Extracellular Proteases from Keratitis Causing Fusarium, Aspergillus and Dematiaceous Species

Purpose Various types of hydrolytic enzymes, like proteases, lipases and phospholipases are important virulence factors produced by pathogenic fungi. In the present study quantification and characterization of proteases produced by different pathogenic Aspergillus, Fusarium and Dematiaceous species isolated from corneal ulcers was done. Method Seven Aspergillus species, twelve Fusarium species and five Dematiaceous species previously isolated from corneal ulcers were used in the present study. All isolates were grown in Sabarouds Dextrose Broth (SDB) and culture filtrate was obtained after 10 days of growth. Acetone precipitated culture filtrate was used as the source of protease. Characterization of proteases was done by assaying the activity at different pH (6-12), with inhibitors (PMSF, Pepstatin and EDTA) and gelatin zymography. Result In Aspergillus spp, maximum specific activity of protease was found in Aspergillus flavus and minimum specific activity was found in Aspergillus niger and Aspergillus tubingensis. In Fusarium spp., proteases from Fusarium solani species complex (FSSC) showed more activity compared to F. delphinoides. Proteases from Aspergillus spp. and FSSC showed inhibition in presence of PMSF, while EDTA inhibited the proteases from F. delphinoides. In Dematiaceous group, Curvularia spp showed highest activity and were inhibited by PMSF.


Introduction
Corneal blindness, in developing countries is predominantly associated with infections. In India, nearly 35-50% of all infectious keratitis are caused by fungi [1]. The term mycotic keratitis refers to a corneal infection caused by fungi. More than 105 species of fungi belonging to 56 genera have been reported to cause fungal keratitis. of the pathogenic potential of any organism [2]. The pathogenecity of the fungus may be due to fungal cell wall components, toxins, enzymes or pigments produced by fungus. The microbial attributes that confer the potential for virulence fall primarily within several categories, including the ability to enter a host; the ability to evade host defenses; to grow in a host environment; to counteract host immune responses; to acquire iron and nutrients from the environment and to sense environmental change. Production and secretion of hydrolytic enzymes, such as proteases, lipases and phospholipases are among the important virulence traits [3].
Proteases, also termed as proteinases or peptidases, are proteolytic enzymes which function as molecular knives which cut long amino acid sequences into fragments, a process that is essential for the synthesis of all proteins, controlling their size,composition, shape, turnover, and ultimate destruction [4]. These enzymes play a role in nutrition, tissue damage, dissemination within the human organism, iron acquisition and overcoming the host immune system and hence strongly contribute to fungal pathogenicity [3].
Proteolytic activities of A. flavus and F. solani in rabbit corneas during active fungal keratitis were investigated in vivo and it was found that the fungal cultures predominantly produced serine proteases in vitro; while, fungal infected corneal homogenates showed the presence of metalloproteases alone [5]. Production of proteases in Aspergillus and Fusarium causing keratitis has also been shown to be correlating significantly with amphotericin B resistance [6].
Further, alkaline protease (Alp1) has also been shown to be one of the abundant proteins in A. flavus exoproteome [7]. Taken together, these studies indicate that proteases can mediate corneal invasion and could also cause corneal melting. However, there is paucity of information regarding their detailed characterization from keratitis causing isolates. Earlier we reported a prospective study to compare different aspects of fungal keratitis such as its clinical features, microbial evaluation, molecular identification, antifungal susceptibility, and clinical outcomes between Fusarium, Aspergillus and Dematiaceous fungi [8]. In the present study detailed analysis of proteases produced by these three groups of fungi is attempted.
(n=12); Aspergillus spp. (n=7) and Dematiaceous spp. (n=5) isolated in the previous study and were available in the lab [8,9]. All isolates were identified using the ITS region sequencing and sequences were deposited in NCBI. Name and GenBank accession no of fungi (n=25) used in the present study are: Fusarium delphinoides

Growth of Fungi
The isolates were first grown on Sabouraud's Dextrose Agar

Extraction and Precipitation of Proteases
After 10 days of fungal growth, the culture filtrate was collected using sterile Whatman filter paper in a sterile bottle. The obtained culture filtrate was then subjected to precipitation using chilled acetone. To 1 ml of fungal culture filtrate 4 ml of chilled acetone was added drop wise. Culture filtrate -acetone mixture was then kept at -26 ºC over night for precipitation. Next day, the precipitate was

Characterization of Proteases
For characterization of proteases; the effect of pH and inhibitors was studied using the precipitated enzyme extracted from all fungi.
Effect of pH: The effect of pH on enzyme activity was studied by incubating the enzyme in azocasein solution of respective pH.
Azocasein activity was studied at 6 different pH ranging from acidic to basic i.e. pH 3, pH 5, pH 7.4, pH 8, pH 10 and pH 12. Effect of enzyme inhibitors: To understand the type of enzyme produced, specific enzyme inhibitors were used and azocasein assay was

Gelatin Zymography
Zymography was performed using gelatin (Sigma Aldrich, USA) as a substrate (0.1%) and 12% of polyacrylamide (Sigma Aldrich, USA). Twelve percent SDS-polyacrylamide gel was prepared with 0.1% gelatin. Enzyme units (0.2) were taken, mixed with 6X loading dye and electrophoresed. After electrophoresis, the gel was incubated in incubation buffer (50mM Tris, 5 mM CaCl2 and 1 µM ZnCl2) containing 2.5 % Triton -X 100 (Himedia Laboratories, India) for 1 hour. The gel was washed twice with deionized water and again kept in same buffer at 37°C for 2 hours. The gel was again washed with deionized water and kept overnight for staining with   (Figure 1d). However, in zymography, inhibition was only in presence of PMSF (Figure 1k). The proteases from A. fumigatus showed maximum inhibition by pepstatin in both in vitro assay and zymography gel (Figures 1g & 1n). However, in A.niger pepstatin showed highest inhibition in tube assay while, PMSF showed maximum inhibition in zymography gel (Figures 1c & 1j). Similar discrepancy was seen in A. tubingensis, the proteases were inhibited maximum in presence of EDTA in tube assay but were inhibited by PMSF in zymography gel (Figures 1e & 1l). These results suggest that the type of proteases produced by Aspergillus is mainly serine proteases, followed by aspartyl and metallo proteases.

Fusarium Proteases
In Fusarium, the proteases from F. delphinoides showed activity at broad pH range between pH 3 to pH 8 (

Dematiaceous Proteases
In Dematiaceous group of fungi, the proteases were studied in three Curvularia lunata, one Phomopsis phoenicicola and one Phaeoacremonium rubrigenum. Proteases from all three C.lunata isolates had activity at all pH, with slight more activity at pH 8 and pH 10 ( Proteases obtained from P.phoenicicola showed high activity at pH 7.4 with the specific activity of 3.15±0.19 units/mg (however, it had activity at broad pH range) and P. rubrigenum proteases presented maximum activity (4.76±0.21 units/mg) at pH 8. The tube inhibition assays from C. lunata isolates (Cc90 and Cc157) showed inhibition in presence of PMSF, followed by pepstatin and no inhibition in presence of EDTA (Figure 4). However, zymography gels showed inhibition only by PMSF (Figure 4d). In tube inhibition assays for proteases from P. phoenicicola and P.rubrigenum, inhibition was noted by pepstatin and EDTA respectively. However, slight inhibition was observed in zymography gels (Figures 4g & h). The results of the specific activity of proteases produced by 24 different isolates suggests that; the number of extracellular proteases was more in Aspergillus and Curvularia species compared to Fusarium species. The overall summary of proteases from different isolates is given in Table 2.

Discussion
In the present study, extracellular protease activity was quantified from three most common keratitis causing fungal pathogens; Aspergillus spp., Fusarium spp., and Dematiaceous fungi.
Among the three, Aspergillus isolates produced the highest amount proteases while, Fusarium and Dematiatous produced moderate amounts. Proteases from Aspergillus spp. causing keratitis to have been reported only from Aspergillus flavus [5,11,12]. The exoproteome analysis of a keratitis causing A. flavus showed that nearly 50% of the exoproteins possess catalytic activity and one of these, an alkaline serine protease (Alp1) is abundant and present in multiple proteoforms [7]. Proteases from none of the other species has been studied from keratitis causing isolates. We have quantified proteases from A. sydowii, A. niger, A. terreus, A.
tubingensis, A. tamarii and A. fumigatus. Proteases from soil isolates of Aspergillus are extensively studied and described from A. niger [13], A. tamarii [14], A. fumigatus [15], A. flavus, A. terreus [16] and A. tubingensis [17]. Total extracellular protease activity for some isolates (A. sydowii, A. tubingensis, A.terreus) was higher than the reported isolates; while, it was less for certain isolates (A. flavus, A niger, A. tamarii). However, these differences could be because of the differences in the isolates used and conditions for their growth.

Large variations in occurrence and abundance of proteases in seven
Aspergillus spp. was shown using genome mining and comparative proteomics [18]. Serine proteases were the largest group in the protease spectrum across Aspergillus spp.  [20], F. oxysporum [21] and F. venenatum [22]. Hence, species of Fusarium are known to produce different types and amounts of extracellular proteases.
Most of these studies report the presence of one major serine protease in Fusarium spp. Zymography results in the present study indicate the presence of more than one type of high molecular weight protease in Fusarium. These results warrant further detailed studies on purification and identification of these proteases.
In Dematiaceous group, our results indicate the presence of serine and aspartyl proteases in Curvularia spp, aspartyl proteases in P. phoenicicola and serine and metallo protease in P. rubrigenum.
Serine protease from C.lunata has been reported [23], but presence of aspartyl proteases was not found. Molecular and immunological characterization of subtilisin like serine proteases was carried out in C. lunata, and the protease was proved as a major allergen responsible for inflammation and asthma [24]. To the best of our knowledge there are no studies on extracellular proteases in P.
phoenicicola species, but P. azadirachtae has been explored for the production of extracellular enzymes like polygalacturonase, laccase, protease and xylanase [25].
Our study showed that serine proteases were the major proteases found in isolates of all the groups (Aspergillus, Fusarium and Dematiaceous) of fungi. Among the three groups; Aspergillus spp. had the highest level of proteases, followed by Fusarium and Dematiaceous group. These results corroborate with our clinical findings of patients from which these fungi were isolated [9].
Aspergillus had the maximum number of worsened cases; followed by Fusarium and then Dematiaceous group; suggesting that Aspergillus spp. are more virulent compared to the other two [9].
The serine proteinases and can degrade elastin, collagen, fibrin and fibrinogen. Many studies have correlated the extracellular proteases production with virulence of fungi [26][27][28]. Hence, collagenase activity may be a mediator of the severe corneal destruction.
Recently, we showed the presence of extracellular serine proteases using the ex vivo goat cornea model using Fusarium as the pathogen [29][30][31]. Further studies to identify the proteases in ongoing.

Conclusion
To conclude, fungal isolates showed the presence of various types of proteases in varying amounts. Proteolytic activity leads to severe corneal destruction and hence, there is a need to identify these proteases. Identification of such proteases could help in identifying molecules which inhibit them. Future strategies should encourage the development of compounds to combat virulence mechanisms as potential targets against drug resistant organisms.