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 conserved and divergent roles of carbonic anhydrases in the filamentous fungi Aspergillus fumigatus and Aspergillus nidulans

Carbon dioxide (CO₂) and its hydration product bicarbonate (HCO₃^‐) are essential molecules in various physiological processes of all living organisms. The reversible interconversion between CO₂ and HCO₃^‐ is in equilibrium. This reaction is slow without catalyst, but can be rapidly facilitated by Zn²⁺‐metalloenzymes named carbonic anhydrases (CAs). To gain an insight into the function of multiple clades of fungal CA, we chose to investigate the filamentous fungi Aspergillus fumigatus and A. nidulans. We identified four and two CAs in A. fumigatus and A. nidulans, respectively, named cafA‐D and canA‐B. The cafA and cafB genes are constitutively, strongly expressed whereas cafC and cafD genes are weakly expressed but CO₂‐inducible. Heterologous expression of the A. fumigatus cafB, and A. nidulans canA and canB genes completely rescued the high CO₂‐requiring phenotype of a Saccharomyces cerevisiaeΔnce103 mutant. Only the ΔcafAΔcafB and ΔcanB deletion mutants were unable to grow at 0.033% CO₂, of which growth defects can be restored by high CO₂. Defects in the CAs can affect Aspergilli conidiation. Furthermore, A. fumigatusΔcafA, ΔcafB, ΔcafC, ΔcafD and ΔcafAΔcafB mutant strains are fully virulent in a low‐dose murine infection.     

Catecholate siderophore esterases Fes,IroD and IroE are required for salmochelins secretion following utilization,but only IroD contributes to virulence of extra‐intestinal pathogenic Escherichia coli

Salmochelins are glucosylated forms of enterobactin (enterochelin) and contribute to the virulence of Salmonella enterica and some extra‐intestinal pathogenic Escherichia coli (ExPEC). Fes, IroD and IroE esterases degrade salmochelins and enterobactin to release iron. We investigated the apparently redundant role of these esterases in virulence and in salmochelin production and utilization of the ExPEC strain χ7122. The ΔiroD, ΔfesΔiroD and ΔfesΔiroDΔiroE mutants displayed attenuated virulence phenotypes in an avian systemic infection model. Growth of ΔfesΔiroD and ΔfesΔiroDΔiroE mutants was severely reduced in the presence of conalbumin, and although enterobactin was produced, no salmochelins were detected in the culture supernatants of these mutants. Elimination of catecholate synthesis via an entA deletion in a ΔfesΔiroDΔiroE restored growth in the presence of conalbumin, but only partially restored the virulence of the strain. Salmochelin production was reestablished by reintroducing active esterases. Intracellular accumulation of cyclic mono‐glucosylated enterobactin was observed in the triple mutant ΔfesΔiroDΔiroE, and deletion of fepC, required for catecholate import into the cytoplasm, restored salmochelin detection in supernatants. These results suggest that in the absence of esterases, cyclic salmochelins are synthesized and secreted, but remain cell‐bound after internalization indicating that esterase‐mediated degradation is required for re‐secretion of catecholate siderophore molecules following their utilization.     

Aspergillus fumigatus MedA governs adherence,host cell interactions and virulence

In medically important fungi, regulatory elements that control development and asexual reproduction often govern the expression of virulence traits. We therefore cloned the Aspergillus fumigatus developmental modifier MedA and characterized its role in conidiation, host cell interactions and virulence. As in the model organism Aspergillus nidulans, disruption of medA in A. fumigatus dramatically reduced conidiation. However, the conidiophore morphology was markedly different between the two species. Further, gene expression analysis suggested that MedA governs conidiation through different pathways in A. fumigatus compared with A. nidulans. The A. fumigatusΔmedA strain was impaired in biofilm production and adherence to plastic, as well as adherence to pulmonary epithelial cells, endothelial cells and fibronectin in vitro. The ΔmedA strain also had reduced capacity to damage pulmonary epithelial cells, and stimulate pro‐inflammatory cytokine mRNA and protein expression. Consistent with these results, the A. fumigatusΔmedA strain also exhibited reduced virulence in both an invertebrate and a mammalian model of invasive aspergillosis. Collectively, these results suggest that the downstream targets of A. fumigatus MedA mediate virulence, and may provide novel therapeutic targets for invasive aspergillosis.     

Trehalose‐6‐phosphate phosphatases from Arabidopsis thaliana: identification by functional complementation of the yeast tps2 mutant

It is currently thought that most flowering plants lack the capacity to synthesize trehalose, a common disaccharide of bacteria, fungi and invertebrates that appears to play a major role in desiccation tolerance. Attempts have therefore been made to render plants more drought-resistant by the expression of microbial genes for trehalose synthesis. It is demonstrated here that Arabidopsis thaliana itself possesses genes for at least one of the enzymes required for trehalose synthesis, trehalose-6-phosphate phosphatase. The yeast tps2 mutant, which lacks this enzyme, is heat-sensitive, and Arabidopsis cDNA able to complement this effect has been screened for. Half of the yeast transformants that grew at 38.6°C were also able to produce trehalose. All of these expressed one of two Arabidopsis cDNA, either AtTPPA or AtTPPB, which are both homologous to the C-terminal part of the yeast TPS2 gene and other microbial trehalose-6-phosphate phosphatases. Yeast tps2 mutants expressing AtTPPA or AtTPPB contained trehalose-6-phosphate phosphatase activity that could be measured both in vivo and in vitro. The enzyme dephosphorylated trehalose-6-phosphate but not glucose-6-phosphate or sucrose-6-phosphate. Both genes are expressed in flowers and young developing tissue of Arabidopsis. The finding of these novel Arabidopsis genes for trehalose-6-phosphate phosphatase strongly indicates that a pathway for trehalose biosynthesis exists in plants.     

Trehalose biosynthesis in Thermus thermophilus RQ-1: biochemical properties of the trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase

The genes for trehalose synthesis in Thermus thermophilus RQ-1, namely otsA [trehalose-phosphate synthase (TPS)], otsB [trehalose-phosphate phosphatase (TPP)], and treS [trehalose synthase (maltose converting) (TreS)] genes are structurally linked. The TPS/TPP pathway plays a role in osmoadaptation, since mutants unable to synthesize trehalose via this pathway were less osmotolerant, in trehalose-deprived medium, than the wild-type strain. The otsA and otsB genes have now been individually cloned and overexpressed in Escherichia coli and the corresponding recombinant enzymes purified. The apparent molecular masses of TPS and TPP were 52 and 26 kDa, respectively. The recombinant TPS utilized UDP-glucose, TDP-glucose, ADP-glucose, or GDP-glucose, in this order as glucosyl donors, and glucose-6-phosphate as the glucosyl acceptor to produce trehalose-6-phosphate (T6P). The recombinant TPP catalyzed the dephosphorylation of T6P to trehalose. This enzyme also dephosphorylated G6P, and this activity was enhanced by NDP-glucose. TPS had an optimal activity at about 98°C and pH near 6.0; TPP had a maximal activity near 70°C and at pH 7.0. The enzymes were extremely thermostable: at 100°C, TPS had a half-life of 31 min, and TPP had a half-life of 40 min. The enzymes did not require the presence of divalent cations for activity; however, the presence of Co²⁺ and Mg²⁺ stimulates both TPS and TPP. This is the first report of the characterization of TPS and TPP from a thermophilic organism.     

MAPK pathway-related tyrosine phosphatases regulate development,secondary metabolism and pathogenicity in fungus Aspergillus flavus

Mitogen-activated protein kinase (MAPK) cascades are highly conserved in eukaryotic cells and are known to play crucial roles in the regulation of various cellular processes. However, compared with kinase-mediated phosphorylation, dephosphorylation catalysed by phosphatases has not been well characterized in filamentous fungi. In this study, we identified five MAPK pathway-related phosphatases (Msg5, Yvh1, Ptp1, Ptp2 and Oca2) and characterized their functions in Aspergillus flavus, which produces aflatoxin B₁ (AFB₁), one of the most toxic and carcinogenic secondary metabolites. These five phosphatases were identified as negative regulators of MAPK (Slt2, Fus3 and Hog1) pathways. Deletion of Msg5 and Yvh1 resulted in significant defects in conidiation, sclerotia formation, aflatoxin production and crop infection. Additionally, double knockout mutants (ΔMsg5/ΔPtp1, ΔMsg5/ΔPtp2 and ΔMsg5/ΔOca2) displayed similar defects to those observed in the ΔMsg5 single mutant, indicating that Msg5 plays a major role in the regulation of development and pathogenicity in A. flavus. Importantly, we found that the active site at C439 is essential for the function of the Msg5 phosphatase. Furthermore, the MAP kinase Fus3 was found to be involved in the regulation of development, aflatoxin biosynthesis and pathogenicity, and its conserved phosphorylation residues (Thr and Tyr) were critical for the full range of its functions in A. flavus. Overall, our results reveal that MAPK related tyrosine phosphatases play important roles in the regulation of development, secondary metabolism and pathogenicity in A. flavus, and could be developed as potential targets for preventing damage caused by this fungal pathogen.     

Cloning and truncation modification of trehalose-6-phosphate synthase gene from Selaginella pulvinata

A homologous sequence was amplified from resurrection plant Selaginella pulvinta by RACE technique, proved to be the full-length cDNA of trehalose-6-phosphate synthase gene by homologous alignment and yeast complementation assay, and nominated as SpTPS1 gene. The open reading frame of this gene was truncated 225 bp at the 5′-end, resulting the N-terminal truncation modification of 75 amino acids for its encoding protein. The TPS1 deletion mutant strain YSH290 of the brewer's yeast transformed by the truncated gene SpTPS1Δ and its original full-length version restored growth on the medium with glucose as a sole carbon source and displayed growth curves with no significant difference, indicating their encoding proteins functioning as TPS enzyme. The TPS activity of the mutant strain transformed by the truncated gene SpTPS1Δ was about six fold higher than that transformed by its original version, reasoning that the extra N-terminal extension of the full-length amino acid sequence acts as an inhibitory domain to trehalose synthesis. However, the trehalose accumulation of the mutant strain transformed by the truncated gene SpTPS1Δ was only 8% higher than that transformed by its original version. This result is explained by the feedback balance of trehalose content coordinated by the comparative activities between trehalose synthase and trehalase. The truncated gene SpTPS1Δ is suggested to be used in transgenic operation, together with the inhibition of trehalase activity by the application of validamycin A or genetic deficiency of the endogenous trehalase gene, for the enhancement of trehalose accumulation and improvement of abiotic tolerance in transgenic plants.