Using In Vivo Expression Technology to Identify Mycobacterium avium Genes Expressed during Intracellular Infection in Dendritic Cells and Mice

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Using In Vivo Expression Technology to Identify
Mycobacterium avium Genes Expressed during
Intracellular Infection in Dendritic Cells and
Mice
Sasha Rose Mentor Dr. Luiz Bermudez
OSU College of Veterinary Medicine
Department of Biomedical Sciences
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Relevance

• Mycobacterium avium
• Closely related to M. leprae and M. tuberculosis
• Common opportunistic infection in AIDS patients
• Usually infects gastrointestinal or respiratory
  system
• Treatment includes a combination of antibiotics

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Background

• Mycobacterium avium
• Common genetic profiles
• Free living in environment
• Invasion of host cell
• Survival in host cell
• Genes turned on while in host cell
• Suppress immune response of the host
• Maintenance of vacuole
• Mineral transport
• Difficult to identify
• What genes do what functions
• Where on the genome the genes are located

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• In vivo expression technology (IVET)
• A technique used to identify the virulence genes
  in a
  bacterium when expressed in a living cell
• Goal To establish an IVET system suitable for
  screening M. avium genes required for survival in
  a host environment, using quinolone resistance as
  a selection marker

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• Quinolones
• Broad spectrum antibiotics
• Inhibit the GyrA subunit of the DNA gyrase enzyme
  
• DNA gyrase enzyme
• Type II topoisomerase
• Crucial for DNA replication
• Relieves tension when DNA
• is wound too tightly
• GyrA subunit
• Binds/breaks DNA
• made from the gyrA gene

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• Mutant gyrA gene
• Single point mutation
• Creates quinolone resistant GyrA subunits
• Previous work
• Genome broken into
  thousands of fragments
• Kanamycin marker
• Transformed into wild type M. avium
• GyrA bacteria

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Hypothesis

• A bacterium that survives the quinolone treatment
  will possess a fragment that contains a promoter
  sequence for a gene that was expressed while in
  the host cell

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Methods

• Part I - using IVET to select bacteria
• Dendritic cells-early infection
• Mice-established infection
• Part II - screening and
  identifying genes

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Obtaining Dendritic Cells
centrifuge
withdraw middle layer
wash 3 times, re-suspend with RPMI medium
whole blood
add cytokines human IL-4 and GM-CSF allow 5 day
growth at 37C
monocytes
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Using IVET in Dendritic Cells
infect with GyrA bacteria 1 well for each time
point infected with wild type MAC 104
incubate for 1 hour
dendritic cell
wash cells and begin 4, 24, or 48 hour time point
lyse cells and plate bacteria on Petri dishes
treat with moxifloxacin at 8µg/mL allow 24 hours
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Using IVET in Mice

• C57BL/6
• 20 total-4 cages
• Bacteria administered orally via
  gavaging
• Cage 1 wild type MAC 104
• Cages 2-4 GyrA

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Using IVET in Mice

• 10 week system
• Kanamycin injections daily for first 3 weeks
• Selecting for plasmid
• Cages 2-4
• Moxifloxacin injections daily for last 7 weeks
• Cages 1-3
• 100mg/kg
• Mice were sacrificed in 3 groups

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Using IVET in Mice

• Necropsies were performed on all of the mice
• Lung, liver, spleen, and mesenteric tissue
  samples were homogenized
• Samples were plated on Petri dishes

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Bacterial Survival

• 2 morphologies
• Yellow
• White
• Each colony should have a unique fragment

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Screening and Identifying Genes
gyrA
fragment
Pick off individual colony isolate plasmid
Use PCR to amplify the fragment
Use gel electrophoresis to screen PCR products
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Results

• Quinolone Selection
• Screened over 60 colonies
• Double band pattern
• No difference between samples
• PCR reagent control negative
• Wild type controls survived treatment

728bp 228bp
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• Mouse toxicity/health
• Multiple mice - fibrinous exudate
• 2 deaths unknown cause
• 1 mouse euthanized early because of severe
  abdominal inflammation
• Ended experiment 1 week early
• Loss of activity
• Abdominal inflammation

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Discussion

• Wild type survival - insufficient selection
  occurred
• Dendritic Cells
• Very short treatment time
• Mice
• Poor absorption of moxifloxacin from the
  intraperitoneal space
• Mouse toxicity/Health
• Health problems not associated with M. avium
• Cage 4 mice received no moxifloxacin

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Acknowledgements

• Dr. Luiz Bermudez and the rest of the lab
• Oregon State University College of Veterinary
  Medicine
• Howard Hughes Medical Institute
• Dr. Kevin Ahern