Aspergillus fumigatus cytochrome c impacts conidial survival during sterilizing immunity

Abstract

Aspergillus fumigatus can cause a life-threatening infection known as invasive pulmonary aspergillosis (IPA), which is marked by fungus-attributable mortality rates of 20%-30%. Individuals at risk for IPA harbor genetic mutations or incur pharmacologic defects that impair myeloid cell numbers and/or function, exemplified by bone marrow transplant recipients, patients that receive corticosteroid therapy, or patients with chronic granulomatous disease (CGD). However, treatments for Aspergillus infections remain limited, and resistance to the few existing drug classes is emerging. Recently, the World Health Organization classified A. fumigatus as a critical priority fungal pathogen. Our cell death research identifies an important aspect of fungal biology that impacts susceptibility to leukocyte killing. Furthering our understanding of mechanisms that mediate the outcome of fungal-leukocyte interactions will increase our understanding of both the underlying fungal biology governing cell death and innate immune evasion strategies utilized during mammalian infection pathogenesis. Consequently, our studies are a critical step toward leveraging these mechanisms for novel therapeutic advances.

Keywords: Aspergillus fumigatus; cytochrome c; fungal virulence; reactive oxygen species; sterilizing immunity.

Fig 1

Cytochrome c sequence annotation in A. fumigatus. (A) Diagram of original annotation of A. fumigatus cycA. Top: diagram of genomic sequence including introns. Bottom: diagram of transcript size. Red arrows indicate the location of primers designed to test transcript size. (B) Diagram of corrected cycA annotation. Top: diagram of genomic sequence including introns. Bottom: diagram of corrected transcript. Red arrows indicate the location of primers designed to test transcript size. (C) Agarose gel displaying PCR products of primers (red arrows in Fig. 1A and B) for cDNA and gDNA. (D) Alignment of A. fumigatus cytochrome c to the sequences of other common organisms. (E) Phyre2 protein structure prediction of A. fumigatus cytochrome c protein (CycA) compared to human cytochrome c (CycS). (F) Representative images of the isogenic set used in this paper: CEA10 H2A:mRFP background strain, ∆cycA, and cycA+ grown on glucose minimal media (GMM).

Fig 2

Loss of cycA alters multiple cell death markers after treatment with H₂O₂. (A) Normalized percent CFUs. Percent untreated = (5 mM treated CFUs/untreated CFUs). Data are representatives of two biological repetitions including three technical repetitions each. Statistical analysis: one-way ANOVA with Bartlett’s test. (B) Schematic of flow cytometry—based cell death assay experimental design. Over 8 hours treatment with 10 mM H₂O₂, H2A:mRFP conidia were analyzed by flow cytometry for mRFP fluorescence and SYTOX Blue fluorescence by flow cytometry. (C) Percent of histone H2A:mRFP positive conidia through H₂O₂ treatment (left) and quantification of histone H2A:mRFP positive cells at 8 hours (right). (D) Percent of SYTOX Blue positive conidia through H₂O₂ treatment (left) and quantification of SYTOX Blue positive cells at 8 hours (right). Data for C and D are representatives of three biological repetitions including 15,000 events each. Statistical analysis: one-way ANOVA with Bartlett’s test.

Fig 3

Aspergillus fumigatus ∆cycA is resistant to lung leukocyte killing. (A) Representative flow plots of lung neutrophil conidial uptake and killing, analyzed based on RFP and AF633 fluorescence, 36 hpi with 3 × 10⁷ AF633+ CEA10:H2A (black), ∆cycA (magenta), or ∆cycA+ (teal) conidia. (B and C) The scatter plots indicate (B) conidial uptake (R1+R2) by and (C) conidial viability [R1/(R1+R2)] in indicated leukocyte subsets. (B and C) Dots represent individual mice and data are expressed as mean ± SEM. Statistical analysis: Kruskal–Wallis test with Dunn’s multiple comparisons test.

Fig 4

∆cycA conidia viability in NADPH oxidase deficient leukocytes. (A) Mice received a 1:1 mixture of p91phox^+/+ and p91phox^−/− bone marrow, were rested for 6 weeks, and then challenged with 3 × 10⁷ AF633-labeled CEA10:H2A (black), ∆cycA (magenta), or ∆cycA+ (teal). (B) Representative flow plots of lung neutrophil conidial uptake and killing from mixed bone marrow chimera mice, analyzed based on RFP and AF633 fluorescence, 36 hpi with 3 × 10⁷ AF633+ CEA10:H2A (black), ∆cycA (magenta), or ∆cycA+ (teal) conidia. (C) The scatter plot indicates conidial viability in p91phox^+/+ (white center) and p91phox^−/− (gray center) neutrophils. Dots represent individual mice with mean ± SEM indicated. Statistical analysis: Ordinary two-way ANOVA with main effects only and Sidak's multiple comparisons test, with a single pooled variance.

Fig 5

Oxidative stress-resistant Bir1^OE-N conidia show baseline transcript abundance variation. Transcriptional profiling of swollen conidia of strain ATCC46645-H2A:mRFP compared with an isogenic strain that features an over-expressed bir1 (Bir1^OE-N) gene (9). (A) Experimental set-up to determine gene expression in swollen conidia from both strains. (B) Differential gene expression comparing Bir1^OE-N to ATCC46645-H2A:mRFP, pink and green highlighting indicates genes with transcripts reduced and increased by more than logFC = 2, respectively. (C) Using FungiDB GO Term analysis, GO ”Mitochondria” gene transcripts were compared to Bir1^OE-N DEGs and visualized by heat map.