The Candida albicans Sur7 protein is needed for proper synthesis of the fibrillar component of the cell wall that confers strength

The Candida albicans plasma membrane plays important roles in interfacing with the environment, morphogenesis, and cell wall synthesis. The role of the Sur7 protein in cell wall structure and function was analyzed, since previous studies showed that this plasma membrane protein is needed to prevent abnormal intracellular growth of the cell wall. Sur7 localizes to stable patches in the plasma membrane, known as MCC (membrane compartment occupied by Can1), that are associated with eisosome proteins. The sur7Δ mutant cells displayed increased sensitivity to factors that exacerbate cell wall defects, such as detergent (SDS) and the chitin-binding agents calcofluor white and Congo red. The sur7Δ cells were also slightly more sensitive to inhibitors that block the synthesis of cell wall chitin (nikkomycin Z) and β-1,3-glucan (caspofungin). In contrast, Fmp45, a paralog of Sur7 that also localizes to punctate plasma membrane patches, did not have a detectable role in cell wall synthesis. Chemical analysis of cell wall composition demonstrated that sur7Δ cells contain decreased levels of β-glucan, a glucose polymer that confers rigidity on the cell wall. Consistent with this, sur7Δ cells were more sensitive to lysis, which could be partially rescued by increasing the osmolarity of the medium. Interestingly, Sur7 is present in static patches, whereas β-1,3-glucan synthase is mobile in the plasma membrane and is often associated with actin patches. Thus, Sur7 may influence β-glucan synthesis indirectly, perhaps by altering the functions of the cell signaling components that localize to the MCC and eisosome domains.     

Members of protein O‐mannosyltransferase family in Aspergillus fumigatus differentially affect growth,morphogenesis and viability

O‐mannosylation is an essential protein modification in eukaryotes. It is initiated at the endoplasmic reticulum by O‐mannosyltransferases (PMT) that are evolutionary conserved from yeast to humans. The PMT family is phylogenetically classified into PMT1, PMT2 and PMT4 subfamilies, which differ in protein substrate specificity and number of genes per subfamily. In this study, we characterized for the first time the whole PMT family of a pathogenic filamentous fungus, Aspergillus fumigatus. Genome analysis showed that only one member of each subfamily is present in A. fumigatus, PMT1, PMT2 and PMT4. Despite the fact that all PMTs are transmembrane proteins with conserved peptide motifs, the phenotype of each PMT deletion mutant was very different in A. fumigatus. If disruption of PMT1 did not reveal any phenotype, deletion of PMT2 was lethal. Disruption of PMT4 resulted in abnormal mycelial growth and highly reduced conidiation associated to significant proteomic changes. The double pmt1pmt4 mutant was lethal. The single pmt4 mutant exhibited an exquisite sensitivity to echinocandins that is associated to major changes in the expression of signal transduction cascade genes. These results indicate that the PMT family members play a major role in growth, morphogenesis and viability of A. fumigatus.     

Aspergillus fumigatus devoid of cell wall β‐1,3‐glucan is viable,massively sheds galactomannan and is killed by septum formation inhibitors

Echinocandins inhibit β‐1,3‐glucan synthesis and are one of the few antimycotic drug classes effective against Aspergillus spp. In this study, we characterized the β‐1,3‐glucan synthase Fks1 of Aspergillus fumigatus, the putative target of echinocandins. Data obtained with a conditional mutant suggest that fks1 is not essential. In agreement, we successfully constructed a viable Δfks1 deletion mutant. Lack of Fks1 results in characteristic growth phenotypes similar to wild type treated with echinocandins and an increased susceptibility to calcofluor white and sodium dodecyl sulfate. In agreement with Fks1 being the only β‐1,3‐glucan synthase in A. fumigatus, the cell wall is devoid of β‐1,3‐glucan. This is accompanied by a compensatory increase of chitin and galactosaminogalactan and a significant decrease in cell wall galactomannan due to a massively enhanced galactomannan shedding. Our data furthermore suggest that inhibition of hyphal septation can overcome the limitations of echinocandin therapy. Compounds inhibiting septum formation boosted the antifungal activity of caspofungin. Thus, development of clinically applicable inhibitors of septum formation is a promising strategy to improve existing antifungal therapy.     

Functional analysis of the fungal/plant class chitinase family in Aspergillus fumigatus

A quintuple mutant was constructed to delete the entire family of the fungal/plant (class III) chitinases of Aspergillus fumigatus. Only a limited reduction in the total chitinolytic activity was seen for the different chitinase mutants including the quintuple mutant. In spite of this reduction in chitinolytic activity, no growth or germination defects were observed in these chitinase mutants. This result demonstrated that the fungal/plant chitinases do not have an essential role in the morphogenesis of A. fumigatus. A slight diminution of the growth during autolysis was seen for the quintuple mutant suggesting that class III chitinases may play only a nutritional role during this phase of the cycle, retarding fungal death.     

Deciphering the role of the chitin synthase families 1 and 2 in the in vivo and in vitro growth of Aspergillus fumigatus by multiple gene targeting deletion

Although chitin is an essential component of the fungal cell wall (CW), its biosynthesis and role in virulence is poorly understood. In Aspergillus fumigatus, there are eight chitin synthase (CHS) genes belonging to two families CHSA‐C, CHSG in family 1 and CHSF, CHSD, CSMA, CSMB in family 2). To understand the function of these CHS genes, their single and multiple deletions were performed using β‐rec/six system to be able to delete all genes within each family (up to a quadruple ΔchsA/C/B/G mutant in family 1 and a quadruple ΔcsmA/csmB/F/D mutant in family 2). Radial growth, conidiation, mycelial/conidial morphology, CW polysaccharide content, Chs‐activity, susceptibility to antifungal molecules and pathogenicity in experimental animal aspergillosis were analysed for all the mutants. Among the family 1 CHS, ΔchsA, ΔchsB and ΔchsC mutants showed limited impact on chitin synthesis. In contrast, there was reduced conidiation, altered mycelial morphotype and reduced growth and Chs‐activity in the ΔchsG and ΔchsA/C/B/G mutants. In spite of this altered phenotype, these two mutants were as virulent as the parental strain in the experimental aspergillosis models. Among family 2 CHS, phenotypic defects mainly resulted from the CSMA deletion. Despite significant morphological mycelial and conidial growth phenotypes in the quadruple ΔcsmA/csmB/F/D mutant, the chitin content was poorly affected by gene deletions in this family. However, the entire mycelial cell wall structure was disorganized in the family 2 mutants that may be related to the reduced pathogenicity of the quadruple ΔcsmA/csmB/F/D mutant strain compared to the parental strain, in vivo. Deletion of the genes encompassing the two families (ΔcsmA/csmB/F/G) showed that in spite of being originated from an ancient divergence of fungi, these two families work cooperatively to synthesize chitin in A. fumigatus and demonstrate the essentiality of chitin biosynthesis for vegetative growth, resistance to antifungal drugs, and virulence of this filamentous fungus.     

The N-terminal Domain of Drosophila Gram-negative Binding Protein 3 (GNBP3) Defines a Novel Family of Fungal Pattern Recognition Receptors

Gram-negative binding protein 3 (GNBP3), a pattern recognition receptor that circulates in the hemolymph of Drosophila, is responsible for sensing fungal infection and triggering Toll pathway activation. Here, we report that GNBP3 N-terminal domain binds to fungi upon identifying long chains of β-1,3-glucans in the fungal cell wall as a major ligand. Interestingly, this domain fails to interact strongly with short oligosaccharides. The crystal structure of GNBP3-Nter reveals an immunoglobulin-like fold in which the glucan binding site is masked by a loop that is highly conserved among glucan-binding proteins identified in several insect orders. Structure-based mutagenesis experiments reveal an essential role for this occluding loop in discriminating between short and long polysaccharides. The displacement of the occluding loop is necessary for binding and could explain the specificity of the interaction with long chain structured polysaccharides. This represents a novel mechanism for β-glucan recognition.The activation of the immune response is energetically costly and may be detrimental to the host, especially when inappropriately triggered. Therefore, the reliable detection of infections is a step of paramount importance in the immune response. To achieve the task of detecting potentially hazardous microorganisms, the innate immune system relies on several strategies. One of them is to sense both pathogenic and nonpathogenic microorganisms thanks to pattern recognition receptors (PRRs)⁴ that recognize intrinsic microbial molecular “signatures” (1). These immune receptors have been selected during evolution for their ability to bind to essential, conserved, structural components of the microorganisms such as flagellins, peptidoglycans of bacteria, lipopolysaccharides of Gram-negative bacteria, lipoteichoic acids of Gram-positive bacteria, and β-glucans of the fungal cell wall (2, 3). Examples of mammalian PRRs include Toll-like receptors (4), intracellular receptors of the NOD family (5), peptidoglycan recognition proteins (PGRPs) (6), and the membrane-bound Dectin-1 receptor, which detects fungal β-glucans (7).One important arm of the innate immunity in Drosophila is a potent systemic response that relies on the synthesis in the fat body (a functional equivalent of the mammalian liver) of potent antimicrobial peptides (AMPs) that are secreted in the hemolymph where they attack invading microorganisms. Genetic analysis has delineated two major regulatory pathways of NF-κB type that control the expression of AMP genes (8). The immune deficiency (imd) pathway is mostly required in the host defense against Gram-negative bacteria (9) and is triggered by PRRs of the PGRP family, namely PGRP-LC (10) and PGRP-LE (11). The Toll pathway is essential for fighting fungal and some Gram-positive bacterial infections (12, 13). Toll, the funding member of the Toll-like receptor family, is not itself a PRR. Rather, it is activated by a ligand of the nerve growth factor family, the Spätzle cytokine. To bind to the Toll receptor, Pro-Spätzle needs to be proteolytically processed by a protease, the Spätzle-processing enzyme (SPE) (14), which is itself activated by upstream proteolytic cascades. One such cascade is activated in response to a Gram-positive bacterial challenge by a complex of PGRP-SA, PGRP-SD, and Gram-negative binding protein 1 (GNBP1) (13, 15, 16). Flies deficient for either PGRP-SA or GNBP1 are deficient in Toll pathway activation and are susceptible to infections by several Gram-positive bacterial species but not to fungal infections. In contrast, flies mutant for GNBP3, another gene encoding a GNBP family member, fail to activate the Toll pathway in response to killed fungi and succumb rapidly to fungal but not bacterial infections (17). GNBP3 is thought to activate a proteolytic cascade, which partially overlaps that triggered by the GNBP1·PGRP-SA complex (18). Even though they belong to the same family and activate the same pathway, GNBP1 and GNBP3 are required for sensing distinct classes of microorganisms.The founding member of the GNBP family, a 50-kDa protein found in hemolymph of Bombyx mori and originally named p50, was characterized as a gram-negative (Escherichia coli) binding protein (19); hence, its name. However, it has become clear that GNBPs belong to the family of β-glucan recognition proteins (βGRP) that had first been purified on their ability to trigger the prophenol oxidase cascade (a wound response that leads to melanization at the injury site) in response to fungal infections (20). Members of the GNBP/βGRP family are extracellular proteins composed of a small N-terminal domain of about 100 residues and a longer C-terminal domain of about 350 residues (21, 22). In the insect Plodia interpunctella, both domains of βGRP bind to laminarin, a soluble β-1,3-glucan with a high affinity (K_A in the 10⁸ m⁻¹ range) (23) which is in the same range as that of the Factor G of the Japanese horseshoe crab (24). The latter factor is used as a diagnostic reagent for the detection of glucans. The C-terminal domain displays sequence similarity to bacterial glucanases, yet the catalytic residues have not been conserved, suggesting that this domain has been selected during evolution for its ability to bind to glucans (21, 22). The N-terminal domain defines a novel β-1,3-glucan binding domain that binds to curdlan, an insoluble linear β-1,3-glucan polymer, a property that the C-terminal glucanase-like domain lacks (21). Full-length recombinant GNBP/βGRPs have been reported to bind to bacteria, lipopolysaccharides, or lipoteichoic acids (19, 22, 23, 25). Although the domain(s) that mediates these interactions has not been thoroughly mapped, it appears that the N-terminal P. interpunctella β-1,3-glucan domain is not required for binding to these bacterial compounds (23).Numerous three-dimensional structures of PGRPs, in some cases complexed with their ligands, have been reported (26–29). In contrast, this knowledge is currently lacking as regarding GNBPs. As a first step toward elucidating the structure/function relationships of GNBPs, we report here that a recombinant polypeptide encoding the N-terminal domain of GNBP3 binds to fungi and to long β-1,3-glucan chains but not to short laminarioligosaccharides. The determination of the crystal structure of GNBP3 N-terminal domain reveals an immunoglobulin fold in which the β-glucan binding site is masked by a lid, which is likely to be displaced by long polysaccharide chains.     

MybA,a transcription factor involved in conidiation and conidial viability of the human pathogen Aspergillus fumigatus

Aspergillus fumigatus, a ubiquitous human fungal pathogen, produces asexual spores (conidia), which are the main mode of propagation, survival and infection of this human pathogen. In this study, we present the molecular characterization of a novel regulator of conidiogenesis and conidial survival called MybA because the predicted protein contains a Myb DNA binding motif. Cellular localization of the MybA::Gfp fusion and immunoprecipitation of the MybA::Gfp or MybA::3xHa protein showed that MybA is localized to the nucleus. RNA sequencing data and a uidA reporter assay indicated that the MybA protein functions upstream of wetA, vosA and velB, the key regulators involved in conidial maturation. The deletion of mybA resulted in a very significant reduction in the number and viability of conidia. As a consequence, the ΔmybA strain has a reduced virulence in an experimental murine model of aspergillosis. RNA‐sequencing and biochemical studies of the ΔmybA strain suggested that MybA protein controls the expression of enzymes involved in trehalose biosynthesis as well as other cell wall and membrane‐associated proteins and ROS scavenging enzymes. In summary, MybA protein is a new key regulator of conidiogenesis and conidial maturation and survival, and plays a crucial role in propagation and virulence of A. fumigatus.