Transcript Using In Vivo Expression Technology to

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
– 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
 Goal

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
random
fragment
Previous work
 Genome
broken into
thousands of fragments
 Kanamycin marker
 Transformed into wild type M.
avium

“GyrA” bacteria
PLDG13-GyrA plasmid
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
centrifuge
withdraw middle
layer
whole blood
wash 3
times, resuspend
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
dendritic
cell
lyse cells and
plate bacteria
on Petri
dishes
infect with
GyrA bacteria;
1 well for each
time point
infected with
wild type MAC
104
treat with
moxifloxacin
at 8µg/mL;
allow 24
hours
incubate for 1
hour
wash cells and begin
4, 24, or 48 hour time
point
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
Pick off
individual
colony; isolate
plasmid
fragment
Use PCR to
amplify the
fragment
Added together equals
228 bp
Use gel
electrophoresis
to screen PCR
products
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
