Abstract
Dicyclomine is a human muscarinic acetylcholine receptor antagonist used for the treatment of abdominal cramps. We are reporting here that dicyclomine can inhibit the in vitro growth and virulence factors of the human pathogen Candida albicans very effectively. Dicyclomine inhibited adhesion, early biofilm, mature biofilm, and planktonic growth. Yeast to hyphal form transition of C. albicans in various inducer media such as serum, proline, glucose, and N-acetylglucosamine was inhibited. Dicyclomine also could kill C. albicans cells within 15 min of exposure. Dicyclomine appears to inhibit the yeast to hyphal conversion by affecting signal transduction pathway. The expression of selected genes associated with yeast to hyphal form transition in serum in presence of dicyclomine was studied using real-time polymerase chain reaction (RtPCR). The RtPCR analysis showed that dicyclomine targets both cAMP pathway as well as MAPK cascade. Eight genes were upregulated. Out of these, three major upregulated genes were Bcy1, Tup1, and Mig1. Dicyclomine downregulated Ume6, Ece1, and Pde2 genes which are involved in cAMP signaling pathway and also downregulated the DNA binding protein gene, Rfg1. Dicyclomine significantly upregulated the master negative regulator of hyphal formation, Tup1. Based on this study we suggest that the muscarinic acetylcholine receptor antagonist, dicyclomine could be repositioned as a potential anti-Candida albicans as well as anti-virulence agent.
Similar content being viewed by others
Introduction
Candida albicans is a part of the normal microbiome of healthy humans. Under immunocompromised conditions, it can become pathogenic [1]. C. albicans can form biofilms on biotic as well as abiotic surfaces [2]. Abiotic surfaces include implanted devices in the body like urinary catheters, venous catheters, voice prostheses, dentures etc [2,3,4]. Candida biofilms are highly resistant to most of the currently used drugs [5]. Seventy percentages of microbial infections in humans involve biofilm formation [3, 6, 7]. As such there is a necessity for developing novel drugs which can target biofilms. One of the strategies for anti-Candida drug development is the inhibition of virulence factors and repositioning of drugs. Repositioning studies have shown promise in the recent years [8, 9]. In this study we are reporting the repositioning of a drug, called dicyclomine. Dicyclomine is an antispasmodic agent [10,11,12]. It is an inexpensive medicine and is commonly used for intestinal hypermotility treatment. Also, it can reduce irritable bowel syndrome. Dicyclomine relieves muscle spasms and cramps in the gastrointestinal tract, which emerges during infections by blocking the activity of acetylcholine on muscarinic receptors located on the surface of smooth muscle cells. Muscarinic acetylcholine receptors are known to play many fundamental roles in human physiology. They are typical G-protein-coupled receptors (GPCRs) which can facilitate signal transduction via heterometric G-protein to downstream second messengers. A number of drugs target muscarinic acetylcholine receptors. Dicyclomine is reported to target human muscarinic acetylcholine M1 receptor as well as M3 receptor [13,14,15]. Many hyphal-specific genes mediate yeast to hyphal transformation through two pathways, Ras1-cAMP-PKA pathway and Ras1-MAPK cascade [16]. In Ras1-cAMP-PKA pathway, when Ras1 is activated, it can stimulate adenylyle cyclase (Cyr1) that leads to increase in cAMP production. This results in the binding of cAMP to Bcy1. Bcy1 is the subunit regulator of protein kinase A (PKA) [17]. Bcy1 may activate either Tpk1 or Tpk2. Tpk1 and Tpk2 are PKA catalytic subunits which can activate the transcription factor Efg1 [18, 19]. The activated Efg1 is required for virulence which can cause systemic candidiasis in mouse model [20]. Ece1 and Ume6 are downstream genes of the activated Efg1 [21, 22]. To know the mechanism through which dicyclomine can inhibit C. albicans morphogenesis, we studied the expression of selected hyphal genes that are involved in Ras1-cAMP-PKA and MAPK signal pathways. Our real time PCR study has identified potential targets of dicyclomine in C. albicans. In this manuscript, we are reporting that dicyclomine can inhibit virulence factors of the human pathogen, C. albicans, raising the possibility of repositioning dicyclomine as an antifungal agent.
Results
Inhibition of yeast to hypha in C. albicans by dicyclomine
C. albicans is affected by many environmental cues which are contributed in yeast to hyphal switching. Here, we tested four factors that can induce yeast to hyphal form transition. Dicyclomine inhibited this transition in two strains of C. albicans (ATCC90028 and GMC-16) induced by 5% serum, 1% proline, 1% glucose, and 1% N-acetlglucosamine at 0.062, 0.062, 0.125, 0.25 mg/ml respectively (Table 1; Figs. 1a, 2a–d).
Dicyclomine inhibits Candida albicans growth
Dicyclomine at concentration of 0.5 mg/ml reduced Candida growth by 50% (Table 1 and Fig. 1b (A)). The minimum fungicidal concentration (MFC) was observed at 1 mg/ml (Table 1 and Fig. 3a). Commercial dicyclomine also reduced Candida albicans growth by 50% [minimun inhibitory concentration (MIC)] at 0.125 mg/ml (Table 1 and Fig. 1b (A)) and MFC was at 0.25 mg/ml (Table 1 and Fig. 3b).
Dicyclomine inhibits adhesion of Candida albicans to polystyrene
The effect of pure dicyclomine on adhesion cells was observed at 0.25 mg/ml where 50% of adhesion was inhibited. This concentration was considered as the minimum inhibitory concentration (MIC). MIC of the commercial dicyclomine on adhesion cells was 0.031 mg/ml (Table 1 and Fig. 1c).
Dicyclomine inhibits biofilm formation of Candida albicans on polystyrene surface
Both pure and commercial dicyclomine inhibited developing biofilm at 0.062 mg/ml after 48 h of incubation at 37 °C (Table 1; Figs. 1d (A), 4a, b). Mature biofilm was inhibited at 0.25 mg/ml and above this concentration after 48 h of incubation at 37 °C (Table 1 and Fig. 1d (B)).
Dicyclomine kills C. albicans cells
Both pure and commercial dicyclomine were highly toxic to Candida albicans (ATCC 90028) cells. Pure dicyclomine killed 100% of inoculum within 15 min of exposure at MFC 1 mg/ml, whereas commercial dicyclomine killed 100% of cells within 15 min of exposure at MFC of 0.25 mg/ml. But in case of C. albicans (GMC-16), both the pure and commercial dicyclomine killed 100% of cells within 4 h of exposure (Fig. 1b (B)).
Dicyclomine inhibits biofilm growth of C. albicans on silicon discs
Candida albicans formed biofilm on silicon rubber air way discs characterized by a thick network of hyphal and yeast cells. Pure and commercial dicyclomine caused considerable inhibition of biofilm (Fig. 4c).
Dicyclomine regulates hyphal genes in yeast to hyphal form conversion in C. albicans
To discover the basic mechanism by which the muscarinic receptor antagonist, dicyclomine inhibited yeast to hyphal form transformation induced by serum, we studied the expression of some genes involved in cAMP-PKA and MAPK pathways using real time-PCR analysis. Dicyclomine affected both these pathways. It altered the expression of genes involved in cAMP-PKA pathway where the subunit regulator of cAMP-dependent PKA, Bcy1 and the transcription factor, Efg1 were upregulated by 42 and 15 folds, respectively. The expression of downstream hyphal genes of cAMP-Efg1 pathway, Ece1 and Ume6 were downregulated by 4.7 and 4.9 folds, respectively. The cAMP regulator, Pde2 also downregulated up to 1.3 fold with dicyclomine treatment. The transcriptional suppressor factor, Tup1 was significantly overexpressed by 38 folds and Tup1-dependent transcriptional repressor, Mig1 was also upregulated by 37 folds. The Tup1-dependent transcriptional repressor, Rfg1 was downregulated up to 3.2 folds. Dicyclomine also could affect the expression of genes involved in MAPK pathway. The expression of Hst7, Cek1, Tec1, and Cst20 was upregulated by 12, 2.8, 2.5, and 1.4 folds, respectively (Figs. 5 and 6).
Discussion
Various drugs are repositioned as anti-Candida albicans agents [9, 23, 24]. These studies suggest that Candida albicans may share a number of targets with humans, since both humans and Candida share a common eukaryotic heritage. The human muscarinic receptors (M1, M2, M3, M4, and M5) are typical GPCRs and play significant roles in signal transduction. In this manuscript, for the first time we are proposing repositioning dicyclomine as an anti-Candida albicans agent. Dicyclomine is an anticholinergic and antispasmodic agent, widely used as a drug for stomach cramps. This drug effectively inhibited the growth and virulence factors of Candida albicans (Table 1; Figs. 1, 2a–d, 3a, b, 4a–c). It also caused considerable inhibition of drug-resistant biofilm of C. albicans (Table 1; Figs. 1d (A, B), 4a–c). Dicyclomine killed Candida cells within 15 min of exposure (Fig. 1b (B)). These results suggest that dicyclomine could be used as a potential anti-Candida agent. Hyphal formation of C. albicans in serum is through the Ras1 signaling pathway [16]. The expression of selected genes involved in Ras1-cAMP-PKA and MAPK signal pathways was studied in presence of serum and dicyclomine. We found that the transcriptional suppressor of hyphal genes, Tup1 was upregulated (Figs. 5 and 6) that can block filamentous growth and repress biofilm formation in C. albicans [25,26,27]. Deletion of Tup1 is reported to cause constitutive hyphal formation [25]. The overexpression of Tup1 leads to the downregulation of hyphal genes, Ece1 Ume6, and the cAMP regulator, Pde [26]. Dicyclomine upregulated Tup1 which led to the downregulation of Ece1, Ume6, and Pde2 expression (Figs. 5 and 6). Ece1 and Ume6 are involved in the extension and growth of hypha, adherence, and biofilm formation [28,29,30,31,32]. The overexpression of Ece1 may be involved mainly in adhesion or biogenesis and may have contributed in hyphal and biofilm formation [33]. In C. albicans, Ume6 encodes a key filament-specific transcriptional regulator of hyphal development and virulence [30]. Ume6 is a downstream target of multisignaling pathways involved in hyphal growth [22]. Mutation of Ume6 causes defects in hyphal extension and biofilm formation [30]. Pde2 encodes a high affinity–phosphodiesterase regulating cAMP level and is required for hyphal growth, virulence and induction of biofilm formation on rat catheter [34,35,36]. Deletion of Pde2 leads to increase cAMP level which causes constitutive activation of cAMP signaling pathway. This results in hyperfilamentation, but without enhancing virulence [35, 37]. Dicyclomine downregulated Pde2 which may cause overexpression of the subunit regulator of cAMP-dependent-PKA, Bcy1 (Figs. 5 and 6) [17]. BCY1 has a crucial role in the regulation of cell differentiation and death. It has an essential role in cell viability in C. albicans [17]. Deletion of Bcy1 leads to multiple cellular morphologies and enhances the development of filaments [17]. Rfg1, a Tup1-dependent suppressor [38] was downregulated by treatment with dicyclomine. This may be due to Mig1 upregulation (Figs. 5 and 6). The key regulator transcription factor Efg1 [18, 19] may be upregulated as a result of Bcy1 overexpression. Dicyclomine altered the expression of Cst20, Hst7, Cek1, and Tec1 that are involved in MAPK pathway (Figs. 5 and 6). In C. albicans, a MAP kinase pathway is involved in controlling of hyphal development [37] and mating processes. The deletion of the genes, Cph1, Cst20, Hst7, and Cek1 cannot block filament growth induced by serum and does not dramatically affect the transcription profile of the yeast to hyphal form transition. Deletion of CPH1 causes delay in hyphal formation [39]. The upregulation of Tec1 may be due to the Efg1 upregulation with dicyclomine treatment (Figs. 5 and 6). The mutation of tec1 exhibits suppressed filament formation in liquid serum-containing media [16]. This study indicates that a protein-like muscarinic receptor may have an important function in the yeast to hyphae formation in C. albicans and it may influence both cAMP and MAPK signaling pathways. Dicyclomine may inhibit yeast to hyphal form conversion by targeting multiple genes.
Methods
Media and chemicals
Dicyclomine hydrochloride (Fig. 7) was purchased from Sigma-Aldrich Chemicals Ltd, Mumbai, India. Dicyclomine hydrochloride injection form was purchased from a local medical store. Menadione and 2, 3-bis (2-methoxy-4-nitro-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) were purchased from Sigma-Aldrich Chemicals Ltd. All other media components and plates were purchased from HiMEDIA Chemicals Ltd, Mumbai, India.
Culture of Candida albicans
A standard strain of Candida albicans ATCC 90028 was obtained from the Institute of Microbial Technology (IMTECH) Chandigarh, India. GMC-16 was obtained from Government Medical College, Nanded, MH, India. The culture was maintained on yeast peptone dextrose (YPD) agar slant at 4 °C and propagated by inoculating a single colony from the YPD agar plates into 50 ml YPD broth in 250-ml conical flask. Flasks were incubated overnight at 30 °C at 100 rpm on an orbital shaking incubator. The cells were harvested by centrifugation at 2000 rpm and washed thrice with sterile 0.1 M phosphate-buffered saline, pH 7.4 and the cell density was determined using a haemocytometer count.
Hyphal formation assay
Yeast to hyphal form transition assay was carried out in 96-well microtitre plates [40]. Candida albicans cells stock was diluted to 1 × 106 cells/ml in several inducer media including 5% serum, 1% v/v N-acetylglucosamine, 1% v/v proline, and 1% v/v glucose. Various concentrations of dicyclomine hydrochloride were prepared and added in each well. Wells without drugs were kept as a control. The final volume was kept at 200 µl in each well. The microtitre plates were incubated at 37 °C at 120 rpm on an orbital shaker incubator for 2 h. After incubation period, cells were observed microscopically by using an inverted light microscope (Metzer, India). Hundred cells were counted and numbers of yeast and hyphal forms were noted. All the experiments were done in triplicates.
Antifungal activity
Minimum inhibitory concentration (MIC)
MIC was determined by using the standard methodology M27 A2 as per CLSI guidelines [23]. Various concentrations of dicyclomine were prepared in RPMI-1640 medium by double dilution manner in the 96-well microtitre plates. 1 × 103 cells/ml from Candida albicans cells stock was prepared. The final volume of RPMI-1640 medium kept in each well was 200 µl. The wells without addition of dicyclomine served as control. The microtitre plates were incubated at 35 °C for 48 h, and the absorbance was recorded spectrophotometrically at 620 nm using a microplate reader (Multiscan Ex, Thermo Electronic Corp, USA). The concentration of dicyclomine which caused a 50% reduction in the absorbance compared to the control was considered as the MIC. All the experiments were done in triplicates.
Minimum fungicidal concentration (MFC)
The wells of MIC of dicyclomine and above MIC against Candida albicans growth were selected for determination of MFC. Ten microliters of cell suspension was taken from the wells and spread on YPD agar plates. The plates were incubated at 30 °C for 24 h and observed for the presence of colonies. The absence of colonies growth was considered as the fungicidal effect. The lowest concentration of dicylomine that showed no growth was considered as the MFC. All the experiments were done in triplicates.
Adhesion assay
The effect of dicyclomine on adhesion of cells on the polystyrene surface was studied using 96-well plates. Double dilutions of various concentrations of dicyclomine were prepared in sterile phospahte-buffered saline (PBS) and put in each well [41]. Fifty microliters of prepared cells was put in each well to obtain 1 × 107 cells/ml and control kept without dicylomine. The plates were incubated at 37 °C for 90 min at 100 rpm in an orbital shaker incubator. After incubation, the wells were washed by sterile PBS to remove non adhered cells. Density of the adhered cells was analyzed by using XTT metabolic assay. The color formation was measured by using microtitre plate reader at 450 nm. More than 50% reduction compared to the control was considered as MIC. All the experiments were done in triplicates.
Biofilm formation
Biofilm formation of Candida albicans was developed in an in vitro on the surface of 96-well polystyrene plates as per standard method [41]. 1 × 107 cells/ml were prepared and 100 µl of cell suspension was added in each well. The microtitre plates were incubated at 37 °C for 90 min at 100 rpm. Non adhered cells were aspirated and removed using sterile PBS gently to avoid biofilm disruption. For early biofilm formation, serial concentrations of dicyclomine were prepared by double dilution in RPMI-1640 and 200 µl was added in each well. Then, the plates were incubated at 37 °C for 48 h to undergo biofilm formation. After incubation period, the plates were washed thrice with sterile PBS and observed under an inverted light microscope (Metzer, India) for the presence or absence of biofilm. Confirmation and quantitation of biofilm formation was studied by the XTT metabolic assay. For mature biofilm formation, 1 × 107cells/l were prepared and 100 µl of cell suspension was added in each well. The microtitre plate was incubated at 37 °C for 90 min at 100 rpm. Non adhered cells were removed by washing with sterile PBS. Then 200 µl of RPMI-1640 was added in each well and microtitre plates incubated at 37 °C for 24 h. After incubation period, the biofilms were washed with sterile PBS. Various concentrations of dicyclomine in RPMI-1640 were prepared by double dilution and 200 µl was added in each well. The plates were incubated at 37 °C for 48 h. The wells were again washed by sterile PBS after incubation period. The biofilm formation was quantitated by the XTT metabolic assay. All the experiments were done in triplicates.
XTT assay for biofilm quantitation
The early and mature biofilm formation was quantitated by using a XTT metabolic assay. One milligram of XTT powder was dissolved in 1 ml distilled water and sterilized by using a bacteriological filter. Menadione was prepared in acetone, then 10 µl of menadione was added to the XTT to the final concentration of 4 µM. The wells were washed with sterile PBS. Eighty microliters of PBS was added in each well followed by adding 20 µl of XTT-menadione reagent. The plates were incubated in dark condition at 37 °C for 3 h to develop color formation. The color formation was measured at 450 nm using a microplate reader (Multiskan EX, Thermo Electron Corp. USA). Wells without test drugs were used as controls. Those without biofilm were kept as blanks. The concentration of dicyclomine which caused ≥50% lowering of growth was considered as the MIC for biofilm formation. All the experiments were done in triplicates.
Kill curve assay
Activity of dicyclomine against the growth of Candida albicans (ATCC 90028 and GMC-16) was studied by time-dependent kill curve assay. 2.5 × 103 cells/ml were prepared in 10 ml sterile RPMI-1640 along with MFC of dicyclomine and were incubated at 30 °C at 100 rpm. From this mixture, 0.5 ml was taken at different time intervals (0, 15, 30 min, 1, 2, 4, 5, 6, 7 and 8 h), and was washed twice with sterile PBS to remove the drug carryover effect. The pellet was re-suspended in 50 µl sterile PBS and spread on YPD agar plates. The plates were incubated at 30 °C for 48 h. The formed colonies on the plates were counted and compared with control plate lacking dicyclomine. Plates were kept in triplicates.
Scanning electronic microscopy of biofilm
Candida albicans biofilm was developed on sterile oropharyngeal silicon rubber airway discs seeding in 12 microtitre plates [40]. A 1 mg/ml concentration of dicyclomine was prepared in RPMI-1640. RPMI-1640 without dicyclomine was kept for control. Two mililiters of 1 × 107 cells/ml was inoculated in each well containing sterile oropharyngeal silicon rubber airway disc and was incubated at 37 °C for 90 min at 100 rpm to allow adhesion of cells on discs. After incubation the discs were washed thrice using sterile PBS. The discs were transferred to sterile wells and dicyclomine –RPMI-1640 solution was added in each well. The discs without dicyclomine were considered as control. The plates were incubated at 37 °C for 48 h at 60 rpm. Then, the discs were washed again using sterile PBS and were fixed in 2.5% glutaraldehyde in 0.1 M phosphate-buffered saline, pH 7.4 for 24 h at 4 °C. After 24 h, the discs were post-fixed in 2% aqueous solution of osmium tetraoxide for 4 h, then dehydrated in a series of graded alcohols and were finally dried to a critical drying point with a critical point dryer unit. The discs were held over stubs and gold coating was performed using an automated gold coater (Model: JOEL JFC-1600, JOEL Limited, Akishima, Tokyo, Japan) for 5 min. Photographs were taken using a scanning electron microscope (Model: JOEL-JSM 5600, JOEL Limited, Akishima, Tokyo, Japan). All the experiments were done in triplicates. Representative pictures are presented.
Real-time PCR Analysis
Hyphal genes expression of yeast to hyphal form transition in serum induction was studied using Real Time PCR. C. albicans yeast cells (1 × 106 cells/ml) were inoculated in 20% serum containing 12.5 µg/ml of dicyclomine and incubated for 90 min. The total RNA was isolated using RNeasy® Mini Kit (QIAGEN). Then, cDNA was built by using SuperScript® III for first strand synthesis (Invitrogen, Life Technologies, USA). PCR reactions were carried out using KAPA SYBR® Fast qPCR Kit Master Mix (2×) (BIOSYSTEMS, South Africa) in 96-well PCR plates with preliminary denaturation for 3 min at 95 °C and were followed by 32 amplifications cycles of denaturation at 95 °C for 30 s. The annealing step was done at 60 °C for 20 s and primer extension at 72 °C for 30 s (CFX 96 Real time System, Bio-Rad, USA). Primers were purchased from Eurofins Genomics India Pvt. Ltd. They are tabulated in Table 2. Actin gene was used as an internal control and the transcript levels of these genes were calculated using the formula 2− ΔΔCT [42]. All the experiments were performed in triplicates.
Statistical analysis
The mean with standard deviations of values were obtained from three different observations. Values in the control and treatment groups were compared using Student’s t test. A value of P < 0.05 was considered statistically significant.
References
Kim J, Sudbery P. Candida albicans, a major human fungal pathogen. J Micro. 2011;49:171.
Lynch AS, Robertson GT. Bacterial and fungal biofilm infections. Annu Rev Med. 2008;59:415–28.
Kojic EM, Darouiche RO. Candida infections of medical devices. Clin Micro Rev. 2004;17:255–67.
Ramage G, Saville SP, Thomas DP, Lopez-Ribot JL. Candidabiofilms: an update. Euk Cel. 2005;4:633–8.
Mukherjee PK, Chandra J. Candida biofilm resistance. Dru Resist Upd. 2004;7:301–9.
Kumamoto CA, Vinces MD. Alternative Candida albicans lifestyles: growth on surfaces. Annu Rev Microbiol. 2005;59:113–33.
Nett J, Lincoln L, Marchillo K, Andes D. β-1, 3 glucan as a test for central venous catheter biofilm infection. J Infect Dise. 2007;195:1705–12.
Sleigh SH, Barton CL. Repurposing strategies for therapeutics. Pharmaceu Med. 2010;24:151–9.
Kathwate GH, Karuppayil SM. Antifungal properties of the anti-hypertensive drug: Aliskiren. Arch Ora Biol. 2013;58:1109–15.
Tilford CH, Campen MV Jr, Shelton RS. Aminoesters of substituted alicylic carboxylic acids1. J Am Chem Soci. 1947;69:2902–6.
Chey WD, Kurlander J, Eswaran S. Irritable bowel syndrome: a clinical review. Jama. 2015;313:949–58.
Robles LAR, Robles RM, Troche JMR, Padilla FJB, Garza HJM, Martinez-Vazquez M. Tu1100 effect of antispasmodic agents, alone or in combination, in the treatment of irritable bowel syndrome: systematic review and meta-analysis. Gastroenterology. 2016;150:S842.
Giachetti A, Giraldo E, Ladinsky H, Montagna E. Binding and functional profiles of the selective M1 muscarinic receptor antagonists trihexyphenidyl and dicyclomine. Brit J Pharm. 1986;89:83–90.
Doods HN, et al. Selectivity of muscarinic antagonists in radioligand and in vivo experiments for the putative M1, M2 and M3 receptors. J Pharm Exp Thera. 1987;242:257–62.
Kilbinger H, Stein A. Dicyclomine discriminates between M1‐and M2‐muscarinic receptors in the guinea‐pig ileum. Brit J Pharm. 1988;94:1270–4.
Biswas S, Van Dijck P, Datta A. Environmental sensing and signal transduction pathways regulating morphopathogenic determinants of Candida albicans. Micro Molec Biol Rev. 2007;71:348–76.
Ding, X, Cao, C, Zheng, Q, Huang, G. The regulatory subunit of protein kinase A (Bcy1) in Candida albicans plays critical roles in filamentation and white-opaque switching but Is not essential for cell growth. Front Micro. 2016;7:2127.
Sharkey LL, McNemar MD, Saporito-Irwin SM, Sypherd PS, Fonzi WA. HWP1 functions in the morphological development of Candida albicans Downstream of EFG1, TUP1, and RBF1. J Bacter. 1999;181:5273–9.
Bockmühl DP, Ernst JF. A potential phosphorylation site for an A-type kinase in the Efg1 regulator protein contributes to hyphal morphogenesis of Candida albicans. Genetics. 2001;157:1523–30.
Rocha CR, et al. Signaling through adenylyl cyclase is essential for hyphal growth and virulence in the pathogenic fungus Candida albicans. Molecu Biol Cel. 2001;12:3631–43.
Lane S, Birse C, Zhou S, Matson R, Liu H. DNA array studies demonstrate convergent regulation of virulence factors by Cph1, Cph2, and Efg1 in Candida albicans. J Biol Chem. 2001;276:48988–96.
Banerjee M, et al. Expression of UME6, a key regulator of Candida albicans hyphal development, enhances biofilm formation via Hgc1-and Sun41-dependent mechanisms. Euk Cel. 2013;12:224–32.
Routh MM, Raut JS, Karuppayil SM. Dual properties of anticancer agents: an exploratory study on the in vitro anti-Candida properties of thirty drugs. Chemotherapy. 2011;57:372–80.
Kathwate GH, Karuppayil SM. Tramadol, an opioid receptor agonist: an inhibitor of growth, morphogenesis, and biofilm formation in the human pathogen, Candida albicans. Ass Dru Devel Tech. 2016;14:567–72.
Braun BR, Johnson AD. Control of filament formation in Candida albicans by the transcriptional repressor TUP1. Science. 1997;277:105–9.
Murad AMA, et al. Transcript profiling in Candida albicans reveals new cellular functions for the transcriptional repressors CaTup1, CaMig1 and CaNrg1. Molecu Micro. 2001;42:981–93.
Zhao R, Lockhart SR, Daniels K, Soll DR. Roles of TUP1 in switching, phase maintenance, and phase-specific gene expression in Candida albicans. Euk Cel. 2002;1:353–65.
Birse CE, Irwin MY, Fonzi WA, Sypherd PS. Cloning and characterization of ECE1, a gene expressed in association with cell elongation of the dimorphic pathogen Candida albicans. Infect Immu. 1993;61:3648–55.
Nobile CJ, et al. Critical role of Bcr1-dependent adhesins in C. albicans biofilm formation in vitro and in vivo. PLOS Pathog. 2006;2:e63.
Banerjee M, et al. UME6, a novel filament-specific regulator of Candida albicans hyphal extension and virulence. Molec Biol Cel. 2008;19:1354–65.
Zeidler U, et al. UME6 is a crucial downstream target of other transcriptional regulators of true hyphal development in Candida albicans. FEMS Yea Res. 2009;9:126–42.
Uppuluri P, Chaturvedi AK, Srinivasan A, Banerjee M, Ramasubramaniam AK, Köhler JR, Lopez-Ribot JL. Dispersion as an important step in the Candida albicans biofilm developmental cycle. PLOS Pathog. 2010;6:e1000828.
Subaran, RL, & Mitchell, AP. Strategic analysis of Candida albicans gene function. Candida: Comparative and Functional Genomics, Hori Scient Pr. 2007;1:349-357.
Jung WH, Stateva LI. The cAMP phosphodiesterase encoded by CaPDE2 is required for hyphal development in Candida albicans. Microbiology. 2003;149:2961–76.
Jung WH, et al. Deletion of PDE2, the gene encoding the high‐affinity cAMP phosphodiesterase, results in changes of the cell wall and membrane in Candida albicans. Yeast. 2005;22:285–94.
Bahn YS, Staab J, Sundstrom P. Increased high‐affinity phosphodiesterase PDE2 gene expression in germ tubes counteracts CAP1‐dependent synthesis of cyclic AMP, limits hypha production and promotes virulence of Candida albicans. Molec Micro. 2003;50:391–409.
Hoyer LL, et al. A Candida albicans cyclic nucleotide phosphodiesterase: cloning and expression in Saccharomyces cerevisiae and biochemical characterization of the recombinant enzyme. Microbiology. 1994;140:1533–42.
Stoldt VR, Sonneborn A, Leuker CE, Ernst JF. Efg1p, an essential regulator of morphogenesis of the human pathogen Candida albicans, is a member of a conserved class of bHLH proteins regulating morphogenetic processes in fungi. EMBO J. 1997;16:1982–91.
Huang H, Harcus D, Whiteway M. Transcript profiling of a MAP kinase pathway in C. albicans. Micro Res. 2008;163:380–93.
Chauhan NM, Raut JS, Karuppayil SM. A morphogenetic regulatory role for ethyl alcohol in Candida albicans. Mycoses. 2011;54:e697–e703.
Raut JS, Shinde RB, Chauhan NM, Mohan Karuppayil S. Terpenoids of plant origin inhibit morphogenesis, adhesion, and biofilm formation by Candida albicans. Biofouling. 2013;29:87–96.
Chang W, Li Y, Zhang L, Cheng A, Lou H. Retigeric acid B attenuates the virulence of Candida albicans via inhibiting adenylyl cyclase activity targeted by enhanced farnesol production. PLOS One. 2012;7:e41624.
Acknowledgements
AA and SMK are thankful to Prof. P Vidyasagar, Vice-chancellor, SRTM University, Nanded, Maharashtra for the facilities. SMK is also thankful to UGC, New Delhi for infrastructural support the UGC-SAP-DRS-II and DST-FIST-1 program. RP thanks to UPE II Phase program and Biotechnology Department Research and Development Program of Savitribai Phule Pune University.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Ali, A., Jadhav, A., Jangid, P. et al. The human muscarinic acetylcholine receptor antagonist, Dicyclomine targets signal transduction genes and inhibits the virulence factors in the human pathogen, Candida albicans. J Antibiot 71, 456–466 (2018). https://doi.org/10.1038/s41429-017-0013-z
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41429-017-0013-z
This article is cited by
-
Novel approach to saturated amino acid derivatives with isolated (hetero)cyclic rings via the hydrogenation of dienes
Chemistry of Heterocyclic Compounds (2023)
-
A semisynthetic borrelidin analogue BN-3b exerts potent antifungal activity against Candida albicans through ROS-mediated oxidative damage
Scientific Reports (2020)
-
Molecular targets of biofabricated silver nanoparticles in Candida albicans
The Journal of Antibiotics (2019)