1. Genetic Analysis of Mycobacterial Susceptibility to Antimicrobial Peptides Nima Motamedi Dr. Luiz Bermudez

2. Antimicrobial peptides Antimicrobial peptides are found in nearly all organisms. Antimicrobial peptides are found in nearly all organisms. Especially common in organisms that eat from the ground due to high bacterial content. 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 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). Antimicrobial peptides are positively charged, so they’re called cationic antimicrobial peptides(CAMPS). Attack bacterial cytoplasmic membrane which is generally negatively charged. Attack bacterial cytoplasmic membrane which is generally negatively charged. CAMPs fight bacterial infections such as Escherichia coli and other pathogens. CAMPs fight bacterial infections such as Escherichia coli and other pathogens.

3. Bacterial Resistance to Antimicrobial Peptides To attack the cytoplasmic membrane antimicrobial peptides modify and traverse outer membrane. 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. 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

4. Relevance Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium avium paratuberculosis are big threats to health and are CAMP resistant. 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. Mycobacterium tuberculosis causes 10% of deaths in the 15-59 age group. 54 million people are infected per year by tuberculosis. 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. 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

5. Relevance Mycobacterium avium paratuberculosis causes Johne’s disease in livestock. Mycobacterium avium paratuberculosis causes Johne’s disease in livestock. 22% of dairy cows, 8% of beef cattle are infected in the United States. 22% of dairy cows, 8% of beef cattle are infected in the United States. Infection caused by contaminated milk. Infection caused by contaminated milk. Only few thousand mycobacteria required for infection. Only few thousand mycobacteria required for infection. 100 million pathogens excreted in 1 gram of infected cow dung. 100 million pathogens excreted in 1 gram of infected cow dung. Each diseased cow must be slaughtered and results in loss of $245.00. 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

6. Disease costs U.S. Cattle Industry $250 million per year Disease costs U.S. Cattle Industry $250 million per year Effects Effects –Hindered development –Lowered milk production –Pathogen distribution Hard to notice: 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 milk. Pasteurization doesn’t fully eradicate the mycobacteria in milk. Connections are being made to Crohn’s disease (Gastrointestinal inflammation). Connections are being made to Crohn’s disease (Gastrointestinal inflammation). Relevance http://www.bvet.admin.ch/info-service/e/publikationen/magazin/2002/3/3_24-25.pdf

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

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

9. 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. Test susceptibility in Polymyxin B. Use transposon mutant library to identify mutants that are more susceptible to Polymyxin B. Use transposon mutant library to identify mutants that are more susceptible to Polymyxin B. Test these mutants regarding survival in macrophages. Test these mutants regarding survival in macrophages.

10. What is a transposon? Segment of DNA that organisms readily incorporate into their genomes. Segment of DNA that organisms readily incorporate into their genomes. Our transposon inserts itself randomly into the genome. Our transposon inserts itself randomly into the genome. Contains Kanamycin resistant gene. Contains Kanamycin resistant gene. Transposon uptake is required for cell survival on Kanamycin plates. Transposon uptake is required for cell survival on Kanamycin plates.

11. Characterization of Transposon Mutants Different clones are placed into each of the 96 wells. Different clones are placed into each of the 96 wells. Each plate is duplicated. Each plate is duplicated. Duplicate plate has Polymyxin B. Duplicate plate has Polymyxin B.

12. Plan of Action Determination of Polymyxin concentration required to kill wild type cells Determination of Polymyxin concentration required to kill wild type cells Determined by turbidity test. Determined by turbidity test. Lethal concentration is 64 ug/mL Lethal concentration is 64 ug/mL Polymyxin B (ug/mL)01248163264128256 Results+++++++++++++--- ++ full turbidity + reduced turbidity - no turbidity

13. Plan of Action Compare control and polymyxin libraries. Compare control and polymyxin libraries. Grow samples of susceptible mutant strains. Grow samples of susceptible mutant strains. Wild type dies at 64 µg/mL. Wild type dies at 64 µg/mL. 45 mutants from 25 plates susceptible at 1/2 of wild-type (die at 32 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. Four mutants susceptible at 16 µg/mL. 4D81A37H128B121A82C66H67C34D75F97B5 ----+++++++ ----------- 2H21A114H94E93C67G57A48B95F68E31C2 +++++++++++ ----------- 16 32 16 32

14. Plan of Action Lyse cells, purify DNA Lyse cells, purify DNA General PCR primed from common sequence General PCR primed from common sequence Specific PCR to amplify transposon sequence Specific PCR to amplify transposon sequence Gel electrophoresis & Plasmid excision/purification. Gel electrophoresis & Plasmid excision/purification.

15. Plan of Action Transformation and digestion of insert. Transformation and digestion of insert. Gel electrophoresis & sequencing. Gel electrophoresis & sequencing. Compare interrupted genes with virulent bacterial genomes in a database. Compare interrupted genes with virulent bacterial genomes in a database.

16. Interrupted Genes DnaB DnaB –Involved in helical structure of DNA LeuS 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. Both genes, if interrupted,* would inhibit growth regardless of antibiotics.

17. Interrupted Genes KasB 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.

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

19. Acknowledgements Howard Hughes Medical Institute Howard Hughes Medical Institute Kevin Ahern Kevin Ahern Luiz Bermudez & vet science laboratory staff Luiz Bermudez & vet science laboratory staff