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

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

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



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

 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

 Mutant gyrA gene  Single point mutation  Creates quinolone resistant GyrA subunits promoterless mutant gyrA gene  Previous work  Genome broken into thousands of fragments  Kanamycin marker  Transformed into wild type

M. avium

 “GyrA” bacteria PLDG13-GyrA plasmid random fragment

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

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

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

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

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

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

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

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

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 Added together equals 228 bp

228bp

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

 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

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

Acknowledgements

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