PKC Signaling Regulates Drug Resistance of the Fungal Pathogen Candida albicans via Circuitry Comprised of Mkc1, Calcineurin, and Hsp90

Fungal pathogens exploit diverse mechanisms to survive exposure to antifungal drugs. This poses concern given the limited number of clinically useful antifungals and the growing population of immunocompromised individuals vulnerable to life-threatening fungal infection. To identify molecules that abrogate resistance to the most widely deployed class of antifungals, the azoles, we conducted a screen of 1,280 pharmacologically active compounds. Three out of seven hits that abolished azole resistance of a resistant mutant of the model yeast Saccharomyces cerevisiae and a clinical isolate of the leading human fungal pathogen Candida albicans were inhibitors of protein kinase C (PKC), which regulates cell wall integrity during growth, morphogenesis, and response to cell wall stress. Pharmacological or genetic impairment of Pkc1 conferred hypersensitivity to multiple drugs that target synthesis of the key cell membrane sterol ergosterol, including azoles, allylamines, and morpholines. Pkc1 enabled survival of cell membrane stress at least in part via the mitogen activated protein kinase (MAPK) cascade in both species, though through distinct downstream effectors. Strikingly, inhibition of Pkc1 phenocopied inhibition of the molecular chaperone Hsp90 or its client protein calcineurin. PKC signaling was required for calcineurin activation in response to drug exposure in S. cerevisiae. In contrast, Pkc1 and calcineurin independently regulate drug resistance via a common target in C. albicans. We identified an additional level of regulatory control in the C. albicans circuitry linking PKC signaling, Hsp90, and calcineurin as genetic reduction of Hsp90 led to depletion of the terminal MAPK, Mkc1. Deletion of C. albicans PKC1 rendered fungistatic ergosterol biosynthesis inhibitors fungicidal and attenuated virulence in a murine model of systemic candidiasis. This work establishes a new role for PKC signaling in drug resistance, novel circuitry through which Hsp90 regulates drug resistance, and that targeting stress response signaling provides a promising strategy for treating life-threatening fungal infections

IL-8 response in AEC co-stimulated with PAF and UV killed SA, or SAF from Δ<i>hla</i> and Δ<i>hlb</i> mutants.

<p><b>A</b>) IL-8 response to UV-killed SA. Beas-2B cells were stimulated with PAF (2.5% v/v) and/or a suspension of UV-killed whole SA bacteria (10⁸ CFU/mL) for 6h. <b>B</b>) IL-8 response to Δ<i>hla</i> and Δ<i>hlb</i> SAF. Beas-2B cells were stimulated with PAF (2.5% v/v) and/or SAF (10% v/v) for 6h. PAF and SAF were prepared from bacterial cultures grown in TSB medium for 24h. WT = 8325–4 SA wild-type parental strain; Δ<i>hla</i> and Δ<i>hlb</i> are its isogenic mutants. In A and B, extracellular IL-8 levels were measured in the AEC supernatant by ELISA after stimulation. Results are shown as mean (±SEM) of four independent biological replicates. *P<0.05, ***P<0.001 compared to PAF alone and to SAF WT, using 1-way ANOVA, followed by multiple comparisons Bonferroni correction test.</p

IL-8 response in AECs stimulated with PAF or SAF alone, or PAF+SAF co-stimulation.

<p>A) Time-dependent IL-8 response to PAF and SAF. Beas-2B cells were stimulated with PAF (2.5% v/v) and/or SAF (10% v/v) for the indicated duration. B) Dose-dependent IL-8 response to PAF+SAF co-stimulation. Beas-2B cells were stimulated for 6h with PAF (2.5% v/v) and SAF at the following concentration: + = 2.5% v/v; ++ = 5% v/v; +++ = 10% v/v. C) Time-dependent IL-8 response to PAF+SAF co-stimulation. Beas-2B cells were stimulated with PAF (2.5% v/v) and/or SAF (10% v/v) for the indicated duration. D) IL-8 mRNA response to PAF, SAF and PAF+SAF co-stimulation. Beas-2B cells were stimulated with PAF (2.5% v/v) and/or SAF (10% v/v) for 1h. Relative mRNA expression of IL-8 / GAPDH are expressed as fold increase. In A, B and C, extracellular IL-8 levels were measured in the AEC conditioned supernatant by ELISA after stimulation. Stimulation with LB medium was used as control. Results represent the mean (±SEM) of n≥3 independent biological replicates. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared to PAF alone at the similar time point using 2-way ANOVA (A and C) and 1-way ANOVA (B and D), followed by multiple comparisons Bonferroni correction test.</p

LasR-deficient Pseudomonas aeruginosa variants increase airway epithelial mICAM-1 expression and enhance neutrophilic lung inflammation.

Pseudomonas aeruginosa causes chronic airway infections, a major determinant of lung inflammation and damage in cystic fibrosis (CF). Loss-of-function lasR mutants commonly arise during chronic CF infections, are associated with accelerated lung function decline in CF patients and induce exaggerated neutrophilic inflammation in model systems. In this study, we investigated how lasR mutants modulate airway epithelial membrane bound ICAM-1 (mICAM-1), a surface adhesion molecule, and determined its impact on neutrophilic inflammation in vitro and in vivo. We demonstrated that LasR-deficient strains induce increased mICAM-1 levels in airway epithelial cells compared to wild-type strains, an effect attributable to the loss of mICAM-1 degradation by LasR-regulated proteases and associated with enhanced neutrophil adhesion. In a subacute airway infection model, we also observed that lasR mutant-infected mice displayed greater airway epithelial ICAM-1 expression and increased neutrophilic pulmonary inflammation. Our findings provide new insights into the intricate interplay between lasR mutants, LasR-regulated proteases and airway epithelial ICAM-1 expression, and reveal a new mechanism involved in the exaggerated inflammatory response induced by lasR mutants

SAF represses PAF and TLR1/2 dependent NF-κB activity.

<p>Beas-2B cells stably expressing a NF-κB promoter-reporter (pGL4.28NF-κB) were stimulated with PAF (2.5% v/v), Pam₃CysSK₄ (10 μg/ml) and/or with SAF (10% v/V) for 3h. Where indicated, Beas-2B cells were pre-treated with the IKKβ inhibitor Bi605906 (7.5 μg/mL) 1h prior to stimulation with Pam₃CysSK₄. Following stimulation, cells were lysed, and the relative luminescence (RLU) was measured in the supernatant using a luciferase assay to determine NF-κB activity. LB media was used as a control. Results are shown as mean (±SEM) of n≥3 independent biological replicates. ****P<0.0001 compared to PAF stimulation alone using 1-way ANOVA, followed by multiple comparisons Bonferroni correction test.</p

AEC IL-8 responses to co-stimulation with different SA and PA strains.

<p><b>A</b>). IL-8 responses to co-stimulation of PAF+SAF from different SA strains. Beas-2B cells were stimulated with SA filtrates (10% v/v) alone or co-stimulated with PAO1 PAF (2.5% v/v) for 6 h. <b>B</b>). IL-8 responses to co-stimulation of PAF+SAF from different PA strains. Beas-2B cells were stimulated with filtrates from different PA strains (2.5% v/v) alone, or co-stimulated with ATCC29213 SAF (10% v/v) for 6 h. Extracellular IL-8 levels were measured in the AEC supernatant by ELISA after stimulation. All results are shown as mean (±SEM) of n≥4 independent biological replicates. *P<0.05, **P<0.01, ***P<0.001 compared to PAF alone using an unpaired two-tailed student’s t-test.</p

IL-8 responses in CFBE41o- cells stimulated with PAF and SAF.

<p>The CFBE41o- airway epithelial cells (CFTR Δ<i>F508</i> homozygotes) were stimulated for 6h with PAF (2.5% v/v) and/or SAF at the following volumes. + = 2.5% v/v; ++ = 5% v/v;. Extracellular IL-8 levels were measured in the AEC supernatant by ELISA after stimulation. Results are shown as mean (±SEM) of four independent biological replicates. *P<0.05, **P<0.01 compared to PAF alone using 2-way ANOVA, followed by multiple comparisons Bonferroni correction test.</p

Relative mRNA expression of IL-8, CXCL2 and ATF3 in response to Pam₃CysSK₄ alone or in co-stimulation with SAF.

<p>Beas-2B cells were stimulated with Pam₃CysSK₄ (10 μg/mL) and/or with SAF (10% v/v) for 3h. All results are shown as mean (±SEM) of three independent biological replicates. *P<0.05, **P<0.01, and ***P<0.001 compared to Pam₃CysSK₄ alone using an unpaired two-tailed student’s t-test.</p

<i>Staphylococcus aureus</i> Inhibits IL-8 Responses Induced by <i>Pseudomonas aeruginosa</i> in Airway Epithelial Cells

<div><p><i>Pseudomonas aeruginosa</i> (PA) and <i>Staphylococcus aureus</i> (SA) are major respiratory pathogens and can concurrently colonize the airways of patients with chronic obstructive diseases, such as cystic fibrosis (CF). Airway epithelial cell signalling is critical to the activation of innate immune responses. In the setting of polymicrobial colonization or infection of the respiratory tract, how epithelial cells integrate different bacterial stimuli remains unknown. Our study examined the inflammatory responses to PA and SA co-stimulations. Immortalised airway epithelial cells (Beas-2B) exposed to bacteria-free filtrates from PA (PAF) induced a robust production of the neutrophil chemoattractant IL-8 while bacteria-free filtrates from SA (SAF) had a minimal effect. Surprisingly, co-stimulation with PAF+SAF demonstrated that SAF strongly inhibited the PAF-driven IL-8 production, showing that SAF has potent anti-inflammatory effects. Similarly SAF decreased IL-8 production induced by the TLR1/TLR2 ligand Pam₃CysSK₄ but not the TLR4 ligand LPS nor TLR5 ligand flagellin in Beas-2B cells. Moreover, SAF greatly dampened TLR1/TLR2-mediated activation of the NF-κB pathway, but not the p38 MAPK pathway. We observed this SAF-dependent anti-inflammatory activity in several SA clinical strains, as well as in the CF epithelial cell line CFBE41o-. These findings show a novel direct anti-inflammatory effect of SA on airway epithelial cells, highlighting its potential to modulate inflammatory responses in the setting of polymicrobial infections.</p></div