strain (DAY286), a hypersensitive cas5Δ/cas5Δ control, and the indicated prototrophic PK mutant strains and their complements were serially diluted onto YPD (−) or YPD+ 125 ng/ml caspofungin (+) and grown for 2 days at 30°C. Data for PSK1 were published in Rauceo, et al . In most cases, complementation fully restored growth on the caspofungin plates, but it should be noted that complementation of the gin4−/− strain with one copy of GIN4 was not sufficient to restore growth on caspofungin.

Figure 2. PK and PK-related mutant strains show a damage response in the absence of cell wall stress.

(A) The expression of six genes upregulated by caspofungin treatment, DDR48, SOD5, STP4, ALS1, RTA4, and ECM331, was analyzed in PK and PK-related mutant strains in the absence of cell well stress. The expression of TDH3, a gene involved in glycolysis, was used to normalize expression between strains and expression values were further normalized to wild type (DAY185) expression for comparison between experiments. Resultant values were log base 2 transformed (wild type expression for all six genes is therefore at 0). (B) A graphical representation of the expression data. Arrows point to targets upregulated in all (solid arrows), most (dashed lines), or half (dotted lines) of the mutants indicated in the clusters.

Figure 3. Septins play an integral role in CWR.

(A) A wild type marker-matched strain (DAY185), a cas5Δ/cas5Δ negative control, a cdc10Δ/cdc10Δ (YAW7), a cdc11Δcdc11/Δ (YAW11), and a sep7Δ/sep7Δ (YAW41) mutant strain were serially diluted onto YPD (−) or YPD+ 125 ng/ml caspofungin (+) and grown for 3 days at 30°C. (B) SEP7-GFP cells (JRB217) were grown to log phase and either treated with 125 ng/ml caspofungin (+ caspofungin) for 30 minutes or left untreated. The cells were then visualized on glass slides. Arrows point to aberrant septin localization.

Figure 4. Septins are mislocalized in some PK mutant strains.

SEP7-GFP tagged wild type (JRB217), gin4−/− (JRB221), kin3−/− (JRB193), cbk1−/− (JRB224), and vps34−/− (JRB216) strains were grown to log phase and imaged at 100× on glass slides. An overlay of DIC and GFP is on the left and GFP alone is on the right. Arrows point to aberrant septin localization.

Figure 5. A model of the role of PKs in septin morphology and cell wall biogenesis.

Based on our observations, an intact septin ring is required for normal cell wall production and a normal cell wall is required for the formation of a septin ring. PK Cluster IV genes have known and predicted roles in septin morphology and signaling and may impact cell wall biogenesis indirectly via this role. We hypothesize PK Cluster I genes have direct roles in biogenesis of the cell wall, while PK Cluster III may indirectly effect cell wall biogenesis by regulating the flow of carbohydrates into cell wall biosynthesis pathways. We hypothesize that the genes in PK Cluster II are involved in cell wall stress response and the upregulation of cell wall integrity genes. PK Clusters I, III, and IV negatively regulate PK Cluster II, either directly or indirectly, based on the observation that cell wall integrity genes are upregulated in the absence of these PKs.