Glutamate dehydrogenase (Gdh2)-dependent alkalization is dispensable for escape from macrophages and virulence of Candida albicans
Fig 5
Competition assay to compare wildtype and gdh2-/- filamentation and survival upon phagocytosis by primary BMDM.
(A) (left panels) Wildtype (WT; PADH1-GFP; SCADH1G4A) or gdh2-/- (PADH1-RFP, CFG275) cells was co-cultured with primary BMDM (MOI of 3:1; C:M) for 30 min in HBSS. Non-phagocytized fungal cells were removed by washing and the co-cultures were monitored by live cell imaging for 4 h (SV3 and SV4 Vids). (right panel) Candidacidal activity of BMDM. Untagged WT (PLC005) and gdh2-/- (CFG279) strains were co-cultured for 2 h with BMDM at MOI of 3:1 (without washing) and then CFUs recovered and compared to the CFUs in the starting inoculum. No significant difference in the co-culture survival between the strains using Student t-test; data presented are average of 4 biological replicates. (B) Competition assay. The same strains as in (A) were mixed 1:1, co-cultured for 30 min in HBSS, washed extensively to remove external cells, and the interactions were followed by TLM for 5 h (S5 Vid). Solid arrows indicate macrophages with phagosomes containing both WT and gdh2-/- cells; open arrows indicate macrophages with phagosomes containing either WT (green) or gdh2-/- (red) cells. The observed growth of WT and gdh2-/- cells within a single macrophage is schematically illustrated (right upper panel). For the WT to gdh2-/- ratio determination following co-culture, mutants were mixed 1:1 and then the mixed cells were used to infect BMDM at MOI of 3:1. After an initial macrophage lysing step, the ratio of WT:gdh2-/- was analyzed by plating serially diluted cells on YPD and then testing at least 50 independent colonies for their ability to alkalinize YNB+CAA medium (pH = 4). The ratio of recovered cells (after 2 h) was compared to the input ratio. No significant difference in the WT:gdh2-/- ratio between input and recovered using Student t-test; data were obtained from six (6) biological replicates.