Vacuolar H(+)-ATPase plays a key role in cell wall biosynthesis of Aspergillus niger.
The identification of suitable targets is crucial for the discovery and development of new antifungals. Since the fungal cell wall is an essential organelle, the identification of genes involved in cell wall biosynthesis is expected to help discover new antifungal targets. From our previously obtained collection of cell wall mutants with a constitutively active cell wall stress response pathway, we selected a thermosensitive, osmotic-remediable mutant with decreased resistance to SDS for complementation analysis. The phenotypes of this mutant were complemented by a gene encoding a protein with high sequence similarity to subunit d of the eukaryotic Vacuolar-H(+)-ATPase (VmaD). Genetic analysis of this thermosensitive mutant revealed that the conditional mutant allele encodes a protein that lacks 12 amino acids at the C-terminus due to a point mutation that introduces a stop codon. Deletion of the entire gene resulted in very poor growth. The conditional mutant displayed several phenotypes that are typical for V-ATPase mutants, including increased sensitivity to zinc ions and reduced acidification of the vacuole as observed by quinacrine staining. Treatment of Aspergillus niger with the V-ATPase inhibitor bafilomycinB(1) induced the expression of agsA and other cell wall related genes. Furthermore genes involved in cell wall reassembly like fksA, agsA and phiA were clearly up-regulated in the conditional mutant. Our results indicate that the ATP-driven transport of protons and acidification of the vacuole is crucial for the strength of the fungal cell wall and that reduced activity of the V-ATPase induces the cell wall stress response pathway.
Institute of Biology Leiden, Leiden University, Molecular Microbiology and Biotechnology, Sylviusweg 72, 2333 BE Leiden, The Netherlands.
Doreen
D
Schachtschabel
Mark
M
Arentshorst
Ellen L
EL
Lagendijk
Arthur F J
AF
Ram
D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology*; D001234:Aspergillus niger / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D000077336:Caspofungin; D002473:Cell Wall / Q000378:metabolism*; D054714:Echinocandins / Q000494:pharmacology; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D005816:Genetic Complementation Test; D056426:Genetic Loci; D025301:Hyphae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D055666:Lipopeptides; D018942:Macrolides / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010641:Phenotype; D017354:Point Mutation; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017384:Sequence Deletion; D013312:Stress, Physiological / Q000187:drug effects; D014617:Vacuoles / Q000201:enzymology / Q000378:metabolism
D000935:Antifungal Agents; D054714:Echinocandins; D005656:Fungal Proteins; D055666:Lipopeptides; D018942:Macrolides; C052501:bafilomycin B1; D006180:Proton-Translocating ATPases; D000077336:Caspofungin