Dataset Information

Kexin-like endoprotease KexB is required for N-glycan processing, morphogenesis and virulence in Aspergillus fumigatus.

ABSTRACT: Kexin-like proteins belong to the subtilisin-like family of the proteinases that cleave secretory proproteins to their active forms. Several fungal kexin-like proteins have been investigated. The mutants lacking of kexin-like protein display strong phenotypes such as cell wall defect, abnormal polarity, and, in case of Candida albicans, diminished virulence. However, only several proteins have been confirmed as the substrates of kexin-like proteases in these fungal species. It still remains unclear how kexin-like proteins contribute to the morphogenesis in these fungal species. In this study, a kexB-null mutant of the human opportunistic fungal pathogen Aspergillus fumigatus was constructed and analyzed. The ?kexB mutant showed retarded growth, temperature-sensitive cell wall defect, reduced conidia formation, and abnormal polarity. Biochemical analyses revealed that deletion of the kexB gene resulted in impaired N-glycan processing, activation of the MpkA-dependent cell wall integrity signaling pathway, and ER-stress. Results from in vivo assays demonstrated that the mutant exhibited an attenuated virulence in immunecompromised mice. Based on our results, the kexin-like endoprotease KexB was involved in the N-glycan processing, which provides a novel insight to understand how kexin-like protein affects the cell-wall modifying enzymes and therefore morphogenesis in fungi.

SUBMITTER: Wang J

PROVIDER: S-EPMC4410318 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Kexin-like endoprotease KexB is required for N-glycan processing, morphogenesis and virulence in Aspergillus fumigatus.

Wang Jingyang J Zhou Hui H Lu Hua H Du Ting T Luo Yuanming Y Wilson Iain B H IB Jin Cheng C

Fungal genetics and biology : FG & B 20150214

Kexin-like proteins belong to the subtilisin-like family of the proteinases that cleave secretory proproteins to their active forms. Several fungal kexin-like proteins have been investigated. The mutants lacking of kexin-like protein display strong phenotypes such as cell wall defect, abnormal polarity, and, in case of Candida albicans, diminished virulence. However, only several proteins have been confirmed as the substrates of kexin-like proteases in these fungal species. It still remains uncl ...[more]

PMID: 25687931

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Project description:The number of patients suffering from fungal diseases has constantly increased during the last decade. Among the fungal pathogens, the airborne filamentous fungus Aspergillus fumigatus can cause chronic and fatal invasive mold infections. So far, only three major classes of drugs (polyenes, azoles, and echinocandins) are available for the treatment of life-threatening fungal infections, and all present pharmacological drawbacks (e.g., low solubility or toxicity). Meanwhile, clinical antifungal-resistant isolates are continuously emerging. Therefore, there is a high demand for novel antifungal drugs, preferentially those that act on new targets. We studied urease and the accessory proteins in A. fumigatus to determine their biochemical roles and their influence on virulence. Urease is crucial for the growth on urea as the sole nitrogen source, and the transcript and protein levels are elevated on urea media. The urease deficient mutant displays attenuated virulence, and its spores are more susceptible to macrophage-mediated killing. We demonstrated that this observation is associated with an inability to prevent the acidification of the phagosome. Furthermore, we could show that a nickel-chelator inhibits growth on urea. The nickel chelator is also able to reverse the effects of urease on macrophage killing and phagosome acidification, thereby reducing virulence in systemic and trachea infection models. IMPORTANCE The development of antifungal drugs is an urgent task, but it has proven to be difficult due to many similarities between fungal and animal cells. Here, we characterized the urease system in A. fumigatus, which depends on nickel for activity. Notably, nickel is not a crucial element for humans. Therefore, we went further to explore the role of nickel-dependent urease in host-pathogen interactions. We were able to show that urease is important in preventing the acidification of the phagosome and therefore reduces the killing of conidia by macrophages. Furthermore, the deletion of urease shows reduced virulence in murine infection models. Taken together, we identified urease as an essential virulence factor of A. fumigatus. We were able to show that the application of the nickel-chelator dimethylglyoxime is effective in both in vitro and in vivo infection models. This suggests that nickel chelators or urease inhibitors are potential candidates for the development of novel antifungal drugs.

Project description:Iron is an essential cofactor for a wide range of cellular processes. Previous studies have shown that siderophore-mediated uptake and intracellular handling of iron are crucial for virulence of Aspergillus fumigatus. Here we show that the bzip-type transcription factor HapX plays a crucial role in the transcriptional remodeling required for adaption to iron starvation in this opportunistic fungal pathogen. HapX was found to be interconnected in a negative feed-back loop with the previously identified iron regulator SreA: SreA repressed expression of hapX during iron sufficiency and HapX repressed sreA during iron starvation. Genome-wide transcriptional profiling and analysis of selected metabolites (protophorphyrine IX, siderophores and amino acids) indicated extensive metabolic remodeling in response to iron starvation. HapX was found to participate in both, repression and activation of genes during iron starvation. HapX was in particular required for repression of iron-dependent and mitochondrial-localized activities including respiration, TCA cycle, amino acid metabolism, iron-sulfur-cluster biosynthesis and heme biosynthesis. Pathways positively affected by HapX included production of siderophores and the ribotoxin and major allergen AspF1. Analysis of the free amino acid pool revealed HapX-dependent coordination of the production of siderophores with the supply of its precursor ornithine. Consistent with the hapX expression pattern, HapX-deficiency was deleterious with respect to growth rate and conidiation during iron depleted but not iron-replete conditions. HapX-deficiency caused significant attenuation of virulence in a murine model aspergillosis underlining that A. fumigatus faces iron starvation in the host and that the HapX-dependent metabolic reprogramming is therefore crucial for virulence. A. fumigatus 293 and hapX mutants were grown in the presence and absence of iron and in cultures shifted from no iron to iron-containing conditions after 1 h incubation. Hybridizations were performed with biological replicates for wt vs hapX +/- iron. For the iron-shift experiments, there were biological replicates for wt in both conditions and for hapX in -iron but there was only a single biological sample for hapX iron-shift sample. All hybs were performed with flip-dye pairs.

Dataset Information

Kexin-like endoprotease KexB is required for N-glycan processing, morphogenesis and virulence in Aspergillus fumigatus.

Publications

Kexin-like endoprotease KexB is required for N-glycan processing, morphogenesis and virulence in Aspergillus fumigatus.

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