Evaluation of catalase activity of clinical and environmental isolates of Aspergillus species.

Background and
Objectives: Catalases are a good scavenger of H2O2 which degrades hydrogen peroxide into water and oxygen. They are considered as a virulence factor that are present in both spores and hypha of fungi. There is limited data regarding catalase activity in Aspergillus species. This study aimed to assess the mycelial catalase activity of clinical and environmental isolates of Aspergillus niger, A. tubingensis, A. flavus, A. luchuensis, A. piperis and A. terreus. Materials and
Methods: Briefly, clinical and environmental Aspergillus species were used in the current study. Catalase activity was assessed for both groups of isolates including 13 A. flavus (12 clinical, 1 environmental), 13 A. terreus (8 clinical, 5 environmental), 26 A. tubingensis (13 clinical, 13 environmental), and 44 A. niger (25 environmental, 19 clinical) species. Fungal balls of mycelia were separated from the liquid culture and were crushed using homogenizer. The supernatants were collected and used for a catalase activity assay.
Results: Totally, in our study 98 Aspergillus including 45 environmental and 53 clinical isolates were assessed for catalase activity. High catalase activity was detected among environmental Aspergillus species (Mean= 1.62 mU/ml) and the mean of mycelial catalase activity among clinical A. terreus isolates was higher than environmental strains.
Conclusion: In summary, mycelial catalase activity varied among species and environmental isolates demonstrated higher catalase activity. Totally a significant difference was found between clinical and environmental Aspergillus isolates.

INTRODUCTION

Catalases are the extracellular and intracellular enzymes existing in bacteria, fungi, plants, and animals. They are a haem-containing antioxidant enzyme and a good scavenger of H2O2 which degrades H2O2 into H2O and O2 and have therapeutics and industrial applications (1–3). Catalase is a putative virulence factor in yeasts, spores, and fungal hypha. Detoxification of hydrogen peroxide by catalase enzyme has been suggested as a way to overcome the host immune response. In vitro studies showed hydrogen peroxide produced by neutrophil play an essential role in the killing of fungal hyphae (4) and this process is blocked by the addition of a commercial catalase (5).

Aspergillus species are saprotrophic fungi that mostly occurring in the soil on decaying organic materials. Aspergillosis mostly occurs as an opportunistic infection in immunocompromised patients. Despite advances in antifungal drugs, aspergillosis remains a major health problem and rapidly grows in high-risk patients. Previous studies suggest that catalase A, primarily is a peroxisomal protein and a key antioxidant protein, is also found in cellular organelles such as mitochondria and the nucleus (6, 7). Catalase activity is mostly reported from Candida albicans (8), Cryptococcus neoformans (9), Aspergillus nidulans (10–12), A. flavus (13) and A. fumigatus (14, 15). However, there is limited data regarding catalase activity in other Aspergillus species. This study aimed to assess the mycelial catalase activity of clinical and environmental isolates of A. niger, A. tubingensis, A. flavus, A. luchuensis, A. piperis and A. terreus.

MATERIALS AND METHODS

Isolates and identification.

Clinical Aspergillus species (53 isolates) were previously isolated from patients with otomycosis, onychomycosis, and environmental Aspergillus species (45 isolates) were previously isolated from air samples. Identification of isolates was assessed according to morphological characteristics and sequencing of the β-Tubulin gene (16, 17) (Table 1). Preserved isolates in sterile distilled water, were cultured on Sabouraud dextrose agar (Scharlau, Spain) and incubated at ambient temperature for one week.

Table 1.

Clinical and environmental isolates of Aspergillus used in this study

Isolates sources		A. flavus	A. terreus	A. tubingensis	A. niger	A. luchuensis	A. piperis	Total
Clinical	Otomycosis	12	1	13	19	1	--	53
	Onychomycosis	--	7	--	--	--	--
Environmental		1	5	13	25	--	1	45
Total		13	13	26	44	1	1	98

Determination of catalase activity.

Catalase activity was assessed for 98 isolates of Aspergillus species including 53 clinical and 45 environmental isolates. Isolates were grown in 1% yeast extract medium (Merck, Germany) in a shaker incubator at 100 rpm for 3 days. After incubation, fungal balls of mycelia were separated from the culture broth by filtration using Whatman filter paper. Harvested mycelia washed twice with 0.05 M Tris-HCl (pH=7.5) and dried in 200 μl acetone. One gram of dried mycelia was resuspended in 500 μl of 10 mM Tris-HCl (PH=7.8) containing acid-washed glass beads. Cell suspensions were crushed a SpeedMill PLUS Homogenizer (Analytikjena, Germany). After centrifugation at 100g for 15 min, the supernatants were collected and used for a catalase activity assay. Catalase activity was determined using a Catalase Assay Kit (Navand Salamat, Iran). After that, the microplate was read by Elisa reader (BioTek, USA) at 540 nm. Finally, the catalase standard curve was drawn based on a serial dilution of the standard catalase solution (13).

Statistical analysis.

All data were analyzed using SPSS 22.0 and statistically, significance (p< 0.05) was calculated. Therefore, a non-parametric t-test was used to analyze the data.

RESULTS

Overall, in our study 98 Aspergillus isolates were tested for catalase activity. These strains were selected from environmental (n=45) and clinical (n=53) sources. The standard curve of catalase activity has been shown in Fig.1. As shown in Fig. 1, according to the regression coefficient (R2= 0.98) curve was linear.

Fig. 1.

The standard curve of catalase activity according to the kit method, as the regression coefficient (R2= 0.98) curve was linear

In the present study, all Aspergillus isolates were showed catalase activity but enzymatic secretion varied among species. High catalase activity was detected among environmental Aspergillus species (Mean = 1.62 mU/ml). Still, the mean of mycelial catalase activity among clinical A. terreus isolates was higher than environmental strains. Also, as shown in table 2 statistical analysis revealed that among all clinical and environmental Aspergillus species only in A. niger isolates difference was significant (p= 0.005844, p< 0.05). Likewise, a significant difference was found between clinical and environmental Aspergillus isolates (p=0. 025813, p< 0.05).

Table 2.

Catalase activity among different clinical and environmental Aspergillus species

Aspergillus species	No.	Catalase activity			p-value
		Lowest	Highest	Mean
A. niger C	19	0.18	2.46	1.34	p = 0.005844.
A. niger E	25	0.97	3	1.88	significant at p < 0.05
A. tubingensis C	13	0.06	2.76	1.46	p = 0.54681.
A. tubingensis E	13	0.49	2.94	1.66	not significant at p < 0.05
A. flavus C	12	0.38	2.84	1.43	-
A. flavus E	1	0.33	-	-	-
A. terreus C	8	0.18	0.85	0.65	p = 0.809634.
A. terreus E	5	0.44	0.69	0.57	not significant at p < 0.05
A. luchuensis C	1	0.69	-	-	-
A. piperis E	1	1.28	-	-	-
Total C isolates	53	0.06	2.84	1.28	P = 0.025813.
Total E isolates	45	0.33	3	1.62	significant at p < 0.05

C: Clinical, E: Environmental

DISCUSSION

Aspergillus species are saprophytic fungi that able to grow on different substrates and secreted a variety of metabolites and enzymes (10). Catalase secretion is one of these enzymes that have been identified in Aspergillus species and produced during invasive disease. Several reports have detected that catalase activity among this genus protects them from cellular damage to the host (10, 18). However, few studies have focused on the secretion of catalase in different clinical species of Aspergillus (13). Our study showed that mycelial catalase activity was detected among different species of Aspergillus isolates. Besides, based on our finding a significant difference was found in catalase activities between clinical and environmental Aspergillus isolates. As a result, the average catalase secretion in environmental isolates was higher than in clinical isolates. However, Rouein et al. reported higher mycelial catalase activity in clinical isolates of A. flavus and A. fumigatus than environmental isolates (13).

Shibuya et al. have believed that mycelial catalases in Aspergillus transiently protect it from the reactions of host defence (19) and the catalase activity of mycelial form is greater than conidial form in Aspergillus (13, 20). Gallin et al. believed that catalase activity is contributing to the pathogenicity of invasive aspergillosis, especially in immunocompromised patients (21). Besides, Paris et al, indicated that the inactivation of catalase secretion in A. fumigatus strains resulted in reduced virulence in mice (20). In our study, only clinical strains of A. terreus had shown an increased level of catalase activity compared to environmental strains. More data are available in catalase secretion by fungi, industrial properties and their activity in literatures (18, 22, 23). However, there is no more data that compared the catalase activity in clinical and environmental isolates of Aspergillus. Generally, similar to other studies, our clinical and environmental strains were secreted variable amount of catalase. However, we found that clinical isolates have a higher catalase activity than environmental isolates.

A recent study suggests that catalase is involved in Aflatoxin B1 biosynthesis (22). Also, Wang et al. showed that catalase activity is an essential factor in fungi for adaptation to environmental stress (18). Therefore, the role of catalase activity in environmental isolates seems to be better growth. In the present study, different species of Aspergillus could secret catalase enzyme and this rate was highest in the Aspergillus niger species complex. It is important to note that the presence of melanin in this complex can play a role similar to the catalase enzyme (24).

CONCLUSION

In summary, mycelial catalase activity varied among species and environmental isolates demonstrated higher catalase activity. Totally a significant difference was found between clinical and environmental Aspergillus isolates. Since catalase activity in Aspergillus isolates is associated with their pathogenesis, this study can help to understand the pathogenesis of different species of Aspergillus in various forms of its disease.