Functions of Candida albicans cell wall glycosidases Dfg5p and Dcw1p in biofilm formation and HOG MAPK pathway

Background Candida albicans is a commensal fungus that inhabits the oral mucosal surface and causes oral and systemic candidiasis. Oral candidiasis most commonly occurs in patients with AIDS, denture wearers and newborn children. Systemic candidiasis occurs mainly in immunocompromised patients and patients admitted to hospitals for prolonged periods. C. albicans homologous genes, DFG5 and DCW1, encode for two closely related cell wall proteins with putative glycosyltransferase enzyme activity and C-terminal GPI-anchors. Past studies have shown that individual DFG5 and DCW1 mutations are viable but simultaneous deletion of DFG5 and DCW1 in C. albicans results in lethality. However, the exact functions of these cell wall based enzymes, which represent potential drug targets, are not understood. Methods C. albicans DFG5/DCW1 heterologous and conditional double mutant strains were assessed for growth and biofilm formation in comparison to wild type and parental strains. Cell wall and heat stress susceptibility of the mutant and control strains were assessed using agar spotting assays. Growth was assessed under normal and osmotic stress conditions along with light microscopy imaging. Biofilm dry weight and microscopic imaging analysis of biofilms was performed. Hypha formation in response to serum was analyzed using light microscopy imaging. Western blot analysis of mutant strains and control strains was performed to assess Hog1 basal levels and phosphorylation status. Results Analysis of the heterologous mutants indicated that Dfg5p is more important for growth while Dcw1p appeared to play a role in cell wall integrity response. The conditional double mutant was observed to be less resistant to cell wall stress. However, growth of the mutants was similar under control and osmotic stress conditions. The mutants were also able to grow similar to wild type under heat stress. Biofilm formation was reduced in the mutants where DFG5 was deleted or suppressed. Hyphal morphogenesis was reduced although germ tube formation was observed in the biofilms of the mutant strains. Basal Hog1 protein levels were reduced or absent in the DFG5 and DCW1 mutants. However, osmotic stress was able to induce Hog1 protein levels comparable to wild type. Hog1 phosphorylation appeared to be slightly reduced although not significantly. In addition to biofilm assays, serum dose response imaging analysis indicated that hyphae formation in DFG5 and DCW1 mutants was defective. Conclusions These data indicate that DFG5 and DCW1 are required for hyphal morphogenesis and biofilm formation in C. albicans. These functions may be regulated via basal Hog1 MAPK which is required for transcriptional regulation of chitin synthesis

<i>Neurospora crassa</i> Female Development Requires the PACC and Other Signal Transduction Pathways, Transcription Factors, Chromatin Remodeling, Cell-To-Cell Fusion, and Autophagy

<div><p>Using a screening protocol we have identified 68 genes that are required for female development in the filamentous fungus <i>Neurospora crassa</i>. We find that we can divide these genes into five general groups: 1) Genes encoding components of the PACC signal transduction pathway, 2) Other signal transduction pathway genes, including genes from the three <i>N. crassa</i> MAP kinase pathways, 3) Transcriptional factor genes, 4) Autophagy genes, and 5) Other miscellaneous genes. Complementation and RIP studies verified that these genes are needed for the formation of the female mating structure, the protoperithecium, and for the maturation of a fertilized protoperithecium into a perithecium. Perithecia grafting experiments demonstrate that the autophagy genes and the cell-to-cell fusion genes (the MAK-1 and MAK-2 pathway genes) are needed for the mobilization and movement of nutrients from an established vegetative hyphal network into the developing protoperithecium. Deletion mutants for the PACC pathway genes <i>palA</i>, <i>palB</i>, <i>palC</i>, <i>palF</i>, <i>palH</i>, and <i>pacC</i> were found to be defective in two aspects of female development. First, they were unable to initiate female development on synthetic crossing medium. However, they could form protoperithecia when grown on cellophane, on corn meal agar, or in response to the presence of nearby perithecia. Second, fertilized perithecia from PACC pathway mutants were unable to produce asci and complete female development. Protein localization experiments with a GFP-tagged PALA construct showed that PALA was localized in a peripheral punctate pattern, consistent with a signaling center associated with the ESCRT complex. The <i>N. crassa</i> PACC signal transduction pathway appears to be similar to the PacC/Rim101 pathway previously characterized in <i>Aspergillus nidulans</i> and <i>Saccharomyces cerevisiae</i>. In <i>N. crassa</i> the pathway plays a key role in regulating female development.</p></div

Perithecia grafting experiments.

<p>Small pieces of cellophane containing “graft” fertilized wild type protoperithecia were placed on mutant “host” vegetative hyphal networks grown on synthetic crossing medium. The “host” shown are: 1) Δ<i>fmf-1</i> (left panel/A) which shows a host supporting the development of the graft perithecia. 2) Δ<i>ada-1</i> (middle panel/B) which shows a host not supporting the development of the graft perithecia. 3) Δ<i>palA</i> (right panel/C) which shows the graft inducing protoperithecia in the Δ<i>palA</i> host. Arrows point to examples of perithecia on the cellophane (left panel/A) and protoperithecia induced in the host vegetative hyphal network (right panel/C).</p

Schematic representation of the <i>N. crassa</i> PACC pathway.

<p>The PACC signal transduction pathway elements found in <i>N. crassa</i>, and the model for how the pathway might function are depicted. The PALH and PALF proteins are thought to be found at the plasma membrane. PALH is a seven transmembrane receptor which is sensitive to environmental cues. PALF is an arrestin type protein that associates with PALH. PALF is phosphorylated and ubiquitinated in response to the environmental cues. These events lead to the endocytosis of the PALH/PALF complex. Following endocytosis, the PALH is directed into an ESCRT compartment, where it enters into a signaling complex containing PALA, PALB, PALC, and PACC. Within the signaling complex, PALB functions as a protease which cleaves PACC. This cleavage event removes a C-terminal inhibitory domain from the PACC transcription factor, and the processed PACC is released from the signaling complex. The activated PACC then enters the nucleus and directs transcriptional activity leading to the formation of the protoperithecium.</p

Signal transduction pathway genes are required for female development.

<p>PP – <u>P</u>reviously<u> p</u>ublished data demonstrated that the gene was needed for female development.</p><p>NA – a deletion strain is <u>n</u>ot <u>a</u>vailable in the single gene deletion library.</p><p>NA(het) – the deletion strain in the single gene deletion library is a <u>het</u>erokaryon and a homokaryon isolate was <u>n</u>ot <u>a</u>vailable during the screening experiments.</p><p>RIP – a RIP experiment was used to verify that the gene is required for female development.</p><p>An * by the NCU number indicates that the gene is needed for CAT (conidia anastomosis tube) formation (a cell fusion phenotype) and is likely to be a component of either the MAK-1 or MAK-2 signal pathway.</p><p>A notation of “This report” in the reference information column indicates that the gene was either newly identified or verified by co-segregation and complementation analysis as being needed for <i>N. crassa</i> development by our experiments.</p><p>Signal transduction pathway genes are required for female development.</p

Miscellaneous genes needed for female development.

<p>PP – <u>P</u>reviously<u> p</u>ublished data demonstrated that the gene was needed for female development.</p><p>NA – a deletion strain is <u>n</u>ot <u>a</u>vailable in the single gene deletion library.</p><p>NA(het) – the deletion strain in the single gene deletion library is a <u>het</u>erokaryon and a homokaryon isolate was <u>n</u>ot <u>a</u>vailable during the screening experiments.</p><p>A notation of “This report” in the reference information column indicates that the gene was identified as being needed for <i>N. crassa</i> development by our experiments.</p><p>Miscellaneous genes needed for female development.</p

PALA is localized to the small intracellular vesicles.

<p>GFP-tagged PALA was expressed under the regulation of the <i>ccg-1</i> promoter in a Δ<i>palA</i> isolate. The GFP fluorescent image of a germling is shown in the panel on the left (A). The DIC image of the same germling is shown in the right panel (B). The bar in the DIC image is 10 µm in length.</p

Autophagy genes required for female development.

<p>RIP – a RIP experiment was used to verify that the gene is required for female development.</p><p>A notation of “This report” in the reference information column indicates that the gene was identified as being needed for <i>N. crassa</i> development by our experiments.</p><p>Autophagy genes required for female development.</p

Constitutively active PACC activates female development.

<p>Cells were inoculated onto agar slants and allowed to grow for 10 days at room temperature. The left panel (A) shows a Δ<i>palC</i> isolate on Vogel’s sucrose medium. The middle panel (B) shows a Δ<i>palC</i> isolate that has been transformed with the constitutively activated PACC construct growing on synthetic crossing medium. The right panel (C) shows a Δ<i>palC</i> isolate that has been transformed with the constitutively activated PACC construct growing on Vogel’s sucrose medium, a medium that represses female development. Note that the constitutively activated PACC caused protoperithecia production in the absence of PALC on both media. The arrows in the middle and right panels point to protoperithecia.</p

Genes from the PacC pathway are required for female development.

<p>A notation of “This report” in the reference information column indicates that the gene was identified as being needed for <i>N. crassa</i> development by our experiments.</p><p>Genes from the PacC pathway are required for female development.</p