Molecular Detection of Secreted Aspartyl Proteinases (Saps) From Dental Isolates of Candida albicans and Targeting With Psidium guajava Biocompounds: An In Vitro and In Silico Analysis

Introduction Candida albicans (C. albicans) is an opportunistic yeast-like fungus and is considered a functional biome of the oral and gut microbiomes. The sap gene and its types play a vital role in the pathogenesis of C. albicans. The emergence of resistance traits is a major problem, and targeting the same with alternative medicines has sparked renewed interest in recent years. Objectives This study is thus aimed at detecting the frequency of sap gene types in the clinical isolates of C. albicans and evaluating the antifungal effect of the crude methanolic extract of Psidium guajava (P. guajava). Further in silico assessments will assess the inhibitory effect of six compounds of P. guajava against the Sap protein. Materials and methods C. albicans was characterized phenotypically in 20 patients with root caries, and the sap gene was detected by PCR. The crude methanolic extract was prepared, and its antifungal efficacy was evaluated by the agar well diffusion method. Auto-docking was performed to assess the best compound based on the docking and overall interactions. Results Six isolates were identified as C. albicans and sap gene types 1-3 were detected in the four strains. P. guajava methanolic extracts showed a promising antifungal effect at varying concentrations. In silico analysis showed myricetin possessing the maximum number of hydrogen bonds and high docking energy with one violation. Conclusion The study concludes that P. guajava has a promising inhibitory effect against C. albicans with myricetin as the best compound to target the sap gene of C. albicans. However, further experimental studies are to be considered for its effectiveness in treating the infections caused by C. albicans.


Introduction
The opportunistic yeast-like fungus Candida albicans (C.albicans) is considered one of the important members of the functional biome of the oral and gut microbiome [1].It contributes to virulence, specifically in immunocompromised patients, affecting the oral-mucosal tissues [2].In dental patients, it is frequently detected in root caries and is also a potent biofilm formed in dental implants [3].Many virulent proteins, such as secreted aspartyl proteinases (Saps), phospholipase B enzymes, and lipases, are associated with the disease establishment by C. albicans.Amidst these, the Saps are encoded by nearly 10 different genetic determinants and exhibit different disease forms.
Saps are considered the most important virulence factor in C. albicans, playing a potent role in adherence to the tooth surfaces [4].The sequence homology of the Saps shows variations, and thus based on the aminoacid sequences, they are categorized into 10 types.They are known to express themselves in a differential manner based on the host and the habitat.The expression and presence of the sap types vary in different patients and in animal models [5,6].The virulence of sap in the establishment of various systemic infections has also been documented [7].
Amidst the virulence of Saps, C. albicans also emerges as resistant traits and is thus a major challenge in treating complicated recalcitrant infections.Plant-based medicines and their bio-compounds have been implemented to tackle the same, and many bioactive compounds have been discovered as novel drugs.Among many plant-based compounds, Psidium guajava (P.guajava), the common guava, is an evergreen shrub or a small tree that is native to tropical countries [8].In traditional medicine, P. guajava has been used to treat a wide variety of systemic and infectious diseases [9].
P. guajava (guava) is a well-known tropical tree with medicinal properties against different systemic illnesses [10].The leaf of P. guajava has wide applications in oral ulcers and gingivitis [11].Its fruit is rich in vitamins and various minerals with potent antioxidant, anti-inflammatory, and anti-cancer properties [12,13].Structural elucidation of P. guajava extracts shows many vital phytocompounds with high medicinal values [14].
With this background, the present investigation is aimed at evaluating the detection of sap from C. albicans in patients with root caries, together with the inhibitory effect of the methanolic crude extracts of P. guajava.
Further in silico evaluation is designed to assess the drug-ligand interactions of the sap with the six bioactive compounds from P. guajava.

Genotypic characterization of sap gene in C. albicans
From the Sabouraud dextrose agar, fresh cultures of C. albicans were retrieved, and the genomic DNA was extracted using the manufacturer's instructions (Qiagen kit, Hilden, Germany).sap types from one to seven were detected by performing PCR with 15 µl of the reaction mixture and specific primers of sap (0.1 µl of 100 pmol/ml concentration) (Table 1).

TABLE 1: Primers used to detect the sap genes from the clinical isolates of C. albicans
Amplification for 35 cycles was performed at an annealing temperature of 58°C.The amplicon sizes were visualized on a 1.5% agarose gel with EtBr, and the size was assessed with a 1.5 Kbp DNA ladder.
Preparation of the P. guajava extract P. guajava fruits were obtained from local market vendors, and using sterile distilled water, the fruits were washed three times properly to remove the dust.Using sterile blades, the fruits were cut into small pieces, shade-dried, and ground into a fine powder.Ten grams of P. guajava powder were then weighed and added to 100 ml of methanol, which was kept for one week at room temperature with intermittent mixing.After one week, the crude filtrate was collected by filtering the mixture on Whatman No. 1 filter paper, followed by evaporation and complete drying.The crude yield was stored at 4°C until further bioassay procedures.

Antifungal bioassay
A varying concentration of P. guajava (100 mg, 50 mg, 25 mg, 12.5 mg, and 6.25 mg) was prepared with 1 ml of dimethylsulfoxide and vortexed.Lawn cultures of C. albicans were made onto the sterile Saboraud dextrose agar, and wells were punctured with a sterile agar cutter.About 50 μl of the diluted extract was added to the appropriate wells, followed by a 48-hour incubation period at 37°C.The bioassay was performed in triplicates, and the mean value of the zone of clearance was recorded.

Sap protein retrieval and optimization
The crystal structure of sap1 was retrieved from the UniProt data bank with added hydrogen atoms to optimize it.Electronic charges were assigned to the Sap protein using the AutoDock tool version 1.5.6 (Center for Computational Structural Biology, California, USA), and the 3D structure of sap was visualized by the RASMOL tool (http://www.openrasmol.org/).

P. guajava ligand selection after optimization
Five bio-compounds from P. guajava, viz., avicularin, apigenin, hyperin, myricetin, chlorogenic acid, and fluconazole as controls, were selected using the ChemSketch software (Advanced Chemistry Development, Ontario, Canada).Using the open babel molecular converter, suitable conformations were done for the ligands to be optimized and were finally stored in PDB and saved in a .molfile.

Molinspiration molecular parameter evaluation of the ligands
The suitability of the selected ligands for their drug properties, bioavailabilities, membrane permeability, molecular weight, and further evaluation of the violations and ADME properties were assessed using a molinspiration tool and based on Lipinski's rule of five.The compounds were assessed for hydrogen donors and acceptors, lipophilic properties such as miLogP and TPSA values, and for the N-atoms.The selected compounds were also evaluated for drug likeliness scores for their ability to bind with the human G protein-coupled receptors, for their ability to modulate the ion channels, and finally their inhibitory properties for kinases, nuclear receptors, proteases, and other enzymes.

Drug ligand interactions of P. guajava compounds with sap1
The chemical affinities via various interactions between the selected compounds and sap1 were evaluated using the AutoDock tool.The chemical interactions formed were visualized using the Discovery studio visualizer (Dassault Systèmes, Vélizy-Villacoublay, France), with further observation of their relative stabilities using the binding energies and the docking scores.

Identification of C. albicans from the clinical samples
Eleven isolates of C. albicans were identified from the specimens based on the typical creamy white moist colonies on the Sabouraud dextrose agar plate and greenish blue-colored colonies on the HiCrome Candida differential agar and gram staining that showed the gram-positive oval cells (Figure 1).Genotypic characterization of sap gene types showed three isolates (27%) with the presence of sap1, two isolates (18%) with sap2, and one isolate (0.09%) with sap3 (Figure 2).None of the strains showed the presence of sap types 4, 5, 6, and 7.

Antifungal activity of P. guajava methanolic extract
The methanolic extract of P. guajava yielded 34 mg of crude extract from 100 gm of the dried fruit powder.The agar well diffusion assay for the antifungal activity showed a promising antifungal effect against all the isolates tested, with a zone size of 19 mm for 100 mg and 12 mm for 50 mg/ml concentrations and no effect with concentrations of 25, 12.5, and 6.25 mg/ml concentrations (Figure 3).

Sap1 structure and molinspiration results
Sap1 from the C. albicans was successfully retrieved with the structure ID 2QZW-A-chain, and the 3D structure retrieved using RASMOL is given in Figure 4.The bioactivity scores assessed >0.03 were also promising for the selected ligands under study (Table 3).

guajava bio-compounds under study
In the molinspiration evaluations, apigenin and fluconazole showed zero violations, followed by myricetin and cholorogenic acid with one violation.Hyperin and avicularin showed two violations.The selected compounds showed a good TPSA value for all the compounds except apigenin.TPSA for fluconazole was also detected less.miLogP is an essential predictor for the lipophilic property, and it was toward the best score.The drug likeliness for the nuclear receptor and the enzyme inhibitor values are >0.3, and the selected compounds showed a drug likeliness score of above 0.2.The larger the drug likeliness values of the compounds, the more active they are, and they are thus considered for further docking analysis.

Results of the sap1 interactions with the P. guajava bio-compounds
The binding energies and docking scores obtained between sap1 and the compounds avicularin, apigenin, hyperin, myricetin, chlorogenic acid, and fluconazole were promising and are shown in Figure 5. Myricetin showed four hydrogen bonds with a docking score of -6.9.It was followed by apigenin at -6.84, hyperin at -6.47, and avicularin at -6.16.The overall interactions for the scores of other interactions like van der Waals, alkyl-, -alkyl, p-sulphur, and p-alkyl were also highly promising for the compounds to be further considered as suitable drug candidates.

Discussion
The opportunistic yeast C. albicans has been documented as the most frequent source of hospital-acquired infections [15].C. albicans has been reported to cause two major types of infections, such as fatal systemic infections and superficial infections.It is documented that 75% of human beings possess C. albicans and other Candida species to a lesser level in the oral cavity, with refractory oral infections in immunecompromised individuals [16].Resistance in C. albicans aids in the progression of the disease and affects the treatment regimens [17].
C. albicans is known for its production of vital virulent factors, and the sap gene family is differentially expressed in the flipped states of the candidal cells and is a suitable target for novel drug design [18].sap was thus chosen for the present investigation, and we aimed to assess the frequency of the sap types in the clinical isolates of C. albicans from root caries.In the present study, we found the association of C. albicans in root caries and the prevalence of sap types 1-3 in the clinical isolates.The other sap types were not detected in the present study.In contrast, a study by Vita et al. documented the specific expression of sap types 4-6 from C. albicans from HIV-positive cases and from the normal flora of healthy individuals [19].At the same time, sap types 1-3 have been shown to be expressed in different candidal species, and their frequency correlates with our study [20].
The emergence of resistant traits is a major challenge for all clinicians, and alternative medicine is being implemented to combat the menace of resistant C. albicans strains.In this context, in the present study, crude extracts of P. guajava were checked against the clinical isolates of Candida species.The antifungal efficacy was moderate in the present study, and many earlier studies have documented only a minimal effect, stating that Psidium compounds may have an inhibitory effect on the virulence of Candida sp.rather than antifungal effects [21].
In silico-based approaches to determining novel compounds from natural plants have sparked renewed interest in recent years.Many computational-based studies have been performed to assess the different bioactivities of various sources and were found to be promising [22][23][24].Health benefits from P. guajava are due to a plethora of phytochemicals, such as quercetin, avicularin, apigenin, guaijaverin, kaempferol, hyperin, myricetin, gallic acid, catechin, epicatechin, chlorogenic acid, epigallocatechin gallate, and caffeic acid.In this note, six compounds were chosen for the docking analysis in the present study, and fluconazole was added as a control.Molinspiration parameters showed the drug properties, where apigenin and fluconazole showed no violations, followed by one violation by myricetin and chlorogenic acid.TPSA is considered a very useful analysis to evaluate the drug absorption and miLopgP for the oral bioavailability of

FIGURE 1 :
FIGURE 1: Phenotypic characterization of C. albicans from the clinical samples.(a) C. albicans growth on HiCrome Candida differential agar.(b) Gram staining showing gram-positive budding cells of C. albicans

FIGURE 2 :
FIGURE 2: PCR amplification of the sap types 1, 2, and 3 from the clinical strains of C. albicans.(a) Electropherogram of sap1 with an amplicon size of 161bp in lanes 1, 2, and 3. (b) sap2 with an amplicon size of 178bp in lanes 1 and 2. (c) sap3 gene product of size 231bp in lane 2 (1.5k bp marker lane (M))

FIGURE 4 :
FIGURE 4: 3D structure of sap1 visualized using RASMOL.Red indicates the Alpha-helix; blue indicates Beta sheets; white indicates the turns

FIGURE 5 :
FIGURE 5: Visualizing hydrogen interactions between sap1 with (a) avicularin, (b) apigenin, (c) hyperin, (d) myricetin, (e) chlorogenic acid, and (f) fluconazole Source: images were created by the author , were selected for the study.The carious scrapings were excavated and collected in sterile Sabouraud dextrose broth and were immediately transferred to the microbiology laboratory.The samples were inoculated onto sterile Sabouraud dextrose agar and also in HiCrome Candida differential agar and incubated at 37°C for 24 hours.After incubation, colonies were identified using the color of the colonies, colony morphology, and gram staining for the preliminary identification of C. albicans.