INTRODUCTION
Candida albicans is a common colonizer of human skin and mucosal surfaces. The balance between colonization and candidiasis depends on changes in the expression of virulence factors in response to environmental changes and the competence of the host immune system1.
Several virulence factors of C. albicans have been discovered or proposed, including adhesion factors, hyphal formation, germ tubes (GTs), phenotypic diversity, and the production of extracellular hydrolytic enzymes such as phospholipases and proteinases2−6.
Hydrolytic enzymes destroy or disorganize elements of host cell membranes, leading to membrane dysfunction and/or physical disruption. Phospholipases act by invading host cells, causing tissue damage, rupturing the epithelial cell membranes, and allowing hyphae to penetrate the cytoplasm. Proteinase production increases the ability of certain organisms to colonize and penetrate host tissue, deceive the host immune system, and disrupt a significant number of proteins important for immunity such as immunoglobulins, complement proteins, and cytokines5,7−9. Yeast can be induced to produce GTs, which develop into mycelia. This morphological transition can be induced by changes in a variety of environmental factors and by blocking the host immune response10.
Because of the increasing incidence of invasive C. albicans infections, interest in their virulence factors has increased. This avenue of research may help to establish strategies for the prevention and control of candidiasis and identify possible targets for the development of new therapeutic interventions7,11. However, studies of the relationship between GT production and hydrolytic enzyme activity in bloodstream isolates are scarce2,12.
Therefore, this study aimed to investigate the known virulence factors, including phospholipase and proteinase activity and GT production, in bloodstream isolates of C. albicans in vitro.
METHODS
One hundred fifty-three human isolates of C. albicans were obtained from blood samples between 1999 and 2009, and the yeast was identified using the ID 32C assay (BioMérieux, Marcy-I’Etoile, France); isolates were stored at -20°C in a freezer at a mycology laboratory (Santa Casa de Porto Alegre, Rio Grande do Sul, Brazil). The isolates were analyzed for GT, phospholipase, and proteinase production. The standard strain of C. albicans, ATCC 28367 (American Type Culture Collection, Rockville, MD, Manassas, USA), was included in the experiments as a control.
The Candida albicans inoculum was prepared from stock cultures, incubated for 24h at 37°C in Sabouraud dextrose agar, and standardized according to the 0.5 McFarland turbidity range using a densitometer (BioMérieux).
The isolates were screened for their extracellular phospholipase activity by growing them on egg yolk medium and measuring the diameter of the zone of precipitation as previously described by Vidotto et al.8. The egg yolk medium consisted of 22.5g of malt agar, 29.2g of NaCl, 0.28g of CaCl2, and 10% sterile egg yolk. Extracellular phospholipase activity was detected by inoculating 10-µL aliquots of C. albicanssuspensions (approximately 1 × 108 cells/mL) into wells punched through the surface of the egg yolk medium. The diameter of the precipitation zone around the well was measured after incubation at 37°C for 6 days. Phospholipase activity (Pz value) was determined as the ratio of the diameter of the colony to the total diameter of the zone of precipitation.
The determination of protease production was performed in agar containing bovine serum albumin, as previously described by Mohan & Ballal2. A solution was prepared by dissolving 11.7g of yeast carbon base, 0.1g of yeast extract, and 2g of bovine serum albumin in 200mL of distilled water. The solution was sterilized by filtration and added to a previously sterilized stock solution of 16g of agar-agar in 800mL of distilled water. After inoculation, plates were incubated at 37°C, and the results were recorded after 6 days of incubation. Enzyme activity was measured as the diameter of the lytic area surrounding the growth area on the serum medium.
The Pz value was determined as the ratio of the diameter of the colony to the total diameter of the colony plus the precipitation zone, and it was scored and categorized as follows: Pz value = 1 (negative); Pz value = 0.75-0.9 (low producers); Pz value = 0.51-0.74 (moderate producers); and Pz value = 0.35-0.5 (high producers). The assay was performed in duplicate, and the enzyme activity values were recorded as the average of the two measurements.
Germ tubes production is defined as a filamentous outgrowth from a blastoconidium at least twice as long as the parent cell without constriction at the junction. Isolated colonies of C. albicans were inoculated in glass tubes with 1mL of human serum and incubated at 37°C for 2h. A drop of the inoculum was then placed on a clean microscope slide, and a coverslip was placed on the slide. Wet mount preparations were examined under the objective lens of a microscope (×40 magnification), and tests were performed in duplicate.
RESULTS
Phospholipase activity was detected for 78% (120/153) of C. albicans isolates, and proteinase activity was detected for 97% (148/153) of isolates.
The mean Pz value was 0.79 (range, 0.48-1.00). The average value for proteinase activity was 0.71 (range, 0.49-1.00).
Isolates were categorized by their levels of phospholipase and proteinase production. The activity of these enzymes is shown in Table 1. A majority of C. albicans isolates with enzymatic activity displayed high proteinase activity and low phospholipase activity. Of the isolates studied, 73% produced both phospholipase and proteinase.
TABLE 1 – Hydrolytic enzyme activity for 153 bloodstream Candida albicans isolates.
| Production of extracellular hydrolytic enzymes (Pz) | Phospholipase (n) | |||||
|---|---|---|---|---|---|---|
| high(0.35-0.5) | moderate(0.51-0.74) | low(0.75-0.99) | negative(1) | total(n) | ||
| Proteinase (n) | High (0.35-0.5) | 10 | 19 | 34 | 18 | 81 |
| Moderate (0.51-0.74) | 4 | 16 | 24 | 12 | 56 | |
| Low (0.75-0.99) | 1 | 2 | 5 | 3 | 11 | |
| Negative (1) | 2 | 1 | 2 | 0 | 5 | |
| Total (n) | 17 | 38 | 65 | 33 | 153 | |
Germ tubes induction was positive in 95% (146/153) of bloodstream C. albicans isolates. The associations of GT production with phospholipase and proteinase activity are described in Table 2.
TABLE 2 – Virulence factor (GT, phospholipase, and proteinase production) associations in bloodstream isolates of Candida albicans.
| Virulence factor associations | Number | Percentage |
|---|---|---|
| Phospholipase + GT | 5 | 3.0 |
| Proteinase + GT | 33 | 22.0 |
| Phospholipase + proteinase | 7 | 5.0 |
| Phospholipase + proteinase + GT | 108 | 70.0 |
| Total | 153 | 100.0 |
DISCUSSION
Yeasts of the genus Candida continue to be among the most important etiologic agents of nosocomial infections. Deep-seated candidemia, the occurrence of which has considerably increased, is most commonly observed in ICU patients with indwelling catheters, individuals receiving oncological treatment, organ transplant recipients, or other immunocompromised individuals subjected to heavy therapeutic protocols2,13.
Although many studies have investigated the exoenzyme activity of Candida spp. isolated from mucosal surfaces3,7−9,12,14,15, few have investigated bloodstream isolates2,12. The virulence of Candida spp. is believed to be caused by the combination of several factors12, including its ability to colonize a variety of different anatomic sites7.
The phospholipases and proteinases of C. albicans are considered important virulence factors, and the absence or decreased expression of these enzymes may indicate reduced virulence for some Candidaspecies2.
In our study, 78 and 97% of the isolates displayed phospholipase and proteinase activity, respectively. Mohan & Ballal2 found a similar percentage of phospholipase activity; however, only 44% of the isolates that they studied exhibited proteinase activity, suggesting that the isolates used in this study were comparatively more virulent.
Few studies have investigated phospholipase and proteinase production simultaneously in the same strains. In the present study, 73% of the isolates produced both phospholipase and proteinase, which is in concordance with the results obtained by D`Eça Júnior et al.7 and Kantarcioglu & Yucel15, who observed concurrent production in 60 and 93% of C. albicans isolates, respectively. However, the isolates used in those studies were isolated from different anatomic sites.
In another similar study, measures of enzymatic activity indicated that all C. albicans isolates were pathogenic2. However, the present study identified no hydrolytic enzyme activity in 38% of the isolates.
Germ tubes production was observed for 95% of the isolates. High levels of phospholipase production were observed in 13 of the GT-positive isolates. Vidotto et al.12 described a correlation between high phospholipase activity and GT production in oral isolates, which suggests that these virulence factors are necessary for C. albicans colonization and infection of the oral cavity. It is possible that high levels of phospholipase activity and GT production can facilitate penetration through the mucosa because phospholipase activity is particularly concentrated in hyphal tips.
Ghannoum4 and D`Eça Júnior et al.7 suggested that the quantity of phospholipase produced by C. albicansvaries with each specific isolate, and it is correlated with the site of infection.
In our study, bloodstream isolates of C. albicans produced extracellular phospholipase and proteinase. In general, bloodstream isolates are known to produce much higher levels of phospholipase and proteinase than wound or urine isolates4,15.
Bloodstream isolates of C. albicans produce virulence factors, including GT and hydrolytic enzymes, making them capable of causing infection under favorable conditions.
