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Identification of sumoylation targets, combined with inactivation of SMT3, reveals the impact of sumoylation upon growth, morphology, and stress resistance in the pathogen Candida albicans.

Leach MD, Stead DA, Argo E, Brown AJ - Mol. Biol. Cell (2011)

Bottom Line: Smt3/smt3 cells also displayed sensitivity to thermal, oxidative, and cell wall stresses as well as to the antifungal drug caspofungin.Furthermore, signaling via the cell integrity pathway was defective in C. albicans smt3/smt3 cells.Clearly sumoylation plays key roles in fundamental cellular processes that underpin the pathogenicity of this medically important fungus.

View Article: PubMed Central - PubMed

Affiliation: School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, United Kingdom.

ABSTRACT
Posttranslational modifications of proteins play critical roles in the control of cellular differentiation, development, and environmental adaptation. In particular, the covalent attachment of the small ubiquitin-like modifier, SUMO, to target proteins (sumoylation) regulates cell cycle progression, transcription, nucleocytoplasmic transport, and stress responses. Here we combine proteomic, molecular, and cellular approaches to examine the roles of sumoylation in the major fungal pathogen of humans, Candida albicans. Using an N-terminally FLAG-tagged SUMO, 31 sumoylated proteins were identified in C. albicans with roles in stress responses (e.g., Hsp60, Hsp70 family members, Hsp104), the cytoskeleton and polarized growth (e.g., Tub1, Cct7, Mlc1), secretion, and endocytosis (e.g., Lsp1, Sec24, Sec7). The output from this proteomic screen was entirely consistent with the phenotypes of C. albicans mutants in which the single SUMO-encoding locus (SMT3) was inactivated or down-regulated. C. albicans smt3/smt3 cells displayed defects in growth, morphology, cell separation, nuclear segregation, and chitin deposition, suggesting important roles for sumoylation in cell cycle control. Smt3/smt3 cells also displayed sensitivity to thermal, oxidative, and cell wall stresses as well as to the antifungal drug caspofungin. Mutation of consensus sumoylation sites in Hsp60 and Hsp104 affected the resistance of C. albicans to thermal stress. Furthermore, signaling via the cell integrity pathway was defective in C. albicans smt3/smt3 cells. These observations provide mechanistic explanations for many of the observed phenotypic effects of Smt3 inactivation upon C. albicans growth and environmental adaptation. Clearly sumoylation plays key roles in fundamental cellular processes that underpin the pathogenicity of this medically important fungus.

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C. albicans smt3 mutants are sensitive to a range of stresses. (A) Sensitivity of an smt3/smt3  mutant to stresses. Serial dilutions of exponentially growing cells were spotted onto YPD plates containing the appropriate stress at the concentrations indicated in Materials and Methods: SMT3/SMT3 (BWP17) and smt3/smt3 (MLC37). (B) Sensitivity of a MET3p-SMT3/smt3 conditional mutant to stresses. Serial dilutions were spotted onto SC plates containing the appropriate stress and containing 2.5 mM methionine and cysteine: SMT3/SMT3 (BWP17) and MET3p-SMT3/smt3 (MLC04). These strains grew at similar rates on control plates that lacked methionine and cysteine (Supplemental Material).
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Figure 7: C. albicans smt3 mutants are sensitive to a range of stresses. (A) Sensitivity of an smt3/smt3 mutant to stresses. Serial dilutions of exponentially growing cells were spotted onto YPD plates containing the appropriate stress at the concentrations indicated in Materials and Methods: SMT3/SMT3 (BWP17) and smt3/smt3 (MLC37). (B) Sensitivity of a MET3p-SMT3/smt3 conditional mutant to stresses. Serial dilutions were spotted onto SC plates containing the appropriate stress and containing 2.5 mM methionine and cysteine: SMT3/SMT3 (BWP17) and MET3p-SMT3/smt3 (MLC04). These strains grew at similar rates on control plates that lacked methionine and cysteine (Supplemental Material).

Mentions: Exponentially growing wild-type (BWP17) and smt3/smt3 cells (MLC37) were spotted onto YPD plates and grown under the appropriate stress conditions. As described above (Figure 4B), smt3/smt3 cells grow more slowly than wild-type cells, and this phenotype was replicated in these stress experiments (Figure 7A). Comparing smt3/smt3 cells in the presence and absence of stress reveals that the inactivation of Smt3 consistently reduced the ability of C. albicans to grow at 37°C and 42°C. This was consistent with the observation that various heat shock proteins are sumoylated in C. albicans (Hsp60, Hsp104, Ssb1, Ssc1, and Sse1; Table 2) as well as in S. cerevisiae (Panse et al., 2004; Wohlschlegel et al., 2004; Zhou et al., 2004; Hannich et al., 2005). Although there was no obvious effect upon osmotic stress resistance, Smt3 inactivation did render C. albicans more sensitive to peroxide. This observation might have physiological significance given the importance of reactive oxygen species in the antimicrobial activity of macrophages, for example (Miller and Britigan, 1997). Furthermore, smt3/smt3 cells consistently displayed cell wall stress phenotypes. They grew more slowly in the presence of Congo red and the antifungal drug caspofungin, which both target glucan biosynthesis. They also showed slight sensitivity to a low concentration of calcofluor white, which affects chitin synthesis (Figure 7A).


Identification of sumoylation targets, combined with inactivation of SMT3, reveals the impact of sumoylation upon growth, morphology, and stress resistance in the pathogen Candida albicans.

Leach MD, Stead DA, Argo E, Brown AJ - Mol. Biol. Cell (2011)

C. albicans smt3 mutants are sensitive to a range of stresses. (A) Sensitivity of an smt3/smt3  mutant to stresses. Serial dilutions of exponentially growing cells were spotted onto YPD plates containing the appropriate stress at the concentrations indicated in Materials and Methods: SMT3/SMT3 (BWP17) and smt3/smt3 (MLC37). (B) Sensitivity of a MET3p-SMT3/smt3 conditional mutant to stresses. Serial dilutions were spotted onto SC plates containing the appropriate stress and containing 2.5 mM methionine and cysteine: SMT3/SMT3 (BWP17) and MET3p-SMT3/smt3 (MLC04). These strains grew at similar rates on control plates that lacked methionine and cysteine (Supplemental Material).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 7: C. albicans smt3 mutants are sensitive to a range of stresses. (A) Sensitivity of an smt3/smt3 mutant to stresses. Serial dilutions of exponentially growing cells were spotted onto YPD plates containing the appropriate stress at the concentrations indicated in Materials and Methods: SMT3/SMT3 (BWP17) and smt3/smt3 (MLC37). (B) Sensitivity of a MET3p-SMT3/smt3 conditional mutant to stresses. Serial dilutions were spotted onto SC plates containing the appropriate stress and containing 2.5 mM methionine and cysteine: SMT3/SMT3 (BWP17) and MET3p-SMT3/smt3 (MLC04). These strains grew at similar rates on control plates that lacked methionine and cysteine (Supplemental Material).
Mentions: Exponentially growing wild-type (BWP17) and smt3/smt3 cells (MLC37) were spotted onto YPD plates and grown under the appropriate stress conditions. As described above (Figure 4B), smt3/smt3 cells grow more slowly than wild-type cells, and this phenotype was replicated in these stress experiments (Figure 7A). Comparing smt3/smt3 cells in the presence and absence of stress reveals that the inactivation of Smt3 consistently reduced the ability of C. albicans to grow at 37°C and 42°C. This was consistent with the observation that various heat shock proteins are sumoylated in C. albicans (Hsp60, Hsp104, Ssb1, Ssc1, and Sse1; Table 2) as well as in S. cerevisiae (Panse et al., 2004; Wohlschlegel et al., 2004; Zhou et al., 2004; Hannich et al., 2005). Although there was no obvious effect upon osmotic stress resistance, Smt3 inactivation did render C. albicans more sensitive to peroxide. This observation might have physiological significance given the importance of reactive oxygen species in the antimicrobial activity of macrophages, for example (Miller and Britigan, 1997). Furthermore, smt3/smt3 cells consistently displayed cell wall stress phenotypes. They grew more slowly in the presence of Congo red and the antifungal drug caspofungin, which both target glucan biosynthesis. They also showed slight sensitivity to a low concentration of calcofluor white, which affects chitin synthesis (Figure 7A).

Bottom Line: Smt3/smt3 cells also displayed sensitivity to thermal, oxidative, and cell wall stresses as well as to the antifungal drug caspofungin.Furthermore, signaling via the cell integrity pathway was defective in C. albicans smt3/smt3 cells.Clearly sumoylation plays key roles in fundamental cellular processes that underpin the pathogenicity of this medically important fungus.

View Article: PubMed Central - PubMed

Affiliation: School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, United Kingdom.

ABSTRACT
Posttranslational modifications of proteins play critical roles in the control of cellular differentiation, development, and environmental adaptation. In particular, the covalent attachment of the small ubiquitin-like modifier, SUMO, to target proteins (sumoylation) regulates cell cycle progression, transcription, nucleocytoplasmic transport, and stress responses. Here we combine proteomic, molecular, and cellular approaches to examine the roles of sumoylation in the major fungal pathogen of humans, Candida albicans. Using an N-terminally FLAG-tagged SUMO, 31 sumoylated proteins were identified in C. albicans with roles in stress responses (e.g., Hsp60, Hsp70 family members, Hsp104), the cytoskeleton and polarized growth (e.g., Tub1, Cct7, Mlc1), secretion, and endocytosis (e.g., Lsp1, Sec24, Sec7). The output from this proteomic screen was entirely consistent with the phenotypes of C. albicans mutants in which the single SUMO-encoding locus (SMT3) was inactivated or down-regulated. C. albicans smt3/smt3 cells displayed defects in growth, morphology, cell separation, nuclear segregation, and chitin deposition, suggesting important roles for sumoylation in cell cycle control. Smt3/smt3 cells also displayed sensitivity to thermal, oxidative, and cell wall stresses as well as to the antifungal drug caspofungin. Mutation of consensus sumoylation sites in Hsp60 and Hsp104 affected the resistance of C. albicans to thermal stress. Furthermore, signaling via the cell integrity pathway was defective in C. albicans smt3/smt3 cells. These observations provide mechanistic explanations for many of the observed phenotypic effects of Smt3 inactivation upon C. albicans growth and environmental adaptation. Clearly sumoylation plays key roles in fundamental cellular processes that underpin the pathogenicity of this medically important fungus.

Show MeSH
Related in: MedlinePlus