Genetic Analysis of Genetic Analysis of Mycobacterial Susceptibility Mycobacterial Susceptibility

to Antimicrobial Peptidesto Antimicrobial Peptides

Nima Motamedi Nima Motamedi Dr. Luiz BermudezDr. Luiz Bermudez

Antimicrobial peptidesAntimicrobial peptides• Antimicrobial peptides are found in nearly all Antimicrobial peptides are found in nearly all

organisms.organisms.• Especially common in organisms that eat Especially common in organisms that eat

from the ground due to high bacterial from the ground due to high bacterial content.content.

• alpha-alpha-defensins produced in Paneth cells in defensins produced in Paneth cells in small intestine are antimicrobial peptides small intestine are antimicrobial peptides

• Antimicrobial peptides are positively Antimicrobial peptides are positively charged, so they’re called cationic charged, so they’re called cationic antimicrobial peptides(CAMPS).antimicrobial peptides(CAMPS).

• Attack bacterial cytoplasmic membrane Attack bacterial cytoplasmic membrane which is generally negatively charged.which is generally negatively charged.

• CAMPs fight bacterial infections such as CAMPs fight bacterial infections such as Escherichia coli and other pathogens.Escherichia coli and other pathogens.

Bacterial Resistance to Bacterial Resistance to Antimicrobial PeptidesAntimicrobial Peptides

• To attack the cytoplasmic membrane To attack the cytoplasmic membrane antimicrobial peptides modify and antimicrobial peptides modify and traverse outer membrane.traverse outer membrane.

• Modifications of lipopolysaccharides Modifications of lipopolysaccharides (LPS) in outer membrane reduce its (LPS) in outer membrane reduce its negative charge and repel negative charge and repel antimicrobial peptides.antimicrobial peptides.

http://www.uni-tuebingen.de/Mikrobiogen/peschel_pdfs/AP-TIM02.pdf

RelevanceRelevance

• Mycobacterium tuberculosis, Mycobacterium Mycobacterium tuberculosis, Mycobacterium avium avium and and Mycobacterium avium Mycobacterium avium paratuberculosis paratuberculosis are big threats to health are big threats to health and are CAMP resistant.and are CAMP resistant.

• Mycobacterium tuberculosis Mycobacterium tuberculosis causes 10% of causes 10% of deaths in the 15-59 age group. deaths in the 15-59 age group.

• 54 million people are infected per year by 54 million people are infected per year by tuberculosis.tuberculosis.

• Tuberculosis is only behind AIDS in deaths Tuberculosis is only behind AIDS in deaths from a single infectious disease, a category from a single infectious disease, a category that is the largest cause of death in the that is the largest cause of death in the world.world.

http://www.molbio.princeton.edu/courses/mb427/2000/projects/0006/TBFACTS.htm

RelevanceRelevance

• Mycobacterium avium paratuberculosis Mycobacterium avium paratuberculosis causes Johne’s disease in livestock.causes Johne’s disease in livestock.

• 22% of dairy cows, 8% of beef cattle are 22% of dairy cows, 8% of beef cattle are infected in the United States.infected in the United States.

• Infection caused by contaminated milk.Infection caused by contaminated milk.• Only few thousand mycobacteria required Only few thousand mycobacteria required

for infection.for infection.• 100 million pathogens excreted in 1 gram 100 million pathogens excreted in 1 gram

of infected cow dung.of infected cow dung.• Each diseased cow must be slaughtered Each diseased cow must be slaughtered

and results in loss of $245.00.and results in loss of $245.00.

http://www.bvet.admin.ch/info-service/e/publikationen/magazin/2002/3/3_24-25.pdf

• Disease costs U.S. Cattle Industry $250 million Disease costs U.S. Cattle Industry $250 million per yearper year

• EffectsEffects– Hindered developmentHindered development– Lowered milk productionLowered milk production– Pathogen distributionPathogen distribution

• Hard to notice:Hard to notice:– Symptoms develop slowly and are unspecificSymptoms develop slowly and are unspecific– Before disease is recognized, more are usually infectedBefore disease is recognized, more are usually infected

• Pasteurization doesn’t fully eradicate the Pasteurization doesn’t fully eradicate the mycobacteria in milk.mycobacteria in milk.

• Connections are being made to Crohn’s disease Connections are being made to Crohn’s disease (Gastrointestinal inflammation).(Gastrointestinal inflammation).

RelevanceRelevance

http://www.bvet.admin.ch/info-service/e/publikationen/magazin/2002/3/3_24-25.pdf

Mycobacterium smegmatisMycobacterium smegmatis

• Good general mycobacteria modelGood general mycobacteria model

• Genetic systems availableGenetic systems available

• Non-pathogenicNon-pathogenic

• 3-4 hour generation time3-4 hour generation time

• Contains large, sequenced genomeContains large, sequenced genome

• CAMP-resistantCAMP-resistant

Polymyxin BPolymyxin B

• Produced by Bacillus Produced by Bacillus Polymyxa.Polymyxa.

• Polymyxin B serves as Polymyxin B serves as a surrogate for a surrogate for antimicrobial peptides.antimicrobial peptides.

• Attacks cytoplasmic Attacks cytoplasmic membrane.membrane.

• Resistances to Resistances to Polymyxin B are Polymyxin B are common.common.

• Cheaper and readily Cheaper and readily available.available.

Plan of ActionPlan of Action

Hypothesis: Hypothesis: Mycobacterium smegmatisMycobacterium smegmatis (and other (and other mycobacteria) has a unique mechanism of mycobacteria) has a unique mechanism of defense against antimicrobial peptides that defense against antimicrobial peptides that involves synthesis of proteins for their outer involves synthesis of proteins for their outer membrane.membrane.

• Test susceptibility in Polymyxin B.Test susceptibility in Polymyxin B.

• Use transposon mutant library to identify Use transposon mutant library to identify mutants that are more susceptible to mutants that are more susceptible to Polymyxin B.Polymyxin B.

• Test these mutants regarding survival in Test these mutants regarding survival in macrophages.macrophages.

What is a transposon?What is a transposon?

• Segment of DNA that organisms Segment of DNA that organisms readily incorporate into their readily incorporate into their genomes.genomes.

• Our transposon inserts itself randomly Our transposon inserts itself randomly into the genome.into the genome.

• Contains Kanamycin resistant gene.Contains Kanamycin resistant gene.

• Transposon uptake is required for cell Transposon uptake is required for cell survival on Kanamycin plates.survival on Kanamycin plates.

Characterization of Transposon Characterization of Transposon MutantsMutants

• Different clones are Different clones are placed into each of placed into each of the 96 wells.the 96 wells.

• Each plate is Each plate is duplicated.duplicated.

• Duplicate plate has Duplicate plate has Polymyxin B.Polymyxin B.

Plan of ActionPlan of Action

• Determination of Polymyxin Determination of Polymyxin concentration required to kill wild type concentration required to kill wild type cellscells

• Determined by turbidity test.Determined by turbidity test.

• Lethal concentration is 64 ug/mLLethal concentration is 64 ug/mLPolymyxin Polymyxin

BB(ug/mL)(ug/mL)

00 11 22 44 88 1616 3232 6464 128128 252566

ResultsResults ++++

++++

++++

++++

++++

++++

++ -- -- --++ full turbidity+ reduced turbidity- no turbidity

Plan of ActionPlan of Action• Compare control and polymyxin libraries.Compare control and polymyxin libraries.

• Grow samples of susceptible mutant Grow samples of susceptible mutant strains.strains.

• Wild type dies at 64 µg/mL.Wild type dies at 64 µg/mL.

• 45 mutants from 25 plates susceptible at 45 mutants from 25 plates susceptible at 1/2 of wild-type (die at 32 µg/mL).1/2 of wild-type (die at 32 µg/mL).

• Four mutants susceptible at 16 µg/mL.Four mutants susceptible at 16 µg/mL.4D84D8 1A31A3 7H17H1

228B18B1

221A81A8 2C62C6 6H66H6 7C37C3 4D74D7 5F95F9 7B57B5

-- -- -- -- ++ ++ ++ ++ ++ ++ ++

-- -- -- -- -- -- -- -- -- -- --

2H22H2 1A11A111

4H94H9 4E94E9 3C63C6 7G57G5 7A47A4 8B98B9 5F65F6 8E38E3 1C21C2

++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++

-- -- -- -- -- -- -- -- -- -- --

16

32

16

32

Plan of ActionPlan of Action

• Lyse cells, purify DNALyse cells, purify DNA

• General PCR primed from common General PCR primed from common sequencesequence

• Specific PCR to amplify transposon Specific PCR to amplify transposon sequencesequence

• Gel electrophoresis & Plasmid Gel electrophoresis & Plasmid excision/purification.excision/purification.

Plan of ActionPlan of Action

• Transformation and digestion of Transformation and digestion of insert.insert.

• Gel electrophoresis & sequencing.Gel electrophoresis & sequencing.

• Compare interrupted genes with Compare interrupted genes with virulent bacterial genomes in a virulent bacterial genomes in a database.database.

Interrupted GenesInterrupted Genes

• DnaBDnaB– Involved in helical structure of DNAInvolved in helical structure of DNA

• LeuSLeuS– Also known as leucyl tRNA synthetaseAlso known as leucyl tRNA synthetase– Required for addition of leucine in protein Required for addition of leucine in protein

synthesissynthesis

• Both genes, if interrupted,* would Both genes, if interrupted,* would inhibit growth regardless of inhibit growth regardless of antibiotics.antibiotics.

Interrupted GenesInterrupted Genes

• KasBKasB– Beta-ketoacyl-ACP synthase.Beta-ketoacyl-ACP synthase.– Involved in meromycolate extension and lipid Involved in meromycolate extension and lipid

biosynthesis.biosynthesis.– Meromycolate is the precursor to mycolic acid.Meromycolate is the precursor to mycolic acid.– Mycolic acid is specific to mycobacteria and Mycolic acid is specific to mycobacteria and

plays a role in envelope permeability.plays a role in envelope permeability.– Tests with mycolic acid deficient Tests with mycolic acid deficient

Mycobacterium tuberculosisMycobacterium tuberculosis in mice have in mice have shown a successful immune response.shown a successful immune response.

Down the RoadDown the Road

• Testing with other anti-microbial peptide Testing with other anti-microbial peptide surrogatessurrogates

• In vitroIn vitro macrophage infection with wild-type macrophage infection with wild-type versus mutant strains.versus mutant strains.

• Testing more virulent mycobacteria: Testing more virulent mycobacteria: Mycobacterium tuberculosisMycobacterium tuberculosis, , Mycobacterium Mycobacterium aviumavium, , Mycobacterium avium Mycobacterium avium paratuberculosisparatuberculosis..

AcknowledgementsAcknowledgements

• Howard Hughes Medical InstituteHoward Hughes Medical Institute

• Kevin AhernKevin Ahern

• Luiz Bermudez & vet science Luiz Bermudez & vet science laboratory stafflaboratory staff