Chitin synthases with a myosin motor-like domain control the resistance of Aspergillus fumigatus to echinocandins

Abstract

Aspergillus fumigatus has two chitin synthases (CSMA and CSMB) with a myosin motor-like domain (MMD) arranged in a head-to-head configuration. To understand the function of these chitin synthases, single and double csm mutant strains were constructed and analyzed. Although there was a slight reduction in mycelial growth of the mutants, the total chitin synthase activity and the cell wall chitin content were similar in the mycelium of all of the mutants and the parental strain. In the conidia, chitin content in the ΔcsmA strain cell wall was less than half the amount found in the parental strain. In contrast, the ΔcsmB mutant strain and, unexpectedly, the ΔcsmA/ΔcsmB mutant strain did not show any modification of chitin content in their conidial cell walls. In contrast to the hydrophobic conidia of the parental strain, conidia of all of the csm mutants were hydrophilic due to the presence of an amorphous material covering the hydrophobic surface-rodlet layer. The deletion of CSM genes also resulted in an increased susceptibility of resting and germinating conidia to echinocandins. These results show that the deletion of the CSMA and CSMB genes induced a significant disorganization of the cell wall structure, even though they contribute only weakly to the overall cell wall chitin synthesis.

Fig 1

(A) Organization of the CSMA and CSMB genes in a head-to-head configuration in A. fumigatus chromosome 2. The orientation of transcription is shown by arrowheads. The distance between both translational start points is 3,221 bp. (B) Structures of CsmA and CsmB proteins. The myosin motor-like domains (MMD) and the chitin synthase domains (CSD) are indicated by solid black and gray boxes, respectively. The P-loop, Switch I, and Switch II motifs are also shown.

Fig 2

Mycelial chitin content (percent in the cell wall [CW] after 24 h of growth; data are means ± standard deviations [SD] from four individual experiments) and zymogenic chitin synthase (CS; means ± SD from three different experiments) activity of the parental strain and the single and double Δcsm mutant strains.

Fig 3

Colony growth of the parental and Δcsm mutant strains in rich media without (A) and with (C) an osmotic stabilizer (KCl) after 48 h at 37°C. (B) Calcofluor white staining of the parental and the Δcsm mutant strains' mycelia after 32 h of growth in liquid Sabouraud culture medium at 37°C.

Fig 4

Morphology of the abnormal conidiophores of the Δcsm mutant strains after 4 days of growth on malt (2%) plus KCl (6%) agar medium at 37°C. Note that in the ΔcsmB strain, of the two conidiophores, one is similar to that of the parental strain.

Fig 5

(A) Conidial germination of the parental and the Δcsm mutant strains showing that the diameters of the mutant conidia are larger (grown in liquid Sabouraud culture medium at 37°C for 7.5 h). (B) Calcofluor white staining of swollen conidia of the parental and the Δcsm mutant strains (grown in liquid Sabouraud culture medium at 37°C for 4.5 h) showing the permeability of swollen conidia to the dye only in the Δcsm mutants.

Fig 6

(A) AFM deflection images in buffer of A. fumigatus conidia of the parental strain and ΔcsmB mutant showing the presence of a rodlet layer on its surface; the ΔcsmB mutant shows an amorphous layer in places. AFM deflection images of A. fumigatus conidia of ΔcsmA mutant and ΔcsmA/ΔcsmB double mutant strains revealing amorphous layers devoid of rodlets. For every strain, the images shown are representative of at least 10 conidia. (B) HF extracts from the ΔcsmA, ΔcsmB, and ΔcsmA ΔcsmB mutant and parental strain conidia showing the presence of RodAp (conidial surface protein, hydrophobin, responsible for the rodlet structure) in all of the strains.

Fig 7

(A) YG plates containing 0.1 μg/ml of caspofungin inoculated with serial 10-fold dilutions of conidia (2 × 10⁶) from all single Δchs mutants of A. fumigatus (but not the ΔcsmB mutant). (B) Ten-fold conidial dilutions (2 × 10⁶) of ΔcsmA, ΔcsmB, and ΔcsmA ΔcsmB mutant strains and the parental strain were spotted on YG plates with 0.1 μg (6 ng/ml) caspofungin. Plates were incubated for 3 days at 28°C. (C) Microscopic analyses of the conidia abnormally swollen on the plates containing 0.1 μg caspofungin at 37°C for 24 h.