University of Lagos

1. University of Lagos Department of Microbiology

2. BIOPROSPECTING FOR ANTIMICROBIAL AND ANTICANCER COMPOUNDS FROM ACTINOMYCETES FROM LAGOS LAGOON SEDIMENTS. Professor I.A. Adeleye Dr. M.O. Akinleye

3. BACKGROUND OF STUDY • About 23,000 bioactive secondary metabolites produced by microorganism have been reported and over 10,000 of these compounds are produced by actinomycetes, representing 45% of all bioactive microbial metabolites discovered. • Among actinomycetes, around 7,600 compounds are produced by Streptomyces spp. (Berdy, 2005).

4. Many of these secondary metabolites are potent antibiotics and clinically useful antitumor drugs. • However, the search for novel drugs is still a priority goal, due to the issue of drug resistance to multiple chemotherapeutics. • In addition, the high toxicity usually associated with cancer chemotherapy drugs and their undesirable side effects has increased the demand for novel antitumor drugs which are active against recalcitrant tumors, with fewer side effects and/or with greater therapeutic efficiency (Demain and Sanchez, 2009).

5. It is therefore imperative to continue the search for novel microbial natural products from underexplored habitats because researchers have hypothesized that since these microorganisms could thrive in the marine environment, fierce competition among the species encourage them to produce novel bioactive compounds to antagonize other competitors to aid their survival.

6. STATEMENT OF THE PROBLEM • Antibiotic resistance in clinical isolates continues to pose formidable challenges in medicine worldwide. • Viral infections exact a great toll on human lives especially in resource limited countries of Africa including Nigeria which has the second largest AIDS burden in Africa with an estimate of 3.8% of her population of 160 million infected with the deadly virus. • There has been an increase in the public health concern about cancer in low-income countries in Africa including Nigeria.

7. AIM OF STUDY This study aimed at isolation, purification and identification of antimicrobial and antitumor agents from actinomycetes in Lagos estuarine sediments and provide information of the actinobacteria community profile and their diversity in these sediments.

8. OBJECTIVES OF THE STUDY • Isolate actinomycetes from Lagos marine sediments, characterize the isolates phenotypically and genotypically by sequencing pertinent strains • Determine the actinobacterial community structure using culture-independent method (DGGE) • Determine the antibacterial, antifungal and antiviral activity of metabolites obtained from the isolated actinomycetes

9. SIGNIFICANCE OF THE STUDY • Determine the antitumor activities of metabolites obtained from the isolated actinomycetes. • Isolate and purify the bioactive metabolites using chromatographic tools • Identify the purified bioactive compounds through structural elucidation using a combination of spectroscopic methods

10. MATERIALS AND METHODS

11. SAMPLES COLLECTION

12. Fig 1. Locations ofsediments obtained from Lagos lagoon, Nigeria

13. Isolation and identification of actinomycetes from Lagos lagoon sediment • Isolation of actinomycetes was done using spread plate method using starch casein, Kuster’s, Gauze 1 and 2, Marine and Actinomycete isolation agar. Plates were incubated at 29°C for 1 to 5weeks • Identification of the isolates was done with API kits and through amplification of 16S rRNA gene and sequencing using actinobacterial-specific primers. (approximately 640 bpof 16S r DNA) • Sequences were identified by NCBI BLAST and submitted to GenBank. • Phylogenetic tree construction. • (Stachet al., 2003)

14. Culture-independent molecular analysis of actinobacterial community structure in Lagos Lagoon sediment samples • Total actinobacterial community DNA was extracted from 12 sediment samples using QIAamp DNA kit (Qiagen, Germany) by enzymatic method (Muyzeret al., 1993) • Nested PCR • PCR products after analysis by agarose gel were separated on 8 % denaturing gradients polyacrylamide gel with a 50 % - 65 % Urea and Formamide denaturants at 140V for 5 hours (Adewumiet al., 2012). • The major DGGE bands of interest were excised from the gels, eluted in 50μl sterile deionized water, amplified and sequenced

15. Production and bioactivity screening of metabolites • Production of metabolites • Standard ATCC Strains E.coli, P. aeruginosa, C. albicans, S. flexneri, S. epidermidis, S. typhi, E. faecaliswere first used to screen the actinomycetes for bioactivity potentials using cross streak method • Pure cultures were fermented in 10ml broth for 3 days at pH7, 28OC and 180rpm and transferred to 200ml broth under same conditions • Fermentation broth was scaled up by transferring to flasks containing 1000 mL sterile culture broth and shaken at 180 rpm for 10 days (Janardhanet al., 2014). • The cells were separated from broth by centrifugation at 6000 rpm and 10OC for 30 minutes and metabolites extracted by liquid-liquid extraction method in ratio 1:1 ethylacetate and cultre broth (Janardhanet al., 2014) • Antibacterial and antifungal assay of the metabolites was determined using agar well diffusion method.

16. Bioactivity screening of metabolites • MIC, MBC and MFC of the crude extracts were determined by broth dilution method (Ramalivhana et al., 2014) • Antiviral activity of Influenza virus strain X-31 (H3N2) using Madin–Darby canine kidney (MDCK) cells using plaque reduction assay technique (Shimizu et al., 2008). • Antitumor activities were determined against five cell lines - K562 (Human acute myelocyticleukemia), HeLa (cervical carcinoma), AGS (Human gastric celline), MCF-7 (breast adenocarcinoma), HL-60 (Human acute promyelocytic leukaemia) cell-lines using CCK8 assay (Ravikumar et al., 2012)

17. Isolation and identification of bioactive metabolites • The actinomycete strain with the best overall antimicrobial activity was selected for the large-scale fermentation by scaling up from 1250mL to 10L fermentation • Extraction of metabolites was achieved using Amberlite XAD7 and Amberlite XAD 16, eluted with acetone and concentrated to dryness yielding 8 grams of crude extract (Kwon et al., 2009) • The crude extract was separated by partitioning Kwon et al. (2009) using the solvents Dichloromethane, water and 10% Dichloromethane in 2-propanol at ratio 1:1:1 (Kwon et al., 2009)

18. Isolation and identification of bioactive metabolites (contd.) • The fraction with highest bioactivity were fractionated further using flash column chromatography using n-hexane/ethylacetate (0-100 % and changed to ethylacetate/methanol (60 % : 40 %) and monitored at UV 254 nm • Structural elucidation was done using IR, ESI-MS and NMR Spectra (1H, 13C, COSY, HMBC) • Statistical analysis • Statistical analysis was done using Microsoft Excel 2013 and GraphPad Prism software • (GraphPad Software Inc., San Diego, CA). Log IC50 calculations were done using algorithms • for dose-response curve with variable slope

19. RESULTS

20. 1. ISOLATION AND IDENTIFICATION OF ACTINOMYCETES FROM LAGOS LAGOON SEDIMENT

21. 1. IDENTIFICATION OF ISOLATED ACTINOBACTERIAL STRAINS

22. Table 1. Physiological characteristics of isolated actinobacterial strains from Lagos Lagoon Key: IND: Indole, URE: Urease, GLU: Glucose, MAN: Mannitol, LAC: Lactose, SAC: Saccharose, MAL: Maltose, SAL: Salicin, XYL: Xylose, ARA:Arabinose, GEL: Gelatin, ESC: Esculin, GLY: Glycerol, CEL: Cellobiose, MNE: Mannose, MLZ: Melezitose, RAF: Raffinose, SOR: Sorbitol, RHA:Rhamnose, TRE: Trehalose, CAT: Catalase, SPO-Spores, GRA: Gram reaction, STA: Starch hydrolysis, CAS: Casein hydrolysis, +: positive, -: negative, +/-: variable

23. Table 2. Identification of actinomycetes isolated from Lagos Lagoon based on 16Sr RNA gene sequences

24. Agromycessp. Figure 2. Phylogenetic tree showing multiple sequence alignment of 16S rRNA gene sequences of actinomycetesActinomycetes isolated in Lagos Lagoon

25. Figure 3. Phylogenetic tree showing multiple sequence alignment of 16S rRNA gene sequences of actinomycetes isolated in Lagos Lagoon and other marine environments worldwide and some other known strains. Pairwise phylogenetic distances were calculated based on 16S rRNA gene.

26. Figure 4. Scanning electron micrograph of Streptomyces bingchenggensisULS14. a) The strain growing on starch casein agar at 100x magnification. b) Strain morphology at 10.00KX magnification

27. 2. IDENTIFICATION OF MAJOR ACTINOBACTERIAL PCR-DGGE BANDS

28. Figure 5. DGGE profiles of PCR-amplified 16S rRNA gene fragments of 12 sampling sites of Lagos Lagoon showing actinobacterial diversity Legend: ID Act- Already Identified actinomycetes strains, ID Micro- Already identified Micromonospora strains. All soil- Combined PCR products for all the soil from sample sites, OK- Okobaba, OFF- Offin, FOL-Folawiyo, IDD-Iddo, ST1-Ejirin, ST2-Imoru, ST3-Itokin, ST4-Imope, ST5-Ikosi, ST6-Egbin, ST7-Ijede, STA 8- Bayeku. Bands a. Micromonosporaaurantica;b. Micromonospora sp.; c. Micromonosporasediminicola; d. Micromonosporahumi; e. Micrococcus luteus; f. Micromonosporasp.; g. Micromonosporasp.; .h. Streptomyces albus; i. Streptomyces avermitilis; j. Streptomyces coelicolor; k. Streptomyces bingchenggensis; l. Arthrobacterphenanthrenivorans; m. Arthrobactersp.; n. Arthrobacterchlorophenolicus

29. 3. DETERMINATION OF ANTIBACTERIAL, ANTIFUNGAL AND ANTIVIRAL ACTIVITY OF METABOLITES OBTAINED FROM THE ISOLATED ACTINOMYCETES

30. Table 4. Antibacterial activity of bioactive actinomycetesstrains (cross-streaking assay)

31. Table 5. Antifungal activity of bioactive crude extracts of actinomycetesstrains (Kirby-Bauer assay)

32. Table 6. Minimum Inhibitory Concentration (MIC) (mg/ml) of bioactive crude extracts against bacterial pathogens

33. Table 7. Minimum Inhibitory Concentration (MIC) (mg/ml) of bioactive crude extracts against fungal pathogens

34. Table 8. Minimum Bactericidal Concentration (MBC) (mg/ml) of bioactive crude extracts of actinomycetes

35. Table 9. Minimum Fungicidal Concentration (MFC) (mg/mL) of bioactive crude extracts of actinomycetes

36. Figure 6. Antiviral activity of crude extracts of actinomyetes against H3N2 influenza virus Legend: C= control (cells) without virus, C+V= control (cells) infected with virus, K2 (0.8-0.3) = concentrations of crude extracts of ULK2, K3 (1-0.3) = concentrations of crude extracts of ULK3 in mg/mL, K10 (1-0.4) = concentrations of crude extracts of ULK10 in mg/mL, K11 (1-0.4) = concentrations of crude extracts of ULK11 in mg/mL

37. 4. DETERMINATION OF THE ANTITUMOR ACTIVITIES OF METABOLITES OF METABOLITES OBTAINED FROM THE ISOLATED ACTINOMYCETES

38. Table 10. Antitumor effect of actinomycete crude extracts on cellines (mg/mL)

39. 5. ISOLATION AND PURIFICATION OF BIOACTIVE METABOLITES USING CHROMATOGRAPHIC TOOLS

40. Figure 7: Isolation of compounds from the crude extract of actinomycete using flash chromatography

41. Table 11. Minimum Inhibitory Concentration (MIC) (µg/mL) of purified bioactive compounds from crude extract of Streptomyces bingchengensisULS14 Legend: na - not applicable

42. Minimum Bactericidal Concentration (MBC) and Minimum Fungicidal Concentration (µg/mL) of purified bioactive compounds from crude extract of Streptomyces bingchengensisULS14

43. (IC50= 0.075µg/mL) Log10 Concentration (µg/mL) Figure 8. Inhibition concentration (IC50) of compound ULDF4 against HeLa cell line

44. (IC50= 0.034µg/mL) Log10 Concentration (µg/mL) Figure 9. Inhibition concentration (IC50) of compound ULDF5 against HeLa cell line

45. 6. IDENTIFICATION OF PURIFIED BIOACTIVE COMPOUNDS THROUGH STRUCTURAL ELUCIDATION USING A COMBINATION OF SPECTROSCOPIC METHODS

46. C34H35NO13 Figure 10. Mass Spectroscopy (ESI-MS) of compound ULDF4

47. Figure 11. Infrared spectroscopy (IR) of compound ULDF4

48. Figure 12. Proton NMR (1H-NMR) of compound ULDF4

49. Figure 13. Carbon NMR (13C-NMR) of compound ULDF4

50. Figure 14. COSY of compound ULDF4