Dic2 and Dic3 loci confer osmotic adaptation and fungicidal sensitivity independent of the HOG pathway in Cochliobolus heterostrophus

Previously, we identified three gene loci, Dic1, Dic2, and Dic3, that confer high-osmolarity adaptation and dicarboximide/phenylpyrrole fungicide sensitivity in Cochliobolus heterostrophus. Dic1 encoded a group III histidine kinase, but the other genes were not characterized. In the present study, we revealed that both Dic2 and Dic3 are involved in the Skn7 pathway. Dic2 encoded an Skn7-type response regulator, ChSkn7. Strain N4502 contained D359N in the response regulator domain of ChSkn7. Strain E4503 contained a deletion of 50 amino acids in the DNA-binding domain. Strain N4507 was a null mutant of the ChSkn7 gene. All of the dic2 mutant strains showed similar levels of sensitivity to high osmolarity and similar levels of resistance to fungicides. These results strongly suggested that both the DNA-binding domain and response regulator domain are essential for Skn7 function in osmotic adaptation and fungicide sensitivity. A western blot analysis revealed that Dic3 is not involved in the regulation of Hog1-type MAPKs. The Chssk1/dic3 double mutant strains clearly showed greater resistance to fungicides than the single mutant strains. An additive effect was also observed in the high-osmolarity experiments. On the other hand, the dic3/dic2 double mutant strains did not show higher levels of resistance to fungicides and greater sensitivity to KCl than the single mutant strains. These results strongly suggested that the dic3 locus confer high-osmolarity adaptation and fungicide sensitivity independently from Ssk1-Hog1 pathway, but not the Skn7 pathway. Moreover, the dic3 strain and all dic2 strains showed similar levels of sensitivity to high-osmolarity stress and similar levels of resistance to fungicides, suggesting Dic3 to have an essential role in the Skn7 pathway. Our results provide new insight into the functions of the Skn7 pathway in filamentous fungi.     

Glycerol-3-Phosphate Acyltransferase Contributes to Triacylglycerol Biosynthesis,Lipid Droplet Formation,and Host Invasion in Metarhizium robertsii

Enzymes involved in the triacylglycerol (TAG) biosynthesis have been well studied in the model organisms of yeasts and animals. Among these, the isoforms of glycerol-3-phosphate acyltransferase (GPAT) redundantly catalyze the first and rate-limiting step in glycerolipid synthesis. Here, we report the functions of mrGAT, a GPAT ortholog, in an insect-pathogenic fungus, Metarhizium robertsii. Unlike in yeasts and animals, a single copy of the mrGAT gene is present in the fungal genome and the gene deletion mutant is viable. Compared to the wild type and the gene-rescued mutant, the ΔmrGAT mutant demonstrated reduced abilities to produce conidia and synthesize TAG, glycerol, and total lipids. More importantly, we found that mrGAT is localized to the endoplasmic reticulum and directly linked to the formation of lipid droplets (LDs) in fungal cells. Insect bioassay results showed that mrGAT is required for full fungal virulence by aiding fungal penetration of host cuticles. Data from this study not only advance our understanding of GPAT functions in fungi but also suggest that filamentous fungi such as M. robertsii can serve as a good model to elucidate the role of the glycerol phosphate pathway in fungal physiology, particularly to determine the mechanistic connection of GPAT to LD formation.     

The response regulator BcSkn7 is required for vegetative differentiation and adaptation to oxidative and osmotic stresses in Botrytis cinerea

The high‐osmolarity glycerol pathway plays an important role in the responses of fungi to various environmental stresses. Saccharomyces cerevisiae Skn7 is a response regulator in the high‐osmolarity glycerol pathway, which regulates the oxidative stress response, cell cycle and cell wall biosynthesis. In this study, we characterized an Skn7 orthologue BcSkn7 in Botrytis cinerea. BcSKN7 can partly restore the growth defects of S. cerevisiae SKN7 mutant and vice versa. The BcSKN7 mutant (ΔBcSkn7‐1) revealed increased sensitivity to ionic osmotic and oxidative stresses and to ergosterol biosynthesis inhibitors. In addition, ΔBcSkn7‐1 was also impaired dramatically in conidiation and sclerotial formation. Western blot analysis showed that BcSkn7 positively regulated the phosphorylation of BcSak1 (the orthologue of S. cerevisiae Hog1) under osmotic stress, indicating that BcSkn7 is associated with the high‐osmolarity glycerol pathway in B. cinerea. In contrast with BcSak1, BcSkn7 is not involved in the regulation of B. cinerea virulence. All of the phenotypic defects of ΔBcSkn7‐1 are restored by genetic complementation of the mutant with the wild‐type BcSKN7. The results of this study indicate that BcSkn7 plays an important role in the regulation of vegetative differentiation and in the response to various stresses in B. cinerea.     

Linkage of autophagy to fungal development,lipid storage and virulence in Metarhizium robertsii

Autophagy is a highly conserved process that maintains intracellular homeostasis by degrading proteins or organelles in all eukaryotes. The effect of autophagy on fungal biology and infection of insect pathogens is unknown. Here, we report the function of MrATG8, an ortholog of yeast ATG8, in the entomopathogenic fungus Metarhizium robertsii. MrATG8 can complement an ATG8-defective yeast strain and deletion of MrATG8 impaired autophagy, conidiation and fungal infection biology in M. robertsii. Compared with the wild-type and gene-rescued mutant, Mratg8Δ is not inductive to form the infection-structure appressorium and is impaired in defense response against insect immunity. In addition, accumulation of lipid droplets (LDs) is significantly reduced in the conidia of Mratg8Δ and the pathogenicity of the mutant is drastically impaired. We also found that the cellular level of a LD-specific perilipin-like protein is significantly lowered by deletion of MrATG8 and that the carboxyl terminus beyond the predicted protease cleavage site is dispensable for MrAtg8 function. To corroborate the role of autophagy in fungal physiology, the homologous genes of yeast ATG1, ATG4 and ATG15, designated as MrATG1, MrATG4 and MrATG15, were also deleted in M. robertsii. In contrast to Mratg8Δ, these mutants could form appressoria, however, the LD accumulation and virulence were also considerably impaired in the mutant strains. Our data showed that autophagy is required in M. robertsii for fungal differentiation, lipid biogenesis and insect infection. The results advance our understanding of autophagic process in fungi and provide evidence to connect autophagy with lipid metabolism.     

Characterization of Regulatory Pathways in Xylella fastidiosa: Genes and Phenotypes Controlled by algU

Many virulence genes in plant bacterial pathogens are coordinately regulated by “global” regulatory genes. Conducting DNA microarray analysis of bacterial mutants of such genes, compared with the wild type, can help to refine the list of genes that may contribute to virulence in bacterial pathogens. The regulatory gene algU, with roles in stress response and regulation of the biosynthesis of the exopolysaccharide alginate in Pseudomonas aeruginosa and many other bacteria, has been extensively studied. The role of algU in Xylella fastidiosa, the cause of Pierce's disease of grapevines, was analyzed by mutation and whole-genome microarray analysis to define its involvement in aggregation, biofilm formation, and virulence. In this study, an algU::nptII mutant had reduced cell-cell aggregation, attachment, and biofilm formation and lower virulence in grapevines. Microarray analysis showed that 42 genes had significantly lower expression in the algU::nptII mutant than in the wild type. Among these are several genes that could contribute to cell aggregation and biofilm formation, as well as other physiological processes such as virulence, competition, and survival.     

Role of CaECM25 in cell morphogenesis, cell growth and virulence in Candida albicans

Candida albicans is the most prominent opportunistic fungal pathogen in humans. Multiple factors are associated with the virulence of C. albicans, including morphogenesis, cell wall organization and growth rate. Here, we describe the identification and functional characterization of CaECM25, a gene that has not been reported before. We constructed Caecm25?/? mutants and investigated the role of the gene in morphogenesis, cell wall organization and virulence. CaECM25 deletion resulted in defects in cell separation, a slower growth rate, reduced filamentous growth and attenuated adherence to plastic surfaces. The Caecm25?/? mutant was also significantly less virulent than wild type when tested for systemic infection in mice. Therefore, CaECM25 plays important roles in morphogenesis, cell wall organization and virulence.     

PASTA kinase-dependent control of peptidoglycan synthesis via ReoM is required for cell wall stress responses,cytosolic survival,and virulence in Listeria monocytogenes

Pathogenic bacteria rely on protein phosphorylation to adapt quickly to stress, including that imposed by the host during infection. Penicillin-binding protein and serine/threonine-associated (PASTA) kinases are signal transduction systems that sense cell wall integrity and modulate multiple facets of bacterial physiology in response to cell envelope stress. The PASTA kinase in the cytosolic pathogen Listeria monocytogenes, PrkA, is required for cell wall stress responses, cytosolic survival, and virulence, yet its substrates and downstream signaling pathways remain incompletely defined. We combined orthogonal phosphoproteomic and genetic analyses in the presence of a β-lactam antibiotic to define PrkA phosphotargets and pathways modulated by PrkA. These analyses synergistically highlighted ReoM, which was recently identified as a PrkA target that influences peptidoglycan (PG) synthesis, as an important phosphosubstrate during cell wall stress. We find that deletion of reoM restores cell wall stress sensitivities and cytosolic survival defects of a ΔprkA mutant to nearly wild-type levels. While a ΔprkA mutant is defective for PG synthesis during cell wall stress, a double ΔreoM ΔprkA mutant synthesizes PG at rates similar to wild type. In a mouse model of systemic listeriosis, deletion of reoM in a ΔprkA background almost fully restored virulence to wild-type levels. However, loss of reoM alone also resulted in attenuated virulence, suggesting ReoM is critical at some points during pathogenesis. Finally, we demonstrate that the PASTA kinase/ReoM cell wall stress response pathway is conserved in a related pathogen, methicillin-resistant Staphylococcus aureus. Taken together, our phosphoproteomic analysis provides a comprehensive overview of the PASTA kinase targets of an important model pathogen and suggests that a critical role of PrkA in vivo is modulating PG synthesis through regulation of ReoM to facilitate cytosolic survival and virulence.     

Glycosylphosphatidylinositol-Anchored Proteins in Fusarium graminearum: Inventory,Variability, and Virulence

The contribution of cell surface proteins to plant pathogenicity of fungi is not well understood. As such, the objective of this study was to investigate the functions and importance of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in the wheat pathogen F. graminearum. GPI-APs are surface proteins that are attached to either the membrane or cell wall. In order to simultaneously disrupt several GPI-APs, a phosphoethanolamine transferase-encoding gene gpi7 was deleted and the resultant mutant characterized in terms of growth, development, and virulence. The Δgpi7 mutants exhibited slower radial growth rates and aberrantly shaped macroconidia. Furthermore, virulence tests and microscopic analyses indicated that Gpi7 is required for ramification of the fungus throughout the rachis of wheat heads. In parallel, bioinformatics tools were utilized to predict and inventory GPI-APs within the proteome of F. graminearum. Two of the genes identified in this screen (FGSG_01588 and FGSG_08844) displayed isolate-specific length variability as observed for other fungal cell wall adhesion genes. Nevertheless, deletion of these genes failed to reveal obvious defects in growth, development, or virulence. This research demonstrates the global importance of GPI-APs to in planta proliferation in F. graminearum, and also highlights the potential of individual GPI-APs as diagnostic markers.     

Functional and proteomic analyses reveal that wxcB is involved in virulence,motility, detergent tolerance,and biofilm formation in Xanthomonas campestris pv. vesicatoria

The bacterial envelope possesses diverse functions, including protection against environmental stress and virulence factors for host infection. Here, we report the function of wxcB in Xanthomonas campestris pv. vesicatoria (Xcv), a causal agent of bacterial leaf spot disease in tomato and pepper. To characterize roles of wxcB, we generated a knockout mutant (XcvΔwxcB) and found that the virulence of the mutant was weaker than that of the wild type in tomato plants. To predict the mechanism affected by wxcB, we compared protein expressions between the wild type and the mutant. Expression of 152 proteins showed a greater than 2-fold difference. Proteins involved in motility and cell wall/membrane were the most abundant. Through phenotypic assays, we further demonstrated that the mutant displayed reduced motility and tolerance to treatment, but it showed increased biofilm formation. Interestingly, the LPS profile was unchanged. These results lead to new insights into the functions of wxcB that is associated with cell wall/membrane functions, which contributes to pathogen virulence.     

A High-Throughput Gene Disruption Methodology for the Entomopathogenic Fungus Metarhizium robertsii

Systematic gene disruption is a direct way to interrogate a fungal genome to functionally characterize the full suite of genes involved in various biological processes. Metarhizium robertsii is extraordinarily versatile, and it is a pathogen of arthropods, a saprophyte and a beneficial colonizer of rhizospheres. Thus, M. robertsii can be used as a representative to simultaneously study several major lifestyles that are not shared by the “model” fungi Saccharomyces cerevisiae and Neurospora crassa; a systematic genetic analysis of M. robertsii will benefit studies in other fungi. In order to systematically disrupt genes in M. robertsii, we developed a high-throughput gene disruption methodology, which includes two technologies. One is the modified OSCAR-based, high-throughput construction of gene disruption plasmids. This technology involves two donor plasmids (pA-Bar-OSCAR with the herbicide resistance genes Bar and pA-Sur-OSCAR with another herbicide resistance gene Sur) and a recipient binary plasmid pPK2-OSCAR-GFP that was produced by replacing the Bar cassette in pPK2-bar-GFP with a ccdB cassette and recombination recognition sites. Using this technology, a gene disruption plasmid can be constructed in one cloning step in two days. The other is a highly efficient gene disruption technology based on homologous recombination using a Ku70 deletion mutant (ΔMrKu70) as the recipient strain. The deletion of MrKu70, a gene encoding a key component involved in nonhomologous end-joining DNA repair in fungi, dramatically increases the gene disruption efficiency. The frequency of disrupting the conidiation-associated gene Cag8 in ΔMrKu70 was 93% compared to 7% in the wild-type strain. Since ΔMrKu70 is not different from the wild-type strain in development, pathogenicity and tolerance to various abiotic stresses, it can be used as a recipient strain for a systematic gene disruption project to characterize the whole suite of genes involved in the biological processes of M. robertsii.     

Identification of the mcpA and mcpM Genes,Encoding Methyl-Accepting Proteins Involved in Amino Acid and l-Malate Chemotaxis,and Involvement of McpM-Mediated Chemotaxis in Plant Infection by Ralstonia pseudosolanacearum (Formerly Ralstonia solanacearum Phylotypes I and III)

Sequence analysis has revealed the presence of 22 putative methyl-accepting chemotaxis protein (mcp) genes in the Ralstonia pseudosolanacearum GMI1000 genome. PCR analysis and DNA sequencing showed that the highly motile R. pseudosolanacearum strain Ps29 possesses homologs of all 22 R. pseudosolanacearum GMI1000 mcp genes. We constructed a complete collection of single mcp gene deletion mutants of R. pseudosolanacearum Ps29 by unmarked gene deletion. Screening of the mutant collection revealed that R. pseudosolanacearum Ps29 mutants of RSp0507 and RSc0606 homologs were defective in chemotaxis to l-malate and amino acids, respectively. RSp0507 and RSc0606 homologs were designated mcpM and mcpA. While wild-type R. pseudosolanacearum strain Ps29 displayed attraction to 16 amino acids, the mcpA mutant showed no response to 12 of these amino acids and decreased responses to 4 amino acids. We constructed mcpA and mcpM deletion mutants of highly virulent R. pseudosolanacearum strain MAFF106611 to investigate the contribution of chemotaxis to l-malate and amino acids to tomato plant infection. Neither single mutant exhibited altered virulence for tomato plants when tested by root dip inoculation assays. In contrast, the mcpM mutant (but not the mcpA mutant) was significantly less infectious than the wild type when tested by a sand soak inoculation assay, which requires bacteria to locate and invade host roots from sand. Thus, McpM-mediated chemotaxis, possibly reflecting chemotaxis to l-malate, facilitates R. pseudosolanacearum motility to tomato roots in sand.     

Hfq Is a Global Regulator That Controls the Pathogenicity of Staphylococcus aureus

The Hfq protein is reported to be an RNA chaperone, which is involved in the stress response and the virulence of several pathogens. In E. coli, Hfq can mediate the interaction between some sRNAs and their target mRNAs. But it is controversial whether Hfq plays an important role in S. aureus. In this study, we found that the deletion of hfq gene in S. aureus 8325-4 can increase the surface carotenoid pigments. The hfq mutant was more resistant to oxidative stress but the pathogenicity of the mutant was reduced. We reveal that the Hfq protein can be detected only in some S. aureus strains. Using microarray and qRT-PCR, we identified 116 genes in the hfq mutant which had differential expression from the wild type, most of which are related to the phenotype and virulence of S. aureus. Among the 116 genes, 49 mRNAs can specifically bind Hfq protein, which indicates that Hfq also acts as an RNA binding protein in S. aureus. Our data suggest that Hfq protein of S. aureus is a multifunctional regulator involved in stress and virulence.     

Systematic Phenotyping of a Large-Scale Candida glabrata Deletion Collection Reveals Novel Antifungal Tolerance Genes

The opportunistic fungal pathogen Candida glabrata is a frequent cause of candidiasis, causing infections ranging from superficial to life-threatening disseminated disease. The inherent tolerance of C. glabrata to azole drugs makes this pathogen a serious clinical threat. To identify novel genes implicated in antifungal drug tolerance, we have constructed a large-scale C. glabrata deletion library consisting of 619 unique, individually bar-coded mutant strains, each lacking one specific gene, all together representing almost 12% of the genome. Functional analysis of this library in a series of phenotypic and fitness assays identified numerous genes required for growth of C. glabrata under normal or specific stress conditions, as well as a number of novel genes involved in tolerance to clinically important antifungal drugs such as azoles and echinocandins. We identified 38 deletion strains displaying strongly increased susceptibility to caspofungin, 28 of which encoding proteins that have not previously been linked to echinocandin tolerance. Our results demonstrate the potential of the C. glabrata mutant collection as a valuable resource in functional genomics studies of this important fungal pathogen of humans, and to facilitate the identification of putative novel antifungal drug target and virulence genes.     

Host-to-Pathogen Gene Transfer Facilitated Infection of Insects by a Pathogenic Fungus

Metarhizium robertsii is a plant root colonizing fungus that is also an insect pathogen. Its entomopathogenicity is a characteristic that was acquired during evolution from a plant endophyte ancestor. This transition provides a novel perspective on how new functional mechanisms important for host switching and virulence have evolved. From a random T-DNA insertion library, we obtained a pathogenicity defective mutant that resulted from the disruption of a sterol carrier gene (Mr-npc2a). Phylogenetic analysis revealed that Metarhizium acquired Mr-npc2a from an insect by horizontal gene transfer (HGT). Mr-NPC2a binds to cholesterol, an animal sterol, rather than the fungal sterol ergosterol, indicating it retains the specificity of insect NPC2 proteins. Mr-NPC2a is an intracellular protein and is exclusively expressed in the hemolymph of living insects. The disruption of Mr-npc2a reduced the amount of sterol in cell membranes of the yeast-like hyphal bodies that facilitate dispersal in the host body. These were consequently more susceptible to insect immune responses than the wild type. Transgenic expression of Mr-NPC2a increased the virulence of Beauveria bassiana, an endophytic insect-pathogenic fungus that lacks a Mr-NPC2a homolog.     

Proteome and phosphoproteome analysis of the serine/threonine protein kinase E mutant of Mycobacterium tuberculosis

Aims

Serine/threonine protein kinases (STPKs) have prominent roles in the survival mechanisms of Mycobacterium tuberculosis (M. tuberculosis). Previous studies from our laboratory underscored the role of PknE, an STPK in virulence, adaptation and the suppression of host cell apoptosis. In this study, two-dimensional gel electrophoresis was used to study the proteome and phosphoproteome profiles of wild type M. tuberculosis and its isogenic pknE deletion mutant (ΔpknE) during growth in Middlebrook 7H9 and nitric oxide stress.

Main methods

Wild-type M. tuberculosis and its isogenic pknE deletion mutant strain were grown in Middlebrook 7H9 as well as subjected to nitric oxide stress using sodium nitroprusside. Whole cell lysates were prepared and analyzed by 2D-gel electrophoresis. Phosphoproteomes were analyzed using phospho serine and phospho threonine antibodies after subjecting the 2D-gels to western blotting. Proteins of interest were identified using mass spectrometry.

Key findings

Our analysis provides insights into the targets that impose pro-apoptotic as well as altered cellular phenotypes on ΔpknE, revealing novel substrates and functions for PknE.

Significance

For the first time, our proteome and phosphoproteome data decipher the function of PknE in cell division, virulence, dormancy, suppression of sigma factor B and its regulated genes, suppression of two-component systems and in the metabolic activity of M. tuberculosis.