Inhibition of EGFR Signaling Protects from Mucormycosis

Watkins, Tonya; Gebremariam, Teclegiorgis; Swidergall, Marc; Shetty, Amol C.; Graf, Karen T.; Alqarihi, Abdullah; Alkhazraji, Sondus; Alsaadi, Abrar I.; Edwards, Vonetta L.; Filler, Scott G.; Ibrahim, Ashraf S.; Bruno, Vincent M.

doi:10.1128/mbio.01384-18

Cited by 52 publications

(58 citation statements)

References 51 publications

(59 reference statements)

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“…To investigate the nature of this cross-talk, we tested the effects of inhibiting EGFR on C. albicans -induced EphA2 activation. When EGFR was either knocked down with siRNA or inhibited with the specific EGFR kinase inhibitor, gefitinib (19), EphA2 phosphorylation was transient, occurring within 30 min of infection, but declining to basal levels by 90 min (Fig. 1C, D; S1).…”

Section: Resultsmentioning

confidence: 99%

Activation of oral epithelial EphA2-EFGR signaling by Candida albicans virulence factors

Swidergall

Millet

et al. 2018

Preprint

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19The induction of antifungal proinflammatory signaling during oropharyngeal candidiasis (OPC) is 20 crucial to limit Candida albicans proliferation and induce fungal clearance. Previously, we 21 determined that the ephrin type-A receptor 2 (EphA2) functions as a β-glucan receptor that senses 22 Importance 39Host cell receptors for fungi have typically been evaluated from one of two different perspectives, 40 their role in inducing the invasion of the organism or their role in stimulating the host inflammatory 41 response. We had found previously that EGFR mediates the endocytosis of C. albicans by oral 42 epithelial cells, and that epithelial cell EphA2 mediates the production of chemokines and 43 proinflammatory cytokines in response to C. albicans. Here, we demonstrate that EGFR and 44EphA2 interact with each other, both physically and functionally. EGFR is required for sustained 45EphA2 activation and for epithelial cells to secrete proinflammatory mediators in response to both 46 C. albicans and IL-17A. Therefore, while activation of EGFR by C. albicans enhances 47 pathogenicity by inducing the endocytosis of the fungus, it also augments the host defense 48 against OPC by stimulating the host inflammatory response. 49 50

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Section: Resultsmentioning

confidence: 99%

Activation of oral epithelial EphA2-EFGR signaling by Candida albicans virulence factors

Swidergall

Millet

et al. 2018

Preprint

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“…24 hours post infection, the supernatant was collected to quantify LDH levels (The CytoTox 96® Non-Radioactive Cytotoxicity Assay, Promega) or FGF-2 protein levels using ELISA (R&D systems). The LDH release was calculated as described previously (82).…”

Section: Methodsmentioning

confidence: 99%

A novel resistance pathway for calcineurin inhibitors in the human pathogenic MucoralesMucor circinelloides

Vellanki

Billmyre

Lorenzen

et al. 2019

Preprint

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24Mucormycosis is an emerging lethal fungal infection in immunocompromised 25 patients. Mucor circinelloides (Mucor) is a causal agent of mucormycosis and serves as 26 a model system to understand genetics in Mucorales. Calcineurin is a conserved 27 virulence factor in many pathogenic fungi and calcineurin inhibition or deletion of the 28 calcineurin regulatory subunit (CnbR) in Mucor results in a shift from hyphal to yeast 29 growth. We analyzed thirty-six calcineurin inhibitor resistant or bypass mutants that 30 exhibited hyphal growth in the presence of calcineurin inhibitors or in the yeast-locked 31 cnbR mutant background without carrying any mutations in known calcineurin 32 components. We found that a majority of the mutants had altered sequence in a gene, 33 named here bycA (bypass of calcineurin A). bycA encodes an amino acid permease. 34 We verified that both bycA, and the bycA cnbR double mutant are resistant to the 35 calcineurin inhibitor FK506, thereby demonstrating a novel resistance mechanism 36 against calcineurin inhibitors. We also found that the expression of bycA was 37 significantly higher in the wild type with FK506 and in the cnbR mutants, but 38 significantly lower in the wild type without FK506. These findings suggest that bycA is a 39 negative regulator of hyphal growth and/or a positive regulator of yeast growth in Mucor 40 and calcineurin suppresses the bycA gene at the mRNA level to promote hyphal growth. 41BycA is involved in the Mucor hyphal-yeast transition as our data demonstrates a 42 positive correlation between bycA expression, protein kinase A activity, and Mucor 43 yeast-growth. Also, calcineurin activity rather than hyphal morphology primarily 44 contributes to virulence traits including phagosome maturation blockade, host cell 45 damages, and pro-angiogenic growth factor induction during interactions with hosts. 46 3 Importance 47 Mucor is intrinsically resistant to most known antifungals, which makes 48 mucormycosis treatment challenging. Calcineurin is a serine/threonine phosphatase 49 widely conserved across eukaryotes. When calcineurin function is inhibited in Mucor, 50 growth shifts to a less-virulent yeast growth form which makes calcineurin an attractive 51 target for development of new antifungal drugs. Previously we identified two distinct 52 mechanisms through which Mucor can become resistant to calcineurin inhibitors 53 involving Mendelian mutations in the gene for FKBP12, calcineurin A or B subunits and 54 epimutations silencing the FKBP12 gene. Here, we identified a third novel mechanism 55 where loss of function mutations in the amino acid permease encoding the bycA gene 56 contribute to resistance against calcineurin inhibitors. When calcineurin activity is 57 absent, BycA can activate PKA to promote yeast growth via a cAMP-independent 58 pathway. Our data also shows that calcineurin activity, not morphology, primarily 59 contributes to host -pathogen interactions in the pathogenesis of Mucor. 60 61 62

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“…At 24 h postinfection, the supernatant was collected to quantify LDH levels (CytoTox 96 nonradioactive cytotoxicity assay; Promega) or FGF-2 protein levels using ELISA (R&D Systems). The level of LDH release was calculated as described previously (83).…”

Section: A Novel Link Between Calcineurin and Byca An Amino Acid Permentioning

confidence: 99%

A Novel Resistance Pathway for Calcineurin Inhibitors in the Human-Pathogenic Mucorales Mucor circinelloides

Vellanki

Billmyre

Lorenzen

et al. 2020

mBio

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Mucormycosis is an emerging lethal fungal infection in immunocompromised patients. Mucor circinelloides is a causal agent of mucormycosis and serves as a model system to understand genetics in Mucorales. Calcineurin is a conserved virulence factor in many pathogenic fungi, and calcineurin inhibition or deletion of the calcineurin regulatory subunit (CnbR) in Mucor results in a shift from hyphal to yeast growth. We analyzed 36 calcineurin inhibitor-resistant or bypass mutants that exhibited hyphal growth in the presence of calcineurin inhibitors or in the yeast-locked cnbRΔ mutant background without carrying any mutations in known calcineurin components. We found that a majority of the mutants had altered sequence in a gene, named here bycA (bypass of calcineurin). bycA encodes an amino acid permease. We verified that both the bycAΔ single mutant and the bycAΔ cnbRΔ double mutant are resistant to calcineurin inhibitor FK506, thereby demonstrating a novel mechanism of resistance against calcineurin inhibitors. We also found that the level of expression of bycA was significantly higher in the wild-type strain treated with FK506 and in the cnbRΔ mutants but was significantly lower in the wild-type strain without FK506 treatment. These findings suggest that bycA is a negative regulator of hyphal growth and/or a positive regulator of yeast growth in Mucor and that calcineurin suppresses expression of the bycA gene at the mRNA level to promote hyphal growth. BycA is involved in the Mucor hypha-yeast transition as our data demonstrate positive correlations among bycA expression, protein kinase A activity, and Mucor yeast growth. Also, calcineurin, independently of its role in morphogenesis, contributes to virulence traits, including phagosome maturation blockade, host cell damages, and proangiogenic growth factor induction during interactions with hosts. IMPORTANCE Mucor is intrinsically resistant to most known antifungals, which makes mucormycosis treatment challenging. Calcineurin is a serine/threonine phosphatase that is widely conserved across eukaryotes. When calcineurin function is inhibited in Mucor, growth shifts to a less virulent yeast growth form, which makes calcineurin an attractive target for development of new antifungal drugs. Previously, we identified two distinct mechanisms through which Mucor can become resistant to calcineurin inhibitors involving Mendelian mutations in the gene for FKBP12, including mechanisms corresponding to calcineurin A or B subunits and epimutations silencing the FKBP12 gene. Here, we identified a third novel mechanism where loss-of-function mutations in the amino acid permease corresponding to the bycA gene contribute to resistance against calcineurin inhibitors. When calcineurin activity is absent, BycA can activate protein kinase A (PKA) to promote yeast growth via a cAMP-independent pathway. Our data also show that calcineurin activity contributes to host-pathogen interactions primarily in the pathogenesis of Mucor.

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Inhibition of EGFR Signaling Protects from Mucormycosis

Cited by 52 publications

References 51 publications

Activation of oral epithelial EphA2-EFGR signaling by Candida albicans virulence factors

Activation of oral epithelial EphA2-EFGR signaling by Candida albicans virulence factors

A novel resistance pathway for calcineurin inhibitors in the human pathogenic MucoralesMucor circinelloides

A Novel Resistance Pathway for Calcineurin Inhibitors in the Human-Pathogenic Mucorales Mucor circinelloides

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