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

Relevance

Mycobacterium avium Closely related to M. leprae

and M. tuberculosis Common opportunistic

infection in AIDS patientsUsually 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

QuinolonesBroad spectrum antibiotics Inhibit the GyrA subunit of the DNA gyrase enzyme

DNA gyrase enzymeType II topoisomeraseCrucial for DNA replicationRelieves tension when DNA is wound too tightlyGyrA subunit

Binds/breaks DNA made from the gyrA gene

Mutant gyrA geneSingle point mutationCreates quinolone resistant

GyrA subunits Previous work

Genome broken into thousands of fragments

Kanamycin markerTransformed into wild type M.

avium “GyrA” bacteria

PLDG13-GyrA plasmid

promoterlessmutant gyrA

generandom

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 bacteriaDendritic cells-early infectionMice-established infection

Part II - screening and identifying genes

http://www.unis.org/UNIScienceNet/DendriticCell_400.jpg

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

Using IVET in Dendritic Cells

incubate for 1 hour

wash cells and begin 4, 24, or 48 hour time pointtreat 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

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 systemKanamycin 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 morphologiesYellowWhite

Each colony should have a unique fragment

Screening and Identifying Genes

Pick off individual

colony; isolate plasmid

Use PCR to amplify the

fragment Use gel electrophoresis to screen PCR products

228bp

gyrA fragment

Added together equals 228 bp

Results

Quinolone SelectionScreened over 60 coloniesDouble band patternNo difference between

samplesPCR reagent control =

negativeWild type controls survived

treatment

728bp

228bp

Mouse toxicity/health

Multiple mice - fibrinous exudate 2 deaths – unknown cause1 mouse euthanized early because of severe

abdominal inflammationEnded experiment 1 week early

Loss of activity Abdominal inflammation

Discussion

Wild type survival - insufficient selection occurredDendritic Cells

Very short treatment time

Mice Poor absorption of moxifloxacin from the intraperitoneal space

Mouse toxicity/HealthHealth problems not associated with M. avium

Cage 4 mice received no moxifloxacin

Dr. Luiz Bermudez and the rest of the lab Oregon State University College of Veterinary

Medicine Howard Hughes Medical Institute Dr. Kevin Ahern

Acknowledgements