Deletion of the sec4 homolog srgA from Aspergillus fumigatus is associated with an impaired stress response, attenuated virulence and phenotypic heterogeneity

Abstract

Small GTPases of the Rab family are master regulators of membrane trafficking, responsible for coordinating the sorting, packaging and delivery of membrane-bound vesicles to specific sites within eukaryotic cells. The contribution of these proteins to the biology of the human pathogenic fungus Aspergillus fumigatus has not been explored. In this study, we characterized the srgA gene, encoding a Rab GTPase closely related to Sec4. We found that a GFP-SrgA fusion protein accumulated preferentially at hyphal tips and mature condiophores. The radial growth of a ΔsrgA mutant was impaired on both rich and minimal medium, consistent with a role for SrgA in filamentous growth. In addition, the ΔsrgA mutant revealed dysmorphic conidiophores that produced conidia with heterogeneous morphology. The ΔsrgA mutant was hypersensitive to brefeldin A-mediated inhibition of vesicular trafficking and showed increased temperature sensitivity relative to wild type A. fumigatus. However, the most striking phenotype of this mutant was its phenotypic heterogeneity. Individual colonies isolated from the original ΔsrgA mutant showed variable morphology with colony sectoring. In addition, each isolate of the ΔsrgA mutant displayed divergent phenotypes with respect to thermotolerance, in vitro stress response and virulence in a Galleria mellonella infection model. Taken together, these results indicate that SrgA contributes to the asexual development and filamentous growth of A. fumigatus. However, the discordant phenotypes observed among individual isolates of the ΔsrgA mutant suggest that the absence of srgA exerts selective pressure for the acquisition of compensatory changes, such as second-site suppressor mutations.

Figure 1. Relationship between A. fumigatus SrgA and Sec4 homologs.

A: Comparison of G-box motifs (G1–G5) and C-termini (C) from fungal Sec4 homologs in Saccharomyces cerevisiae (Sc), Schizosaccharomyces pombe (Sp), Candida albicans (Ca), Neurospora crassa (Nc)), Aspergillus niger (An), and Aspergillus fumigatus (Af). B: Intracellular localization of A. fumigatus SrgA. The SrgA protein was tagged at its N-terminus with GFP and expressed in A. fumigatus under the control of the gpdA promoter. Scale bar  = 10 µm.

Figure 2. Deletion of srgA from A. fumigatus.

Southern blot analysis of HindIII-digested genomic DNA using a probe located upstream of the srgA coding region (probe A) identified the predicted 2.8 kb fragment in wt A. fumigatus, which was lengthened to 10.3 kb in the ΔsrgA mutant due to replacement of srgA with the phleomycin-resistance cassette (PHLEO).

Figure 3. Loss of SrgA is associated with diverse colony morphology.

A: Conidia harvested from the initial monoconidial ΔsrgA mutant produced a heterogeneous population of colonies when spread for isolation. B: Colony sectoring was observed in ΔsrgA isolates (shown here, isolate-A). C: Three individual ΔsrgA isolates (A–C) were selected from the heterogeneous population shown in panel A. D: Southern blot analysis of HindIII-digested genomic DNA using a probe located upstream of the srgA coding region (Figure 2, probe B) identified the predicted 2.8 kb fragment in wt A. fumigatus, which was lengthened to 10.3 kb in the ΔsrgA isolates due to replacement of srgA with the phleomycin-resistance cassette.

Figure 4. Loss of SrgA impairs conidiation.

A: All three ΔsrgA isolates have attenuated conidophores and dysmorphic phialides (normal phialides are shown by the arrow in wt). B: All three ΔsrgA isolates release conidia that are heterogeneous in both size and shape. Some of the elongated conidia may be abnormal phialides that are released along with the conidia (arrow) (Scale bar  = 20 µm).

Figure 5. GFP-SrgA localizes to conidiophores.

GFP-SrgA localizes to the apex of both hyphae and conidiophores. A punctate accumulation at the tip is seen in both hyphae and the early stages of vesicle swelling (top and middle rows, respectively), but a more diffuse localization is evident in mature conidiophores (bottom row). Left column: brightfield; middle column: GFP fluorescence; bottom column: Merged image.

Figure 6. Loss of SrgA impairs hyphal growth.

Equal numbers of conidia were plated on the center of a plate of solid AMM and colony diameter was measured every day during a four-day incubation period at the indicated temperatures. The experiment was performed in triplicate and the values represent the mean ± SEM.

Figure 7. Sensitivity of ΔsrgA to ER stress.

A: Equal numbers of conidia were added to individual wells of a 24-well plate containing liquid AMM media and the indicated concentrations of dithiothreitol (DTT). Plates were incubated at 37°C for three days, after which the mycelial biomass that was adhered to the plate surface was stained with methylene blue and photographed. B: Equal numbers of conidia were inoculated onto solid AMM media containing either the vehicle control (DMSO) or 100 µg/ml tunicamycin (TM) and incubated for three days at 37°C.

Figure 8. Sensitivity of ΔsrgA to brefeldin A.

Equal numbers of conidia were inoculated onto solid AMM media containing the indicated concentrations of brefeldin A (BFA) and incubated for three days at 37°C.

Figure 9. Analysis of ΔsrgA virulence in an insect model of A. fumigatus infection.

Groups of 12 G. mellonella larvae were infected with conidia from the indicated strains. Larvae were incubated at 37°C and mortality was monitored over a five day period. Kaplan-Meier survival curves were compared using a log-rank test, followed by a pairwise multiple comparison test (Holm-Sidak). The ΔsrgA isolate C survival curve is statistically different from wt, but isolates A and B were indistinguishable from wt.