PRESENTED BY: LAUREN SHINMENTOR: DR. LUIZ BERMUDEZMICROBIOLOGY DEPARTMENT

Determining the Role of the luxR homolog in Mycobacterium avium subsp. paratuberculosis in Bacterial Invasion of Bovine Epithelial Cells

http://microbewiki.kenyon.edu/index.php/File:EM_Scan_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 milkMAP exposed to milk greater efficiency of

invasionluxR homolog gene also significantly up-

regulated when exposed to milkLuxR regulates transcription of many other

genesThese 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 pLDG13A 5-step process

1. PCR

2. Digestion

3. Ligation

4. Transformation

5. Screening/

Sequencing

Designing Our Primers and PCR

Forward Primer:

Reverse Primer:

Restriction Site (HindIII)

Ribosomal Binding

Site

HIS-Tag

Forward Sequence

Reverse Sequence

Restriction Site (KpnI)

http://www.mun.ca/biology/scarr/PCR_simplified.html

Cloning

Digest with restriction enzymesLigate with T4 DNA ligaseTransformation by electroporation into E. coliPlate on Kanamycin platesScreen by colony PCR or digestion to

visualize clonesVerify 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-well-cell-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

Lyse cells with detergent to

release bacteria

Serial dilute lysates and

plate

Difficulties with Cloning…

PCR Amplification?

Problem: No amplification of genes

Possible Explanation: HIS-tag primers contain unspecific sequences; cannot anneal with such a large template of genomic DNA

4088 0482 0483Ladder

PCR Amplification?

Proposed Solution: 2-step PCR amplification

Outcome: Still faint or inconsistent bands

Gene gDNA

His-tag

Forward Primer

Reverse Primer

Gene 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

Outcome: Genes amplified!

_____0483______L

Continued Cloning

Problem: After digesting and ligating the amplified genes, the screens showed empty vectors with no insert.

Possible Explanations: 1. The restriction enzymes may not be cutting

completely, leaving uncut plasmid2. The plasmid may be re-ligating together

Still Troubleshooting

Proposed Solutions:1. Shrimp Alkaline Phosphatase (SAP)

dephosphorylate plasmid to prevent self ligation

2. Include controls for ligations; without insert3. Make sure digestion and ligation is working

correctly with pLDG13

Another Important Gene

MAP1203 LuxR regulated geneAlready cloned into pLDG13Attempting to verify expressionContinued 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

Smeg PLDG13 WT 1203 ∆RGD0

0.1

0.2

0.3

0.4

0.5

0.6

Percent Invasion of M. smegmatis in MDBK Cells at 1 hour and at 3 hours

1h3h

% I

nvas

ion

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 BermudezDr. Kevin AhernJamie EvermanBermudez Lab

Howard Hughes Medical InstituteUniversity Honors College

Cripps