Cell Wall Perturbing Research Articles

A novel Ras GTPase (Ras3) regulates conidiation, multi-stress tolerance and virulence by acting upstream of Hog1 signaling pathway in Beauveria bassiana

Two Ras ATPases (Ras1 and Ras2) are well known to regulate antagonistically or cooperatively various cellular events in many fungi. Here we show the significance of a novel Ras homolog (Ras3) for Beauveria bassiana. Ras3 possesses five domains and two GTP/GDP switches typical for Ras family and was proven to localize to plasma membrane despite the position change of a membrane-targeting cysteine in C-terminal CAAX motif. Deletion of ras3 altered temporal transcription pattern of ras1 instead of ras2. Compared with wild-type, Δras3 grew significantly faster in a rich medium but slower in some minimal media, and produced far fewer conidia with impaired quality, which was evident with slower germination, attenuated virulence, reduced thermotolerance and decreased UV-B resistance. Moreover, Δras3 was much more sensitive to the oxidative stress of menadione than of H2O2 and to the stress of high osmolarity than of cell wall perturbation during growth. The high sensitivity of Δras3 to menadione was concurrent with reductions in both gene transcripts and total activity of superoxide dismutases. Intriguingly, the high osmosensitivity was concurrent with not only reduced transcripts of a critical transcription factor (Msn2) and most signaling proteins in the high-osmolarity-glycerol pathway of Δras3 but nearly undetectable phosphorylation signal of Hog1 hallmarking the pathway. All the changes were restored by ras3 complementation. Taken together, Ras3 is involved in the Hog1 pathway required for osmoregulation and hence can positively regulate conidiation, germination, multi-stress tolerance and virulence linked to the biological control potential of the filamentous insect pathogen.

Loss of Ypk1, the Yeast Homolog to the Human Serum- and Glucocorticoid-induced Protein Kinase, Accelerates Phospholipase B1-mediated Phosphatidylcholine Deacylation

Ypk1, the yeast homolog of the human serum- and glucocorticoid-induced kinase (Sgk1), affects diverse cellular activities, including sphingolipid homeostasis. We now report that Ypk1 also impacts the turnover of the major phospholipid, phosphatidylcholine (PC). Pulse-chase radiolabeling reveals that a ypk1Δ mutant exhibits increased PC deacylation and glycerophosphocholine production compared with wild type yeast. Deletion of PLB1, a gene encoding a B-type phospholipase that hydrolyzes PC, in a ypk1Δ mutant curtails the increased PC deacylation. In contrast to previous data, we find that Plb1 resides in the ER and in the medium. Consistent with a link between Ypk1 and Plb1, the levels of both Plb1 protein and PLB1 message are elevated in a ypk1Δ strain compared with wild type yeast. Furthermore, deletion of PLB1 in a ypk1Δ mutant exacerbates phenotypes associated with loss of YPK1, including slowed growth and sensitivity to cell wall perturbation, suggesting that increased Plb1 activity buffers against the loss of Ypk1. Because Plb1 lacks a consensus phosphorylation site for Ypk1, we probed other processes under the control of Ypk1 that might be linked to PC turnover. Inhibition of sphingolipid biosynthesis by the drug myriocin or through utilization of a lcb1-100 mutant results in increased PLB1 expression. Furthermore, we discovered that the increase in PLB1 expression observed upon inhibition of sphingolipid synthesis or loss of Ypk1 is under the control of the Crz1 transcription factor. Taken together, these results suggest a functional interaction between Ypk1 and Plb1 in which altered sphingolipid metabolism up-regulates PLB1 expression via Crz1.

Combination of Pretreatment with White Rot Fungi and Modification of Primary and Secondary Cell Walls Improves Saccharification

Plant cell walls have protective and structural functions conferring resistance to degradation. The lignin and hemicellulose network surrounding the cellulose microfibrils is insoluble unless subjected to harsh treatments. As lignin, pectin and xylan are effective barriers to cellulose extraction and hydrolysis, reducing their presence in cell walls improves saccharification. Microorganisms that can depolymerise lignin are of extreme interest to the biofuel industry. White rot fungi can be effective in pretreatment of lignocellulosic biomass prior to saccharification. Here, we show the cumulative effects of pretreating biomass with two white rot fungi, Phanerochaete chrysosporium and Trametes cingulata, on tobacco lines with reduced lignin or xylan, caused by suppression of the CINNAMOYL-CoA REDUCTASE, CINNAMATE-4-HYDROXYLASE, TOBACCO PEROXIDASE 60 or UDP-GLUCURONATE DECARBOXYLASE and on Arabidopsis thaliana with reduced de-esterified homogalacturonan content, obtained by overexpressing a pectin methyl esterase inhibitor or constitutively expressing the Aspergillus niger POLYGALACTURONASE II gene. Tests were extended to fresh material from an Arabidopsis mutant for a cell wall peroxidase. We demonstrate that fungal pretreatment is a reliable method of improving cellulose accessibility in biofuel feedstocks, fresh material and cell wall residues from different plants. These results contribute to the understanding of the consequences of primary and secondary cell wall perturbations on lignocellulosic biomass accessibility to white rot fungi and on saccharification yield. A comparison of the effects of P. chrysosporium and T. cingulata on tobacco saccharification also highlights the limitation of current knowledge in this research field and the necessity to systematically test culture conditions to avoid generalisations.

Three α-1,2-mannosyltransferases contribute differentially to conidiation, cell wall integrity, multistress tolerance and virulence of Beauveria bassiana

Members of α-1,2-mannosyltransferase (Ktr) family are required for protein O-mannosylation for the elongation of Ser/Thr mannose residues in yeasts but functionally unknown in most filamentous fungi. Here we characterized the functions of the Ktr orthologues Ktr1, Ktr4 and Kre2/Mnt1 in Beauveria bassiana, a filamentous enotmopathogen, and found that they were positive, but differential, mediators of many biological traits. Inactivation of Ktr4 and Kre2 resulted in 92% reduction of conidial yield on a standard medium and growth defects on substrates with altered carbon or nitrogen sources and availability, accompanied with reduced conidial size and complexity. This contrasts to the dispensability of Ktr1 for fungal growth and conidiation. More cell wall damage occurred in Δktr4 and Δkre2 than in Δktr1, including altered contents of the cell wall components mannoproteins, α-glucans and chitin, more carbohydrate epitopes changed on conidial surfaces, much lower conidial hydrophobicity, and thinner cell walls. Consequently, Δktr4 and Δkre2 became more sensitive to oxidation and cell wall perturbation than Δktr1 during colony growth or conidial germination despite less difference in their sensitivities to two osmotic agents. Conidial thermotolerance, UV-B resistance and virulence were all lowered greatly in Δktr4 and Δkre2 but only the thermotolerance decreased in Δktr1. All the phenotypical changes were well restored to wild-type levels by the complementation of each target gene. Our results indicate that Ktr4 and Kre2 contribute more to the biocontrol potential of B. bassiana than Ktr1 although all of them are significant contributors.

The Neurospora crassa CPS-1 polysaccharide synthase functions in cell wall biosynthesis

The Neurospora crassa cps-1 gene encodes a polysaccharide synthase with homology to the Cryptococcus neoformans hyaluronic acid synthase Cps1p. Homologs of the cps-1 gene are found in the genomes of many fungi. Loss of CPS-1 results in a cell wall defect that affects all stages of the N. crassa life cycle, including vegetative growth, protoperithecia (female mating structure) development, and conidia (asexual spore) development. The cell wall of cps-1 deletion mutants is sensitive to cell wall perturbation reagents. Our results demonstrate that CPS-1 is required for the incorporation of cell wall proteins into the cell wall and plays a critical role in cell wall biogenesis. We found that the N. crassa cell wall is devoid of hyaluronic acid, and conclude that the polysaccharide produced by the CPS-1 is not hyaluronic acid.

The connection of protein O-mannosyltransferase family to the biocontrol potential of Beauveria bassiana, a fungal entomopathogen.

O-Mannosylation dependent on the protein O-mannosyltransferase (Pmt) family is an essential post-translational modification process in eukaryotes, but their connection to the biocontrol potential of a filamentous entomopathogen against arthropod pests is not understood. Here, we characterized the functions of three Pmt orthologues (Pmt1, Pmt2 and Pmt4) in the Pmt family of Beauveria bassiana and found that they were positive, but differential, regulators of the fungal growth, conidiation, multi-stress tolerance and virulence. Three Pmt2 knockdown mutants (ΔPmt2 was lethal), ΔPmt1 and ΔPmt4 grew 20-79% slower on nutrition-rich and limited media. Their conidial yields on a standard medium were reduced by 17-62%, accompanied with delayed germination. All the mutants became significantly less tolerant to most stresses of cell wall perturbation, high osmolarity, oxidation, wet heat and UV-B irradiation during colony growth and conidial germination and lost virulence by 53-62% via cuticle infection, although their virulence via hemocoel injection was not affected. Strikingly, these phenotypic defects were accompanied with remarkable cell wall damage, including thinner cell wall, lower conidial hydrophobicity and altered cell wall composition. All the changes were well restored to wild-type levels by targeted Pmt1 or Pmt4 complementation. Our results indicate for the first time that Pmt1, Pmt2 and Pmt4 are all required for the full biocontrol potential of B. bassiana despite differential contributions.

Adenylate cyclase orthologues in two filamentous entomopathogens contribute differentially to growth, conidiation, pathogenicity, and multistress responses

Adenylate cyclase (AC) is a core element of cAMP signalling network. Here we show functional diversity and differentiation of Beauveria bassiana AC (BbAC) and Metarhizium robertsii AC (MrAC). Severe growth defects occurred in ΔBbAC and ΔMrAC grown on nutrition-rich SDAY and several minimal media but were largely alleviated by adding cAMP to SDAY. Conidial yield increased greatly in ΔBbAC but decreased in ΔMrAC. During colony growth, ΔBbAC was highly sensitive to oxidation, high osmolarity, cell wall perturbation, carbendazim fungicide, Mn2+, Zn2+, Fe3+, and EDTA but more tolerant to Cu2+ while ΔMrAC showed higher osmotolerance, decreased sensitivity to Fe3+, and null response to carbendazim or cell wall stress despite similar responses to oxidation and other metal ions. Conidial UV-B resistance decreased by 32 % in ΔBbAC and 22 % in ΔMrAC despite little change in their theromotolerance. Median lethal time (LT50) estimates of ΔBbAC and ΔMrAC against susceptible insects were 10.9 and 1.4 d longer than those from wild-type strains respectively. All the phenotypic changes were restored to wild-type levels by each gene complementation. Taken together, BbAC and MrAC regulated differentially conidiation, pathogenicity, and multistress responses in B. bassiana and M. robertsii, thereby making different contributions to their biocontrol potential.

Three Mitogen-Activated Protein Kinases Required for Cell Wall Integrity Contribute Greatly to Biocontrol Potential of a Fungal Entomopathogen

Bck1, Mkk1 and Slt2 are three mitogen-activated protein (MAP) kinases constituting cell wall integrity (CWI) pathway that may control multi-stress responses via crosstalk with high-osmolarity glycerol (HOG) pathway in budding yeast. In this study, Bck1, Mkk1 and Slt2 orthologues in Beauveria bassiana were confirmed as the three-module cascade essential for CWI because cell wall impairment occurred in the hyphae and conidia of Δbck1, Δmkk1 and Δslt2 examined in multiple experiments. Strikingly, all the deletion mutants became more sensitive to hyperosmotic NaCl and sorbitol with the Western blot of Hog1 phosphorylation being weakened in Δbck1 and absent in Δmkk1 and Δslt2. Apart from crossing responses to cell wall perturbation and high osmolarity, three deletion mutants exhibited faster growth and conidiation on nutrition-rich medium, much less virulence to Galleria mellonella larvae, and higher sensitivity to nutritional, fungicidal, thermal and UV-B irradiative stresses, accompanied with less accumulation of intracellular mannitol and trehalose. Moreover, Δmkk1 and Δslt2 were equally more sensitive to all the stresses of different types except wet-heat stress than wild type and more or less different from Δbck1 in sensitivity to most of the stresses despite their null responses to two oxidants. All the changes in three deletion mutants were restored by each targeted gene complementation. Taken together, the CWI-required Bck1, Mkk1 and Slt2 are all positive, but differential, regulators of multi-stress tolerance and virulence perhaps due to interplay with the HOG pathway essential for osmoregulation, thereby contributing greatly to the biocontrol potential of the fungal entomopathogen.

NikA/TcsC Histidine Kinase Is Involved in Conidiation, Hyphal Morphology, and Responses to Osmotic Stress and Antifungal Chemicals in Aspergillus fumigatus

The fungal high osmolarity glycerol (HOG) pathway is composed of a two-component system (TCS) and Hog1-type mitogen-activated protein kinase (MAPK) cascade. A group III (Nik1-type) histidine kinase plays a major role in the HOG pathway of several filamentous fungi. In this study, we characterized a group III histidine kinase, NikA/TcsC, in the life-threatening pathogenic fungus, Aspergillus fumigatus. A deletion mutant of nikA showed low conidia production, abnormal hyphae, marked sensitivity to high osmolarity stresses, and resistance to cell wall perturbing reagents such as congo red and calcofluor white, as well as to fungicides such as fludioxonil, iprodione, and pyrrolnitrin. None of these phenotypes were observed in mutants of the SskA response regulator and SakA MAPK, which were thought to be downstream components of NikA. In contrast, in response to fludioxonil treatment, NikA was implicated in the phosphorylation of SakA MAPK and the transcriptional upregulation of catA, dprA, and dprB, which are regulated under the control of SakA. We then tested the idea that not only NikA, but also the other 13 histidine kinases play certain roles in the regulation of the HOG pathway. Interestingly, the expression of fos1, phkA, phkB, fhk5, and fhk6 increased by osmotic shock or fludioxonil treatment in a SakA-dependent manner. However, deletion mutants of the histidine kinases showed no significant defects in growth under the tested conditions. Collectively, although the signal transduction network related to NikA seems complicated, NikA plays a crucial role in several aspects of A. fumigatus physiology and, to a certain extent, modulates the HOG pathway.

Cytokinesis-required Cdc14 is a signaling hub of asexual development and multi-stress tolerance in Beauveria bassiana

A dual-specificity, paralogue-free Cdc14 phosphatase was located in the nuclei of Beauveria bassiana (filamentous entomopathogen) and functionally characterized. Inactivation of cdc14 caused defective cytokinesis due to multinucleate cells formed in Δcdc14 and 89% decrease of blastospore production, followed by slower growth and a loss of ≥ 96% conidial yield under normal conditions. These defects coincided well with drastic down-regulation of 25 genes required for mitosis and conidiation. Moreover, Δcdc14 became hypersensitive to oxidative, osmotic, and cell wall and mitosis perturbing stresses, and lost 41−70% of conidial thermotolerance, UV-B resistance and virulence, accompanied with transcriptional down-regualtion of various signaling factors and stress-responsive effectors and depressed phosphorylation signals of Hog1 and Slt2 in high-osmolarity glycerol and cell-wall integrity pathways. All changes were well restored by rescuing cdc14. Our findings indicate that Cdc14 vital for the fungal cytokinesis acts as a signaling hub in regulating not only asexual development but multi-stress responses and virulence.

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

Curcumin (CUR) shows antifungal activity against a range of pathogenic fungi, including Candida 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 in C. 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 in C. 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 in C. albicans. Together, these data provide strong evidence that CUR disrupts cell wall integrity in C. albicans. This new information on the mechanistic action of CUR could be employed in improving treatment strategies and in combinatorial drug therapy.

Nuclear proteome response to cell wall removal in rice (Oryza sativa)

Plant cells are routinely exposed to various pathogens and environmental stresses that cause cell wall perturbations. Little is known of the mechanisms that plant cells use to sense these disturbances and transduce corresponding signals to regulate cellular responses to maintain cell wall integrity. Previous studies in rice have shown that removal of the cell wall leads to substantial chromatin reorganization and histone modification changes concomitant with cell wall re-synthesis. But the genes and proteins that regulate these cellular responses are still largely unknown. Here we present an examination of the nuclear proteome differential expression in response to removal of the cell wall in rice suspension cells using multiple nuclear proteome extraction methods. A total of 382 nuclear proteins were identified with two or more peptides, including 26 transcription factors. Upon removal of the cell wall, 142 nuclear proteins were up regulated and 112 were down regulated. The differentially expressed proteins included transcription factors, histones, histone domain containing proteins, and histone modification enzymes. Gene ontology analysis of the differentially expressed proteins indicates that chromatin & nucleosome assembly, protein-DNA complex assembly, and DNA packaging are tightly associated with cell wall removal. Our results indicate that removal of the cell wall imposes a tremendous challenge to the cells. Consequently, plant cells respond to the removal of the cell wall in the nucleus at every level of the regulatory hierarchy.

Cell Wall Perturbation Sensitizes Fungi to the Antimalarial Drug Chloroquine

Chloroquine (CQ) has been a mainstay of antimalarial drug treatment for several decades. Additional therapeutic actions of CQ have been described, including some reports of fungal inhibition. Here we investigated the action of CQ in fungi, including the yeast model Saccharomyces cerevisiae. A genomewide yeast deletion strain collection was screened against CQ, revealing that bck1Δ and slt2Δ mutants of the cell wall integrity pathway are CQ hypersensitive. This phenotype was rescued with sorbitol, consistent with cell wall involvement. The cell wall-targeting agent caffeine caused hypersensitivity to CQ, as did cell wall perturbation by sonication. The phenotypes were not caused by CQ-induced changes to cell wall components. Instead, CQ accumulated to higher levels in cells with perturbed cell walls: CQ uptake was 2- to 3-fold greater in bck1Δ and slt2Δ mutants than in wild-type yeast. CQ toxicity was synergistic with that of the major cell wall-targeting antifungal drug, caspofungin. The MIC of caspofungin against the yeast pathogen Candida albicans was decreased 2-fold by 250 μM CQ and up to 8-fold at higher CQ concentrations. Similar effects were seen in Candida glabrata and Aspergillus fumigatus. The results show that the cell wall is critical for CQ resistance in fungi and suggest that combination treatments with cell wall-targeting drugs could have potential for antifungal treatment.

Light Sensing in Aspergillus fumigatus Highlights the Case for Establishing New Models for Fungal Photobiology

ABSTRACTMicrobes inhabit diverse environmental locations, and many species need to shift their physiology between different niches. To do this effectively requires the accurate sensing of and response to the environment. For pathogens, exposure to light is one major change between a free-living saprophyte lifestyle and causation of disease within the host. However, how light may act as a signal to influence pathogenesis, on the side of either the host or the pathogen, is poorly understood. Research during the last 2 decades has uncovered aspects about the machinery for light sensing in a small number of fungi. Now, Fuller et al. have initiated studies into the role that light and two photosensor homologs play in the behavior of the ubiquitous fungal pathogen Aspergillus fumigatus [K. K. Fuller, C. S. Ringelberg, J. J. Loros, and J. C. Dunlap, mBio 4(2):e00142-13, 2013, doi:10.1128/mBio.00142-13]. Light represses the germination of A. fumigatus spores and enhances resistance to ultraviolet light, oxidative stresses, and cell wall perturbations. The phenotypes of the strains with mutations in the LreA and FphA homologs revealed that these sensors control some, but not all, responses to light. Furthermore, interactions occur between blue and red light signaling pathways, as has been described for a related saprophytic species, Aspergillus nidulans. Genome-wide transcript analyses found that about 2.6% of genes increase or decrease their transcript levels in response to light. This use of A. fumigatus establishes common elements between model filamentous species and pathogenic species, underscoring the benefits of extending photobiology to new species of fungi.

The Fungal Pathogen Aspergillus fumigatus Regulates Growth, Metabolism, and Stress Resistance in Response to Light

ABSTRACTLight is a pervasive environmental factor that regulates development, stress resistance, and even virulence in numerous fungal species. Though much research has focused on signaling pathways in Aspergillus fumigatus, an understanding of how this pathogen responds to light is lacking. In this report, we demonstrate that the fungus does indeed respond to both blue and red portions of the visible spectrum. Included in the A. fumigatus light response is a reduction in conidial germination rates, increased hyphal pigmentation, enhanced resistance to acute ultraviolet and oxidative stresses, and an increased susceptibility to cell wall perturbation. By performing gene deletion analyses, we have found that the predicted blue light receptor LreA and red light receptor FphA play unique and overlapping roles in regulating the described photoresponsive behaviors of A. fumigatus. However, our data also indicate that the photobiology of this fungus is complex and likely involves input from additional photosensory pathways beyond those analyzed here. Finally, whole-genome microarray analysis has revealed that A. fumigatus broadly regulates a variety of metabolic genes in response to light, including those involved in respiration, amino acid metabolism, and metal homeostasis. Together, these data demonstrate the importance of the photic environment on the physiology of A. fumigatus and provide a basis for future studies into this unexplored area of its biology.

Insight into the transcriptional regulation of Msn2 required for conidiation, multi-stress responses and virulence of two entomopathogenic fungi

Msn2/4 transcription factors in some fungi have null effects on virulence and cellular stress responses. Here we found that the transcriptional regulation of Msn2 orthologs is vital for the conidiation, virulence and multi-stress responses of Beauveria bassiana (Bb) and Metarhizium robertsii (Mr), which lack Msn4 orthologs. Compared to wild-type and complemented strains of each fungus with all similar phenotypes, ΔBbmsn2 and ΔMrmsn2 showed remarkable defects in conidial yield (∼40% decrease) and virulence (∼25% decrease). Both delta mutants lost 20–65% of their tolerances to hyperosmolarity, oxidation, carbendazim, cell wall perturbing and high temperature at 34°C during colony growth. Their conidia were also significantly (18–41%) less tolerant to oxidation, hyperosmolarity, wet-heat stress at 45°C and UV-B irradiation. Accompanied with the defective phenotypes, several conidiation- and virulence-associated genes were greatly repressed in ΔBbmsn2 and ΔMrmsn2. Moreover, differentially expressed genes in the transcriptomes of ΔBbmsn2 versus wild type were ∼3% more under oxidative stress, but ∼12% fewer under heat shock, than those in the ΔMrmsn2 counterparts. Many stress-responsive effector genes and cellular signaling factors were remarkably downregulated. Taken together, the two entomopathogens could have evolved somewhat distinct stress-responsive mechanisms finely tuned by Msn2, highlighting the biological significance of Msn2 orthologs for filamentous fungi.

The Crz1/Sp1 Transcription Factor of Cryptococcus neoformans Is Activated by Calcineurin and Regulates Cell Wall Integrity

Cryptococcus neoformans survives host temperature and regulates cell wall integrity via a calcium-dependent phosphatase, calcineurin. However, downstream effectors of C. neoformans calcineurin are largely unknown. In S. cerevisiae and other fungal species, a calcineurin-dependent transcription factor Crz1, translocates to nuclei upon activation and triggers expression of target genes. We now show that the C. neoformans Crz1 ortholog (Crz1/Sp1), previously identified as a protein kinase C target during starvation, is a bona fide target of calcineurin under non-starvation conditions, during cell wall stress and growth at high temperature. Both the calcineurin-defective mutant, Δcna1, and a CRZ1/SP1 mutant (Δcrz1) were susceptible to cell wall perturbing agents. Furthermore, expression of the chitin synthase encoding gene, CHS6, was reduced in both mutants. We tracked the subcellular localization of Crz1-GFP in WT C. neoformans and Δcna1 in response to different stimuli, in the presence and absence of the calcineurin inhibitor, FK506. Exposure to elevated temperature (30–37°C vs 25°C) and extracellular calcium caused calcineurin-dependent nuclear accumulation of Crz1-GFP. Unexpectedly, 1M salt and heat shock triggered calcineurin-independent Crz1-GFP sequestration within cytosolic and nuclear puncta. To our knowledge, punctate cytosolic distribution, as opposed to nuclear targeting, is a unique feature of C. neoformans Crz1. We conclude that Crz1 is selectively activated by calcium/calcineurin-dependent and independent signals depending on the environmental conditions.

Absence of Yps7p, a putative glycosylphosphatidylinositol-linked aspartyl protease inPichia pastoris, results in aberrant cell wall composition and increased osmotic stress resistance

Recently, studies performed on Saccharomyces cerevisiae and Candida albicans have confirmed the importance of fungal glycosylphosphatidylinositol (GPI)-anchored aspartyl proteases (yapsins) for cell-wall integrity. Genome sequence annotation of Pichia pastoris also revealed seven putative GPI-anchored aspartyl protease genes. The five yapsin genes assigned as YPS1, YPS2, YPS3, YPS7 and MKC7 in P. pastoris were disrupted. Among these putative GPI-linked aspartyl proteases, disruption of PpYPS7 gene confers the Ppyps7Δ mutant cell increased resistance to cell wall perturbing reagents congo red, calcofluor white (CW) and sodium dodecyl sulfate. Quantitative analysis of cell wall components shows lower content of chitin and increased amounts of β-1,3-glucan. Further staining of the cell with CW demonstrates that disruption of PpYPS7 gene causes a reduction of the chitin content in lateral cell wall. Consistently, transmission electron micrographs show that the inner layer of mutant cell wall, mainly composed of chitin and β-1, 3-glucan, is much thicker than that in parental strain GS115. Additionally, Ppyps7Δ mutant also exhibits increased osmotic resistance compared with parental strain GS115. This could be due to the dramatically elevated intracellular glycerol level in Ppyps7Δ mutant. These results suggest that PpYPS7 is involved in cell wall integrity and response to osmotic stress.

Deciphering cell wall integrity signalling in Aspergillus fumigatus: identification and functional characterization of cell wall stress sensors and relevant Rho GTPases

The fungal cell wall, a conserved and highly dynamic structure, is essential for virulence and viability of fungal pathogens and is an important antifungal drug target. The cell wall integrity (CWI) signalling pathway regulates shape and biosynthesis of the cell wall. In this work we identified, localized and functionally characterized four putative CWI stress sensors of Aspergillus fumigatus, an airborne opportunistic human pathogen and the cause of invasive aspergillosis. We show that Wsc1 is specifically required for resistance to echinocandin antifungals. MidA is specifically required for elevated temperature tolerance and resistance to the cell wall perturbing agents congo red and calcofluor white. Wsc1, Wsc3 and MidA additionally have overlapping functions and are redundantly required for radial growth and conidiation. We have also analysed the roles of three Rho GTPases that have been implicated in CWI signalling in other fungi. We show that Rho1 is essential and that conditional downregulation of rho1 or deletion of rho2 or rho4 results in severely impaired CWI. Our data indicate significant functional differences between the CWI stress sensors of yeasts and moulds.

Metabolism of free oligosaccharides is facilitated in the och1 mutant of Saccharomyces cerevisiae

In eukaryotic cells, it is known that N-glycans play a pivotal role in quality control of carrier proteins. Although "free" forms of oligosaccharides (fOSs) are known to be accumulated in the cytosol, the precise mechanism of their formation, degradation and biological relevance remains poorly understood. It has been shown that, in budding yeast, almost all fOSs are formed from misfolded glycoproteins. Precise structural analysis of fOSs revealed that several yeast fOSs bear a yeast-specific modification by Golgi-resident α-1,6-mannosyltransferase, Och1. In this study, structural diversity of fOSs in och1Δ cells was analyzed. To our surprise, several fOSs in och1Δ cells have unusual α-1,3-linked mannose residues at their non-reducing termini. These mannose residues were not observed in wild-type cells, suggesting that the addition of these unique mannoses occurred as a compensation of Och1 defect. A significant increase in the amount of fOSs modified by Golgi-localized mannosyltransferases was also observed in och1Δ cells. Moreover, the amount of processed fOSs and intracellular α-mannosidase (Ams1) both increased in this mutant. Up-regulation of Ams1 activity was also apparent for cells treated with cell wall perturbation reagent. These results provide an insight into a possible link between catabolism of fOSs and cell wall stress.