Mechanism of validamycin A inhibiting DON biosynthesis and synergizing with DMI fungicides against Fusarium graminearum.

Deoxynivalenol (DON) is a vital virulence factor of Fusarium graminearum, which causes Fusarium head blight (FHB). We recently found that validamycin A (VMA), an aminoglycoside antibiotic, can be used to control FHB and inhibit DON contamination, but its molecular mechanism is still unclear. In this study, we found that both neutral and acid trehalase (FgNTH and FgATH) are the targets of VMA in F. graminearum, and the deficiency of FgNTH and FgATH reduces the sensitivity to VMA by 2.12- and 1.79-fold, respectively, indicating that FgNTH is the main target of VMA. We found FgNTH is responsible for vegetative growth, FgATH is critical to sexual reproduction, and both of them play an important role in conidiation and virulence in F. graminearum. We found that FgNTH resided in the cytoplasm, affected the localization of FgATH, and positively regulated DON biosynthesis; however, FgATH resided in vacuole and negatively regulated DON biosynthesis. FgNTH interacted with FgPK (pyruvate kinase), a key enzyme in glycolysis, and the interaction was reduced by VMA; the deficiency of FgNTH affected the localization of FgPK under DON induction condition. Strains with a deficiency of FgNTH were more sensitive to demethylation inhibitor (DMI) fungicides. FgNTH regulated the expression level of FgCYP51A and FgCYP51B by interacting with FgCYP51B. Taken together, VMA inhibits DON biosynthesis by targeting FgNTH and reducing the interaction between FgNTH and FgPK, and synergizes with DMI fungicides against F. graminearum by decreasing FgCYP51A and FgCYP51B expression.

The secreted acid trehalase encoded by the CgATH1 gene is involved in Candida glabrata virulence

BACKGROUND Candida glabrata yeast is the second cause of candidiasis worldwide. Differs from other yeasts since assimilates only glucose and trehalose (a characteristic used in rapid identification tests for this pathogen) by secreting into the medium a highly active acid trehalase encoded by the CgATH1 gene. OBJECTIVE This study aimed to characterise the function of the acid trehalase in the physiopathology of C. glabrata. METHODS Gene deletion was performed to obtain a mutant ath1Δ strain, and the ability of the ath1Δ strain to grow in trehalase, or the presence of trehalase activity in the ath1Δ yeast cells, was verified. We also tested the virulence of the ath1Δ strain in a murine model of infection. FINDINGS The ath1Δ mutant strain grows normally in the presence of glucose, but loses its ability to grow in trehalose. Due to the high acid trehalase activity present in wild-type cells, the cytoplasmic neutral trehalase activity is only detected in the ath1Δ strain. We also observed a significantly lower virulence of the ath1Δ strain in a murine model of infection with either normal or immunocompromised mice. MAIN CONCLUSIONS The acid trehalase is involved in the hydrolysis of external trehalose by C. glabrata, and the enzyme also plays a major virulence role during infectivity.

The Evolutionary History and Functional Divergence of Trehalase (treh) Genes in Insects.

Trehalases (treh) have been found in different organisms, such as bacteria, fungi, yeast, nematodes, insects, vertebrates, and plants. Their biochemical properties are extremely variable and not yet fully understood. Gene expression patterns have shown differences among insect species suggesting a potential functional diversification of trehalase enzymes during their evolution. A second gene family encoding for enzymes with hypothetical trehalase activity has been repeatedly annotated in insect genome as acid trehalases/acid trehalase-like (ath), but its functional role is still not clear. The currently available large amount of genomic data from many insect species may enable a better understanding of the evolutionary history, phylogenetic relationships and possible roles of trehalase encoding genes in this taxon. The aim of the present study is to infer the evolutionary history of trehalases and acid trehalase genes in insects and analyze the trehalase functional divergence during their evolution, combining phylogenetic and genomic synteny/colinearity analyses.

Effect of acid trehalase (ATH) on impaired yeast vacuolar activity.

In this study, this protein was overexpressed in yeast cells grown on trehalose-containing medium to assess its impact on yeast vacuolar activity. ATH was confirmed to be located in both cell surface and vacuoles and the overexpression of ATH was observed to decrease vacuolar activity. Therefore, an assumption was suggested to explain this phenomenon as follows: when grown on containing trehalose medium, the ATH localization at cellular periplasm, but not the vacuole, is prioritized to utilize the extracellular trehalose for cell growth. The multivesicular body pathway (MVB pathway) via which ATH is transported into vacuoles is believed to be down-regulated to favor the accumulation of ATH at cell surface area. By extension, other vacuolar proteins travelling through MVB pathway to reach yeast vacuoles likely also suffer the down regulation. It can be concluded that acid trehalase may contribute down regulation of other vacuolar proteins through MVB pathway. This study suggests that it is a potential of acid trehalase (ATH) on impaired activity of yeast vacuolar.

Secretion of the acid trehalase encoded by the CgATH1 gene allows trehalose fermentation by Candida glabrata.

The emergent pathogen Candida glabrata differs from other yeasts because it assimilates only two sugars, glucose and the disaccharide trehalose. Since rapid identification tests are based on the ability of this yeast to rapidly hydrolyze trehalose, in this work a biochemical and molecular characterization of trehalose catabolism by this yeast was performed. Our results show that C. glabrata consumes and ferments trehalose, with parameters similar to those observed during glucose fermentation. The presence of glucose in the medium during exponential growth on trehalose revealed extracellular hydrolysis of the sugar by a cell surface acid trehalase with a pH optimum of 4.4. Approximately ∼30% of the total enzymatic activity is secreted into the medium during growth on trehalose or glycerol. The secreted enzyme shows an apparent molecular mass of 275 kDa in its native form, but denaturant gel electrophoresis revealed a protein with ∼130 kDa, which due to its migration pattern and strong binding to concanavalin A, indicates that it is probably a dimeric glycoprotein. The secreted acid trehalase shows high affinity and activity for trehalose, with Km and Vmax values of 3.4 mM and 80 U (mg protein)(-1), respectively. Cloning of the CgATH1 gene (CAGLOK05137g) from de C. glabrata genome, a gene showing high homology to fungal acid trehalases, allowed trehalose fermentation after heterologous expression in Saccharomyces cerevisiae.

Enhancing the utilization of host trehalose by fungal trehalase improves the virulence of fungal insecticide.

Entomopathogenic fungi proliferate in insect hemolymph by using host nutrients after penetrating the cuticle. To improve the virulence of the locust specific fungus, Metarhizium acridum, we genetically modified the fungus to overexpress ATM1, an endogenous hydrolase of trehalose, which is the main carbon source in insect hemolymph. Compared with the wild-type strain, Metarhizium acridum overexpressing ATM1 gene secreted more acid trehalase into locust hemolymph. The trehalose concentrations in locusts infected with the ATM1-overexpressing strain were 5.5 and 6.1 mmol/l, lower than that in locusts infected with the wild-type strain at 3 and 5 days post-inoculation, representing 44.5 and 60.7 % reduction, respectively. Correspondingly, overexpressing ATM1 accelerated the growth of Metarhizium acridum in host hemolymph, and the dose causing 50 % mortality (LD50) of the ATM1-overexpressing strain was reduced by 8.3-fold compared with the wild-type strain, suggesting that increasing the utilization of host nutrients by pathogens could be a promising way to improve the virulence of biopesticides based on parasites of pests.

The acid trehalase, ATM1, contributes to the in vivo growth and virulence of the entomopathogenic fungus, Metarhizium acridum.

For pathogens, the ability to acquire available nutrients in a host is a key to their survival and replication. Entomopathogenic fungi of the genus Metarhizium secrete trehalase, which enables them to use trehalose, the predominant sugar in insects. Here, the roles of the acid trehalase gene (ATM1) in the in vivo growth and virulence of Metarhizium acridum were investigated. Phenotypic analysis showed that disruption of ATM1 severely reduced fungal growth on exogenous trehalose as the sole carbon source. Bioassays showed that ATM1 disruption impaired the virulence of M. acridum against the host insect Locusta migratoria. The ATM1-disruption strain (ΔATM1) grown more slowly than the wild-type strain (WT) and complemented transformant (CP) in locust blood, which was consistent with the activity of acid trehalase in the hemolymph of infected locusts. Correspondingly, the trehalose concentration in locusts infected by ΔATM1 was significantly higher than in those infected by WT or CP. Thus, ATM1 disruption led to a significant reduction in virulence by adversely affecting the fungal growth in insect hemolymph, which resulted from the inability of the mutant strain to use trehalose.