الفهرس 
IntroductionOnly 14 pages are availabe for public view
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Abstract
Natural products derived from marine microbes play a pivotal role in drug discovery and development due to their diverse molecular and chemical scaffolds, which cannot be matched by any synthetic or combinatorial libraries. Marine actinobacteria are gaining importance not only for their taxonomic and ecological perspectives, but also for their production of novel bioactive compounds with novel molecular scaffolds like antibiotics, antitumor agents, immunosuppressive agents, enzymes, enzyme inhibitors and pigments. On the other hand, the re-isolation of known metabolites has been increasing in recent decades. This is one of the major challenges that pharmaceutical and agricultural research faces in the discovery of novel bioactive compounds, emphasising the need for more efficient approaches. Among the various approaches to address this issue, the focus on screening microbes residing in less assessable and extreme environments such as hyper-arid deserts, permafrost soils, deep-sea sediments, highly acidic, and hypersaline habitats, is most highlighted. Additionally, competition between different microorganisms for food and space is considered to be one of the major ecological forces that induces silent biogenetic gene clusters which triggers the accumulation of cryptic secondary metabolites (SMs) that are not traced in their axenic cultures . The increase in the current problems of resistance of numerous pathogens together with the new emerging infectious diseases as well as the adverse effects of some of the currently used drugs necessitate the searching for new anti-infective drugs. So, the main goal of the present study is to allocate novel anti-infective compounds from marine-associated actinomycetes.
The present study was divided into five chapters :
Chapter I: sponge collection, actinomycetes isolation and molecular identification of actinomycetes.
Callyspongia sp. and Spheciosponge vagabunda were collected from the Red Sea (Ras Mohamed, Sinai, in August 2006. The collected sponges, identified by R.W.M. van Soest (University of Amsterdam, Netherlands.(
Different media were used for isolation of actinomycetes. Micromonospora sp. UR56 and
Actinokinospora sp. EG49 were cultivated on ISP2 medium. The inoculated agar plates
were incubated at 30 ◦C for a long time, ranging from 6 to 8 weeks. Distinct colony were selected and re-streaked many times until free of any contaminants.
Micromonospora sp. UR56 and Actinokinospora sp. EG49 were isolated and taxonomically identified according to morphology and its 16S rRNA genome sequence and phylogentic analyses.
Chapter II: Microbial fermentation, co-fermentation and extract preparation as modern approaches for elicitation of secondary metabolites.
Micromonospora sp. UR 56 and Actinokineospora sp. EG49 were isolated from Red Sea sponges Callyspongia sp and Spheciospongia vagabunda, respectively. Each microbial strain was fermented and co-fermented. After fermentation of axenic cultures and co-culture, the supernatant was extracted with ethyl acetate to yield ethyl acetate soluble fraction 700 mg (co-culture), 250mg Micromonospora sp. UR56 (axenic culture ) and 200mg Actinokineospora sp. EG49 ( axenic culture). All fractions were subjected to metabolomics analysis using LC-HRESI-MS technique.
Chapter III: Metabolomic profile of axenic and co-culture extracts
The metabolomic profile of the co-culture extract displayed induced diverse metabolites from different chemical classes compared to that of the two axenic cultures. Twelve metabolites were putatively identified in the Micromonospora sp. UR56-derived extract where phenazine derivatives were found to prevail. Most of these dereplicated phenazines e.g. Phenazine -1-carboxylic acid, aestivophoenin C and methyl saphenate have been reported to possess antimicrobial and cytotoxic properties. The remaining identified compounds were found to belong to the N-containing and polyketide classes. Within the axenic Actinokineospora sp. EG49 culture, no phenazine derivatives were traced in the LC-HRMS analysis of its extract. Additionally, its chemical profile revealed a poor diversity with a few identified N-containing and polyketide metabolites. On the other hand, the mixed fermentation of both actinomycetes has elicited Micromonospora sp. UR56 to accumulate diverse phenazine derivatives. Such induction could be due to environmental competition or chemical defense mechanisms. Based on the metabolomic profiling of the co-culture, the major induced metabolites were targeted and isolated using Sephadex LH20 followed by silica gel column chromatography and identified using different spectroscopic approaches. Subsequently, they were subjected to antibacterial, antibiofilm, and cytotoxicity testing.
Chapter IV: Isolation and structural elucidation of isolated pure compounds.
The crude ethyl acetate (700 mg), obtained from co-fermentation) was chromatographed on Sephadex LH20 MeOH / H2O (80:20%), to afford five main fractions .Fr.2 (200 mg) was chromatographed over silica gel column (CC). Gradient elution was performed using gradient mixture DCM : EtOAc followed by 100%MeOH to afford 13 sub fractions. Sub Fr (5) (30 mg) was further chromatographed on Sephadex LH20 using n-Hexane : DCM (50:50) to yield Phenazine-1-carboxylic acid; tubermycin. On the other hand, sub -Fr (7) was crystallized by DCM to afford N-(2-hydroxyphenyl)-acetamide. Fr.3 (175 mg) was chromatographed over silica gel column with n-hexane: EtOAc (100:0 to 0:100) to yield 9 sub- fractions. Fr 3-7 and Fr 3-8 (30mg and 20mg) respectively were further chromatographed over Sephadex LH20 using n-hexane : CH2CL2 (50:50) as mobile phase to afford pure compounds dimethyl phenazine-1,6-dicarboxylate and phenazine-1,6-dicarboxylic acid mono methyl ester; phencomycin respectively. Fr.5 (75mg) was further chromatographed on Sephadex LH20 using MeOH as mobile phase to afford pure compound p-anisamide. The structure elucidation of all isolated compounds
were determined based on detailed spectroscopic data including 1D and 2D nuclear magnetic resonance (NMR) experimental analyses in combination with high resolution electrospray ionization mass spectrometry (HR-ESI-MS).
Chapter V: Biological studies of isolated compounds were divided into:
In-vitro antimicrobial activity
The antibacterial activity of all isolated pure compounds (1-5) was evaluated on Gram-positive pathogenic bacteria such as Bacillus subtilis (ATCC29212) and Staphylococcus aureus (ATCC9144( and Gram-negative pathogenic bacteria such as Escherichia coli (ATCC25922) and Pseudomonas aeruginosa (ATCC27853 ). Compounds 3 and 5 displayed potent antibacterial activity against P. aeruginosa while compounds 1, 2 and 4 showed considerable antibacterial activity against S. aureus .Depending on these results together with reported ones, we could conclude that the phenazine-1-carboxylic acid scaffold is essential for the antibacterial activity against Gram-negative bacteria and has no observed activity towards Gram-positive ones. Although the addition of another carboxylic acid or carboxyl ester at C-6 significantly decreased the inhibitory activity
against Gram-negative bacteria, it converted these phenazine derivatives to be active against Gram-positive strains.
In-vitro antibiofilm activity
The biofilm-inhibitory activities of the isolated pure compounds (1–5) were measured using 96 well flat polystyrene plates against four clinical microbes comprising Gram-positive (Bacillus subtilis and Staphylococcus aureus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) pathogenic bacteria. Compounds 3 and 5 displayed potent antibiofilm activity against P. aeruginosa, while compounds 1, 2 and 4 showed a mild to moderate inhibitory activity against E. coli. Both compounds 1 and 2 showed potent to moderate inhibitory activity against S. aureus. Similar to the antibacterial results, the presence of carboxylic acids on both C-1 and C-6 of the phenazine ring system decreased the antibiofilm effect towards Gram-negative strains, though made these derivatives active against Gram-positive ones, particularly, S. aureus.
In-vitro cytotoxic activity by MTT assay
This assay was performed using different human cancer cell lines such as Mammary gland Breast cancer (MCF-7), Colorectal carcinoma (HCT-116), Human lung cancer cell line (WI38) and Hepatocellular carcinoma (HePG-2). Doxorubicin was used as a positive control drug. Only compound 4 was able to induce moderate cytotoxicity towards the tested cell lines with IC50 values ranged from 10-36 µg/mL. While, the activity of phenazine derivatives (1 and 3) showed no cytotoxic activity against tested cell lines. On the other hand, compound 2 showed mild cytotoxic activity against different cell lines.
In-vitro enzyme assay
Testing against Staphylococcal DNA gyrase-B and pyruvate kinase as possible molecular targets, together with their binding mode studies showed that compounds 1-3 could exert their bacterial inhibitory activities through inhibition of both enzymes. Moreover, their structural differences, particularly, the substitution at C-1 and C-6 played a crucial role in the determination of their inhibitory spectrum and potency.