Parallel beta-helix proteins required for accurate capsule polysaccharide synthesis and virulence in the yeast Cryptococcus neoformans

The principal capsular polysaccharide of the opportunistic fungal pathogen Cryptococcus neoformans consists of an alpha-1,3-linked mannose backbone decorated with a repeating pattern of glucuronyl and xylosyl side groups. This structure is critical for virulence, yet little is known about how the polymer, called glucuronoxylomannan (GXM), is faithfully synthesized and assembled. We have generated deletions in two genes encoding predicted parallel beta-helix repeat proteins, which we have designated PBX1 and PBX2. Deletion of either gene results in a dry-colony morphology, clumpy cells, and decreased capsule integrity. Two-dimensional nuclear magnetic resonance spectroscopy of purified GXM from the mutants indicated that both the wild-type GXM structure and novel, aberrant linkages were present. Carbohydrate composition and linkage analysis determined that these aberrant structures are correlated with the incorporation of terminal glucose residues that are not found in wild-type capsule polysaccharide. We conclude that Pbx1 and Pbx2 are required for the fidelity of GXM synthesis and may be involved in editing incorrectly added glucose residues. PBX1 and PBX2 knockout mutants showed severely attenuated virulence in a murine inhalation model of cryptococcosis. Unlike acapsular strains, these mutant strains induced delayed symptoms of cryptococcosis, though the infected animals eventually contained the infection and recovered.     

Comparative Secretome Analysis of Magnaporthe oryzae Identified Proteins Involved in Virulence and Cell Wall Integrity

Plant fungal pathogens secrete numerous proteins into the apoplast at the plant–fungus contact sites to facilitate colonization. However, only a few secretory proteins were functionally characterized in Magnaporthe oryzae, the fungal pathogen causing rice blast disease worldwide. Asparagine-linked glycosylation 3 (Alg3) is an α-1,3-mannosyltransferase functioning in the N-glycan synthesis of N-glycosylated secretory proteins. Fungal pathogenicity and cell wall integrity are impaired in Δalg3 mutants, but the secreted proteins affected in Δalg3 mutants are largely unknown. In this study, we compared the secretomes of the wild-type strain and the Δalg3 mutant and identified 51 proteins that require Alg3 for proper secretion. These proteins were predicted to be involved in metabolic processes, interspecies interactions, cell wall organization, and response to chemicals. Nine proteins were selected for further validation. We found that these proteins were localized at the apoplastic region surrounding the fungal infection hyphae. Moreover, the N-glycosylation of these proteins was significantly changed in the Δalg3 mutant, leading to the decreased protein secretion and abnormal protein localization. Furthermore, we tested the biological functions of two genes, INV1 (encoding invertase 1, a secreted invertase) and AMCase (encoding acid mammalian chinitase, a secreted chitinase). The fungal virulence was significantly reduced, and the cell wall integrity was altered in the Δinv1 and Δamcase mutant strains. Moreover, the N-glycosylation was essential for the function and secretion of AMCase. Taken together, our study provides new insight into the role of N-glycosylated secretory proteins in fungal virulence and cell wall integrity.     

Adenylyl cyclase functions downstream of the Galpha protein Gpa1 and controls mating and pathogenicity of Cryptococcus neoformans

The signaling molecule cyclic AMP (cAMP) is a ubiquitous second messenger that enables cells to detect and respond to extracellular signals. cAMP is generated by the enzyme adenylyl cyclase, which is activated or inhibited by the Gα subunits of heterotrimeric G proteins in response to ligand-activated G-protein-coupled receptors. Here we identified the unique gene (CAC1) encoding adenylyl cyclase in the opportunistic fungal pathogen Cryptococcus neoformans. The CAC1 gene was disrupted by transformation and homologous recombination. In stark contrast to the situation for Saccharomyces cerevisiae, in which adenylyl cyclase is essential, C. neoformans cac1 mutant strains were viable and had no vegetative growth defect. Furthermore, cac1 mutants maintained the yeast-like morphology of wild-type cells, in contrast to the constitutively filamentous phenotype found upon the loss of adenylyl cyclase in another basidiomycete pathogen, Ustilago maydis. Like C. neoformans mutants lacking the Gα protein Gpa1, cac1 mutants were mating defective and failed to produce two inducible virulence factors: capsule and melanin. As a consequence, cac1 mutant strains were avirulent in animal models of cryptococcal meningitis. Reintroduction of the wild-type CAC1 gene or the addition of exogenous cAMP suppressed cac1 mutant phenotypes. Moreover, the overexpression of adenylyl cyclase restored mating and virulence factor production in gpa1 mutant strains. Physiological studies revealed that the Gα protein Gpa1 and adenylyl cyclase controlled cAMP production in response to glucose, and no cAMP was detectable in extracts from cac1 or gpa1 mutant strains. These findings provide direct evidence that Gpa1 and adenylyl cyclase function in a conserved signal transduction pathway controlling cAMP production, hyphal differentiation, and virulence of this human fungal pathogen.     

Transposon-Induced Mutations in Two Loci of Listeria monocytogenes Serotype 1/2a Result in Phage Resistance and Lack of N-Acetylglucosamine in the Teichoic Acid of the Cell Wall

Teichoic acid-associated N-acetylglucosamine and rhamnose have been shown to serve as phage receptors in Listeria monocytogenes serotype 1/2a. We generated and characterized two single-copy Tn916ΔE mutants which were resistant to phage A118 and several other serotype 1/2a-specific phages. In one mutant the insertion was immediately upstream of the recently identified ptsHI locus, which encodes two proteins of the phosphoenolpyruvate-dependent carbohydrate uptake system, whereas in the other the insertion was immediately upstream of an operon whose most distal gene was clpC, involved in stress responses and virulence. Transduction experiments confirmed the association of the phage-resistant phenotype of these mutants with the transposon insertion. Phage A118 resistance of the mutants could be attributed to inability of the phage to adsorb onto the mutant cells, and biochemical analysis of cell wall composition showed that the teichoic acids of both mutants were deficient in N-acetylglucosamine. Rhamnose and other teichoic acid and cell wall components were not affected.     

Characterization of the PMT Gene Family in Cryptococcus neoformans

Background

Protein-O-mannosyltransferases (Pmt''s) catalyze the initial step of protein-O-glycosylation, the addition of mannose residues to serine or threonine residues of target proteins.

Methodology/Principal Findings

Based on protein similarities, this highly conserved protein family can be divided into three subfamilies: the Pmt1 sub-family, the Pmt2 sub-family and the Pmt4 sub-family. In contrast to Saccharomyces cerevisiae and Candida albicans, but similar to filamentous fungi, three putative PMT genes (PMT1, PMT2, and PMT4) were identified in the genome of the human fungal pathogen Cryptococcus neoformans. Similar to Schizosaccharomyces pombe and C. albicans, C. neoformans PMT2 is an essential gene. In contrast, the pmt1 and pmt4 single mutants are viable; however, the pmt1/pmt4 deletions are synthetically lethal. Mutation of PMT1 and PMT4 resulted in distinct defects in cell morphology and cell integrity. The pmt1 mutant was more susceptible to SDS medium than wild-type strains and the mutant cells were enlarged. The pmt4 mutant grew poorly on high salt medium and demonstrated abnormal septum formation and defects in cell separation. Interestingly, the pmt1 and pmt4 mutants demonstrated variety-specific differences in the levels of susceptibility to osmotic and cell wall stress. Delayed melanin production in the pmt4 mutant was the only alteration of classical virulence-associated phenotypes. However, the pmt1 and pmt4 mutants showed attenuated virulence in a murine inhalation model of cryptococcosis.

Conclusion/Significance

These findings suggest that C. neoformans protein-O-mannosyltransferases play a crucial role in maintaining cell morphology, and that reduced protein-O-glycosylation leads to alterations in stress resistance, cell wall composition, cell integrity, and survival within the host.     

Abnormalities of caudal pharyngeal pouch development in Pbx1 knockout mice mimic loss of Hox3 paralogs

Pbx1 is a TALE-class homeodomain protein that functions in part as a cofactor for Hox class homeodomain proteins. Previous analysis of the in vivo functions of Pbx1 by targeted mutagenesis in mice has revealed roles for this gene in skeletal patterning and development and in the organogenesis of multiple systems. Both RNA expression and protein localization studies have suggested a possible role for Pbx1 in pharyngeal region development. As several Hox mutants have distinct phenotypes in this region, we investigated the potential requirement for Pbx1 in the development of the pharyngeal arches and pouches and their organ derivatives. Pbx1 homozygous mutants exhibited delayed or absent formation of the caudal pharyngeal pouches, and disorganized patterning of the third pharyngeal pouch. Formation of the third pouch-derived thymus/parathyroid primordia was also affected, with absent or hypoplastic primordia, delayed expression of organ-specific differentiation markers, and reduced proliferation of thymic epithelium. The fourth pouch and the fourth pouch-derived ultimobranchial bodies were usually absent. These phenotypes are similar to those previously reported in Hoxa3(-/-) single mutants and Hoxa1(-/-);Hoxb1(-/-) or Hoxa3(+/-);Hoxb3(-/-);Hoxd3(-/-) compound mutants, suggesting that Pbx1 acts together with multiple Hox proteins in the development of the caudal pharyngeal region. However, some aspects of the Pbx1 mutant phenotype included specific defects that were less severe than those found in known Hox mutant mice, suggesting that some functions of Hox proteins in this region are Pbx1-independent.     

A prl Mutation in SecY Suppresses Secretion and Virulence Defects of Listeria monocytogenes secA2 Mutants

The bulk of bacterial protein secretion occurs through the conserved SecY translocation channel that is powered by SecA-dependent ATP hydrolysis. Many Gram-positive bacteria, including the human pathogen Listeria monocytogenes, possess an additional nonessential specialized ATPase, SecA2. SecA2-dependent secretion is required for normal cell morphology and virulence in L. monocytogenes; however, the mechanism of export via this pathway is poorly understood. L. monocytogenes secA2 mutants form rough colonies, have septation defects, are impaired for swarming motility, and form small plaques in tissue culture cells. In this study, 70 spontaneous mutants were isolated that restored swarming motility to L. monocytogenes secA2 mutants. Most of the mutants had smooth colony morphology and septated normally, but all were lysozyme sensitive. Five representative mutants were subjected to whole-genome sequencing. Four of the five had mutations in proteins encoded by the lmo2769 operon that conferred lysozyme sensitivity and increased swarming but did not rescue virulence defects. A point mutation in secY was identified that conferred smooth colony morphology to secA2 mutants, restored wild-type plaque formation, and increased virulence in mice. This secY mutation resembled a prl suppressor known to expand the repertoire of proteins secreted through the SecY translocation complex. Accordingly, the ΔsecA2prlA1 mutant showed wild-type secretion levels of P60, an established SecA2-dependent secreted autolysin. Although the prl mutation largely suppressed almost all of the measurable SecA2-dependent traits, the ΔsecA2prlA1 mutant was still less virulent in vivo than the wild-type strain, suggesting that SecA2 function was still required for pathogenesis.     

A Novel Protein,RafX, Is Important for Common Cell Wall Polysaccharide Biosynthesis in Streptococcus pneumoniae: Implications for Bacterial Virulence

Teichoic acid (TA), together with peptidoglycan (PG), represents a highly complex glycopolymer that ensures cell wall integrity and has several crucial physiological activities. Through an insertion-deletion mutation strategy, we show that ΔrafX mutants are impaired in cell wall covalently attached TA (WTA)-PG biosynthesis, as evidenced by their abnormal banding patterns and reduced amounts of WTA in comparison with wild-type strains. Site-directed mutagenesis revealed an essential role for external loop 4 and some highly conserved amino acid residues in the function of RafX protein. The rafX gene was highly conserved in closely related streptococcal species, suggesting an important physiological function in the lifestyle of streptococci. Moreover, a strain D39 ΔrafX mutant was impaired in bacterial growth, autolysis, bacterial division, and morphology. We observed that a strain R6 ΔrafX mutant was reduced in adhesion relative to the wild-type R6 strain, which was supported by an inhibition assay and a reduced amount of CbpA protein on the ΔrafX mutant bacterial cell surface, as shown by flow cytometric analysis. Finally, ΔrafX mutants were significantly attenuated in virulence in a murine sepsis model. Together, these findings suggest that RafX contributes to the biosynthesis of WTA, which is essential for full pneumococcal virulence.     

Systematic Mutagenesis of Genes Encoding Predicted Autotransported Proteins of Burkholderia pseudomallei Identifies Factors Mediating Virulence in Mice,Net Intracellular Replication and a Novel Protein Conferring Serum Resistance

Burkholderia pseudomallei is the causative agent of the severe tropical disease melioidosis, which commonly presents as sepsis. The B. pseudomallei K96243 genome encodes eleven predicted autotransporters, a diverse family of secreted and outer membrane proteins often associated with virulence. In a systematic study of these autotransporters, we constructed insertion mutants in each gene predicted to encode an autotransporter and assessed them for three pathogenesis-associated phenotypes: virulence in the BALB/c intra-peritoneal mouse melioidosis model, net intracellular replication in J774.2 murine macrophage-like cells and survival in 45% (v/v) normal human serum. From the complete repertoire of eleven autotransporter mutants, we identified eight mutants which exhibited an increase in median lethal dose of 1 to 2-log₁₀ compared to the isogenic parent strain (bcaA, boaA, boaB, bpaA, bpaC, bpaE, bpaF and bimA). Four mutants, all demonstrating attenuation for virulence, exhibited reduced net intracellular replication in J774.2 macrophage-like cells (bimA, boaB, bpaC and bpaE). A single mutant (bpaC) was identified that exhibited significantly reduced serum survival compared to wild-type. The bpaC mutant, which demonstrated attenuation for virulence and net intracellular replication, was sensitive to complement-mediated killing via the classical and/or lectin pathway. Serum resistance was rescued by in trans complementation. Subsequently, we expressed recombinant proteins of the passenger domain of four predicted autotransporters representing each of the phenotypic groups identified: those attenuated for virulence (BcaA), those attenuated for virulence and net intracellular replication (BpaE), the BpaC mutant with defects in virulence, net intracellular replication and serum resistance and those displaying wild-type phenotypes (BatA). Only BcaA and BpaE elicited a strong IFN-γ response in a restimulation assay using whole blood from seropositive donors and were recognised by seropositive human sera from the endemic area. To conclude, several predicted autotransporters contribute to B. pseudomallei virulence and BpaC may do so by conferring resistance against complement-mediated killing.     

Two cyclophilin A homologs with shared and distinct functions important for growth and virulence of Cryptococcus neoformans

Cyclophilin A is the target of the immunosuppressant cyclosporin A (CsA) and is encoded by a single unique gene conserved from yeast to humans. In the pathogenic fungus Cryptococcus neoformans, two homologous linked genes, CPA1 and CPA2, were found to encode two conserved cyclophilin A proteins. In contrast to Saccharomyces cerevisiae, in which cyclophilin A mutations confer CsA resistance but few other phenotypes, cyclophilin A mutations conferred dramatic phenotypes in C. neoformans. The Cpa1 and Cpa2 cyclophilin A proteins play a shared role in cell growth, mating, virulence and CsA toxicity. The Cpa1 and Cpa2 proteins also have divergent functions. cpa1 mutants are inviable at 39°C and attenuated for virulence, whereas cpa2 mutants are viable at 39°C and fully virulent. cpa1 cpa2 double mutants exhibited synthetic defects in growth and virulence. Cyclophilin A active site mutants restored growth of cpa1 cpa2 mutants at ambient but not at higher temperatures, suggesting that the prolyl isomerase activity of cyclophilin A has an in vivo function.     

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 analyses of regulators of G protein signaling in Gibberella zeae

Regulators of G protein signaling (RGS) proteins make up a highly diverse and multifunctional protein family that plays a critical role in controlling heterotrimeric G protein signaling. In this study, seven RGS genes (FgFlbA, FgFlbB, FgRgsA, FgRgsB, FgRgsB2, FgRgsC, and FgGprK) were functionally characterized in the plant pathogenic fungus, Gibberella zeae. Mutant phenotypes were observed for deletion mutants of FgRgsA and FgRgsB in vegetative growth, FgFlbB and FgRgsB in conidia morphology, FgFlbA in conidia production, FgFlbA, FgRgsB, and FgRgsC in sexual development, FgFlbA and FgRgsA in spore germination and mycotoxin production, and FgFlbA, FgRgsA, and FgRgsB in virulence. Furthermore, FgFlbA, FgRgsA, and FgRgsB acted pleiotropically, while FgFlbB and FgRgsC deletion mutants exhibited a specific defect in conidia morphology and sexual development, respectively. Amino acid substitutions in Gα subunits and overexpression of the FgFlbA gene revealed that deletion of FgFlbA and dominant active GzGPA2 mutant, gzgpa2(Q207L), had similar phenotypes in cell wall integrity, perithecia formation, mycotoxin production, and virulence, suggesting that FgFlbA may regulate asexual/sexual development, mycotoxin biosynthesis, and virulence through GzGPA2-dependent signaling in G. zeae.     

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.     

Temperature-sensitive mutants of hsp82 of the budding yeast Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae has two HSP90-related genes per haploid genome, HSP82 and HSC82. Random mutations were induced in vitro in the HSP82 gene by treatment of the plasmid with hydroxylamine. Four temperature-sensitive (ts) mutants and one simultaneously is and cold-sensitivie (cs) mutant were then selected in a yeast strain in which HSC82 had previously been disrupted. The mutants were found to have single base changes in the coding region, which caused single amino acid substitutions in the HSP82 protein. All of these mutations occurred in amino acid residues that are well conserved among HSP90-related proteins of various species from Escherichia coli to human. Various properties including cell morphology, macromolecular syntheses and thermosensitivity were examined in each mutant at both the permissive and nonpermissive temperatures. The mutations in HSP82 caused pleiotropic effects on these properties although the phenotypes exhibited at the nonpermissive temperature varied among the mutants.     

KRE genes are required for β‐1,6‐glucan synthesis,maintenance of capsule architecture and cell wall protein anchoring in Cryptococcus neoformans

The polysaccharide β‐1,6‐glucan is a major component of the cell wall of Cryptococcus neoformans, but its function has not been investigated in this fungal pathogen. We have identified and characterized seven genes, belonging to the KRE family, which are putatively involved in β‐1,6‐glucan synthesis. The H99 deletion mutants kre5Δ and kre6Δskn1Δ contained less cell wall β‐1,6‐glucan, grew slowly with an aberrant morphology, were highly sensitive to environmental and chemical stress and were avirulent in a mouse inhalation model of infection. These two mutants displayed alterations in cell wall chitosan and the exopolysaccharide capsule, a primary cryptococcal virulence determinant. The cell wall content of the GPI‐anchored phospholipase B1 (Plb1) enzyme, which is required for cryptococcal cell wall integrity and virulence, was reduced in kre5Δ and kre6Δskn1Δ. Our results indicate that KRE5, KRE6 and SKN1 are involved in β‐1,6‐glucan synthesis, maintenance of cell wall integrity and retention of mannoproteins and known cryptococcal virulence factors in the cell wall of C. neoformans. This study sets the stage for future investigations into the function of this abundant cell wall polymer.     

Genetic analysis of Hsp70 phosphorylation sites reveals a role in Candida albicans cell and colony morphogenesis

Heat shock proteins are best known for their role as chaperonins involved in general proteostasis, but they can also participate in specific cellular regulatory pathways, e.g. via their post-translational modification. Hsp70/Ssa1 is a central cytoplasmic chaperonin in eukaryotes, which also participates in cell cycle regulation via its phosphorylation at a specific residue. Here we analyze the role of Ssa1 phosphorylation in the morphogenesis of the fungus Candida albicans, a common human opportunistic pathogen. C. albicans can assume alternative yeast and hyphal (mold) morphologies, an ability that contributes to its virulence. We identified 11 phosphorylation sites on C. albicans Ssa1, of which 8 were only detected in the hyphal cells. Genetic analysis of these sites revealed allele-specific effects on growth or hyphae formation at 42 °C. Colony morphology, which is normally wrinkled or crenellated at 37 °C, reverted to smooth in several mutants, but this colony morphology phenotype was unrelated to cellular morphology. Two mutants exhibited a mild increase in sensitivity to the cell wall-active compounds caspofungin and calcofluor white. We suggest that this analysis could help direct screens for Ssa1-specific drugs to combat C. albicans virulence. The pleiotropic effects of many Ssa1 mutations are consistent with the large number of Ssa1 client proteins, whereas the lack of concordance between the phenotypes of the different alleles suggests that different sites on Ssa1 can affect interaction with specific classes of client proteins, and that modification of these sites can play cellular regulatory roles, consistent with the “chaperone code” hypothesis.     

Two α(1-3) Glucan Synthases with Different Functions in Aspergillus fumigatus

α(1-3) glucan is a main component of the Aspergillus fumigatus cell wall. In spite of its importance, synthesis of this amorphous polymer has not been investigated to date. Two genes in A. fumigatus, AGS1 and AGS2, are highly homologous to the AGS genes of Schizosaccharomyces pombe, which encode putative α(1-3) glucan synthases. The predicted Ags proteins of A. fumigatus have an estimated molecular mass of 270 kDa. AGS1 and AGS2 were disrupted in A. fumigatus. Both Δags mutants have similar altered hyphal morphologies and reduced conidiation levels. Only Δags1 presented a reduction in the α(1-3) glucan content of the cell wall. These results showed that Ags1p and Ags2p were functionally different. The cellular localization of the two proteins was in agreement with their different functions: Ags1p was localized at the periphery of the cell in connection with the cell wall, whereas Ags2p was intracellularly located. An original experimental model of invasive aspergillosis based on mixed infection and quantitative PCR was developed to analyze the virulence of A. fumigatus mutant and wild-type strains. Using this model, it was shown that the cell wall and morphogenesis defects of Δags1 and Δags2 were not associated with a reduction in virulence in either mutant. This result showed that a 50% reduction in the content of the cell wall α(1-3) glucan does not play a significant role in A. fumigatus pathogenicity.     

Cell wall mutants of Saccharomyces cerevisiae with increased digestibility by cell wall lytic enzymes and protein extractability

Yeast cell wall mutants were obtained by mutagenesis of Saccharomyces cerevisiae X2180-1A, a haplid strain, with N-methyl-N′-nitro-N-nitrosoguanidine. The two S. cerevisiae mutants showed considerable morphological changes and digestibilities by lytic enzymes. Sequential extractions of proteins and polysaccharides from the mutant and wild type cells indicate that the mutants have high protein extractability and lack some wall proteins as well as some polysaccharide fractions.