Targeted action and molecular interactions of sarcin and thionin with aspergillosis causing Aspergillus fumigatus

Arabia. * Correspondence: kavitha_bio@avinuty.ac.in Abstract : Fungal infections are more predominant in agricultural and clinical fields. Aspergillosis caused by Aspergillus fumigatus leads to respiratory failure in patients along with various illnesses. Due to the limitation of antifungal therapy and antifungal drugs, there is an emergence to develop efficient antifungal compounds from natural sources to cure and prevent fungal infections. The present study deals with the investigation of the mechanism of active compounds from our candidate agonist Aspergillus giganteus for aspergillosis. The integrity of treated Aspergillus fumigatus cell membrane and nuclear membrane was analyzed by determining the release of cellular materials. The antagonistic potential of antifungal compounds on the pathogen was confirmed by SEM analysis. The effective concentration of antifungal compounds (AFCs) was found to be 250μg/ml. The GC-MS profiling has revealed the bioactive metabolites responsible for the antagonistic nature of Aspergillus giganteus. The bioavailability and toxicological properties of pathogenesis related proteins have proved the efficiency of pharmacokinetic properties of selected compounds. Interaction of sarcin, thionin, chitinase and its derivatives from Aspergillus giganteus with the virulence proteins of UDP-N-acetylglucosamine pyrophosphorylase, N-myristoyl transferase and Chitinase have proved the druggable nature of the antifungal compounds.

noticed in plates treated with AFCs of Aspergillus giganteus. In control plates, regular-shaped homogenous hyphae were observed and it was confirmed under different resolutions. AFCs in the culture filtrates also targets RNA, another important genetic material. Fig. 2B shows the results of cell membrane integrity assay for the release of RNA components. Maximum leakage of RNA components was noted at 120 mins of treated pathogenic strain with the concentration of AFCs with the range of 50, 100, 150, 200 and 250µg, and the amount of released constituents was observed as 75±0.01 µg/ml, 141.6±0.01 µg/ml, 158.3±0.02 µg/ml, 198.7±0.02 µg/ml and 258.3±0.01 µg/ml, respectively.
The AFCs in the culture filtrates of Aspergillus giganteus proved to be an effective tool to disrupt and damage the pathogenic fungal cell membrane. The amount of released protein and glucose were measured by their absorbance at 670 nm and 630 nm, respectively. Figure 3 shows the action of AFCs on the pathogenic cell membrane by estimating the released protein and glucose contents.
It explains that the release of cellular compounds has increased in a concentration-time-dependent manner.
The enhancement of released protein contents was observed in the treated Aspergillus fumigatus with AFCs (50,100,150,200 and 250 µg/ml). Indeed, the released contents were found to be maximum at 60 mins and 120 mins of treatment with AFCs which reflects that AFCs in Aspergillus giganteus has potential effect on the pathogen and causes damage to the cell membrane thus, releasing the protein contents within a short period.
Leakage of glucose in the treated sample was found to be increased with exposure to increased concentration of AFCs with increased time. Release of increased glucose contents was observed at OD530 with exposure of AFCs to Aspergillus fumigatus at 120 mins of treatment. The amount of glucose leaked by the AFCs (50,100,150,200 and 250 µg/ml) at 120 mins treated sample was 30.6±0.01 µg/ml, 47.6±0.01 µg/ml, 53.8±0.01 µg/ml, 82.68±0.01 µg/ml and 109.8±0.01 µg/ml. The leakage of components (DNA, RNA, protein and glucose) were released into the medium at a higher level in the treated pathogen than that of control (without AFCs treatment). Examination of pH changes observed in the AFCs treated pathogenic strain was conducted using a pH meter. Results of pH variation are represented in Figure 5, where pH was shown to decrease gradually with time exposure and AFCs concentration. The pH level began to fall after 30 minutes of treatment compared to that of control (without AFCs). The decreased pH level in the treated pathogenic cell has revealed that might be some acids or any other acidic metabolites in the pathogenic strain may be leaked into the medium, thus changes in the pH were observed.  Table 2. The list of fatty acids and methyl esters identified are also given as supplementary figure S1.
Several fatty acid metabolites, have been identified as antifungal compounds which are biodegradable with high specificity for many pathogenic fungi. It has also been found that pathogenic fungi do not become resistant with constant exposure to these antifungal fatty acids. These fatty acids incorporate themselves into the cell membranes of target pathogens and increase membrane fluidity and disruptions in the arrangement of membrane proteins which eventually releases the cellular constituents.

Cytotoxicity of antifungal compounds
The hemolytic activity of antifungal compounds in cell free supernatant of Aspergillus giganteus was measured and used for the determination of its toxicity level.
The result of cytotoxicity of AFCs in antagonistic fungus is given in Figure 7. The  Results are expressed in mean±SD (n=3)

ADMET profile of sarcin, thionin, chitinase and its derivatives of A. giganteus
Supplementary Table S2 explains the profile of the selected compounds such as molecular weight, log P value, rotatable bonds, acceptors, donars, and surface area. The ADME property of the fluconazole, sarcin, thionin, chitinase and its derivatives is listed in Table 3. The all the selected compounds were found to have a good absorption level except the chitinase derivative. The highest absorption was found with the sarcin and thionin derivatives. The intestinal absorption and skin permeability profile represent the selected compounds that have a good absorption profile compared with the standard drug, fluconazole. Sarcin from Aspergillus giganteus possesses to be distributed into the blood-brain barrier and central nervous system. The compounds were observed as neither substrate nor inhibitor for the cytochrome P450, which is an important detoxification enzyme found in the liver. The studies have given the way to conclude that the sarcin and its derivatives of the Aspergillus giganteus shown a good ADME profile while comparing with the standard antifungal drug. The toxicological properties of the sarcin, thionin, chitinase and its derivatives showing fewer toxic properties and it is contemplating that the selected compounds are safe for use to treat the fungal infection (Table 4).

Molecular interaction of bioactive compounds with the virulence proteins
Molecular simulation studies were performed to identify the best interaction of ligand molecules with the pathogenic target proteins which leads to the development of a wide range of drugs after undergone several layers of screening. The best selected three ligand molecules with the selected target proteins were docked at its active site.
The missing hydrogen atoms were incorporated and proper ionization states were generated using the OPLS-2005 force field. For optimal molecular interactions, the centered on the centroid of the co-crystallized ligand has been created. The weak force, Van der Waals radius scaling factor was set 4 to 1.0 Å and the partial charge of cut off was maintained at 0.25 with no constraints. The sarcin, thionin, chitinase from Aspergillus 5 giganteus and fluconazole was docked with the virulence proteins of pathogenic Aspergillus fumigatus. Table 6   potential on the pathogenic cell membrane and cell wall. proved that the active metabolites in the Aspergillus giganteus is secreted extracellularly 129 [19]. In this regard, the cell-free supernatant was chosen to assess its potential 130 mechanism on the pathogenic cell wall. Lowry's method [27].    Anthrone method [27,30]. The treatment was given as same as in the above method 175