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

2. 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 http://www.hain-lifescience.com/images/avium.jpg

3. 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

4.  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

5.  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

6.  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 PLDG13-GyrA plasmid promoterless mutant gyrA gene random fragment

7. 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

8. Methods  Part I - using IVET to select bacteria  Dendritic cells-early infection  Mice-established infection  Part II - screening and identifying genes http://www.unis.org/UNIScienceNet/DendriticC ell_400.jpg

9. Obtaining Dendritic Cells whole blood centrifuge withdraw middle layer wash 3 times, re- suspend with RPMI medium add cytokines human IL-4 and GM-CSF; allow 5 day growth at 37°C mature dendritic cells monocytes

10. Using IVET in Dendritic Cells incubate for 1 hour wash cells and begin 4, 24, or 48 hour time point treat with moxifloxacin at 8µg/mL; allow 24 hours lyse cells and plate bacteria on Petri dishes dendritic cell infect with GyrA bacteria; 1 well for each time point infected with wild type MAC 104

11. 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

12. 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

13. 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

14. Bacterial Survival  2 morphologies  Yellow  White  Each colony should have a unique fragment

15. Screening and Identifying Genes Pick off individual colony; isolate plasmid Use PCR to amplify the fragment Use gel electrophoresis to screen PCR products 228bp gyrAfragment Added together equals 228 bp

16. 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

17.  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

18. 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

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