Mucor circinelloides Thrives inside the Phagosome through an Atf-Mediated Germination Pathway

Abstract

Mucormycosis is an emerging fungal infection that is often lethal due to the ineffectiveness of current therapies. Here, we have studied the first stage of this infection-the germination of Mucor circinelloides spores inside phagocytic cells-from an integrated transcriptomic and functional perspective. A relevant fungal gene network is remodeled in response to phagocytosis, being enriched in crucial functions to survive and germinate inside the phagosome, such as nutritional adaptation and response to oxidative stress. Correspondingly, the phagocytic cells induced a specific proinflammatory and apoptotic response to the pathogenic strain. Deletion of fungal genes encoding putative transcription factors (atf1, atf2, and gcn4), extracellular proteins (chi1 and pps1), and an aquaporin (aqp1) revealed that these genes perform important roles in survival following phagocytosis, germination inside the phagosome, and virulence in mice. atf1 and atf2 play a major role in these pathogenic processes, since their mutants showed the strongest phenotypes and both genes control a complex gene network of secondarily regulated genes, including chi1 and aqp1 These new insights into the initial phase of mucormycosis define genetic regulators and molecular processes that could serve as pharmacological targets.IMPORTANCE Mucorales are a group of ancient saprophytic fungi that cause neglected infectious diseases collectively known as mucormycoses. The molecular processes underlying the establishment and progression of this disease are largely unknown. Our work presents a transcriptomic study to unveil the Mucor circinelloides genetic network triggered in fungal spores in response to phagocytosis by macrophages and the transcriptional response of the host cells. Functional characterization of differentially expressed fungal genes revealed three transcription factors and three extracellular proteins essential for the fungus to survive and germinate inside the phagosome and to cause disease in mice. Two of the transcription factors, highly similar to activating transcription factors (ATFs), coordinate a complex secondary gene response involved in pathogenesis. The significance of our research is in characterizing the initial stages that lead to evasion of the host innate immune response and, in consequence, the dissemination of the infection. This genetic study offers possible targets for novel antifungal drugs against these opportunistic human pathogens.

Keywords: emerging pathogens; host-pathogen interaction; innate immunity; mucormycosis; transcriptomics.

FIG 1

Mucor response to mouse macrophages. (A) Heat map showing Mucor genes that are differentially expressed (Q ≤ 0.05) in virulent R7B and avirulent NRRL3631 spores phagocytosed by mouse macrophages (Mφ) (cell line J774A.1) during coculture for 5 h in L15 cell culture medium. Differential expression values with respect to noninteraction cultures (without macrophages) were calculated for each strain and hierarchically clustered according to similarities. Red and blue represent up- and downregulated genes, respectively. Clusters 1 (C1) and 4 (C4) contain genes up- and downregulated, respectively, in both Mucor strains during phagocytosis, which was defined as the general response. Clusters 2 (C2) and 3 (C3) contain genes up- and downregulated, respectively, in the virulent Mucor strain R7B during phagocytosis, comprising the virulence-specific response. (B) Functional enrichment analysis showing the percentages of genes for each KOG class found in the Mucor genome (gray) and in the general (green) and virulence-specific (red) responses. Asterisks (*) indicate a significant enrichment (P ≤ 0.05, Fisher’s exact test) in the corresponding KOG class with respect to the expected number of genes found in the Mucor genome. FC, fold change.

FIG 2

Macrophages respond exclusively to the virulent Mucor strain. (A) Differential expression (Q ≤ 0.05) of upregulated (red) and downregulated (blue) mouse macrophage (Mφ) (cell line J774A.1) genes induced after phagocytosing spores from each depicted Mucor strain in L15 cell culture medium for 5 h. Differential expression values were calculated by comparison with expression in noninteraction cultures (without spores). (B) Percentage of genes for each significantly enriched (adjusted P value, ≤0.01) KEGG pathway found in the macrophage response to the virulent Mucor R7B strain compared to the expected percentage of genes found in the mouse genome. (C) Differential expression values (Q ≤ 0.05) of selected mouse macrophage (Mφ) (cell line J774A.1) genes induced after phagocytosing spores from each depicted Mucor strain for 5 h. The selected genes are involved in two enriched GO Biological Processes (adjusted P value, ≤0.05), Immune System Process and Apoptotic Process. FC, fold change.

FIG 3

In vitro and in vivo gene expression analysis of selected Mucor genes. (A) RT-qPCR-determined differences in the expression of selected Mucor genes during mouse macrophage phagocytosis. Transcript levels were quantified in cDNA obtained from virulent Mucor strain R7B spores cocultured with mouse macrophages (Mφ) (cell line J774A.1) in L15 cell culture medium for 5 h and from the same spores cultured in noninteraction controls (without macrophages). Values were normalized using 18S rRNA as an internal control, and the differential expression (log₂-fold change) was calculated with respect to expression in noninteraction samples. (B) RT-qPCR-determined differences in the expression of the selected genes during interaction with peritoneal macrophages. Transcript levels were quantified in cDNA obtained from virulent Mucor strain R7B spores that were injected into the peritoneal cavity of OF-1 mice and recovered after 5 h, as well as from the same spores incubated in L15 cell culture medium as a control. Values were normalized using 18S rRNA as an internal control, and the differential expression (log₂-fold change) was calculated with respect to the expression in L15 control samples. (C) Comparison of differential expression of selected genes during in vitro interaction with mouse macrophages, quantified by both RNA-seq and RT-qPCR, and during in vivo interaction with peritoneal macrophages, quantified by RT-qPCR. Error bars correspond to the standard deviations (SD) of the results from technical triplicates, and asterisks indicate a significant difference determined by unpaired t test (*, P ≤ 0.05; **, P ≤ 0.005; ***, P < 0.0001).

FIG 4

Phenotypic analyses of Mucor deletion mutants in selected genes during phagocytosis and mouse infection. (A) Spore fitness of strains with the indicated mutations after 5 h of macrophage phagocytosis, assessed by their ability to develop healthy colonies on MMC medium. Control plates contain spores obtained after growing in cell culture medium for 5 h without macrophages. R7B was used as the wild-type (WT) strain. All MMC plates were incubated for 48 h. (B) Polarity index measures after phagocytosis for all deletion strains generated compared to those of the wild-type control (R7B). Error bars correspond to the standard errors of the means (SEM) from technical replicates (n = 50 spores), and statistical significance was analyzed by the unpaired t test. (C) Virulence assays with immunosuppressed mice. Each color shows the survival curve for a group of 10 mice infected with 1 × 10⁶ spores from one of the deletion mutant strains. Survival rates were compared to the results for mice infected with a virulent control strain (R7B) and statistically analyzed by a Mantel-Cox test. NRRL3631 was used as an avirulent control strain. Asterisks (*) indicate a significant difference determined by the unpaired t test (*, P ≤ 0.05; **, P ≤ 0.005; ***, P < 0.0001).

FIG 5

Atf1- and Atf2-regulated genes. (A) Venn diagram showing the relationships between differentially expressed genes (Q ≤ 0.05) in Mucor atf1Δ and atf2Δ mutants. Differential expression values were calculated as the ratio between the expression in virulent wild-type strain R7B and in each depicted mutant, either cocultured with mouse macrophages (cell line J774A.1) in L15 cell culture medium for 5 h (Mφ) or grown in liquid MMC, pH 4.5, for 24 h (c24h). Overlapping genes shared similar expression values (up- or downregulation) under the depicted conditions. (B) Percentages of genes regulated by Atf1 (red and orange) and Atf2 (dark and light blue) and of all genes found in the genome (gray) for each KOG class. Asterisks (*) indicate a significant enrichment (P ≤ 0.05, Fisher’s exact test) in the corresponding KOG class with respect to the expected number of genes found in the Mucor genome. (C) Analysis of genes upregulated (Q ≤ 0.05, fold change of ≥2.0) in the virulent wild-type strain R7B compared to their expression in either the Mucor atf1Δ or atf2Δ mutant cocultured with mouse macrophages (cell line J774A.1) for each enriched KOG class found in panel B. Z score was calculated as the deviation from the mean FPKM (fragments per kilobase of transcript per million mapped reads) value in standard deviation units for Mucor atf1Δ and atf2Δ mutants and strain R7B cocultured with mouse macrophages (Atf1+Mφ, Atf2+Mφ, and R7B+Mφ) and for R7B without mouse macrophages (R7Bc). Red gene IDs appear in more than one KOG class.