Aspergillus fumigatus RasA Regulates Asexual Development and Cell Wall Integrity

ABSTRACT The Ras family of proteins is a large group of monomeric GTPases. Members of the fungal Ras family act as molecular switches that transduce signals from the outside of the cell to signaling cascades inside the cell. A. fumigatus RasA is 94% identical to the essential RasA gene of Aspergillus nidulans and is the Ras family member sharing the highest identity to Ras homologs studied in many other fungi. In this study, we report that rasA is not essential in A. fumigatus, but its absence is associated with slowed germination and a severe defect in radial growth. The ΔrasA hyphae were more than two times the diameter of wild-type hyphae, and they displayed repeated changes in the axis of polarity during hyphal growth. The deformed hyphae accumulated numerous nuclei within each hyphal compartment. The ΔrasA mutant conidiated poorly, but this phenotype could be ameliorated by growth on osmotically stabilized media. The ΔrasA mutant also showed increased susceptibility to cell wall stressors, stained more intensely with calcofluor white, and was refractory to lysing enzymes used to make protoplasts, suggesting an alteration of the cell wall. All phenotypes associated with deletion of rasA could be corrected by reinsertion of the wild-type gene. These data demonstrate a crucial role for RasA in both hyphal growth and asexual development in A. fumigatus and provide evidence that RasA function is linked to cell wall integrity.

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The DNA Binding Protein Rfg1 Is a Repressor of Filamentation inCandida albicans

Genetics ◽

10.1093/genetics/157.4.1503 ◽

2001 ◽

Vol 157 (4) ◽

pp. 1503-1512 ◽

Cited By ~ 1

Author(s):

Roy A Khalaf ◽

Richard S Zitomer

Keyword(s):

Dna Binding ◽

Binding Protein ◽

Hyphal Growth ◽

Dna Binding Protein ◽

Homozygous Deletion ◽

Wild Type ◽

Pathogenic Yeast ◽

Repression Complex ◽

Type Gene ◽

Repression Domain

AbstractWe have identified a repressor of hyphal growth in the pathogenic yeast Candida albicans. The gene was originally cloned in an attempt to characterize the homologue of the Saccharomyces cerevisiae Rox1, a repressor of hypoxic genes. Rox1 is an HMG-domain, DNA binding protein with a repression domain that recruits the Tup1/Ssn6 general repression complex to achieve repression. The C. albicans clone also encoded an HMG protein that was capable of repression of a hypoxic gene in a S. cerevisiae rox1 deletion strain. Gel retardation experiments using the purified HMG domain of this protein demonstrated that it was capable of binding specifically to a S. cerevisiae hypoxic operator DNA sequence. These data seemed to indicate that this gene encoded a hypoxic repressor. However, surprisingly, when a homozygous deletion was generated in C. albicans, the cells became constitutive for hyphal growth. This phenotype was rescued by the reintroduction of the wild-type gene on a plasmid, proving that the hyphal growth phenotype was due to the deletion and not a secondary mutation. Furthermore, oxygen repression of the hypoxic HEM13 gene was not affected by the deletion nor was this putative ROX1 gene regulated positively by oxygen as is the case for the S. cerevisiae gene. All these data indicate that this gene, now designated RFG1 for Repressor of Filamentous Growth, is a repressor of genes required for hyphal growth and not a hypoxic repressor.

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Yeast Nap1-Binding Protein Nbp2p Is Required for Mitotic Growth at High Temperatures and for Cell Wall Integrity

Genetics ◽

10.1093/genetics/165.2.517 ◽

2003 ◽

Vol 165 (2) ◽

pp. 517-529

Author(s):

Kentaro Ohkuni ◽

Asuko Okuda ◽

Akihiko Kikuchi

Keyword(s):

Cell Death ◽

Cell Wall ◽

High Temperature ◽

High Temperatures ◽

Hybrid System ◽

Mapk Pathway ◽

Binding Protein ◽

Cell Wall Integrity ◽

Calcofluor White ◽

Two Hybrid

AbstractNbp2p is a Nap1-binding protein in Saccharomyces cerevisiae identified by its interaction with Nap1 by a two-hybrid system. NBP2 encodes a novel protein consisting of 236 amino acids with a Src homology 3 (SH3) domain. We showed that NBP2 functions to promote mitotic cell growth at high temperatures and cell wall integrity. Loss of Nbp2 results in cell death at high temperatures and in sensitivity to calcofluor white. Cell death at high temperature is thought not to be due to a weakened cell wall. Additionally, we have isolated several type-2C serine threonine protein phosphatases (PTCs) as multicopy suppressors and MAP kinase-kinase (MAPKK), related to the yeast PKC MAPK pathway, as deletion suppressors of the nbp2Δ mutant. Screening for deletion suppressors is a new genetic approach to identify and characterize additional proteins in the Nbp2-dependent pathway. Genetic analyses suggested that Ptc1, which interacts with Nbp2 by the two-hybrid system, acts downstream of Nbp2 and that cells lacking the function of Nbp2 prefer to lose Mkk1, but the PKC MAPK pathway itself is indispensable when Nbp2 is deleted at high temperature.

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Identification and Characterization of a Peptidoglycan Hydrolase, MurA, of Listeria monocytogenes, a Muramidase Needed for Cell Separation

Journal of Bacteriology ◽

10.1128/jb.185.23.6801-6808.2003 ◽

2003 ◽

Vol 185 (23) ◽

pp. 6801-6808 ◽

Cited By ~ 56

Author(s):

Shannon A. Carroll ◽

Torsten Hain ◽

Ulrike Technow ◽

Ayub Darji ◽

Philippos Pashalidis ◽

...

Keyword(s):

Cell Wall ◽

Listeria Monocytogenes ◽

Cell Separation ◽

Bacterial Species ◽

Surface Protein ◽

Wild Type ◽

Terminal Domain ◽

Cell Wall Hydrolase ◽

Characteristic Features ◽

Type Gene

ABSTRACT A novel cell wall hydrolase encoded by the murA gene of Listeria monocytogenes is reported here. Mature MurA is a 66-kDa cell surface protein that is recognized by the well-characterized L. monocytogenes-specific monoclonal antibody EM-7G1. MurA displays two characteristic features: (i) an N-terminal domain with homology to muramidases from several gram-positive bacterial species and (ii) four copies of a cell wall-anchoring LysM repeat motif present within its C-terminal domain. Purified recombinant MurA produced in Escherichia coli was confirmed to be an authentic cell wall hydrolase with lytic properties toward cell wall preparations of Micrococcus lysodeikticus. An isogenic mutant with a deletion of murA that lacked the 66-kDa cell wall hydrolase grew as long chains during exponential growth. Complementation of the mutant strain by chromosomal reintegration of the wild-type gene restored expression of this murein hydrolase activity and cell separation levels to those of the wild-type strain. Studies reported herein suggest that the MurA protein is involved in generalized autolysis of L. monocytogenes.

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Aspergillus nidulans Protein O-Mannosyltransferases Play Roles in Cell Wall Integrity and Developmental Patterning

Eukaryotic Cell ◽

10.1128/ec.00040-09 ◽

2009 ◽

Vol 8 (10) ◽

pp. 1475-1485 ◽

Cited By ~ 33

Author(s):

Thanyanuch Kriangkripipat ◽

Michelle Momany

Keyword(s):

Cell Wall ◽

Aspergillus Nidulans ◽

Double Mutant ◽

Elevated Temperatures ◽

Cell Wall Integrity ◽

Wild Type ◽

Spatial Cues ◽

Developmental Patterning ◽

In The Wild ◽

Increased Sensitivity

ABSTRACT Protein O-mannosyltransferases (Pmts) initiate O-mannosyl glycan biosynthesis from Ser and Thr residues of target proteins. Fungal Pmts are divided into three subfamilies, Pmt1, -2, and -4. Aspergillus nidulans possesses a single representative of each Pmt subfamily, pmtA (subfamily 2), pmtB (subfamily 1), and pmtC (subfamily 4). In this work, we show that single Δpmt mutants are viable and have unique phenotypes and that the ΔpmtA ΔpmtB double mutant is the only viable double mutant. This makes A. nidulans the first fungus in which all members of individual Pmt subfamilies can be deleted without loss of viability. At elevated temperatures, all A. nidulans Δpmt mutants show cell wall-associated defects and increased sensitivity to cell wall-perturbing agents. The Δpmt mutants also show defects in developmental patterning. Germ tube emergence is early in ΔpmtA and more frequent in ΔpmtC mutants than in the wild type. In ΔpmtB mutants, intrahyphal hyphae develop. All Δpmt mutants show distinct conidiophore defects. The ΔpmtA strain has swollen vesicles and conidiogenous cells, the ΔpmtB strain has swollen conidiophore stalks, and the ΔpmtC strain has dramatically elongated conidiophore stalks. We also show that AN5660, an ortholog of Saccharomyces cerevisiae Wsc1p, is modified by PmtA and PmtC. The Δpmt phenotypes at elevated temperatures, increased sensitivity to cell wall-perturbing agents and restoration to wild-type growth with osmoticum suggest that A. nidulans Pmts modify proteins in the cell wall integrity pathway. The altered developmental patterns in Δpmt mutants suggest that A. nidulans Pmts modify proteins that serve as spatial cues.

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Proliferation of Intrahyphal Hyphae Caused by Disruption ofcsmA, Which Encodes a Class V Chitin Synthase with a Myosin Motor-Like Domain in Aspergillus nidulans

Journal of Bacteriology ◽

10.1128/jb.181.12.3721-3729.1999 ◽

1999 ◽

Vol 181 (12) ◽

pp. 3721-3729 ◽

Cited By ~ 96

Author(s):

Hiroyuki Horiuchi ◽

Makoto Fujiwara ◽

Shuichi Yamashita ◽

Akinori Ohta ◽

Masamichi Takagi

Keyword(s):

Cell Wall ◽

Aspergillus Nidulans ◽

Wild Type Strain ◽

Hyphal Growth ◽

Chitin Synthase ◽

Calcofluor White ◽

Class V ◽

Myosin Motor ◽

Fungal Morphogenesis ◽

Novel Protein

ABSTRACT We have found that the Aspergillus nidulans csmA gene encodes a novel protein which consists of an N-terminal myosin motor-like domain and a C-terminal chitin synthase domain (M. Fujiwara, H. Horiuchi, A. Ohta, and M. Takagi, Biochem. Biophys. Res. Commun. 236:75–78, 1997). To clarify the roles of csmA in fungal morphogenesis, we constructed csmA null mutants. The growth rate of the mutant colonies was almost the same as that of the wild-type strain, but hyphal growth was severely inhibited when a chitin-binding reagent, Calcofluor white or Congo red, was added to the medium. Moreover, morphological abnormalities in tip growth and septum formation were identified microscopically. Proliferation of intracellular new hyphae, called intrahyphal hyphae, which behaved as intrinsic hyphae, was the most striking phenotypic feature among them. These phenotypes were not suppressed when the only chitin synthase domain of csmA was expressed under the control of thealcA promoter, whereas they were suppressed when the intact form of csmA was expressed. Therefore, it was concluded that the product of csmA (CsmA) has important roles in polarized cell wall synthesis and maintenance of cell wall integrity and that the myosin motor-like domain is indispensable for these functions.

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Cross Talk between the Cell Wall Integrity and Cyclic AMP/Protein Kinase A Pathways in Cryptococcus neoformans

mBio ◽

10.1128/mbio.01573-14 ◽

2014 ◽

Vol 5 (4) ◽

Cited By ~ 18

Author(s):

Maureen J. Donlin ◽

Rajendra Upadhya ◽

Kimberly J. Gerik ◽

Woei Lam ◽

Laura G. VanArendonk ◽

...

Keyword(s):

Cell Wall ◽

Protein Kinase ◽

Cyclic Amp ◽

Protein Kinase A ◽

Cryptococcus Neoformans ◽

Signaling Pathway ◽

Cell Wall Integrity ◽

Wild Type ◽

Content Type ◽

Kinase A

ABSTRACTCryptococcus neoformans is a fungal pathogen of immunocompromised people that causes fatal meningitis. The fungal cell wall is essential to viability and pathogenesis ofC. neoformans, and biosynthesis and repair of the wall is primarily controlled by the cell wall integrity (CWI) signaling pathway. Previous work has shown that deletion of genes encoding the four major kinases in the CWI signaling pathway, namely,PKC1,BCK1,MKK2, andMPK1results in severe cell wall phenotypes, sensitivity to a variety of cell wall stressors, and for Mpk1, reduced virulence in a mouse model. Here, we examined the global transcriptional responses to gene deletions ofBCK1,MKK2, andMPK1compared to wild-type cells. We found that over 1,000 genes were differentially expressed in one or more of the deletion strains, with 115 genes differentially expressed in all three strains, many of which have been identified as genes regulated by the cyclic AMP (cAMP)/protein kinase A (PKA) pathway. Biochemical measurements of cAMP levels in the kinase deletion strains revealed significantly less cAMP in all of the deletion strains compared to the wild-type strain. The deletion strains also produced significantly smaller capsules than the wild-type KN99 strain did under capsule-inducing conditions, although the levels of capsule they shed were similar to those shed by the wild type. Finally, addition of exogenous cAMP led to reduced sensitivity to cell wall stress and restored surface capsule to levels near those of wild type. Thus, we have direct evidence of cross talk between the CWI and cAMP/PKA pathways that may have important implications for regulation of cell wall and capsule homeostasis.IMPORTANCECryptococcus neoformans is a fungal pathogen of immunocompromised people that causes fatal meningitis. The fungal cell wall is essential to viability and pathogenesis ofC. neoformans, and biosynthesis and repair of the wall are primarily controlled by the cell wall integrity (CWI) signaling pathway. In this study, we demonstrate that deletion of any of three core kinases in the CWI pathway impacts not only the cell wall but also the amount of surface capsule. Deletion of any of the kinases results in significantly reduced cellular cyclic AMP (cAMP) levels, and addition of exogenous cAMP rescues the capsule defect and some cell wall defects, supporting a direct role for the CWI pathway in regulation of capsule in conjunction with the cAMP/protein kinase A pathway.

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A Chitin Synthase with a Myosin-Like Motor Domain Is Essential for Hyphal Growth, Appressorium Differentiation, and Pathogenicity of the Maize Anthracnose Fungus Colletotrichum graminicola

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-20-12-1555 ◽

2007 ◽

Vol 20 (12) ◽

pp. 1555-1567 ◽

Cited By ~ 69

Author(s):

Stefan Werner ◽

Janyce A. Sugui ◽

Gero Steinberg ◽

Holger B. Deising

Keyword(s):

Cell Wall ◽

Filamentous Fungi ◽

Hyphal Growth ◽

Motor Domain ◽

Plant Colonization ◽

Colletotrichum Graminicola ◽

Wild Type ◽

Chitin Synthesis ◽

Cell Wall Biogenesis ◽

Host Cell Wall

Chitin synthesis contributes to cell wall biogenesis and is essential for invasion of solid substrata and pathogenicity of filamentous fungi. In contrast to yeasts, filamentous fungi contain up to 10 chitin synthases (CHS), which might reflect overlapping functions and indicate their complex lifestyle. Previous studies have shown that a class VI CHS of the maize anthracnose fungus Colletotrichum graminicola is essential for cell wall synthesis of conidia and vegetative hyphae. Here, we report on cloning and characterization of three additional CHS genes, CgChsI, CgChsIII, and CgChsV, encoding class I, III, and V CHS, respectively. All CHS genes are expressed during vegetative and pathogenic development. ΔCgChsI and ΔCgChsIII mutants did not differ significantly from the wild-type isolate with respect to hyphal growth and pathogenicity. In contrast, null mutants in the CgChsV gene, which encodes a CHS with an N-terminal myosin-like motor domain, are strongly impaired in vegetative growth and pathogenicity. Even in osmotically stabilized media, vegetative hyphae of ΔCgChsV mutants exhibited large balloon-like swellings, appressorial walls appeared to disintegrate during maturation, and infection cells were nonfunctional. Surprisingly, ΔCgChsV mutants were able to form dome-shaped hyphopodia that exerted force and showed host cell wall penetration rates comparable with the wild type. However, infection hyphae that formed within the plant cells exhibited severe swellings and were not able to proceed with plant colonization efficiently. Consequently, ΔCgChsV mutants did not develop macroscopically visible anthracnose disease symptoms and, thus, were nonpathogenic.

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Curcumin Targets Cell Wall Integrity via Calcineurin-Mediated Signaling in Candida albicans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01385-13 ◽

2013 ◽

Vol 58 (1) ◽

pp. 167-175 ◽

Cited By ~ 48

Author(s):

Awanish Kumar ◽

Sanjiveeni Dhamgaye ◽

Indresh Kumar Maurya ◽

Ashutosh Singh ◽

Monika Sharma ◽

...

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Critical Role ◽

Pathogenic Fungi ◽

Cell Wall Integrity ◽

Ergosterol Biosynthesis ◽

Ros Generation ◽

Calcofluor White ◽

Content Type ◽

Cell Wall Damage

ABSTRACTCurcumin (CUR) shows antifungal activity against a range of pathogenic fungi, includingCandida albicans. The reported mechanisms of action of CUR include reactive oxygen species (ROS) generation, defects in the ergosterol biosynthesis pathway, decrease in hyphal development, and modulation of multidrug efflux pumps. Reportedly, each of these pathways is independently linked to the cell wall machinery inC. albicans, but surprisingly, CUR has not been previously implicated in cell wall damage. In the present study, we performed transcriptional profiling to identify the yet-unidentified targets of CUR inC. albicans. We found that, among 348 CUR-affected genes, 51 were upregulated and 297 were downregulated. Interestingly, most of the cell wall integrity pathway genes were downregulated. The possibility of the cell wall playing a critical role in the mechanism of CUR required further validation; therefore, we performed specific experiments to establish if there was any link between the two. The fractional inhibitory concentration index values of 0.24 to 0.37 show that CUR interacts synergistically with cell wall-perturbing (CWP) agents (caspofungin, calcofluor white, Congo red, and SDS). Furthermore, we could observe cell wall damage and membrane permeabilization by CUR alone, as well as synergistically with CWP agents. We also found hypersusceptibility in calcineurin and mitogen-activated protein (MAP) kinase pathway mutants against CUR, which confirmed that CUR also targets cell wall biosynthesis inC. albicans. Together, these data provide strong evidence that CUR disrupts cell wall integrity inC. albicans. This new information on the mechanistic action of CUR could be employed in improving treatment strategies and in combinatorial drug therapy.

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Deletion of YLR358C Contributes to Reduce Cell Wall Integrity in Saccharomyces Cerevisiae

10.21203/rs.3.rs-1235133/v1 ◽

2022 ◽

Author(s):

Yu Zhang ◽

Mengyan Li ◽

Hanying Wang ◽

Juqing Deng ◽

Jianxing Liu ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Sodium Dodecyl ◽

Wall Stress ◽

Pathogenic Fungi ◽

Cell Wall Integrity ◽

Calcofluor White ◽

Fungal Cell ◽

Reading Frame ◽

Stress Signals

Abstract The mechanism of fungal cell wall synthesis and assembly is still unclear. Saccharomyces cerevisiae (S. cerevisiae) and pathogenic fungi are conserved in cell wall construction and response to stress signals, and often respond to cell wall stress through activated cell wall integrity (CWI) pathways. Whether the YLR358C open reading frame regulates CWI remains unclear. This study found that the growth of S. cerevisiae with YLR358C knockout was significantly inhibited on the medium containing different concentrations of cell wall interfering agents Calcofluor White (CFW), Congo Red (CR) and sodium dodecyl sulfate (SDS). CFW staining showed that the cell wall chitin was down-regulated, and transmission electron microscopy also observed a decrease in cell wall thickness. Transcriptome sequencing and analysis showed that YLR358C gene may be involved in the regulation of CWI signaling pathway. It was found by qRT-PCR that WSC3, SWI4 and HSP12 were differentially expressed after YLR358C was knocked out. The above results suggest that YLR358C may regulate the integrity of the yeast cell walls and has some potential for application in fermentation.

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SUN-Family Protein UvSUN1 Regulates the Development and Virulence of Ustilaginoidea virens

Frontiers in Microbiology ◽

10.3389/fmicb.2021.739453 ◽

2021 ◽

Vol 12 ◽

Author(s):

Mina Yu ◽

Junjie Yu ◽

Huijuan Cao ◽

Tianqiao Song ◽

Xiayan Pan ◽

...

Keyword(s):

Cell Wall ◽

Hyphal Growth ◽

Cell Wall Integrity ◽

Signal Recognition ◽

Phenotypic Analysis ◽

Ustilaginoidea Virens ◽

Group I ◽

Family Protein ◽

False Smut

Ustilaginoidea virens, the causal agent of rice false smut disease, is an important plant pathogen that causes severe quantitative and qualitative losses in rice worldwide. UvSUN1 is the only member of Group-I SUN family proteins in U. virens. In this work, the role of UvSUN1 in different aspects of the U. virens biology was studied by phenotypic analysis of Uvsun1 knockout strains. We identified that UvSUN1 was expressed during both conidial germination and the infection of rice. Disruption of the Uvsun1 gene affected the hyphal growth, conidiation, morphology of hyphae and conidia, adhesion and virulence. We also found that UvSUN1 is involved in the production of toxic compounds, which are able to inhibit elongation of the germinated seeds. Moreover, RNA-seq data showed that knockout of Uvsun1 resulted in misregulation of a subset of genes involved in signal recognition and transduction system, glycometabolism, cell wall integrity, and secondary metabolism. Collectively, this study reveals that Uvsun1 is required for growth, cell wall integrity and pathogenicity of U. virens, thereby providing new insights into the function of SUN family proteins in the growth and pathogenesis of this pathogen.

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