Repurposing Pilocarpine Hydrochloride for Treatment of Candida albicans Infections

Abstract

Acetylcholine modulates the virulence of Candidaalbicans and regulates an appropriate immune response to infection in a Galleria mellonella infection model. Indeed, the evidence suggests that C. albicans possesses a functional cholinergic receptor that can regulate filamentous growth and biofilm formation. Furthermore, G. mellonella immune cell subsets possess repertories of cholinergic receptors which regulate an effective and appropriate cellular immune response to C. albicans infection. This study aimed to investigate the cholinergic receptor subtype involved in regulation of filamentous growth and biofilm formation by C. albicans and determine the roles of cholinergic receptors in modulation of G. mellonella immune cell subsets. The general muscarinic receptor agonist, pilocarpine hydrochloride, inhibited C. albicans biofilm formation and pathogenicity, a phenomenon that could be reversed using the general muscarinic receptor antagonist, scopolamine. Pilocarpine hydrochloride protected G. mellonella larvae from C. albicans infection via inhibition of C. albicans filamentation and appropriate regulation of cellular immunity. However, scopolamine abrogated the capacity of pilocarpine hydrochloride to protect G. mellonella larvae from C. albicans infection. Furthermore, acetylcholine and pilocarpine hydrochloride exhibited differential modulatory capabilities on Galleria mellonella hemocyte responses to C. albicans The data in this article demonstrate that a muscarinic receptor modulates C. albicans filamentation and biofilm formation. Furthermore, the results suggest that G. mellonella hemocyte subsets possess unique repertoires of cholinergic receptors that regulate their differentiation, activation, and function in contrasting manners. Therefore, targeting cholinergic receptors by repurposing currently licensed cholinergic drugs may offer novel therapeutic solutions for the prevention or treatment of fungal infections.IMPORTANCECandida albicans is the most common human fungal pathogen with an estimated crude mortality rate of 40%. The ability of the organism to switch from the yeast to hyphal form and produce biofilms are important virulence factors. C. albicans infections are combatted by the host immune system. However, Candida triggers a strong inflammatory response that, if not appropriately regulated, can damage host tissues. Therefore, it is important that the host immune response eliminates the fungus but limits tissue damage. This study provides evidence that targeting cholinergic receptors cannot only curb the virulence of C. albicans by inhibiting filamentous growth and biofilm formation but can also appropriately regulate the host immune response to induce rapid clearance with limited damage to vital tissues. This article provides evidence that repurposing licensed drugs that target cholinergic receptors may offer novel therapeutic solutions for the prevention or treatment of fungal infections.

Keywords: Candida albicans; Galleria mellonella; biofilm; muscarinic; pilocarpine hydrochloride; repurposing.

FIG 1

The general muscarinic receptor agonist pilocarpine hydrochloride inhibits Candida albicans biofilm formation in vitro. (A and B) Biofilm biomass was assessed using the crystal violet assay after C. albicans was cultured for 24 h in RPMI 1640 containing different concentrations of SIBm (0 to 10 mM) (A) and PHCl (0 to 50 mM) (B). Data are expressed as raw OD₅₉₅ values, and the bars represent the mean values (plus standard deviations [SD] [error bars]) from triplicate wells of six independent experiments (n = 6). (C and D) Biofilm metabolic activity was assessed using the XTT assay after C. albicans was cultured for 24 h in RPMI 1640 containing different concentrations of SIBm (0 to 10 mM) (C) and PHCl (0 to 50 mM) (D). Data are expressed as percent metabolic activity compared to untreated controls (0 mM PHCl), and the bars represent the mean values (+SD) from triplicate wells of six independent experiments (n = 6). For panels A to D, values that are significantly different compared to the control values (0 mM SIBm or PHCl) are indicated by asterisks as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. (E and F) The effect of PHCl on the permeability of the C. albicans cell wall was investigated using a propidium iodide (PI) uptake (E) and ATP release assay (F). For the PI uptake assay, data are shown as fluorescence intensity units, and the bars represent the mean values (+SD) from triplicate wells of three independent experiments (n = 3). Heat-killed (HK) and chlorhexidine (CHX) (0.2%)-treated C. albicans were included as positive controls, and cells in RPMI 1640 alone were included as a negative control. For the ATP release assays, data are shown as nanomolar concentrations of ATP release, and the bars represent the mean values (+SD) from triplicate wells of three independent experiments (n = 3). Chlorhexidine (0.2%)- and fulvic acid (2.0%)-treated C. albicans cells were included as positive controls, and cells in RPMI 1640 alone were included as a negative control. *, P < 0.05; **, P < 0.01 compared to the control (cells in RPMI 1640 alone).

FIG 2

The general muscarinic receptor agonist pilocarpine hydrochloride inhibits filamentation and biofilm formation in vitro without affecting cell viability. (A to J) To assess the effects of PHCl on C. albicans morphology and viability, microscopy was employed. Light microscopy (LM) (A to E) and scanning electron microscopy (SEM) (F to J) was performed to visualize changes in biofilm biomass and C. albicans cell morphology after culture for 24 h in RPMI 1640 containing different concentrations of PHCl (0 to 50 mM). Representative images are shown from duplicate coverslips of three independent experiments. YC, yeast cells; PH, pseudohyphae; TH, true hyphae. Bars, 100 µm (A to E) and 700 µm (F to J). (K to O) Fluorescence microscopy (FM) was performed to assess cell viability. C. albicans was cultured for 24 h in RPMI 1640 containing different concentrations of PHCl, and viability was assessed using calcofluor white (blue) and propidium iodide (red) staining. Representative images are shown from duplicate coverslips of three independent experiments. Bars, 100 µm.

FIG 3

Pilocarpine hydrochloride acts through a specific muscarinic-like receptor to inhibit biofilm formation and modulate cell wall hydrophobicity. (A) Biofilm biomass was assessed using the crystal violet assay after C. albicans was cultured for 24 h in RPMI 1640 containing 25 mM PHCl and different concentrations of the nonspecific muscarinic receptor antagonist scopolamine (SCP) (0 to 128 µM). Data are expressed as raw OD₅₉₅ values, and the bars represent the mean values (+SD) from triplicate wells of three independent experiments (n = 3). Candida albicans was cultured in the absence of any compound, in the presence of PHCl alone, and in the presence of SCP alone as controls. #, significantly different from cells cultured in the absence of any compound; *, significantly different from cells cultured in PHCl alone; * or #, P < 0.05; ** or ##, = P < 0.01; *** or ###, P < 0.001. (B) Biofilm metabolic activity was assessed using the XTT assay after C. albicans was cultured for 24 h in RPMI 1640 containing 25 mM PHCl and different concentrations of the nonspecific muscarinic receptor antagonist scopolamine (SCP) (0 to 128 µM). Data are expressed as percent metabolic activity compared to untreated controls (0 mM PHCl or SCP), and the bars represent the mean values (+SD) from triplicate wells of three independent experiments (n = 3). Candida albicans cultured in the presence of PHCl and SCP alone acted as controls. *, significantly different from cells cultured in SCP alone; ***, P < 0.001. (C) Cell wall hydrophobicity was assessed using the MATH assay (29). The bars represent the mean values (+SD) from duplicate samples of five independent experiments (n = 5). #, significantly different from cells cultured planktonically; *, significantly different from cells cultured as a biofilm in the absence of PHCl.

FIG 4

Pilocarpine hydrochloride acts through muscarinic-like receptors to prolong survival of Candida albicans-infected Galleria mellonella by inhibiting biofilm formation and modulating host immunity in vivo. The effect of PHCl on the outcome of systemic candidiasis was investigated using a G. mellonella infection model. (A) A Kaplan-Meier plot shows the effects of different concentrations of PHCl on the survival of Candida albicans-infected larvae. The data are derived from three independent experiments with groups of 16 larvae (n = 48). ***, P < 0.001; *, P < 0.05, as determined by the log rank test in comparison to larvae inoculated with C. albicans alone. PBS alone and PHCl alone-injected larvae were used as controls and had no effect on larvae survival. (B) To verify specificity, C. albicans-infected larvae were also inoculated with PHCl and SCP in combination. The data are derived from three independent experiments with groups of 16 larvae (n = 48). *, P < 0.05, as determined by the log rank test in comparison to larvae inoculated with C. albicans. Larvae injected with PBS alone and SCP alone were used as controls, and PBS and SCP alone had no effect on larva survival. (C) Histological analysis of larvae was performed at 24 and 48 h postinoculation using hematoxylin and eosin (HE) and periodic acid-Schiff (PAS) staining. (i and vi; HE) (ii and vii; HE) larva inoculated with PHCl (6.25 mM) alone. (iii and viii; PAS) larvae infected with C. albicans. (iv and ix; HE) larvae infected with C. albicans in the presence of PHCl (6.25 mM). (v and x; PAS). Larvae infected with C. albicans in the presence of PHCl (6.25 mM) and SCP (6.25 mM). Asterisks highlight melanized nodules, whereas arrows show C. albicans cells and hyphae. Representative images are shown from histological analysis of two larvae for each condition from three independent experiments. Fb, fat body; Ct, cuticle; GI, gastrointestinal tract; T, trachea; Nd, nodule. Bars, 250 μm (panels i, v, and ix) and 100 μm (panels ii, iii, iv, vi, vii, viii, and x).

FIG 5

Effects of pilocarpine hydrochloride and acetylcholine on hemocyte responses to C. albicans in vitro and in vivo. (A) In the in vitro study, hemocytes isolated from untreated larvae were either left unstimulated (control) (i) or stimulated with C. albicans (ii), ACh (iii), PHCl (iv), C. albicans plus ACh (v), and C. albicans plus PHCl for 24 h. (B) In the in vivo study, larvae were bled 24 h after sham inoculation with PBS (control) (i) or inoculation with C. albicans (ii), ACh (iii), PHCl (iv), C. albicans plus ACh (v), and C. albicans plus PHCl. Representative images are shown from hematoxylin and eosin staining of hemocytes from three larvae for each condition from three independent experiments. Bars, 100 μm.