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4 Publications visible to you, out of a total of 4
The zinc cluster transcription factor Czf1 regulates cell wall architecture and integrity in Candida albicans.

Abstract (Expand)

The fungal cell wall is essential for the maintenance of cellular integrity and mediates interactions of the cells with the environment. It is a highly flexible organelle whose composition and organization … is modulated in response to changing growth conditions. In the pathogenic yeast Candida albicans, a network of signaling pathways regulates the structure of the cell wall, and mutants with defects in these pathways are hypersensitive to cell wall stress. By harnessing a library of genetically activated forms of all C. albicans zinc cluster transcription factors, we found that a hyperactive Czf1 rescued the hypersensitivity to cell wall stress of different protein kinase deletion mutants. The hyperactive Czf1 induced the expression of many genes with cell wall-related functions and caused visible changes in the cell wall structure. C. albicans czf1Delta mutants were hypersensitive to the antifungal drug caspofungin, which inhibits cell wall biosynthesis. The changes in cell wall architecture caused by hyperactivity or absence of Czf1 resulted in an increased recognition of C. albicans by human neutrophils. Our results show that Czf1, which is known as a regulator of filamentous growth and white-opaque switching, controls the expression of cell wall genes and modulates the architecture of the cell wall.

Authors: A. Mottola, B. Ramirez-Zavala, K. Hunniger, O. Kurzai, J. Morschhauser

Date Published: 15th Apr 2021

Publication Type: Not specified

Generation of Viable Candida albicans Mutants Lacking the "Essential" Protein Kinase Snf1 by Inducible Gene Deletion.

Abstract (Expand)

The protein kinase Snf1, a member of the highly conserved AMP-activated protein kinase family, is a central regulator of metabolic adaptation. In the pathogenic yeast Candida albicans, Snf1 is considered … to be essential, as previous attempts by different research groups to generate homozygous snf1Delta mutants were unsuccessful. We aimed to elucidate why Snf1 is required for viability in C. albicans by generating snf1Delta null mutants through forced, inducible gene deletion and observing the terminal phenotype before cell death. Unexpectedly, we found that snf1Delta mutants were viable and could grow, albeit very slowly, on rich media containing the preferred carbon source glucose. Growth was improved when the cells were incubated at 37 degrees C instead of 30 degrees C, and this phenotype enabled us to isolate homozygous snf1Delta mutants also by conventional, sequential deletion of both SNF1 alleles in a wild-type C. albicans strain. All snf1Delta mutants could grow slowly on glucose but were unable to utilize alternative carbon sources. Our results show that, under optimal conditions, C. albicans can live and grow without Snf1. Furthermore, they demonstrate that inducible gene deletion is a powerful method for assessing gene essentiality in C. albicans IMPORTANCE Essential genes are those that are indispensable for the viability and growth of an organism. Previous studies indicated that the protein kinase Snf1, a central regulator of metabolic adaptation, is essential in the pathogenic yeast Candida albicans, because no homozygous snf1 deletion mutants of C. albicans wild-type strains could be obtained by standard approaches. In order to investigate the lethal consequences of SNF1 deletion, we generated conditional mutants in which SNF1 could be deleted by forced, inducible excision from the genome. Unexpectedly, we found that snf1 null mutants were viable and could grow slowly under optimal conditions. The growth phenotypes of the snf1Delta mutants explain why such mutants were not recovered in previous attempts. Our study demonstrates that inducible gene deletion is a powerful method for assessing gene essentiality in C. albicans.

Authors: A. Mottola, S. Schwanfelder, J. Morschhauser

Date Published: 19th Aug 2020

Publication Type: Not specified

An Intragenic Recombination Event Generates a Snf4-Independent Form of the Essential Protein Kinase Snf1 in Candida albicans.

Abstract (Expand)

The heterotrimeric protein kinase SNF1 plays a key role in the metabolic adaptation of the pathogenic yeast Candida albicans It consists of the essential catalytic alpha-subunit Snf1, the gamma-subunit … Snf4, and one of the two beta-subunits Kis1 and Kis2. Snf4 is required to release the N-terminal catalytic domain of Snf1 from autoinhibition by the C-terminal regulatory domain, and snf4Delta mutants cannot grow on carbon sources other than glucose. In a screen for suppressor mutations that restore growth of a snf4Delta mutant on alternative carbon sources, we isolated a mutant in which six amino acids between the N-terminal kinase domain and the C-terminal regulatory domain of Snf1 were deleted. The deletion was caused by an intragenic recombination event between two 8-bp direct repeats flanking six intervening codons. In contrast to truncated forms of Snf1 that contain only the kinase domain, the Snf4-independent Snf1(Delta311 - 316) was fully functional and could replace wild-type Snf1 for normal growth, because it retained the ability to interact with the Kis1 and Kis2 beta-subunits via its C-terminal domain. Indeed, the Snf4-independent Snf1(Delta311 - 316) still required the beta-subunits of the SNF1 complex to perform its functions and did not rescue the growth defects of kis1Delta mutants. Our results demonstrate that a preprogrammed in-frame deletion event within the SNF1 coding region can generate a mutated form of this essential kinase which abolishes autoinhibition and thereby overcomes growth deficiencies caused by a defect in the gamma-subunit Snf4.IMPORTANCE Genomic alterations, including different types of recombination events, facilitate the generation of genetically altered variants and enable the pathogenic yeast Candida albicans to adapt to stressful conditions encountered in its human host. Here, we show that a specific recombination event between two 8-bp direct repeats within the coding sequence of the SNF1 gene results in the deletion of six amino acids between the N-terminal kinase domain and the C-terminal regulatory domain and relieves this essential kinase from autoinhibition. This preprogrammed deletion allowed C. albicans to overcome growth defects caused by the absence of the regulatory subunit Snf4 and represents a built-in mechanism for the generation of a Snf4-independent Snf1 kinase.

Authors: A. Mottola, J. Morschhauser

Date Published: 19th Jun 2019

Publication Type: Not specified

The Snf1-activating kinase Sak1 is a key regulator of metabolic adaptation and in vivo fitness of Candida albicans.

Abstract (Expand)

The metabolic flexibility of the opportunistic fungal pathogen Candida albicans is important for colonisation and infection of different host niches. Complex regulatory networks, in which protein kinases … play central roles, link metabolism and other virulence-associated traits, such as filamentous growth and stress resistance, and thereby control commensalism and pathogenicity. By screening a protein kinase deletion mutant library that was generated in the present work using an improved SAT1 flipper cassette, we found that the previously uncharacterised kinase Sak1 is a key upstream activator of the protein kinase Snf1, a highly conserved regulator of nutrient stress responses that is essential for viability in C. albicans. The sak1Delta mutants failed to grow on many alternative carbon sources and were hypersensitive to cell wall/membrane stress. These phenotypes were mirrored in mutants lacking other subunits of the SNF1 complex and partially compensated by a hyperactive form of Snf1. Transcriptional profiling of sak1Delta mutants showed that Sak1 ensures basal expression of glyoxylate cycle and gluconeogenesis genes even in glucose-rich media and thereby contributes to the metabolic plasticity of C. albicans. In a mouse model of gastrointestinal colonisation, sak1Delta mutants were rapidly outcompeted by wild-type cells, demonstrating that Sak1 is essential for the in vivo fitness of C. albicans.

Authors: B. Ramirez-Zavala, A. Mottola, J. Haubenreisser, S. Schneider, S. Allert, S. Brunke, K. Ohlsen, B. Hube, J. Morschhauser

Date Published: 25th Mar 2017

Publication Type: Not specified

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