Biofilm Formation and Phospholipase and Proteinase Production in Cryptococcus neoformans Clinical Isolates and Susceptibility towards Some Bioactive Natural Products

Background Cryptococcosis is one of the most common fungal infections in immunocompromised patients, which is caused by Cryptococcus neoformans. However, relatively little is known about the virulence factors of C. neoformans and the incidence of antifungal drug resistance in C. neoformans is rapidly increasing. This study was undertaken to investigate the virulence factors in C. neoformans, thymol, curcumin, piperine, gallic acid, eugenol, and plumbagin for their potential antimicrobial activity against C. neoformans. Methods The production of phospholipase and proteinase was detected using standard methods. Biofilm formation was determined using the microtiter plate method. The broth microdilution method was used to determine the antifungal activity. The antibiofilm activity was assessed using the safranin staining method. Results All isolates of C. neoformans produced biofilms with optical density values ranging from 0.16 to 0.89. A majority of C. neoformans isolates that were tested exhibited strong phospholipase (7/8) and proteinase (5/8) production. Plumbagin (with minimum inhibitory concentration values ranging from 4 to 16 μg/mL) showed the highest antifungal activity followed by thymol (with minimum biofilm inhibitory concentration values ranging from 8 to 64 μg/mL). In addition, plumbagin showed the highest antibiofilm activity with minimum biofilm inhibitory concentration and minimum biofilm eradication concentration values ranging from 4 to 16 μg/mL and 32 to 256 μg/mL, respectively. Conclusion Plumbagin, compared to other natural products studied, was the most efficient in terms of antifungal and antibiofilm activities. Hence, plumbagin could be used in combination with antifungals for the development of new anticryptococcal drugs.


Introduction
Cryptococcus neoformans is a major opportunistic pathogen causing cryptococcosis. Cryptococcosis occurs when yeast is inhaled into the lungs, which proliferates and disseminates to the central nervous system mostly in individuals with immune system defciencies leading to meningitis that represents the major cause of death in people living with HIV/AIDS [1]. Approximately, one million cases of cryptococcosis meningitis have been reported in HIV/AIDS patients with over 600 000 deaths per year worldwide [2]. In sub-Saharan Africa, the global annual incidence estimate for cryptococcosis cases exceeds 957 900 with 180 000 deaths [3,4]. In fact, several virulence factors, such as bioflm formation, and the secretion of phospholipases and proteases contribute to the success of fungal infection [5]. C. neoformans grows as a bioflm. Bioflms are communities of aggregated microbial cells embedded in an extracellular matrix composed of water, polysaccharides, lipids, proteins, and extracellular DNA [6]. Bioflm formation confers increased resistance of cells inside bioflms to antimicrobials and hosts immune mechanisms compared with their planktonic cells [7]. Te phospholipases hydrolyse the ester linkages on membrane phospholipids, releasing fatty acids as a potential energy source of C. neoformans [8]. Proteases can interfere with the host defense mechanisms by cleaving essential immunological components and directly damaging the efector cells [8,9]. In addition, serine protease secreted by C. neoformans promotes increased blood-brain barrier permeability [8,10].
Terapeutic options for the treatment of cryptococcosis are limited to polyenes, azoles, and 5-fucytosine. However, these classes of antifungal drugs are hampered by host toxicity and pathogen resistance. In addition, the lack of access to drugs and the high cost of efective treatment hamper therapeutic options [11,12]. Terefore, the development of new therapeutic strategies is urgent.
Natural products from plants can have the potential for the development of new antimicrobials with high activity, low toxicity, and the capacity to enhance the antimicrobial activity of conventional drugs [13]. Tymol is a monoterpene phenol compound that is a main constituent of the essential oils of thymus. Piperine is an alkaloid present in Piper nigrum and Piper longum vulgaris [14]. Gallic acid, curcumin, eugenol, and plumbagin are natural phenolic compounds found in Caesalpinia mimosoides, Curcuma longa, Eugenia caryophyllata, and Plumbago zeylanica, respectively. Tese natural products have been shown to possess several pharmacological properties such as antioxidant, anticancer, anti-infammatory, immunomodulatory, antibacterial, antifungal, antibioflm, and antiviral properties [15][16][17][18][19][20]. However, up to now, the antifungal and antibioflm activities of these natural products against C. neoformans have remained poorly studied. Te present study aimed to investigate the virulence factors in C. neoformans and the potential antifungal and antibioflm activities of thymol, curcumin, piperine, gallic acid, eugenol, and plumbagin against C. neoformans.

Microorganisms and Culture Media.
A total eight clinical isolates, namely, Cn46, Cn47, Cn91, Cn96, Cn118, Cn158, Cn169, and Cn173, were used in this work. Te clinical isolates of C. neoformans were obtained from the HIVpositive patients at the Yaoundé Central Hospital and identifed by serotyping by multiplex PCR in a previous study [21]. Sabouraud Dextrose agar (SDA, Lioflchem) and Sabouraud Dextrose broth (SDB, Lioflchem) were used for the activation of yeasts and antimicrobial assays, respectively.

Bioflm Formation
Assay. Te bioflm formation assay was performed using the microtiter plate method as previously described by Bisso et al. with some modifcations [6]. Standard cultures (1.5 × 10 6 CFU/mL) were diluted 100-fold in SDB supplemented with 2% glucose. From each culture, 200 μL of the solution was introduced into a 96-wellfatbottomed sterile polystyrene plate and incubated at 37°C for 48 h. After incubation, the wells were gently discarded and washed three times with sterile phosphate-bufered saline (PBS, pH 7.2) to remove the planktonic cells. Ten, 150 μL of methanol was transferred into each well to fx adherent cells, and the plates were incubated at room temperature for 20 min. After incubation, the wells were discarded and 150 μL of safranin (1%) was added to each well to stain the bioflm. After incubation at room temperature for 20 min, the wells were discarded and 150 μL of 95% ethanol was added to each well to solubilise the dye bound to adherent cells. Te optical density (OD) of plates was spectrophotometrically read at 570 nm. Te wells flled with SDB supplemented with 2% glucose were used as a blank. Each isolate was tested in triplicate. To determine the bioflm formation capacity of yeasts, the cut-of optical density (ODc) was defned as the sum of the mean OD value for the blank and three times the standard deviation. Te intensity of bioflm formation was categorized as follows: nonbioflm producer (OD < ODc), weak bioflm producer (ODc < OD < 2 × ODc), moderate bioflm producer (2 × ODc < OD ≤ 4 × ODc), and strong bioflm producer (OD > 4 × ODc).

Production of Phospholipase and Proteinase Assays.
Te production of phospholipase and proteinase was detected using the method described by Lahkar et al. with minor modifcations [22]. For the production of phospholipase, 10 μL of yeast inoculum (1.5 × 10 4 CFU/mL) was transferred onto egg yolk plates and incubated at 37°C for 2 days. An opaque zone around the yeast colony was characterized as the production of phospholipase (Pz) and expressed as Pz � colony diameter/(colony diameter-+ precipitation zone). Te phospholipase was categorized as strong when Pz ≤ 0.63, moderate when 0.64 ≤ Pz ≤ 0.99, and no activity when Pz � 1 [23].
For the production of proteinase, 10 μL of yeast inoculum (1.5 × 10 4 CFU/mL) was transferred onto agar plates containing bovine serum albumin and incubated at 37°C for 2 days. Te production of proteinase (Prz) was observed by a white dense zone of hydrolysis around yeast colonies. Prz was calculated and interpreted as described above for the production of phospholipase. Te experiment was performed in triplicate.

Determination of Minimum Inhibitory Concentrations (MICs) and Minimum Fungicidal Concentrations (MFCs).
Te antifungal activity was determined by the broth microdilution method [24]. In brief, serial twofold dilutions of natural products and antifungals were made using SDB at a total volume of 100 μL per well in 96-well microplates. Ten, 100 μL of fungal inoculum (1.5 × 10 4 CFU/mL) was added to each well, and the plates were incubated at 37°C for 48 h. Te fnal concentrations of natural products and antifungals ranged from 0.5 to 1024 μg/mL and from 0.125 to 256 μg/mL, respectively. Te MIC endpoint is the lowest concentration of natural product or antifungal where no growth was observed in the microplate. For MFC determination, 50 μL of the solution from the wells that showed no visible fungal growth was cultured on SDA plates. Ten, the plate was incubated at 37°C for 48 h. Te MFC was recorded as the lowest concentration that killed all yeast with no visible viable colonies on an agar plate. Te experiment was performed in triplicate and repeated three times. Te antifungal activity of natural products was considered as follows: most active (MIC ≤ 10 μg/mL), active (MIC ≤ 25 μg/ mL), moderate (25 < MIC ≤ 100 μg/mL), and inactive (MIC > 100 μg/mL) [25]. Te epidemiological cut-of values of antifungals described by the Clinical Laboratory Standard Institute were used for C. neoformans. For fuconazole, yeast with MIC ≤ 8 μg/mL was considered susceptible while yeast with MIC ≥ 64 μg/mL was considered resistant. For amphotericin B, the MIC value ≤ 1 μg/mL indicated that the yeast was susceptible, while MIC value > 4 μg/mL indicated that the yeast was resistant [26].

Bioflm Inhibition Assay.
Te activities of natural products and antifungals against the bioflm formation of C. neoformans were determined by the microtiter plate method as previously described by Kuaté et al. [14] with slight modifcations. In brief, 100 μL of serially double diluted solution concentrations from 0.5 to 1024 μg/mL for natural products or antifungals were added into the wells of the microplate. Ten, 100 μL of yeast inoculum (1.5 × 10 6 CFU/mL) was added to each well, and the microplate was incubated at 37°C for 24 h. After incubation, the planktonic cells were removed by aspiration of the medium and the wells were washed three times with 200 μL of sterile PBS. Ten, the microplate was treated as described above for bioflm formation. Untreated wells were used as a positive control while wells containing SDB supplemented with 2% glucose were used as a blank. Te percentage of bioflm inhibition of each compound was calculated using the formula (1 − (OD 570 test − OD 570 blank)/(OD 570 control − OD 570 blank)) × 100. Te minimum bioflm inhibitory concentration (MBIC) was defned as the lowest concentration of natural products or antifungals that reduced 100% of the bioflm biomass.

Bioflm Eradication Assay.
Te capacity of natural products and antifungals to eliminate mature bioflms was determined by using the method described by Kuaté et al. [14]. In brief, after bioflm formation as described above in the bioflm formation assay, the microplate was washed thrice with PBS to remove the nonadherent cells. Ten, each well of the microplate was flled with 200 μL of the serial two dilutions at concentrations ranging from 0.5 to 1024 μg/mL, and the plate was incubated at 37°C for 24 h. After incubation, the microplate was treated as described above and the minimum bioflm eradication concentration (MBEC) was recorded as the lowest concentration of natural products or antifungals that eradicated 100% of the preformed bioflm. Te test was performed in triplicate and repeated three times.

Statistical Analysis.
Statistical analyses were performed using GraphPad Prism version 8.0. Te correlation between bioflm formation and phospholipase/proteinase production was determined by Pearson's correlation coefcient (r). Tere is a perfect correlation when r � 1, no correlation when r � 0, and a negative correlation when r � −1. Te signifcance level was set at p < 0.05. Table 1 shows the virulence factors in C. neoformans. For bioflm formation, all isolates were able to form bioflms. Based on the cut-of value of ODc � 0.121, four isolates of C. neoformans (Cn46, Cn91, Cn96, and Cn118) were classifed as strong bioflm producers with OD values ranging from 0.523 ± 0.045 to 0.899 ± 0.057, while the four other isolates of C. neoformans (Cn47, Cn158, Cn169, and Cn173) were classifed as moderate bioflm producers with OD values ranging from 0.292 ± 0.05 to 0.45 ± 0.018.

Virulence Factors.
All isolates of C. neoformans showed strong production of phospholipase except the Cn46 and Cn47 isolates. Strong production of proteinase was observed in C. neoformans isolates except for the Cn47, Cn118, Cn169, and Cn173 isolates.

Correlation between Bioflm Formation and Extracellular
Enzymes. Correlation analysis results showed a negative correlation between bioflm formation and the following extracellular enzymes: phospholipase (r � −0.156, p � 0.738) and proteinase (r � −0.695, p � 0.085) ( Table 2). Table 3 shows the antifungal and antibioflm potencies of natural products against C. neoformans. For antifungal activity, the most active natural product tested in this study was plumbagin with MIC values below 25 μg/mL. Tymol showed moderate antifungal activity against all isolates of C. neoformans with MIC values below 100 μg/mL. However, eugenol, piperine, curcumin, and gallic acid were inactive against the majority of C. neoformans isolates with MIC values greater than 100 μg/mL. Te MIC values of eugenol and piperine ranged from 32 to 256 μg/mL, while the MIC values of curcumin and gallic acid ranged from 64 to 256 μg/mL and 64 to 512 μg/mL, respectively. According to the epidemiological cut-of values of antifungals, all C. neoformans isolates were resistant to amphotericin B and fuconazole.

Antifungal and Antibioflm Activities.
For antibioflm activity, the MBIC and MBEC values of the natural products and antifungals as well as the MBIC/ MIC and MBEC/MBC were determined. Te MBIC/MIC and MBEC/MBC ratios demonstrate the increased resistance in yeast living in bioflms compared to their planktonic cells. Plumbagin showed the highest antibioflm activity with MBIC and MBEC values ranging from 4 to 16 μg/mL and 32 to 256 μg/mL, respectively. Tymol and eugenol exhibited antibioflm activity, with MBIC values ranging from 32 to 512 μg/mL, whereas the MBIC values of curcumin and gallic acid ranged from 128 to 512 μg/mL. Te mature bioflms of C. neoformans isolates were destroyed by thymol, eugenol, piperine, and gallic acid with MBEC values ranging from 512 to 1024 μg/mL. Te MBIC values of amphotericin B and fuconazole varied from 64 to 128 μg/mL and 16 to 256 μg/ mL, respectively, while their MBEC values ranged from 256 to 512 μg/mL. Te MBIC/MIC and MBEC/MFC ratios of natural products (1 to 16 and 1 to 8, respectively) were higher than those obtained with antifungals (MBIC/MIC and MBEC/MFC ratios ranged from 0.25 to 2 and 1 to 4, respectively).

Discussion
In recent years, the emergence of antifungal drug resistance in C. neoformans has increased rapidly [27]. Hence, the morbidity and mortality rates due to cryptococcosis caused by C. neoformans are increasing continuously. Terefore, new therapeutic strategies need to be explored [11]. In the present study, C. neoformans virulence factors such as bioflm formation and the production of phospholipase and proteinase were studied. Te diferent levels of bioflm formation observed in C. neoformans isolates could be attributed to the diferent gene expression levels involved in bioflm formation in these isolates. In particular, a strong bioflm formation was reported in C. neoformans isolates [28]. Te strong production of phospholipase and proteinase observed in C. neoformans isolates would refect their more virulent character compared to the isolates of C. neoformans that produce moderate or weak virulence factors. Our results are similar to those reported earlier [29].
In addition, in the present study, the antifungal activity of thymol, curcumin, piperine, gallic acid, eugenol, and plumbagin was investigated. Tese natural products showed varying degrees of antifungal activity against C. neoformans isolates that could be attributed to their diferent mechanisms of action. In fact, thymol displayed an antifungal activity against C. neoformans by reducing the expression levels of calcium transporter genes in a calcineurin-dependent manner, and the expression of ergosterol biosynthesis genes in a high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway that responds to a variety of stimuli [30]. Curcumin can bind to the ergosterol present in the membrane, which leads to fungal cell disruption and a loss of intracellular content [31]. Gallic acid and eugenol decreased the activity of sterol 14α demethylase P450 (CYP51) and squalene epoxidase, resulting in the inhibition of ergosterol biosynthesis [32,33]. Plumbagin disrupts the cell membrane integrity and reduces the metabolic activity of C. neoformans [15]. Few studies have reported similar results of the antifungal activity of the studied natural products against C. neoformans. Kumari et al. reported the antifungal activity of thymol at a concentration of 16 μg/mL against C. neoformans [34]. Hassanpour et al. showed that eugenol at concentrations of 250 μg/mL and 500 μg/mL displayed 90% and 100% growth inhibition in C. neoformans, respectively [35]. Qian et al. reported the antifungal activity of plumbagin with an MIC value of 8 μg/ mL against C. neoformans [15].
Te antibioflm activity of these natural products was also investigated. It was observed that the MBIC values of natural products and antifungals were up to 16 times and 2 times higher than their MIC values, respectively. In addition, the MBEC values of natural products and antifungals were up to 8 times and 4 times higher than their MFC values, respectively. Tese results showed that the bioflm was more resistant to antimicrobial agents than planktonic cells, and this could be explained by the limited difusion of antimicrobial agents through the bioflm matrix or the lower growth rate of cells in the bioflm [6,36]. Tese results corroborated those reported earlier [14,24]. Kumari et al. reported the antibioflm activity of thymol and eugenol

Conclusion
Te natural products studied in this study were found to possess antifungal and antibioflm activities against C. neoformans. In particular, plumbagin was found to be the most active against