Determining the Role of the luxR homolog in
Mycobacterium avium subsp.
paratuberculosis in Bacterial Invasion of
Bovine Epithelial Cells
PRESENTED BY: LAUREN SHIN
MENTOR: DR. LUIZ BERMUDEZ
MICROBIOLOGY DEPARTMENT
http://microbewiki.kenyon.edu/index.php/File:EM_S
can_Paratuberculosis.jpg
Mycobacterium avium subsp. paratuberculosis
(MAP) and Johne’s Disease
 MAP  Agent of Johne’s




disease in cattle and other
ruminants
Infects and grows within
lining of intestine
Passed through the milk of
infected animals
Mortality rate = 100%
No treatment or efficient
vaccine
http://www.johnes.org/dairy/_Holstein_front.html
Significance of Our Research
 Provides useful information for characterizing and
determining more desirable vaccine targets for
Johne’s disease
 2007 study by the USDA estimated that Johne’s
disease has an approximately $200 million/year
economical impact on beef and dairy industry
 Further research on how LuxR contributes to
invasion in the early stages of the disease
Background Research
 MAP can be delivered to host by milk
 MAP exposed to milk  greater efficiency of
invasion
 luxR homolog gene also significantly up-regulated
when exposed to milk
 LuxR regulates transcription of many other genes
 These gene homologues alter bacterial cell wall
composition  may assist in invasion
Our Research Project
 Aim: To determine whether or not the luxR homolog
gene plays a direct role in invasion of MAP into
epithelial cells
 Approach: Overexpress LuxR and its dependent
genes in normally non-invasive Mycobacterium
smegmatis and observe its effect on invasion.
Our Research Project
 Hypothesis: The luxR homolog gene and its
dependent genes in MAP play a direct role in the
invasion of MAP into epithelial cells.
 Prediction: If LuxR and its dependent genes are
overexpressed in M. smegmatis, the mycobacterium
will invade epithelial cells with greater efficiency
than a wild-type invasion.
Our Genes
 Cloning three genes: MAP0482, MAP0483, and
MAP4088
 MAP0482 and MAP0483 make up luxR homolog in
M. avium subsp. avium
 LuxR regulates the transcription of MAP4088 and
MAP1203, both hypothetical invasion proteins
Methods
 Step 1: Clone luxR related genes into pLDG13
 A 5-step process
1. PCR
2. Digestion
3. Ligation
4. Transformation
5. Screening/
Sequencing
Designing Our Primers and PCR
 Forward Primer:
Ribosomal
Binding Site
Forward
Sequence
HIS-Tag
Restriction Site
(HindIII)
 Reverse Primer:
Reverse
Sequence
Restriction
Site (KpnI)
http://www.mun.ca/biology/scarr/PCR_simplified.html
Cloning
 Digest with restriction enzymes
 Ligate with T4 DNA ligase
 Transformation by electroporation into E. coli
 Plate on Kanamycin plates
 Screen by colony PCR or digestion to visualize clones
 Verify sequence
Methods
Step 2: Transform
plasmid with inserted
gene into M. smegmatis
by electroporation
Step 3: Protein Gel and
Western Blotting to verify
expression of genes in M.
smegmatis
Methods
 Step 4: Perform invasion assays in which
transformed M. smegmatis is allowed to infect
epithelial cells
http://www.sz-wholesaler.com/p/893/905-1/24-wellcell-culture-plate-406360.html
Invasion Assay Protocol
Add bacteria
to epithelial
cells
Incubate (1h, 3h)
in 37°C to allow
invasion
Add
antibiotics and
wash off
extracellular
bacteria
Serial dilute
lysates and
plate
Lyse cells with
detergent to
release bacteria
Difficulties with Cloning…
PCR Amplification?
 Problem: No amplification of genes
Ladder
4088
0482
0483
 Possible Explanation: HIS-tag primers contain
unspecific sequences; cannot anneal with such a
large template of genomic DNA
PCR Amplification?
 Proposed Solution: 2-step PCR amplification
Forward
Primer
His-tag
gDNA
Gene
Gene
Reverse
Primer
 Outcome: Still faint or inconsistent bands
Gene
PCR Amplification?
 Possible Explanation #2: Genes 4088, 0482, and
0483 are GC rich; difficult to PCR because they can
form secondary structures like hairpins and have
higher melting temperatures
 Proposed Solution: Use GC-RICH PCR System
 Contains DMSO, Polymerase from GC-rich organism
L
 Outcome: Genes amplified!
_____0483______
Continued Cloning
 Problem: After digesting and ligating the amplified
genes, the screens showed empty vectors with no
insert.
 Possible Explanations:
The restriction enzymes may not be cutting
completely, leaving uncut plasmid
2. The plasmid may be re-ligating together
1.
Still Troubleshooting
 Proposed Solutions:
Shrimp Alkaline Phosphatase (SAP) 
dephosphorylate plasmid to prevent self ligation
2. Include controls for ligations; without insert
3. Make sure digestion and ligation is working
correctly with pLDG13
1.
Another Important Gene
 MAP1203  LuxR regulated gene
 Already cloned into pLDG13
 Attempting to verify expression
 Continued with invasion assay
 Untransformed M. smegmatis
 M. smegmatis transformed with empty vector, PLDG13
 M. smegmatis transformed with wild type 1203 clone
 M. smegmatis transformed with ΔRGD 1203 mutant
Invasion Assay Results
Percent Invasion of M. smegmatis in MDBK Cells at 1
hour and at 3 hours
0.6
% Invasion
0.5
0.4
1h
0.3
3h
0.2
0.1
0
Smeg
PLDG13
WT 1203
∆RGD
Future Direction
 Continue troubleshooting to obtain clones
 Verify expression of genes
 Invasion Assay
 Binding Assay
 Yeast Two-Hybrid System
 Identify receptor protein to which the bacterium binds
Acknowledgements
Dr. Luiz Bermudez
Dr. Kevin Ahern
Jamie Everman
Bermudez Lab
Howard Hughes Medical Institute
University Honors College
Cripps