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Talaromyces marneffei simA Encodes a Fungal Cytochrome P450 Essential for Survival in Macrophages

  • Boyce K
  • De Souza D
  • Dayalan S
  • et al.
mSphere (2018) 3(2)
DOI: 10.1128/msphere.00056-18
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Abstract

This study in a dimorphic fungal pathogen uncovered a role for a yeast-specific cytochrome P450 (CYP)-encoding gene in the ability of T. marneffei to grow as yeast cells within the host macrophages. This report will inspire further research into the role of CYPs and secondary metabolite synthesis during fungal pathogenic growth. Fungi are adept at occupying specific environmental niches and often exploit numerous secondary metabolites generated by the cytochrome P450 (CYP) monoxygenases. This report describes the characterization of a yeast-specific CYP encoded by simA (" s urvival i n m acrophages"). Deletion of simA does not affect yeast growth at 37°C in vitro but is essential for yeast cell production during macrophage infection. The Δ simA strain exhibits reduced conidial germination and intracellular growth of yeast in macrophages, suggesting that the enzymatic product of SimA is required for normal fungal growth in vivo . Intracellular Δ simA yeast cells exhibit cell wall defects, and metabolomic and chemical sensitivity data suggest that SimA may promote chitin synthesis or deposition in vitro . In vivo , Δ simA yeast cells subsequently lyse and are degraded, suggesting that SimA may increase resistance to and/or suppress host cell biocidal effectors. The results suggest that simA synthesizes a secondary metabolite that allows T. marneffei to occupy the specific intracellular environmental niche within the macrophage. IMPORTANCE This study in a dimorphic fungal pathogen uncovered a role for a yeast-specific cytochrome P450 (CYP)-encoding gene in the ability of T. marneffei to grow as yeast cells within the host macrophages. This report will inspire further research into the role of CYPs and secondary metabolite synthesis during fungal pathogenic growth.

Figures

  • FIG 1 Expression of simA. (A) Reverse transcriptase PCR (RT-PCR) of simA and a benA loading control performed on RNA isolated from wild-type vegetative hyphae grown for 2 days in liquid medium at 25°C, asexual development (conidiation) cultures grown for 7 days on solid medium at 25°C, and yeast cells grown for 6 days in liquid medium at 37°C. A simA transcript could not be detected during vegetative hyphal growth at 25°C. simA is expressed during asexual development at 25°C and highest during yeast growth at 37°C. Relative intensity values, adjusted using the loading controls, are indicated below the lanes. (B) RNA was isolated from wild-type T. marneffei incubated in macrophage medium alone for 24 h at 37°C (- ) or from infected LPS-activated J774 murine macrophages 24 h postinfection at 37°C ( ). The amount of simA transcript was increased in cells isolated from infected macrophages. Relative intensity values, adjusted using the loading controls, are indicated below the lanes.
  • FIG 2 SimA localization in yeast cells during macrophage infection. Anti-HA immunostaining was performed on macrophages infected with simA HA conidia 24 h postinfection. (A) Calcofluor (CAL) staining of T. marneffei yeast cells during infection. Anti-HA immunostaining shows SimA localized in the cytoplasm. (B) Costaining with Hoechst 33258 (Hoescht) shows that the localization is perinuclear, consistent with localization in the ER. Images were captured using differential interference contrast (DIC) or with epifluorescence to observe calcofluor-stained fungal cell walls (CAL) or Hoechst 33258-stained nuclei (Hoescht). Scale bars, 10 m.
  • FIG 3 SimA is required for yeast growth in vivo but not in vitro. (A) simA and ΔsimA strains grown in vitro on BHI medium for 5 days at 37°C. Numerous ΔsimA yeast cells were observed after 5 days, and the cells were morphologically indistinguishable from the wild-type (simA ) cells. (B) Macrophages infected with simA and ΔsimA conidia 24 h postinfection. After 24 h, numerous yeast cells dividing by fission were observed in macrophages infected with wild-type (simA ) conidia. In contrast, ΔsimA conidia remained mostly ungerminated after 24 h. A proportion of those conidia did not stain with calcofluor (indicated by arrows) and appeared to have lost cellular integrity (double-headed arrow). Images were captured using differential interference contrast (DIC) or with epifluorescence to observe calcofluorstained fungal cell walls (CAL). Scale bars, 10 m (A) and 20 m (B).
  • FIG 4 The ΔsimA strain shows less cellular proliferation during macrophage infection and ruptured yeast cells. (A) Macrophages infected with simA , ΔsimA, and ΔsimA simA conidia 48 h postinfection. After 48 h, numerous yeast cells dividing by fission were observed in macrophages infected with wild-type (simA ) or ΔsimA simA conidia. The ΔsimA strain showed less cellular proliferation within macrophages than the wild-type (simA ) and ΔsimA simA strains. Some ΔsimA conidia remained ungerminated (black arrowhead), but a number had germinated into yeast cells (black double arrowhead). Yeast cells appeared ruptured and to be releasing cellular contents (black double arrowhead). (B) Unlike wild-type (simA ) and ΔsimA simA yeast cells, ΔsimA yeast cells were often ruptured (white arrowhead) and leaking their cellular contents (black arrowhead). Cells were not clearly visible under DIC conditions (black double arrowhead), and degraded fungal cellular material was observed (white double arrowhead). Images were captured using differential interference contrast (DIC) or with epifluorescence to observe calcofluor-stained fungal cell walls (CAL). Scale bars, 10 m.
  • FIG 5 Conidia of the ΔsimA mutant display cell wall defects. (A and B) Transmission electron microscopy of wild-type (simA ) and ΔsimA conidia (ANMmedium for 12 days at 25°C) (A) and yeast cells grown in vitro (BHI medium for 5 days at 37°C) (B). (A) The cell wall of wild-type (simA ) conidia appears as three layers: a thin dense layer which lies directly adjacent to the lipid bilayer, a thick nondense layer in the middle layer, and an outer electron-dense layer which is slightly uneven. Compared to the wild-type results, the middle and outer layers of ΔsimA conidia are unevenly distributed. (B) The cell wall of wild-type (simA ) yeast cells grown in vitro appears as two distinct layers: a thick dense layer lying directly adjacent to the lipid bilayer and a denser, unevenly distributed outer layer. The cell wall of ΔsimA yeast cells in vitro appears indistinguishable from the wild-type (simA ) cell wall. Scales bars, 100 nm (A) and 0.2 m (B).
  • FIG 6 ΔsimA is sensitive to cell wall-disrupting agents and resistant to oxidizing agents at 37°C. Serial dilutions of wild-type, ΔsimA, and ΔsimA simA conidial suspensions were dropped onto SD medium(NH4)2SO4 plates containing 30 g/ l calcofluor (CAL), 10 g/ l Congo red (CR), 1 mM H2O2, or 0.5 mM NO2 and incubated for 5 days at 37°C.
  • FIG 7 ΔsimA yeast cells appear degraded in vivo. Transmission electron microscopy images of the wild-type (simA ) and ΔsimA strains 24 h postinfection of macrophages are shown. (A) After 24 h postinfection of macrophages, wild-type (simA ) yeast cells were observed within a macrophage phagosome. In contrast, the ungerminated ΔsimA conidia were not encompassed within an intracellular macrophage compartment. (B) In contrast to macrophages infected with the wild-type strain, macrophages infected with ΔsimA conidia contained numerous degraded cells. (C) The cell wall of wild-type (simA ) yeast cells appeared as at least two distinct defined layers: a thick dense layer lying directly adjacent to the lipid bilayer and a denser, unevenly distributed outer layer. Some ΔsimA conidia lacked clearly defined cell wall layers. Scale bars, 5 m (A), 2 m (B), and 0.5 m (C).
  • FIG 8 Deletion of simA does not result in increased phagolysosomal maturation. LPS-activated J774 murine macrophages infected with wild-type or ΔsimA conidia 24 h postinfection were labeled with the lysosomal marker LAMP1. Wild-type or ΔsimA conidia did not colocalize with LAMP1. The arrowhead and double arrowheads indicate a ΔsimA conidium that stained with calcofluor white and ΔsimA conidia that were not stained, respectively. Images were captured using differential interference contrast (DIC) or with epifluorescence to observe calcofluor-stained fungal cell walls (CAL) or LAMP1. Scale bars, 20 m.

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Boyce, K. J., De Souza, D. P., Dayalan, S., Pasricha, S., Tull, D., McConville, M. J., & Andrianopoulos, A. (2018). Talaromyces marneffei simA Encodes a Fungal Cytochrome P450 Essential for Survival in Macrophages. MSphere, 3(2). https://doi.org/10.1128/msphere.00056-18

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