Transcript Genetic Profiling and Antibacterial Screening of Indonesian Actinomycete Bacteria By Lisa Ching Dept. of Pharmaceutical Sciences Mentor: Dr.

Genetic Profiling and Antibacterial
Screening of Indonesian
Actinomycete Bacteria
By Lisa Ching
Dept. of Pharmaceutical Sciences
Mentor: Dr. Mark Zabriskie
The Antibiotic Challenge
- An Urgent Need for New Anti-infective Agents •
•
Infectious diseases are the leading cause of illness
worldwide and the third leading cause of death in the U.S.
New drug therapies are needed for drug-resistant infections
and emerging infectious diseases.
Emerging
infectious diseases
Infections leading to
chronic diseases
SARS, West Nile virus
HIV, hepatitis B & C, H.
pylori
Drug-resistant
Infections
Infectious
Disease
Threats
Resurgence of
‘old’ diseases
TB, malaria, dengue
MRSA, VRE, MDR-TB
Bioterrorism
anthrax, plague, ebola
ICBB/OSU Project
• A collaborative effort with the Indonesian Center for
Biodiversity & Biotechnology (ICBB)
• Studying microorganisms from the Black Water Ecosystem
(BWE) in Kalimantan, Indonesia for new anti-infective agents
Objective
The objective of this research is to combine genetic
profiling with antibacterial assays to screen soil bacteria
from the BWE for new antibiotic compounds.
Hypothesis
The ability of microbes to flourish in such extreme
conditions as the BWE suggest that the likelihood of
finding novel organisms, capable of producing new
anti-infective agents, is high.
Example of colored
secondary metabolites
secreted from BWE
actinomycetes
Strategy for Finding Novel Compounds
Novel antibacterial and anticancer
compounds isolated from BWE
bacteria
Isolate Actinomycetes
O
Genomic DNA
H
PCR product
amplification of NRPS gene
“Genome
profiling”
Examples
OH of
Examples: targeting NonRibosomal
primers used:
Peptide
Synthetase
(NRPS)
genes
&
OH O
Small-scale cultures
OH OH
Polyketide
• Halo
Panglimycin D
in multipleSynthase
media (PKS) genes
• HMG
O• NRPS- A3F
• ValA-S1
• PKS-I
HO NH
2
H3 C
16S rDNA
Analysis
PCR profiling for
targeted 2°
metabolite genes
O
CH3
OH
O
O
O
Nonribosomal
OH
Peptide
OH
O
O
Cl
Bioactivity
HO
OH
O
O
O
PCR
product
amplification
of
PKS
H
H
H gene O HO
N
N
screening
N
N
N
N
O
H
O
NH
Taxonomy
OH
OH
HO
Bioassay guided
isolation
CH3
CH3
H
Limamycin B
H
O
O
vancomycin
OH
O
N
O
HO
OH
N
OH
OH
O
OH
CH3
Et
HO
Chemodiversity
HO
O
CH3
O
O
Polyketides
New anti-infective agents
O
O
O
NH
H3C
HO
O
OCH2COOH
Rifamycin
O
Limazepine D
NMe2
O
OMe
O
CH3
H
CH3
AcO
H3CO
H
NH2
O
Biodiversity
N
Cl
HO
Metabolic capacity
O
Erythromycin
Dr. Serge Fotso
OH
http://www.flmnh.ufl.edu/cowries/PCR.gif
Summer Research
• Isolates 8380, 8379, and 8394
• Cultured on six different media:
Kings, Phytone, RARE3, M-17, M-18, M-29
• Crude methanolic extracts were prepared and screened for
bioactivity.
• Thin Layer Chromatography (TLC) and bioautography of active
extracts were conducted.
• High Performance Liquid Chromatography (HPLC) was used to
purify compounds.
• Spectroscopic analysis (e.g. NMR, Mass Spectrometry) was
used to identify compounds.
Bioactivity Results
8380
8394
8379
Sa
K
P
R M17 M18 M29
K
P
R M17 M29 M18
K
P M29 M18 M17 R
Ms
Ca
Pa
Sa: Staphylococcus aureus
Ms: Mycobacterium smegmatis
Ca: Candida albicans
Pa: Pseudomonas aeruginosa
• The three isolates exhibit varying
activities against each microbe.
• Isolate 8379 grown in M-17 & RARE3
media is active against P. aeruginosa
• When grown in Kings media, isolate
8379 is active against S. aureus & M.
smegmatis.
HPLC Metabolite
Comparisons
8379-K.ss
8379-M17.ss
The difference in HPLC
chromatograms suggest
that isolate 8379
produces different
secondary metabolites
in different media.
8379-R.ss
Conditions:
C18, 20-100% ACN
30 min, 5 mL/min.
254 nm
Gram-negative Activity
- Isolate 8379 • Gram-negative bacteria are harder to kill.
- complex outer membrane
- efflux pumps
- excel at making enzymes that can inactivate drugs
• There is a greater need for new antibiotics to treat Gramnegative bacteria compared to Gram-positive bacteria.
Pa
M17 R
TLC & Bioautography Results
8379-M17.ls
TLC
• Normal silica gel plates
8379-R.ls
Bioautography
• Solvent system:
CH2Cl2: MeOH (9:1)
• Stain:
vanillin (TLC)
MTT (bioautography)
•Test organism: P. aeruginosa
(bioautography)
• Isolate 8379 produces different active compounds in M-17 & RARE3 media.
• The active compound produced in M-17 medium has greater polarity.
Chromatography Results
- Reverse Phase HPLC 8379-M17.ls
8379-R.ls
active HPLC fractions
• Two active components from 8379-M17 elute around 15 min.
• A single active component from 8379-R elutes at 23 min.
Spectroscopy Results
Deducing the structure of 8379-M17.ls.9 :
• known bioactive compound
• precursor to & metabolite of
tryptophan
• precursor to various natural
products
1H
NMR
anthranilic acid
13C
NMR
Mass Spectrometry (MS)
Current and Future Work
• Complete spectroscopic analyses for all active HPLC
fractions, both small scale and large scale
• Apply spectroscopy data to determine the structure of
each compound
• Determine minimum inhibitory concentrations (MICs)
• Identify the taxonomy of remaining isolates & complete
PCR profiling
Acknowledgments
• Howard Hughes Medical Institute
• Dr. Kevin Ahern
• Dr. Mark Zabriskie
• Dr. Noer Kasanah
• Zabriskie Lab
• Mahmud Lab