FsFKS1, the 1,3-beta-glucan synthase from the caspofungin-resistant fungus Fusarium solani

Abstract

The cell wall, a mesh of carbohydrates and proteins, shapes and protects the fungal cell. The enzyme responsible for the synthesis of one of the main components of the fungal wall, 1,3-beta-glucan synthase, is targeted by the antifungal caspofungin acetate (CFA). Clinical isolates of Candida albicans and Aspergillus fumigatus are much more sensitive to CFA than clinical isolates of Fusarium species. To better understand CFA resistance in Fusarium species, we cloned and sequenced FsFKS1, which encodes the Fusarium solani f. sp. pisi beta(1,3)-D-glucan synthase, used RNA interference to reduce its expression and complemented deletion of the essential fks gene of the CFA-sensitive fungus A. fumigatus with FsFKS1. Reduction of the FsFKS1 message in F. solani f. sp. pisi reduced spore viability and caused lysis of spores and hyphae, consistent with cell wall defects. Compensating for the loss of A. fumigatus fks1 with FsFKS1 caused only a modest increase in the tolerance of A. fumigatus for CFA. Our results suggest that FsFKS1 is required for the proper construction of F. solani cell walls and that the resistance of F. solani to CFA is at best only partially due to resistance of the FsFKS1 enzyme to this antifungal agent.

FIG. 1.

F. solani f. sp. pisi FKS1 genomic region. (A) Southern hybridization of F. solani f. sp. pisi 156-30-6 genomic DNA with conserved FsFKS1 probe 2. F. solani f. sp. pisi 156-30-6 DNA was digested with BamHI (B), EcoRI (E), EcoRV (EV), and XhoI (X); separated along with 1-kb ladder (M) on a 0.8% agarose gel; and probed with radiolabeled FsFKS1 probe 2. (B) Restriction map of the chromosome region containing the FsFKS1 gene. PCR probes used for screening library are numbered 1 and 2. White boxes after ATG and before TAA indicate introns.

FIG. 2.

Spores, germ tubes, and hyphal morphology of wild-type and FsFKS1/RNAi transformants. (A and D) Wild type; (B, C, E, and F) FsFKS1 RNAi transformants. (A to C) Spores were inoculated into liquid induction medium and incubated for 18 h. (D to F) Cultures were grown in liquid repression medium for 48 h and transferred to induction medium for 24 h. Arrows indicate swollen cells and empty compartments.

FIG. 3.

Northern analysis of FsFKS1 in wild-type and FsFKS1/RNAi transformants. Cultures were grown in liquid repression medium for 48 h and transferred to liquid induction medium. Lanes: 1, wild-type strain with empty vector; 2 and 3, RNAi transformants showing moderate phenotypes (FsFKS1/RNAi 4 and FsFKS1/RNAi 5, respectively); 4 and 5, transformants showing severe phenotypes (FsFKS1/RNAi 3 and FsFKS1/RNAi 9, respectively).

FIG. 4.

A. fumigatus wild-type strain and FsFKS1 transformants on caspofungin-containing medium. (A) Colony phenotype and (B) Northern hybridization of FsFKS1 in wild-type A. fumigatus and FsFKS1 transformants. (A) Wild-type A. fumigatus and FsFKS1 transformants were inoculated in medium containing hygromycin as a selection marker. After colonies were visible, plates were overlaid with agar containing caspofungin. 1, A. fumigatus containing empty vector pAN7-1; 2 to 6, A. fumigatus transformed with FsFKS1, 2, A. fumigatus/FsFKS1-12; 3, A. fumigatus/FsFKS1-16; 4, A. fumigatus/FsFKS1-18; 5, A. fumigatus/FsFKS1-19; 6, A. fumigatus/FsFKS1-20; 7, A. fumigatus/FsFKS1-pF4 ectopic integrant 13 (A. fumigatus/FsFKS1-18 in which an Affks gene disruption construct was ectopically integrated); 8, A. fumigatus/FsFKS1-Affks1 disruptant 29 (A. fumigatus/FsFKS1-18 in which the Affks gene is disrupted); 7 and 8, colonies incubated on minimal medium containing hygromycin and caspofungin. (B) Lanes 1 to 6, as in panel A. The lower panel shows total RNA stained with ethidium bromide.

FIG. 5.

Affks genomic region. (A) Diagram and (B) PCR analysis of Affks genomic region in wild type and in gene disruptant. (A) The KpnI fragment of Affks (stippled box) was ligated into a vector carrying selectable markers to produce pF4. fks1^a and fks1^b are identical primers annealing to endogenous Affks (fks1^a) or to the identical sequence derived from pF4 (fks1^b). (B) Upper panel, 2.4-kb PCR products amplified with fks1 and fks2 primers. Lower panel, 2.6-kb PCR products generated by T7 and primer fks2, indicating disruption of the Affks gene. M, 1-kb marker; WT, A. fumigatus AF293.1; Ectopic, A. fumigatus/FsFKS1-pF4 ectopic integrant 13; Disruptant, A. fumigatus/FsFKS1-Affks1 disruptant 29.