Identification and deletion of Tft1, a predicted glycosyltransferase necessary for cell wall β-1,3;1,4-glucan synthesis in Aspergillus fumigatus

Abstract

Aspergillus fumigatus is an environmental mold that causes severe, often fatal invasive infections in immunocompromised patients. The search for new antifungal drug targets is critical, and the synthesis of the cell wall represents a potential area to find such a target. Embedded within the main β-1,3-glucan core of the A. fumigatus cell wall is a mixed linkage, β-D-(1,3;1,4)-glucan. The role of this molecule or how it is synthesized is unknown, though it comprises 10% of the glucans within the wall. While this is not a well-studied molecule in fungi, it has been studied in plants. Using the sequences of two plant mixed linkage glucan synthases, a single ortholog was identified in A. fumigatus (Tft1). A strain lacking this enzyme (tft1Δ) was generated along with revertant strains containing the native gene under the control of either the native or a strongly expressing promoter. Immunofluorescence staining with an antibody against β-(1,3;1,4)-glucan and biochemical quantification of this polysaccharide in the tft1Δ strain demonstrated complete loss of this molecule. Reintroduction of the gene into the knockout strain yielded reappearance in amounts that correlated with expected expression of the gene. The loss of Tft1 and mixed linkage glucan yielded no in vitro growth phenotype. However, there was a modest increase in virulence for the tft1Δ strain in a wax worm model. While the precise roles for β-(1,3;1,4)-glucan within A. fumigatus cell wall are still uncertain, it is clear that Tft1 plays a pivotal role in the biosynthesis of this cell wall polysaccharide.

Fig 1. Immunostaining of hyphae with a specific β-1,3;1,4 glucan antibody.

A. No primary antibody Af293(WT) control. β-1,3;1,4-glucan staining of B. WT.; C. β-1,3;1,4-glucanase digested WT(only WT shown; other digested strains were also negative for staining); D. tft1Δ; E. Revtft1; and F. SSA1Revtft1.

Fig 2. The relative glucose released when insoluble cell wall fractions were digested with A. endo β-1,3;1,4-glucanase or B. β-1,3 glucanase.

tft1Δ shown to release zero glucose when exposed to endo β-1,3;1,4-glucan hydrolase. The box represents calculated error based on replicates of 3 samples for each digestion. WT vs tft1Δ, p<0.001; WT vs Revtft1, p<0.001; WT vs SSA1-Revtft1, p<0.001.

Fig 3. In vitro growth of the Tft1 transformant strains.

Strains grown on, A. Aspergillus Minimal Media (AMM); B. AMM+Farnesol; C. AMM+Congo Red; or D AMM-Calcofluor white. No apparent differences in phenotype were observed between the strains and WT.

Fig 4. Log rank survival analysis of each strain after 1 x 10⁶ spore injection into Galleria mellonella.

tft1Δ was shown to have a slight, yet significant (p value: .000187), increase in virulence when compared to its parent Af293. This hyper-virulence was lost upon re-introduction of the gene, either under native or strong promoter (Revtft1 and SSA1Revtft1).