Repurposing Pilocarpine Hydrochloride for Treatment of Candida albicans Infections

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.

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.

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).