Genetic Analysis of Mycobacterial
Susceptibility to Antimicrobial
Peptides
Nima Motamedi
Dr. Luiz Bermudez
Antimicrobial peptides
• Antimicrobial peptides are found in nearly all organisms.
• Especially common in organisms that eat from the ground due to high
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bacterial content.
alpha-defensins produced in Paneth cells in small
intestine are antimicrobial peptides
Antimicrobial peptides are positively charged, so
they’re called cationic antimicrobial
peptides(CAMPS).
Attack bacterial cytoplasmic membrane which is
generally negatively charged.
CAMPs fight bacterial infections such as
Escherichia coli and other pathogens.
Bacterial Resistance to
Antimicrobial Peptides
• To attack the cytoplasmic membrane
antimicrobial peptides modify and traverse
outer membrane.
• Modifications of lipopolysaccharides (LPS)
in outer membrane reduce its negative
charge and repel antimicrobial peptides.
http://www.uni-tuebingen.de/Mikrobiogen/peschel_pdfs/AP-TIM02.pdf
Relevance
• Mycobacterium tuberculosis, Mycobacterium
avium and Mycobacterium avium
paratuberculosis are big threats to health and
are CAMP resistant.
• Mycobacterium tuberculosis causes 10% of
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deaths in the 15-59 age group.
54 million people are infected per year by
tuberculosis.
Tuberculosis is only behind AIDS in deaths from
a single infectious disease, a category that is the
largest cause of death in the world.
http://www.molbio.princeton.edu/courses/mb427/2000/projects/0006/TBFACTS.htm
Relevance
• Mycobacterium avium paratuberculosis causes
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Johne’s disease in livestock.
22% of dairy cows, 8% of beef cattle are
infected in the United States.
Infection caused by contaminated milk.
Only few thousand mycobacteria required for
infection.
100 million pathogens excreted in 1 gram of
infected cow dung.
• Each diseased cow must be slaughtered and
results in loss of $245.00.
http://www.bvet.admin.ch/info-service/e/publikationen/magazin/2002/3/3_24-25.pdf
Relevance
• Disease costs U.S. Cattle Industry $250 million per year
• Effects
– Hindered development
– Lowered milk production
– Pathogen distribution
• Hard to notice:
– Symptoms develop slowly and are unspecific
– Before disease is recognized, more are usually infected
• Pasteurization doesn’t fully eradicate the mycobacteria in
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milk.
Connections are being made to Crohn’s disease
(Gastrointestinal inflammation).
http://www.bvet.admin.ch/info-service/e/publikationen/magazin/2002/3/3_24-25.pdf
Mycobacterium smegmatis
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Good general mycobacteria model
Genetic systems available
Non-pathogenic
3-4 hour generation time
Contains large, sequenced genome
CAMP-resistant
Polymyxin B
• Produced by Bacillus
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Polymyxa.
Polymyxin B serves as a
surrogate for
antimicrobial peptides.
Attacks cytoplasmic
membrane.
Resistances to Polymyxin
B are common.
Cheaper and readily
available.
Plan of Action
Hypothesis: Mycobacterium smegmatis (and other
mycobacteria) has a unique mechanism of defense
against antimicrobial peptides that involves synthesis
of proteins for their outer membrane.
• Test susceptibility in Polymyxin B.
• Use transposon mutant library to identify mutants
that are more susceptible to Polymyxin B.
• Test these mutants regarding survival in
macrophages.
What is a transposon?
• Segment of DNA that organisms readily
incorporate into their genomes.
• Our transposon inserts itself randomly into
the genome.
• Contains Kanamycin resistant gene.
• Transposon uptake is required for cell
survival on Kanamycin plates.
Characterization of Transposon
Mutants
• Different clones are
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placed into each of
the 96 wells.
Each plate is
duplicated.
Duplicate plate has
Polymyxin B.
Plan of Action
• Determination of Polymyxin concentration
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required to kill wild type cells
Determined by turbidity test.
Lethal concentration is 64 ug/mL
Polymyxin B
(ug/mL)
Results
0
1
2
4
8
16 32
++ ++ ++ ++ ++ ++ +
++ full turbidity
+ reduced turbidity
- no turbidity
64
128
256
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-
Plan of Action
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4D8
Compare control and polymyxin libraries.
Grow samples of susceptible mutant strains.
Wild type dies at 64 µg/mL.
45 mutants from 25 plates susceptible at 1/2 of
wild-type (die at 32 µg/mL).
Four mutants susceptible at 16 µg/mL.
1A3 7H12 8B12 1A8
2C6
6H6
7C3
4D7
5F9
7B5
16
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+
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+
32
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4E9
3C6
7G5
7A4
8B9
5F6
8E3
1C2
2H2 1A11 4H9
16
32
+
+
+
+
+
+
+
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+
+
-
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Plan of Action
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Lyse cells, purify DNA
General PCR primed from common sequence
Specific PCR to amplify transposon sequence
Gel electrophoresis & Plasmid
excision/purification.
Plan of Action
• Transformation and digestion of insert.
• Gel electrophoresis & sequencing.
• Compare interrupted genes with virulent
bacterial genomes in a database.
Interrupted Genes
• DnaB
– Involved in helical structure of DNA
• LeuS
– Also known as leucyl tRNA synthetase
– Required for addition of leucine in protein
synthesis
• Both genes, if interrupted,* would inhibit
growth regardless of antibiotics.
Interrupted Genes
• KasB
– Beta-ketoacyl-ACP synthase.
– Involved in meromycolate extension and lipid
biosynthesis.
– Meromycolate is the precursor to mycolic acid.
– Mycolic acid is specific to mycobacteria and plays a
role in envelope permeability.
– Tests with mycolic acid deficient Mycobacterium
tuberculosis in mice have shown a successful immune
response.
Down the Road
• Testing with other anti-microbial peptide
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surrogates
In vitro macrophage infection with wild-type
versus mutant strains.
Testing more virulent mycobacteria:
Mycobacterium tuberculosis, Mycobacterium
avium, Mycobacterium avium paratuberculosis.
Acknowledgements
• Howard Hughes Medical Institute
• Kevin Ahern
• Luiz Bermudez & vet science laboratory
staff