GRP78 and Integrins Play Different Roles in Host Cell Invasion during Mucormycosis

Abstract

Mucormycosis, caused by Rhizopus species, is a life-threatening fungal infection that occurs in patients immunocompromised by diabetic ketoacidosis (DKA), cytotoxic chemotherapy, immunosuppressive therapy, hematologic malignancies, or severe trauma. Inhaled Rhizopus spores cause pulmonary infections in patients with hematologic malignancies, while patients with DKA are much more prone to rhinoorbital/cerebral mucormycosis. Here, we show that Rhizopus delemar interacts with glucose-regulated protein 78 (GRP78) on nasal epithelial cells via its spore coat protein CotH3 to invade and damage the nasal epithelial cells. Expression of the two proteins is significantly enhanced by high glucose, iron, and ketone body levels (hallmark features of DKA), potentially leading to frequently lethal rhinoorbital/cerebral mucormycosis. In contrast, R. delemar CotH7 recognizes integrin β1 as a receptor on alveolar epithelial cells, causing the activation of epidermal growth factor receptor (EGFR) and leading to host cell invasion. Anti-integrin β1 antibodies inhibit R. delemar invasion of alveolar epithelial cells and protect mice from pulmonary mucormycosis. Our results show that R. delemar interacts with different mammalian receptors depending on the host cell type. Susceptibility of patients with DKA primarily to rhinoorbital/cerebral disease can be explained by host factors typically present in DKA and known to upregulate CotH3 and nasal GRP78, thereby trapping the fungal cells within the rhinoorbital milieu, leading to subsequent invasion and damage. Our studies highlight that mucormycosis pathogenesis can potentially be overcome by the development of novel customized therapies targeting niche-specific host receptors or their respective fungal ligands.IMPORTANCE Mucormycosis caused by Rhizopus species is a fungal infection with often fatal prognosis. Inhalation of spores is the major route of entry, with nasal and alveolar epithelial cells among the first cells that encounter the fungi. In patients with hematologic malignancies or those undergoing cytotoxic chemotherapy, Rhizopus causes pulmonary infections. On the other hand, DKA patients predominantly suffer from rhinoorbital/cerebral mucormycosis. The reason for such disparity in disease types by the same fungus is not known. Here, we show that the unique susceptibility of DKA subjects to rhinoorbital/cerebral mucormycosis is likely due to specific interaction between nasal epithelial cell GRP78 and fungal CotH3, the expression of which increases in the presence of host factors present in DKA. In contrast, pulmonary mucormycosis is initiated via interaction of inhaled spores expressing CotH7 with integrin β1 receptor, which activates EGFR to induce fungal invasion of host cells. These results introduce a plausible explanation for disparate disease manifestations in DKA versus those in hematologic malignancy patients and provide a foundation for development of therapeutic interventions against these lethal forms of mucormycosis.

Keywords: GRP78; Rhizopus; cell invasion; epithelial cells; integrin β1; mice; mucormycosis.

FIG 1

R. delemar-mediated invasion and damage of nasal and alveolar epithelial cells. R. delemar invasion of nasal (A) or alveolar (B) epithelial cells was determined using differential fluorescence assays by staining with 1% Uvitex for 1 h, while the damage assay was performed using the ⁵¹Cr release method. ***, P < 0.0001; **, P < 0.001 compared to the first time point in each panel. Data are presented as medians ± interquartile ranges from 3 independent experiments.

FIG 2

GRP78 is a nasal epithelial cell receptor, while integrin α3β1 is an alveolar epithelial cell receptor during Mucorales interaction. Biotinylated nasal (A) or alveolar (B) epithelial cells were incubated with R. delemar germlings, and unbound proteins were removed with repeated washing. Bound proteins were separated by SDS-PAGE and identified by Western blotting using an anti-biotin monoclonal antibody (Ab; top); the identity of the proteins was confirmed to be GRP78 (78 kDa) for nasal (A) or integrin β1 (130 kDa) (B) by using anti-GRP78 or anti-integrin α3β1 antibodies, respectively (bottom). Affinity purification of GRP78 (C) or integrin β1 (D) by other Mucorales. Anti-GRP78 and anti-integrin antibodies block R. delemar-mediated invasion and subsequent damage of nasal (E) and alveolar (F) epithelial cells, respectively, compared to that with the isotype matched-IgG. Both antibodies had no effect on adherence of the fungus to host cells. Data in panels E and F are expressed as medians ± interquartile ranges from 3 independent experiments. Different colors were used to simplify the graph: purple, isotype IgG; green, anti-GRP78 Ab; and yellow, anti-integrin β1 Ab.

FIG 3

Integrin α3β1 is required for R. delemar-mediated host cell invasion and damage. (A) R. delemar has reduced invasion and damage of GD25 fibroblast cell line lacking integrin β1, compared to β1GD25, an integrin β1-restored fibroblast cell line. Adhesion and invasion of GD25 and β1GD25 fibroblast cell lines were assessed using differential fluorescence assays, while host cell damage was assessed using the ⁵¹Cr release method. (B) Confocal microscopy images showing the accumulation of integrin α3β1 around R. delemar during infection of alveolar epithelial cells. Images were taken after 2.5 h of incubation of the fungus with the host cells (C). Anti-integrin α3β1 monoclonal antibody blocks R. delemar-mediated invasion of alveolar epithelial cells. Alveolar epithelial cells were incubated with 5 μg/ml of different anti-integrin antibodies or isotype-matched IgG for 1 h prior to infection with R. delemar. Data in panels A and C are expressed as medians ± interquartile ranges from 3 independent experiments.

FIG 4

Anti-integrin antibodies block activation of alveolar epithelial cell EGFR. Representative immunoblots (A) and densitometric analysis (B) show that R. delemar infection induced phosphorylation of EGFR on tyrosine residue 1068 compared to that in the control and that anti-integrin β1 antibody blocked it. Data in panel B are means ± standard deviations from three independent experiments.

FIG 5

CotH3 is the R. delemar cell-surface ligand to GRP78 on nasal epithelial cells. (A) Far-Western blot of R. delemar surface proteins that bound to GRP78. (B) Affinity purification of nasal cell GRP78 by S. cerevisiae cells expressing CotH3 identified by anti-GRP78 antibody. Dashed line represents cropped image from GRP78 blot shown in Fig. 2A. (C) Confocal microscopy images showing interaction of nasal epithelial GRP78 and R. delemar CotH3 after a 2.5-h incubation shown by proximity ligation assay (PLA). DAPI staining was used to identify host cells. (D) Inhibition of CotH3 expression by RNAi reduced the ability of R. delemar to invade (by differential fluorescence) and damage (by ⁵¹Cr release method) nasal epithelial cells compared to that with empty plasmid-transformed R. delemar. Anti-CotH3 antibody blocked R. delemar-mediated invasion of and damage to nasal epithelial cells. Data in panels D and E are expressed as medians ± interquartile ranges from 3 independent experiments.

FIG 6

CotH7 is the R. delemar cell surface ligand to integrin α3β1. (A) Far-Western blot of R. delemar surface proteins that bound to integrin. (B) Inhibition of CotH7 expression by RNAi reduced the ability of R. delemar to damage alveolar epithelial cells compared to that with empty plasmid-transformed R. delemar. Data in panel B are expressed as medians ± interquartile ranges from 3 independent experiments.

FIG 7

DKA host factors increase nasal epithelial cell GRP78 expression and host cell susceptibility to R. delemar-mediated invasion and damage. (A) Nasal epithelial cells were incubated with physiologically elevated concentrations of glucose, iron, or BHB for 5 h, and GRP78 gene expression was determined by quantitative reverse transcription-PCR (qRT-PCR). Elevated concentrations of glucose, iron, or BHB significantly enhanced R. delemar-mediated nasal epithelial cell invasion (B) and damage (C). Fold changes were calculated by comparison to the lowest concentration of the exogenous factors used. Data are expressed as medians ± interquartile ranges from 3 independent experiments.

FIG 8

DKA host factors have no effect on integrin β1 expression levels and did not affect R. delemar interactions with alveolar epithelial cells. (A) Alveolar epithelial cells were incubated with physiologically elevated concentrations of glucose, iron, or BHB for 5 h, and integrin β1 gene expression was determined by qRT-PCR. Elevated concentrations of glucose, iron, or BHB had no effect on R. delemar-mediated alveolar epithelial cell invasion (B) or subsequent damage (C). Data are expressed as medians ± interquartile ranges from 3 independent experiments.

FIG 9

Anti-integrin β1 antibodies protect immunosuppressed mice from invasive pulmonary mucormycosis due to R. delemar. ICR mice (n = 10 [5 female and 5 male]/group with no difference in survival among the two sexes]) were immunosuppressed on days −2, +3, and +8 with cyclophosphamide and cortisone acetate and infected on day 0 intratracheally with R. delemar (actual inhaled inoculum of 2.8 × 10³/mouse). Twenty-four hours postinfection, mice were treated with a single dose of 100 μg of either an isotype-matched IgG (control) or an anti-integrin β1 antibody. P = 0.0006 by log rank test.

FIG 10

A diagram showing the molecular pathogenesis of the two main manifestations of mucormycosis. (A) R. delemar inhaled spores are trapped in the sinus cavities of patients with DKA due to the overexpression of GRP78 on nasal epithelial cells, and the interaction with fungal CotH3 results in rhinoorbital/cerebral mucormycosis. Colored circles represent elevated levels of glucose, iron, and ketone bodies. (B) In immunosuppressed patients, inhaled spores reach the alveoli and bind to integrin α3β1 via fungal CotH7, thereby triggering activation of EGFR and subsequent invasion and pulmonary infection.