1. PRESENTED BY: LAUREN SHIN MENTOR: DR. LUIZ BERMUDEZ MICROBIOLOGY 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_S can_Paratuberculosis.jpg

2. 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

3. 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

4. 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

5. 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.

6. 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.

7. 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

8. Methods Step 1: Clone luxR related genes into pLDG13 A 5-step process 1. PCR 2. Digestion 3. Ligation 4. Transformation 5. Screening/ Sequencing

9. 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

10. 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

11. 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

12. 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

13. 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

14. Difficulties with Cloning…

15. 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 408804820483Ladder

16. PCR Amplification? Proposed Solution: 2-step PCR amplification Outcome: Still faint or inconsistent bands Gene gDNA His-tagForward Primer Reverse Primer Gene

17. 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

18. 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 plasmid 2. The plasmid may be re-ligating together

19. Still Troubleshooting Proposed Solutions: 1. 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

20. 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

21. Invasion Assay Results

22. 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

23. Acknowledgements Dr. Luiz Bermudez Dr. Kevin Ahern Jamie Everman Bermudez Lab Howard Hughes Medical Institute University Honors College Cripps