A Comparative Evaluation of Exoenzyme Production, Biofilm Development, and Cell Surface Hydrophobicity in Dominant Genotypes of Candida albicans

ABSTRACT

Background

Candida albicans exhibits significant genotypic diversity, and genotypes A and C are the most predominant clinical isolates. The aim of this study was to evaluate the virulence traits of these genotypes, focusing on exoenzyme production, biofilm formation, and cell surface hydrophobicity (CSH) property.

Methods

A total of 15 genotype A and 15 genotype C clinical C. albicans isolates were evaluated for proteinase, phospholipase, and esterase activities using standard methods. Biofilm formation was quantified using a microtiter plate assay, and CSH was measured using a water‐octane two‐phase assay.

Results

The study found that Genotype C had higher proteinase and phospholipase activity but no esterase production, while 40% of Genotype A isolates showed strong esterase activity. Biofilm formation and CSH did not differ significantly, though Genotype A trended toward stronger biofilm formation.

Conclusion

This study highlights how genotypic variation in C. albicans influences virulence, with Genotype C exhibiting a distinct profile (high proteinase/phospholipase, no esterase) that may enhance pathogenicity, while Genotype A shows adaptability through variable enzyme production and stronger biofilm trends. These differences underscore the need for genotype‐specific diagnostics and targeted therapies to improve candidiasis treatment.

Schematic overview of virulence trait assessment in Candida albicans clinical isolates.

1. Introduction

Candida albicans is an opportunistic yeast that exists as a human microbiome that mostly colonizes the oral cavity, gastrointestinal tract, and vaginal mucosa [1]. It gains its significance as it has the capacity to coexist with the host while being able to switch into a pathogen form. C. albicans causes infections from mild to high‐severe manifestations ranging from superficial to potentially life‐threatening systemic candidiasis [2]. The clinical importance of C. albicans is the fact that it is able to capitalize on various host vulnerabilities and escape from immune responses, making it the most common fungal pathogen worldwide [3]. C. albicans has three genotypes characterized as A, B, and C. The genotypes A and C are typically the most predominant genotypes isolated from different patients [4]. Numerous studies have investigated the relationship between different genotypes and their virulence factors; however, they have reported conflicting results, thus emphasizing the complicated interactions between fungal genetics, host immunity, and environmental circumstances [4, 5, 6]. Such a conditional conclusion draws a line of ambiguity in directly associating C. albicans' genotype with virulence or pathogenicity in further studies directed to confer the complexity of their interactions. C. albicans pathogenicity is through secretion of hydrolytic exoenzymes (the proteases, phospholipases, and esterase, etc.), a high‐strength biofilm formation, the presence of cell surface hydrophobicity (CSH), and phenotypic switching [7]. Tissue invasion and nutrient gain by exoenzymes include breaking down host cell membranes and extracellular matrix components. On the other hand, biofilm development provides a protective niche for the yeast, and thus its resistance to antifungal drugs and host immunity is increased [8]. Furthermore, CSH also induces adhesion to biotic and abiotic surfaces that are necessary for colonization and infection [9]. Polymorphism is a central strategy for C. albicans virulence. It directly enables invasion, enhances other virulence factors, regulates their expression, helps the yeast adapt to harsh host environments, and provides ways to evade the immune system [10]. Though the importance of these virulence traits in C. albicans infections has been well documented, the impact of genetic diversity among strains on their expression is less known. Candida albicans exhibits significant genotypic variation, with certain dominant genotypes being more frequently associated with clinical isolates. These genotypes might have differential phenotypic profiles that may facilitate increased virulence and adaptability and thus make contributions to the phenotypic differences in severity, recurrence, and therapeutic endpoints. Knowledge of the correlation between genotypic variation and virulence factor expression is fundamental for separating causal from accidental contributions to the pathogenic potential of C. albicans and genome‐guided therapeutic approaches. The aim of this study was to assess exoenzyme activity, the severity of biofilm formation, and cell surface hydrophobicity between dominant genotypes of clinical C. albicans isolates.

2. Materials and Methods

2.1. Specimen's Collection

In this study, 30 C. albicans isolates, comprised of 15 genotype A and 15 genotype C, from candidiasis patients were investigated. These isolates have been previously identified to genotypes using molecular methods in an earlier study [6]. Isolates were subcultured onto Sabouraud dextrose agar before use.

2.2. Enzyme Activities

2.2.1. Proteinase Activity

The proteinase activity was measured using a bovine serum albumin (BSA) medium, following the method described by Dagdeviren et al. [11]. Each 6 mm blank disk was soaked in a solution with a concentration of 10⁷ cells/mL. Then, the disk was placed onto the BSA agar plates. The plates were kept in an incubator at 35°C for 6 days. After this time, the opacity around the disks was measured. The formation of a precipitant zone around the colonies was evaluated, and the precipitation zone (Pz) for proteinase activity was categorized as follows: Pz negative [1], Pz 1+ (0.9–0.99), Pz 2+ (0.8–0.89), Pz 3+ (0.7–0.79), and Pz 4+ (< 0.7).

2.2.2. Phospholipase Activity

Phospholipase activity was measured using the egg yolk agar method, a technique developed by Price et al. [12]. The medium was composed of 5.5 g of CaCl₂ and 58.4 g of NaCl added to Sabouraud dextrose agar (Merck, Germany). A sterile egg yolk was centrifuged at 5000 × g for 30 min, and 20 mL of the supernatant was incorporated into the cooled medium. A yeast suspension with 2 McFarland turbidity was prepared, and 10 μL of this suspension was spot‐inoculated onto the plate medium. The plates were then incubated at 35°C for up to 5 days; the precipitant zones around the colonies were measured, and the results were reported as Pz, according to the categories defined above.

2.2.3. Esterase Activity

For evaluation of esterase activity, Tween agar plate (Sigma‐Aldrich) was used. The medium was made by combining 1 g peptone, 0.5 g NaCl, and 0.01 g CaCl₂ with 100 mL of distilled water, pH adjusted to 7.0. After adding 1.5 g of agar, the medium was autoclaved and cooled to around 50°C. Later on, 0.5 mL of autoclaved Tween was added to the medium. A yeast suspension adjusted to 2 McFarland turbidity was prepared, and 10 μL of the suspension was spot‐inoculated onto the plate medium. The hydrolysis of Tween by esterase activity resulted in the release of fatty acids, which then chelated with calcium, forming an opaque precipitation zone (Pz) around the fungal colonies. The sizes of these zones were measured [13].

2.3. Biofilm Formation Assay

Biofilm formation was measured using a protocol of Jin et al. [14] and modified by Melo et al. [15]. In brief, 100 μL of 10⁷ cells/mL suspension was inoculated into flat‐bottom 96‐well microtiter plates and shaken at 37°C for 1.5 h using a shaker at 75 rpm. Eight wells per plate, without Candida spp., were considered as negative controls. After incubation, the cell suspensions were aspirated gently, and each well was washed twice with 150 μL PBS (phosphate‐buffered saline). Next, 100 μL of Yeast Nitrogen Base (YNB) medium (Sigma‐Aldrich) supplemented with 50 μM glucose (D‐glucose monohydrate) was added to each well, and the plates were incubated at 37°C with a shaker at 75 rpm for 66 h. After incubation, the wells were washed twice with 150 μL of PBS and air‐dried. The biofilms were stained using 110 μL of 0.4% aqueous crystal violet solution for 45 min. The excess stain was removed by washing each well four times with 350 μL of sterile distilled water. The biofilms were destained by 95% ethanol. Next, 100 μL of the destain solution was added to a new well, and the crystal violet stain absorbance was measured in a microtiter plate reader (BMG LABTECH) at 570 nm. Density of biofilm was classified into four levels according to defined cut‐off values as follows (ODc) [16].

• –OD ≤ ODc: Negative.
• –ODc < OD ≤ 2 × ODc: Weakly positive.
• –2 × ODc < OD ≤ 4 × ODc: Moderately positive.
• –OD > 4 × ODc: Strongly positive.

All the experiments were done in triplicate.

2.4. Cell Surface Hydrophobicity Property

Measurement of cell surface hydrophobicity was carried out in a water‐octane two‐phase assay [17]. In this method, 1.2 mL of yeast cells, washed with PBS and adjusted to a concentration of 10⁸ cells/mL, were mixed vigorously with 0.3 mL of octane. The mixture was left at room temperature to separate for 15 min. Negative controls were set up by measuring the absorbance at 600 nm (ABS600) of PBS‐octane‐free fungal cells. The relative cell hydrophobicity was calculated using the following formula to determine the percentage of fungal cells excluded from the aqueous phase.

To calculate the relative cell hydrophobicity, the percentage of yeast cells excluded from the aqueous phase was determined using the following formula:

$$\mathrm{CHS}\left(\%\right)=\left(\frac{\mathrm{ABS}600\text{of the control}-\mathrm{ABS}600\text{after octane overlay}}{\mathrm{ABS}600\text{of the control}}\right)\times 100$$

Based on the ABS600 changes, CSH levels were classified into three categories:

• –High CSH: 80%–100%.
• –Moderate CSH: 20%–80%.
• –Low CSH: 0%–20%.

2.5. Ethics Approval

This project was found to be in accordance with the ethical principles and the national norms and standards for conducting Medical Research in Iran and has been approved by the research ethics (IR.SUMS.MED.REC.1403.339).

2.6. Statistical Analysis

The statistical analyses for the enzyme tests consisted of the chi‐squared and Fisher's exact test using SPSS software (version 22) and GraphPad Prism 6, and the statistical significance level was considered at p value ≤ 0.05.

3. Results

The comparative analysis of dominant Candida albicans genotypes (A and C) revealed significant differences in biofilm formation, cell surface hydrophobicity (CSH), and exoenzyme secretion, including proteinase, phospholipase, and esterase activities.

3.1. Exoenzyme Production

3.1.1. Proteinase Activity

Genotype A isolates displayed variability, with 4 isolates (26.7%) being negative and 11 isolates (73.3%) showing strong activity (4+). All 15 isolates of genotype C (100%) exhibited strong proteinase activity (4+). Proteinase activity demonstrated a statistically significant difference between the two genotypes (p value = 0.05) (Figure 1).

FIGURE 1.

Differential extracellular enzymatic profiles of C. albicans genotypes A and C. (Top row) Genotype A: Positive proteinase, phospholipase, and esterase activities. (Bottom row) Genotype C: Positive proteinase, negative phospholipase, and negative esterase activities.

3.1.2. Phospholipase Activity

Phospholipase activity further differed significantly between the genotypes (p value = 0.007). In genotype A isolates, the majority, 11 (73.3%), were negative, while 4 (26.7%) had strong activity (4+). In contrast, it was observed that among the 15 genotype C isolates, 9 (60%) exhibited strong phospholipase activity (4+), while the remaining 6 isolates (40%) were phospholipase negative.

3.1.3. Esterase Activity

Esterase activity was significantly different between genotypes A and B (p value = 0.008). The genotype A had variation, of which 9 isolates (60%) were esterase negative, and the prevalence of 6 isolates (40%) had a strong activity (4+), respectively. All 15 isolates (100%) of genotype C were esterase negative, suggesting that this genotype does not produce esterase altogether (Table 1).

TABLE 1.

Enzymatic activity of isolates in different genotypes.

	Proteinase activity		Phospholipase activity		Esterase activity
	Negative	4+	Negative	4+	Negative	4+
Genotype
A	4 (26.7%)	11 (73.3%)	11 (73.3%)	4 (26.7%)	9 (60%)	6 (40%)
C	0 (0%)	15 (100%)	6 (40%)	9 (60%)	15 (100%)	0 (0%)

3.2. Biofilm Formation

Biofilm formation between the two genotypes did not show any statistical difference with a p value equal to 0.2. Biofilm formation in genotype A isolates ranged as follows: 4 isolates (26.7%) were negative, 5 isolates (33.3%) were mildly positive, 3 isolates (20%) were moderately positive, and 3 isolates (20%) were intensely positive. In contrast, biofilm formation in genotype C isolates was predominantly mildly positive (53.3%), with 1 isolate (6.7%) being intensely positive and 6 isolates (40.0%) being negative.

3.3. Cell Surface Hydrophobicity (CSH)

There were no statistically significant differences in the CSH between the two genotypes (p value = 0.5). Genotype A and C exhibited low hydrophobicity, accounting for 60% and more than 66.7% of isolates, respectively. Moderate hydrophobicity can be found in 40% of genotype A and 33.3% of genotype C. No observations of high hydrophobicity were reported for either genotype. More details were demonstrated in Table 2.

TABLE 2.

Frequency of Biofilm formation among the isolates.

	Biofilm formation				Hydrophobicity
	Negative	Mild	Moderate	Intense	Low	Moderate
Genotype
A	4 (26.7%)	5 (33.3%)	3 (20%)	3 (20%)	9 (60%)	6 (40%)
C	6 (40%)	8 (53.3%)	0 (0%)	1 (6.7%)	10 (66.7%)	15 (33.3%)

4. Discussion

Although numerous studies have been conducted on enzymatic activity and biofilm formation capabilities in Candida albicans species, it remains unclear which genotype specifically possesses these abilities. According to existing research, the dominant genotypes are primarily A and C [4, 18, 19]. These genotypes are frequently associated with different sources of clinical isolates and have been the focus of many studies on the prevalence and potential impact on virulence [20]. Further complementary studies are needed to elucidate the mechanisms underlying these differences and to determine whether genotypic variation significantly influences the expression of virulence factors such as exoenzyme production and biofilm formation.

In the present study, we carried out the comparative analysis of the exoenzyme activities and biofilm formation, along with cell surface hydrophobicity, among dominant C. albicans genotypes (A and C). Overall, in this study, results demonstrate that genotype C exhibited higher phospholipase and proteinase activities compared to genotype A, whereas esterase activity was more noticeable in genotype A than in genotype C, which has significant differences in exoenzyme production.

Proteinase activity, a major virulence factor enabling tissue invasion for nutrient uptake, was consistently positive in all isolates of genotype C (100% with strong activities), while genotype A displayed heterogeneity, being 73.3% positive and the rest being negative. One of the assumptions suggested based on our results is that genotype C has a genotypic susceptibility for high proteinase production, and this probably underlies high pathogenicity. In a study conducted in 2022 by Gharghani et al. [5] the most prevalent genotypes were A (88.3%) and B (7.4%). The enzymatic profiles of these strains reveal that the highest enzyme activity was for proteinase (97.1%). In our study, isolates from genotype C exhibited a significantly higher strong phospholipase activity compared to those from genotype A, aligning with the findings reported by Gharghani et al. [5]. The difference may be due to the amplification of host cell membrane degradation and tissue invasion that phospholipases can do, and that genotype C isolates could be more adept at breaching host defenses. The wide range of phospholipase activity among the genotype A isolates further highlights the genetic diversity associated with this genotype.

In contrast, the esterase activity indeed presented a sharp contrast between both genotypes. While 40% of genotype A isolates were esterase producers, all genotype C isolates were esterase negative. In another study, Ali et al. [21] reported that 45.6% of genotype A isolates were esterase producers, whereas, in contrast to our findings, 86.9% of genotype C isolates were positive for esterase activity. The negative esterase activity in genotype C isolates indicates either genotypic constraint or a potential trade‐off for virulence factor expression. The p value for esterase activity (p value = 0.008), being significant, demonstrates the genotypic disparity in determining exoenzyme profiles and hence pathogenicity tendencies.

One of the fundamental virulence determinants that contributes to antifungal resistance and immune evasion is biofilm formation [22], which in our study data did not demonstrate statistically significant differences (p value = 0.2) between the two genotypes. However, our data suggested that with some trends, genotype A isolates may have stronger biofilm formation. This was in line with earlier studies on the marked variation in biofilm‐forming capacity of C. albicans genotypes [21, 23]. The lack of statistical significance may reflect the impact of environmental and host‐dependent factors in inducing biofilm formation, which needs further investigation focusing on genotype–environment interaction. These contrasting findings highlight the complexity of genotype–phenotype interactions in C. albicans and underscore the need for further research to elucidate the mechanisms underlying biofilm formation in different genotypes.

CSH property, which facilitates adhesion to host tissues and medical devices, did not differ significantly between the two genotypes (p value = 0.5). Both genotypes were predominantly characterized by low hydrophobicity, with moderate hydrophobicity observed in a subset of isolates. The absence of high hydrophobicity in either genotype suggests that this trait may not be a primary or basic determinant of virulence in these isolates [9]. However, the role of CSH in specific clinical contexts, such as catheter‐associated infections, warrants further exploration.

Overall, the significant differences in exoenzyme production between genotype A and genotype C highlight the potential impact of genotypic variation on C. albicans pathogenicity. Genotype C's uniformly high proteinase and phospholipase activities, coupled with its lack of esterase activity, suggest a distinct virulence profile that may contribute to its clinical success. In contrast, genotype A's variability in exoenzyme production and biofilm formation may reflect a broader adaptive capacity, enabling it to thrive in diverse host environments. These findings have important implications for understanding the mechanisms underlying C. albicans infections and developing targeted therapeutic strategies. For instance, the high proteinase and phospholipase activities observed in genotype C isolates could serve as potential causes of clinical manifestation and targets for antifungal and supplementary therapies [23]. Conversely, the absence of esterase activity in genotype C may represent a vulnerability that could be exploited in future treatment approaches. While the variability in exoenzyme production and biofilm formation among genotype A may represent a broader adaptive capacity, demonstrating the ability to succeed in many hosts.

Differences in exoenzymes may be caused by polymorphisms in the secreted aspartic proteinase family (SAP), phospholipase (PL), and triacylglycerol lipase (TGL) gene families. The variation in genes encoding enzymes can significantly influence the severity of their activity, especially under specific in vivo conditions. For example, the pH level plays a crucial role in regulating the activity of SAP genes, either increasing or decreasing the enzyme's production. The absence of esterase in Genotype C may be attributed to TGL mutations or epigenetic silencing, necessitating transcriptomic analysis [24, 25]. Genotype C's elevated proteinase/phospholipase activity indicates possible diagnostic markers (e.g., genotype‐specific PCR) for severe infections. Therapies aimed at SAP/PL genes (such as protease inhibitors like pepstatin A) could enhance outcomes. On the other hand, the variability of esterase in genotype A shows how important it is to have personalized antifungal regimens (like azoles + biofilm disruptors). While there is a lot of research on comparing the genotypes of C. albicans , this study moves the field forward by showing links between different genotypes and different phenotypic virulence traits. While this contribution represents incremental rather than transformative progress, it provides a critical foundation for delineating the role of esterases in host colonization (e.g., nutrient assimilation, tissue barrier compromise) and their clinical utility (e.g., as prognostic biomarkers for strain virulence or antifungal resistance). Subsequent studies proposed to emphasize the mechanistic elucidation of the molecular pathways regulating these adaptations and the validation of their translational significance in candidiasis patients to enhance targeted diagnostic and therapeutic approaches.

These findings hold significant implications for enhancing our understanding of host tissue adaptations during C. albicans infections. They also provide valuable insights that can guide the exploration of invasion mechanisms and inform the design of targeted therapeutic agents. Our study limitations include a modest sample size, restrictions to a single institution, and possible undetected co‐infections by bacteria. Thus, future multi‐center studies with longitudinal surveillance are essential to confirm these trends.

5. Conclusion

This study has revealed the differences in virulence factors in different genotypes of C. albicans , particularly in exoenzyme activity. The significant differences in proteinase, phospholipase, and esterase activities between genotype A and genotype C highlight the importance of genotypic variation in shaping virulence traits. While biofilm formation and CSH did not show significant genotypic differences, trends in the data suggest potential influences that warrant further investigation. These data provide insight into the pathogenic mechanisms of C. albicans and add to the urgent need for a genotype‐specific approach in the diagnosis and treatment of candidiasis. Data from this study may inform treatment strategies and elucidate genetic adaptations and invasion mechanisms that enable C. albicans to colonize and infect host environments more effectively.

Author Contributions

Conceptualization: Keyvan Pakshir; methodology: Keyvan Pakshir, Hasti Nouraei; formal analysis and investigation: Hasti Nouraei, Neda Amirzadeh, Fatemeh Ahmadi; writing – original draft preparation: Keyvan Pakshir, Hasti Nouraei; writing – review and editing: Keyvan Pakshir and Hasti Nouraei; funding acquisition: Keyvan Pakshir; resources: Keyvan Pakshir; supervision: Keyvan Pakshir.

Consent

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.