<i>KRE5</i> Suppression Induces Cell Wall Stress and Alternative ER Stress Response Required for Maintaining Cell Wall Integrity in <i>Candida glabrata</i>

<div><p>The maintenance of cell wall integrity in fungi is required for normal cell growth, division, hyphae formation, and antifungal tolerance. We observed that endoplasmic reticulum stress regulated cell wall integrity in <i>Candida glabrata</i>, which possesses uniquely evolved mechanisms for unfolded protein response mechanisms. Tetracycline-mediated suppression of <i>KRE5</i>, which encodes a predicted UDP-glucose:glycoprotein glucosyltransferase localized in the endoplasmic reticulum, significantly increased cell wall chitin content and decreased cell wall β-1,6-glucan content. <i>KRE5</i> repression induced endoplasmic reticulum stress-related gene expression and MAP kinase pathway activation, including Slt2p and Hog1p phosphorylation, through the cell wall integrity signaling pathway. Moreover, the calcineurin pathway negatively regulated cell wall integrity, but not the reduction of β-1,6-glucan content. These results indicate that <i>KRE5</i> is required for maintaining both endoplasmic reticulum homeostasis and cell wall integrity, and that the calcineurin pathway acts as a regulator of chitin-glucan balance in the cell wall and as an alternative mediator of endoplasmic reticulum stress in <i>C</i>. <i>glabrata</i>.</p></div

Calcineurin inhibitor FK-506 upregulated cell wall chitin synthesis by accelerating Slt2p phosphorylation.

<p>(A) Relative mRNA levels of <i>CHS1</i> and <i>GFA1</i> were calculated from the ratio of the signal intensities of <i>GFA1</i> and <i>CHS1</i> mRNAs compared with that of <i>PGK1</i> mRNA, which was used as a reference. Values are represented as an average of three independent experiments, and error bars indicate S.D.; *, p < 0.05, compared with DOX-untreated tet-<i>KRE5</i> cells (Student´s <i>t</i>-test). (B and C) Cell wall composition of <i>C</i>. <i>glabrata</i> strains is shown. Alkali-insoluble chitin (B) and β-1, 6-glucan (C) contents in <i>C</i>. <i>glabrata</i> cells were measured as the quantity of glucosamine or glucose substituted for the standard curve. Data shown represent the results of at least three independent experiments. Error bars represent standard deviations; *, p < 0.05; n.s, not significant. (D) Phosphorylation of Slt2p and Hog1p was determined by performing western blotting analysis with antibodies against p-Slt2p and p-Hog1p, respectively. Anti-Pgk1p antibody was used as the loading control. Representative data are shown of independent experiments is shown. (E) Microscopic analysis of tet-<i>KRE5</i> cells. A representative image of three independent experiments is shown; scale bar: 10 μm; arrow, disrupted <i>C</i>. <i>glabrata</i> cells.</p

<i>CgKRE5</i> repression enhances the sensitivity of <i>C</i>. <i>glabrata</i> to micafungin.

<p>Inhibition ring test by using micafungin or fluconazole is shown. <i>C</i>. <i>glabrata</i> cells were streaked on YPD solid medium, and indicated concentrations of micafungin or fluconazole were spotted onto paper discs, which were then placed on the YPD solid medium inoculated with <i>C</i>. <i>glabrata</i>.</p

Repression of <i>CgKRE5</i> induces ER stress in <i>C</i>. <i>glabrata</i>.

<p>(A) Spot dilution assay was performed using the ER stress indicator. Five-microliter suspensions at an OD of 0.1 and serially diluted (1: 5) cells were spotted on YPD plates with the indicated concentration of reagents incubated at 37°C. A representative image of three independent experiments is shown. (B) Real-time RT-PCR was performed to measure the mRNA expression levels of ER stress-associated genes. Amplification efficiencies were validated and were normalized using that of <i>PGK1</i>. Relative mRNA levels were calculated as the ratio of normalized mRNA level to the mRNA level of <i>PGK1</i>. Values are represented as the average of three independent experiments, and error bars indicate S.D.; *, p < 0.05 compared with DMSO-treated tet-<i>KRE5</i> cells (Student´s <i>t</i>-test).</p

<i>CgKRE5</i> repression increased cell wall chitin content by activating the CWI-regulating MAP kinase pathway.

<p>(A) Phosphorylation of Slt2p and Hog1p was determined by performing western blotting analysis with antibodies against phosphorylated Slt2p and Hog1p (p-Slt2p and p-Hog1p, respectively). Anti-Pgk1p antibody was used as a loading control. Representative data of three independent experiments are shown; TM, tunicamycin. (B and C) Alkali-insoluble chitin (B) and β-1,6-glucan (C) content in <i>C</i>. <i>glabrata</i> cells were measured as the quality of glucose or glucosamine substituted for the standard curve. Data shown represent the results of at least three independent experiments. Error bars represent standard deviations; *, p < 0.05. (D) Spot dilution assay was performed using ER stress indicator. For this, 5 μL suspensions at an OD of 0.1 and serially diluted (1: 5) cells were spotted on YPD plates with the indicated concentration of reagents incubated at 37°C. A representative image of three independent experiments is shown.</p