Genetic Analysis of Mycobacterial Susceptibility to Antimicrobial Peptides

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Genetic Analysis of Mycobacterial Susceptibility
to Antimicrobial Peptides

• Nima Motamedi Dr. Luiz Bermudez

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Antimicrobial peptides

• Antimicrobial peptides are found in nearly all
  organisms.
• Especially common in organisms that eat from the
  ground due to high bacterial content.
• alpha-defensins produced in Paneth cells in small
  intestine are antimicrobial peptides
• Antimicrobial peptides are positively charged, so
  theyre 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.

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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_pd
fs/AP-TIM02.pdf
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Relevance

• Mycobacterium tuberculosis, Mycobacterium avium
  and Mycobacterium avium paratuberculosis are big
  threats to health and are CAMP resistant.
• Mycobacterium tuberculosis causes 10 of 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
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Relevance

• Mycobacterium avium paratuberculosis causes
  Johnes 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/publikatio
nen/magazin/2002/3/3_24-25.pdf
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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 doesnt fully eradicate the
  mycobacteria in milk.
• Connections are being made to Crohns disease
  (Gastrointestinal inflammation).

http//www.bvet.admin.ch/info-service/e/publikatio
nen/magazin/2002/3/3_24-25.pdf
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Mycobacterium smegmatis

• Good general mycobacteria model
• Genetic systems available
• Non-pathogenic
• 3-4 hour generation time
• Contains large, sequenced genome
• CAMP-resistant

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Polymyxin B

• Produced by Bacillus Polymyxa.
• Polymyxin B serves as a surrogate for
  antimicrobial peptides.
• Attacks cytoplasmic membrane.
• Resistances to Polymyxin B are common.
• Cheaper and readily available.

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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.

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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.

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Characterization of Transposon Mutants

• Different clones are placed into each of the 96
  wells.
• Each plate is duplicated.
• Duplicate plate has Polymyxin B.

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Plan of Action

• Determination of Polymyxin concentration required
  to kill wild type cells
• Determined by turbidity test.
• Lethal concentration is 64 ug/mL

full turbidity reduced turbidity - no
turbidity
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Plan of Action

• 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.

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Plan of Action

• Lyse cells, purify DNA
• General PCR primed from common sequence
• Specific PCR to amplify transposon sequence
• Gel electrophoresis Plasmid excision/purificatio
  n.

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Plan of Action

• Transformation and digestion of insert.
• Gel electrophoresis sequencing.
• Compare interrupted genes with virulent bacterial
  genomes in a database.

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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.

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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.

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Down the Road

• Testing with other anti-microbial peptide
  surrogates
• In vitro macrophage infection with wild-type
  versus mutant strains.
• Testing more virulent mycobacteria Mycobacterium
  tuberculosis, Mycobacterium avium, Mycobacterium
  avium paratuberculosis.

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Acknowledgements

• Howard Hughes Medical Institute
• Kevin Ahern
• Luiz Bermudez vet science laboratory staff