Red Sea Sponges of the Genus Hyrtios as a Source of Symbiotic Fungi with Antimicrobial Activities

Red Sea sponges Genus Hyrtios Symbiotic fungi antimicrobial activity Abstract The diversity of the symbiotic fungi associated with two Red Sea sponges of the genus Hyrtios (H. erecta and H. erectus) was investigated. A total of 20 isolates were purified from these sponges. Using the morphological properties, 10 fungal strains were identified to the genus level from each sponge. Strains isolated from H. erecta were identified to the species level using the Internal Transcribed Spacer ITS-rDNA sequences. Most of the identified fungal strains belong to the genera Penicillium and Aspergillus. The antibacterial activities of organic extracts of these fungi were investigated against three pathogenic microbes including E. coli, S. aureus and C. albicans. All of the fungal extracts displayed moderate to high levels of antimicrobial activities against the three pathogens. Several fungal strains of the genera Penicillium and Aspergillus displayed strong antibacterial activities provide potential for future investigation of these strains to identify the bioactive leads in these fungal extracts.


Introduction
The quest to exploit factors leading to the production of diverse molecular structures from cultured microorganisms represents a continuing challenge for natural products research (Firn and Jones, 2003) Filter-feeding marine invertebrates, such as sponges, have been shown to host a variety of microorganisms that do not merely reflect the microbial communities present in the surrounding seawater but appear to constitute a more specialized association between sponge hosts and microbial associates (Friedrich et al, 1999).An early, conservative estimate based upon thousands of assayed sponge species suggested that as many as 11% produce cytotoxic compounds (Garson, 1994).This percentage is high compared to other organisms and possibly may result from the intimate association between sponges and diverse microbial communities (Sipkema, 2009).Sponge-derived fungal cultures have been shown to be interesting sources of bioactive secondary metabolites previously unknown from terrestrial strains of the same species.The genus Aspergillus has been repeatedly been isolated from marine sources such as sponges or algae (Hiort et al, 2004).Although their ecological roles in the sea are not well defined, the genus Aspergillus has been identified as the causative agents for mass mortalities in marine invertebrates (Smith, 1996) providing evidence that these fungi can produce significant ecological effects (Garo, 2003).The probability of finding useful metabolites from marine organisms is obviously dependent on the number and quantity of samples screened.Therefore working on symbionts instead of marine macroorganisms has further expanded the diversity of structures that can be obtained without spending great efforts to collect vast amounts of the marine macro creatures.In addition, the increased awareness of the potential of marine with sterile artificial sea water (ASW).Small pieces of pre-washed sponge tissues were inoculated onto different agar media; malt Agar (MA, Difco), Sabouraud dextrose Agar (SDA, Difco), Czapek's Dox yeast Agar (CZYA (g/L each) sucrose (30), yeast (5), NaNo 3 (3), K 2 HPo 4 (1), KCl (0.5), MgSo 4 .7H 2 O (0.5), FeSo 4 .7H 2 O (0.01)), yeast peptone dextrose Agar (YPDA (g/L each) yeast (10), peptone (20), dextrose (20)).

Identification of fungal isolates
Fungal DNA was prepared using DNeasy Plant Mini Kit (Qiagen).The prepared DNA was amplified with polymerase chain reactions (PCR) followed by agarose gel electrophoresis.DNA was then retrieved from the agarose gel with QIAquick gel extraction kit (Qiagen).DNA sequencing combined with WWW searchable databases was used for fungi identification.

Extraction and isolation
A. flavus was chosen for large scale culture.20 L of CZYB in ASW were prepared and inoculated with the studied fungus.The prepared cultures were kept at 27 ºC with shaking at 120 rpm for 14 days.Extraction of the filtered broth with EtOAc gave 3.1 g crude extract which was further partitioned between 10% aqueous MeOH and hexane.The hexane fraction was separated and evaporated to give 1 mL of red oil (586.6 mg).The polarity of the aqueous portion was raised to 50 % aqueous MeOH and partitioned with dichloromethane (DCM).DCM fraction was separated and dried under vacuum to give 794 mg.DCM extract (794 mg) was applied to a column of sephadex-LH 20 and eluted with MeOH:DCM (1:1) to give three different fractions (F 1 -F 3 ).F 2 showed the most intense biological activities against the tested bacteria and fungi.F 2 was applied to the top of several silica gel columns (A-C) and step gradient elution with hexane, chloroform and EtOAc was used.Two fractions from columns A and C were crystallized from saturated chloroform solution to give 1 (2.5 mg) and 2 (1.5 mg), respectively.Bioassay guided fractionation led to the identification of two major fractions from column C. FC-1 (12 mg) was cytotoxic to brine shrimps in a concentration of 150 µg.RP-HPLC of FC-1 resulted in the separation of 3 (1.5 mg), 4 (1mg), 5 (5 mg) and 6 (1mg).FC-2 (6 mg) showed antimicrobial properties.Further purification of FC-2 with RP-HPLC gave rise to the separation of 7 (800 µg) and 8 (1.2 mg).The structures of the isolated compounds were elucidated using 1D and 2D NMR spectroscopic data together with ESI-TOF-MS biological resources has highlighted the need for nations to become aware of biological diversity and the importance of conservation of these resources.In our study we report the isolation of fungi associated with the sponge Haliclona (Gellius) bubastens.Several marine derived fungi were identified by the use of DNA sequencing combined with World Wide Web (WWW) searchable databases.
The sponge derived A. flavus was chosen for chemical and biological investigation of its secondary metabolites in saline cultures.In addition, the effect of changing the carbon source in culture media of the studied fungus on metabolites production and diversity was also studied.Eight saline culture media were prepared; czapek's dox yeast broth (CZYB), four modified czapek's dox yeast (M.CZYB), Sabouraud dextrose broth (SDB), malt broth (MB) and yeast peptone dextrose broth (YPDB) in order to find the optimal culture composition for A. flavus to produce its biologically active metabolites in respect to concentration and diversity.

General
NMR experiments were carried out at 25 °C using either a 400 MHz or 500 MHz Varian INOVA-NMR spectrometer with standard pulse sequences.UV spectra were recorded on an hp-8552A diode array spectrophotometer.Liquid chromatography-mass spectrometry (LC/MS) with electrospray ionization mass spectrometry (ESI-MS) was run at the University of Utah Mass Spectrometry and Proteomics Core Facility.DNA sequencing was done using high throughput capillary electrophoresis instrument (ABI 3730XL) at the University of Utah DNA sequencing and genomics core facility.HPLC-Hitachi Ez lachrome equipped with C 18 columns (10 x 250 mm and 4.6 x 150 mm) and photodiode array detector was used for compounds isolation and analysis.

Sponge material
The host sponge was collected in the Red Sea and was identified as Haliclona (Gellius) bubastens (Row, 1911).

Isolation of sponge associated fungi
In order to ensure fungal isolates to be spongeassociated or symbiotic, surface sterilization of the collected sponge had to be made.A piece of sponge tissues (1 g) was washed with 5% sodium hypochlorite followed by 70% ethanol and finally MeOH for 48 h while the broth was extracted with EtOAc (3 x 500 mL).These extracts were evaporated to dryness under vacuum and weighed (Table 1).

Preparation of standard mixture and HPLC analysis
A standard mixture of the compounds 1, 2, 3, 5 and 8 was prepared (1 µg/µL each, in 100 µL MeOH-H 2 O) and analyzed by RP-HPLC using a C 18 column (4.6 mm x 150 mm).Compounds were eluted with a linear gradient from 95% solvent A (0.05% trifluoroacetic acid in water) to 95% solvent B (0.05% trifluoroacetic acid in acetonitrile) over 70 min at 1 mL/min (Figure 2).Each compound was applied separately to C 18 HPLC analytical column using the proposed method and purity of each compound was checked and found to be more than 98%.EtOAc extract of each of the eight cultures (100 µg/µL) was analyzed using the previously described method.Quantitative data (Figure 1).

Biological assays
The bactericidal and fungicidal activitities of EtOAc and MeOH extracts were tested against E.coli, Bacillus subtilis, Staphylococcus aureus, Candida albicans, Saccharomyces pombe and Saccharomyces cervisiae using agar disc diffusion assay (Anderson, 1978).The cytotoxic properties of these extracts were examined using brine shrimp lethality assay (Kapadia et al, 2002;Pimenta et al, 2003).The lethal dose required to kill 50 % of brine shrimps population (LD 50 ) was calculated.

Aflatoxin B1 (2)
UV/Vis λ max (EtOH) nm (log ε): 223 (1.4), 265 (1.1), and 362 (1.3).Analysis of HPLC data using the proposed method with peak area measurement revealed that A. flavus synthesized higher concentration of compound 3 in SDB than it did with any other substrate tested.Although compound 3 is produced only in three of the eight studied culture media, its concentration differs significantly between their extracts.It was found in SDB culture extract in forty and ten folds its concentration in CZYB and M.CZYB 1 , respectively.The concentration of compound 1 in EtOAc extract of MB was found to be more than two and thirty fold its concentration in M.CZYB 3 , M.CZYB 4 and CZYB, respectively (Table 2).Compounds 1 and 2 were synthesized in the highest concentrations in MB.The production of compound 2 was three fold more in MB than in CZYB and M.CZYB 4 while it is not produced in cultures of M.CZYB 3 and YPDB (Table 2).Compound 5 is present in cultures of M.CZYB 2 with the highest percentage while it is not produced in CZYB, MB and YPDB culture media.Compound 5 was found at the lowest concentration in M.CZYB 4 and at different proportions in each of M.CZYB 1 , M.CZYB 3 and SDB (Table 2).Compound 8 is produced in all of the eight studied cultures with the highest percentage in M.CZYB 3 .No significant variation in the production of compound 8 in all of the studied culture media.The biological activities of the methanolic (MeOH) and EtOAc extracts of the studied cultures were tested for antimicrobial and cytotoxic properties.Analysis of results of the antimicrobial assays revealed that some extracts of the studied cultures showed antibacterial and antifungal activities as potent as or even better than chloramphenicol and nystatin, respectively, at the same tested concentration.EtOAc extract of SDB showed the highest activity against B. subtilis and S. aureus which is higher than the activity of chloramphenicol at the same tested concentration (Figure 4).400 µg of EtOAc extracts of CZYB and M.CZYB 3 showed the same fungicidal activity against C. albicans while they were one and half fold more potent than nystatin against S. pombe.Moreover, EtOAc extract of SDB gave the same inhibition zone against both C. albicans and S. cervisiae as nystatin at the same tested concentration (Figure 4).Close analysis of the results of antimicrobial assays of extracts of all studied cultures revealed that sponge-derived A. flavus favored the synthesis of both antibacterial and antifungal metabolites in saline SDB.Cytotoxic activities of the prepared extracts were tested with the rapid and efficient brine shrimp lethality assay.Results showed a significant difference of their LD 50 .The EtOAc extract of M.CZYB 1 showed the lowest LD 50 (as low as 2 µg), while more than 1 mg of its MeOH extract 25.5 (CH 3 ), 26.0 (CH 2 ), 35.5 (CH), 52.5 (CH), 71.9 (CH), 108.4 (CH), 115.9 (CH), 120.4 (CH), 122.8 (CH).
of the sponge-associated A. flavus in eight culture media was extensively studied to find the optimal culture composition for their production.Large scale culture of A. flavus led to isolation of eight compounds.The isolated compounds represent a wide diversity in their chemical structures and sizes.Their complexity varies from Kojic acid, γ-pyrone derivative, to the tetramic acid, cyclopiazonic acid.Analysis of HPLC data and bioassays results of extracts of the eight studied culture media showed a significant effect of the nutritional environment on the production of secondary metabolites in respect to concentration and diversity.

Acknowledgments
This work was supported by the U.S.-Egypt Joint Fund for Science and Technology under project number (BIO10-002-003).The authors would like to thank Rob van Soest for the taxonomic identification of the sponge specimen.

Conflict of interest
The authors report no declaration of conflict of interest.was required to kill 50% of brine shrimps population.20 µg was calculated for LD 50 of the MeOH extract of YPDB, while no cytotoxic activity was noticed for the MeOH extract of M.CZYB 2 (Figure 5).

References
A good explanation of these results is that the secondary metabolites responsible for the cytotoxic properties are released extensively in the culture media of M.CZYB 1 and SDB.This situation is clearly opposite in case of YPDB, where the cytotoxic compounds are present extensively inside the mycelia of the studied fungus (Table 2, Figure 5).A good explanation of these results is that the secondary metabolites responsible for the cytotoxic properties are released extensively in the culture media of M.CZYB 1 and SDB.This situation is clearly opposite in case of YPDB, where the cytotoxic compounds are present extensively inside the mycelia of the studied fungus (Table 2, Figure 5).

Conclusions
In conclusion, marine fungi are considered an environmentally friendly source of pharmacologicallyactive natural products that allow the conservation of marine biological resources.Therefore, chemical and biological investigation of the secondary metabolites

Figure 4 :
Figure 4: Results of antimicrobial assays of extracts of the eight studied cultures (400 µg each); A: antibacterial assay using chloramphenicol as a reference standard, B: antifungal assay using nystatin as a reference standard.

Figure 5 :
Figure 5: Results of brine shrimp lethality assay of extracts of the eight studied cultures