Assessing pH-dependent activities of virulence factors secreted by Candida albicans

Abstract

Candida albicans is an opportunistic pathogen that can thrive under adverse conditions including suboptimal pH, nutrient scarcity, and low levels of oxygen. Its pathogenicity is associated with the production of virulence factors such as extracellular hydrolytic enzymes and toxins. This study was aimed at determining the effect of external pH, substrate nature, and strain origin on protease, lipase, and hemolysin production. To achieve this objective, agar plate assays were performed at pH 5.0, 6.5, and 7.5 with substrates suitable for the detection of each family of enzymes. Moreover, the study was conducted with 20 clinical C. albicans isolates from blood, oral cavity, skin, urine, and vagina. The hydrolytic zones formed around the colonies were further measured to calculate the Ez (enzymatic zone) indexes. We found that detection of proteases in skim milk agar plates was possible for most isolates only at pH 5 (80%) and pH 6.5 (75%), whereas BSA plates could confer protease detection exclusively at pH 5 (80%). Similarly, the percentage of isolates possessing lipolytic activities was higher at pH 5 (90%) than at pH 6.5 (70%) and pH 7.5 (35%). In contrast, hemolytic activities were detected in all isolates at pH 6.5 and 7.5 but not at pH 5. Further analysis revealed that some differences in the detected activities could potentially be attributed to the anatomical origin of these isolates. Collectively, these findings suggest that the pH of the site of infection might be critical for mimicking the microenvironment employed to experimentally discover the key virulence factors.

Keywords: Candida pathogenicity; clinical isolates; hydrolytic zones; secreted enzymes.

Figure 1

Detection of proteases secreted by oral Candida albicans isolates 08‐052 (O1), 18‐034 (O2), 19‐078 (O3), and 19‐010 (O4). Assays were carried out using BSA‐containing agar plates at pH 5 (a) and skim milk‐containing agar plates at pH 6.5 (b) and pH 5 (c).

Figure 2

Dependence of the enzymatic protease activity coefficient (Ez) of Candida albicans isolates on the tested pH of milk agar. Scattered red (pH 5), green (pH 6.5), and blue (pH 7.5) circles represent the studied strains. Horizontal bars represent the statistically reliable (ɑ = 0.05) mean (longer bar) and standard deviation (shorter bars) values. Brackets indicate statistically reliable sets of data characterized by two thresholds: **p ≤ 0.01 and ***p ≤ 0.001.

Figure 3

Dependence of the protease activity coefficient (Ez) of Candida albicans isolates on pH. Assays with Candida albicans isolates from the oral cavity (a), urine (b), vagina (c), skin (d), and blood (e) were carried out on milk agar plates at pH 5, 6.5, and 7.5. Scattered red (pH 5), green (pH 6.5), and blue (pH 7.5) circles represent the studied strains. Horizontal bars represent the mean (longer bars) and standard deviation (shorter bars) values.

Figure 4

Detection of lipases secreted by oral Candida albicans isolates 08‐052 (O1), 18‐034 (O2), 19‐010 (O3), 19‐078 (O4), and C. parapsilosis (ATCC 22019) (C). The assay was carried out at pH 5 (a), pH 6.5 (b), and pH 7.5 (c).

Figure 5

Dependence of the enzymatic activity coefficient (Ez) of Candida albicans isolates on the tested pH. Scattered red (pH 5), green (pH 6.5), and blue (pH 7.5) circles represent the studied strains. Horizontal bars represent the statistically reliable (ɑ = 0.05) mean (longer bar) and standard deviation (shorter bars) values. Brackets indicate statistically distinct sets of data with two thresholds (*p ≤ 0.05 and ****p ≤ 0.0001).

Figure 6

Dependence of the lipase activity coefficient (Ez) of Candida albicans isolates on pH. Assays with Candida albicans isolates from the oral cavity (a), urine (b), vagina (c), skin (d), and blood (e) were carried out on malt agar plates at pH 5, 6.5, and 7.5. Scattered red (pH 5), green (pH 6.5), and blue (pH 7.5) circles correspond to the studied isolates. Horizontal bars represent the statistically reliable (ɑ = 0.05) mean (longer bar) and standard deviation (shorter bars) values. Brackets indicate statistically distinct sets of data (*p ≤ 0.05).

Figure 7

Detection of hemolysins secreted by oral Candida albicans isolates 08‐052 (O1), 18‐034 (O2), 19‐078 (O3), and 19‐010 (O4). Assays were carried out at pH 5.0 (a), pH 6.5 (b), and pH 7.5 (c).

Figure 8

Dependence of the β‐hemolytic activity coefficient (Ez) of Candida albicans isolates on the tested pH. Scattered red (pH 5), green (pH 6.5), and blue (pH 7.5) circles represent the studied strains. Horizontal bars represent the statistically reliable (ɑ = 0.05) mean (longer bar) and standard deviation (shorter bars) values. Brackets indicate statistically comparable sets of data and thresholds (*p ≤ 0.05,**p ≤ 0.01, and ****p ≤ 0.0001).

Figure 9

Dependence of the β‐hemolytic activity coefficient (Ez) of Candida albicans isolates on pH. Assays with Candida albicans strains isolated oral cavity (a), urine (b), vagina (c), skin (d), and blood (e) were carried out on blood agar plates at pH 5, 6.5, and 7.5. Scattered red (pH 5), green (pH 6.5), and blue (pH 7.5) circles correspond to the studied strains. Horizontal bars represent the statistically reliable (ɑ = 0.05) mean (longer bar) and standard deviation (shorter bars) values. Brackets indicate statistically distinct sets of data (*p ≤ 0.05).