Inhibition of EGFR Signaling Protects from Mucormycosis

Abstract

Mucormycosis is a life-threatening, invasive fungal infection that is caused by various species belonging to the order Mucorales. Rhizopus species are the most common cause of the disease, responsible for approximately 70% of all cases of mucormycosis. During pulmonary mucormycosis, inhaled Rhizopus spores must adhere to and invade airway epithelial cells in order to establish infection. The molecular mechanisms that govern this interaction are poorly understood. We performed an unbiased survey of the host transcriptional response during early stages of Rhizopus arrhizus var. delemar (R. delemar) infection in a murine model of pulmonary mucormycosis using transcriptome sequencing (RNA-seq). Network analysis revealed activation of the host's epidermal growth factor receptor (EGFR) signaling. Consistent with the RNA-seq results, EGFR became phosphorylated upon in vitro infection of human alveolar epithelial cells with several members of the Mucorales, and this phosphorylated, activated form of EGFR colocalized with R. delemar spores. Inhibition of EGFR signaling with cetuximab or gefitinib, specific FDA-approved inhibitors of EGFR, significantly reduced the ability of R. delemar to invade and damage airway epithelial cells. Furthermore, gefitinib treatment significantly prolonged survival of mice with pulmonary mucormycosis, reduced tissue fungal burden, and attenuated the activation of EGFR in response to pulmonary mucormycosis. These results indicate EGFR represents a novel host target to block invasion of alveolar epithelial cells by R. delemar, and inhibition of EGFR signaling provides a novel approach for treating mucormycosis by repurposing an FDA-approved drug.IMPORTANCE Mucormycosis is an increasingly common, highly lethal fungal infection with very limited treatment options. Using a combination of in vivo animal models, transcriptomics, cell biology, and pharmacological approaches, we have demonstrated that Mucorales fungi activate EGFR signaling to induce fungal uptake into airway epithelial cells. Inhibition of EGFR signaling with existing FDA-approved drugs significantly increased survival following R. arrhizus var. delemar infection in mice. This study enhances our understanding of how Mucorales fungi invade host cells during the establishment of pulmonary mucormycosis and provides a proof-of-concept for the repurposing of FDA-approved drugs that target EGFR function.

Keywords: EGFR; Rhizopus; gefitinib; mucormycosis.

FIG 1

Host response to R. delemar infection in vivo and in vitro. (A) Mouse upstream regulators that are predicted to be changed in R. delemar-infected lungs in a mouse DKA model of mucormycosis. Red indicates predicted activation (Z score of >2). Teal indicates predicted repression (Z score of < −2). Black indicates no predicted effect. (B) Expression of known mouse EGFR targets in lungs 14 h postinoculation of the in vivo DKA model. (C) Expression of known human EGFR targets in A549 cells at 6 and 16 h following inoculation of an in vitro infection. Plotted for panels B and C are the log-transformed reads per kilobase per million (RPKM) values that have been normalized across all samples. Red indicates high gene expression; blue indicates low expression. Each column in panels B and C represents an individual sample from a different mouse.

FIG 2

Phosphorylation and localization of EGFR in A549 cells infected with Mucorales. (A) Representative immunoblot examining tyrosine phosphorylation of EGFR residue Y1068 in response to individual infection with five different Mucorales fungi. (B) Densitometric analysis of the immunoblots. ctrl, control.

FIG 3

Localization of phosphor-EGFR in A549 cells infected with R. delemar. A549 cells were infected for 30 min with 2 × 10⁵ R. delemar spores that had been germinated for 1 h. Cells were then stained for phospho-EGFR (red) and host cell nuclei using Hoechst (blue). DIC, differential inference contrast.

FIG 4

Effects of EGFR inhibition on invasion of airway epithelial cells by R. delemar. A549 alveolar epithelial cells were pretreated with vehicle, 25 µM gefitinib, or 25 µg/ml cetuximab for 1 h followed by (top) 3 h of infection in the presence of inhibitor with 2 × 10⁵ R. delemar spores or (bottom) 24 h of infection (in the presence of inhibitors) with 2 × 10⁶ R. delemar spores that were germinated for 1 h. *, P < 0.0001, and **, P < 0.01, versus control by Wilcoxon rank-sum test. Data are expressed as median ± interquartile range and represent at least two independent experiments.

FIG 5

Gefitinib inhibits infection-induced damage of airway epithelial cells by four different species of Mucorales. A549 alveolar epithelial cells were pretreated with vehicle or gefitinib (25 or 50 µM) for 1 to 2 h followed by 24 h of infection (in the presence of inhibitors) with 2 × 10⁶ (C. bertholletiae and R. oryzae) or 3 × 10⁶ (L. corymbifera and M. circineollides) spores that were germinated for 1 h *, P < 0.0001, and **, P < 0.01, versus the control by Wilcoxon rank sum test. Data are expressed as median ± interquartile range and represent at least two independent experiments done in triplicate.

FIG 6

Gefitinib inhibits EGFR phosphorylation in vivo and protects mice from pulmonary mucormycosis. (A) Survival of neutropenic mice (n = 20/group from two independent experiments with similar results) infected intratracheally with R. delemar (average inoculum of 3.8 × 10³ spores per mouse) and treated with vehicle control (placebo) or 10 mg/kg gefitinib 4 h postinfection for 5 consecutive days. *, P = 0.0084 versus placebo-treated mice by log rank test. (B) Tissue fungal burden of lungs and brains of mice (n = 10 per group) infected intratracheally with R. delemar (5.6 × 10³ spores per mouse of confirmed inoculum) and treated with vehicle control (placebo) or gefitinib. Data are presented as median ± interquartile range. (C) Twenty-four hours post-intratracheal infection, lungs of gefitinib (GEF)-treated or untreated mice were harvested, sectioned, and stained with calcofluor white (CFW) and anti-pY1068 antibody. Scale bars, 20 µm. Dashed boxes in the top row of panels indicate the sections shown enlarged in the bottom row. ctrl, control.