Regulation of Salmonella Enterica by: Laurel Kovach

1. Regulation of Salmonella Entericaby: Laurel Kovach Fahlen, T., Wilson, R., Boddicker, J., and, Jones, B. 2001. Hha is a negative modulator of transcription of hilA, the Salmonella enterica serovar Typhimurium invasion gene transcriptional activator. J. Bacteriol. 183:6620-6629.

2. Questions for Experimenting • What genes negatively regulate the hilA gene? • How do different growth conditions affect Salmonella? • What will decrease the expression of the hilA gene? • How will the mutation of different genes affect the growth of Salmonella? • What are the similarities between Salmonella and E. coli. • What hemolytic characteristics do the two bacteria posses? • What is the effect of overexpression of the hha gene on other invasive genes.

3. Background • Salmonella is known to cause gastroenteritis and enteric fever. • It can be caused by the ingestion of any contaminated meat, vegetable, or liquid. • There are many forms of Salmonella, some of which are multidrug resistant. • It is important to find other ways to control Salmonella.

4. Pathway of Salmonella enterica • Small intestine • Specialized M cells of the Peyers Patches • Regional nodes • Liver • Spleen

5. How does Salmonella gain access to the small intestine? • The most critical step for Salmonella is the ability to invade the small intestine. • First, the cellular membrane rearranges to form actin ruffles. • The actin ruffles phagocytize the bacteria.

6. Were does the bacteria go? • Salmonella enterica localizes at the 40 kb region of the chromosome. • This is located at the 63rd centromere. • This area is termed Salmonella pathogenicity island 1 (SPI-1).

7. Conditions that repress Salmonella invasion • High oxygen • Low osmolarity • Low pH • Stationary phase growth

8. Rationale • The factors that regulate the hilA gene are the same as those that regulate the invasive phenotype of Salmonella. • Thus, if you can regulate the hilA gene, then you can regulate the invasive phenotype of Salmonella.

9. Explanation • Repressing the hilA gene will also repress the invasive phenotype of Salmonella. • On the other hand, overexpression of the hilA gene causes a hyperinvasive phenotype.

10. Purpose and reasoning of the experiment • hupB and ams genes were found to be negative for hilA expression. • Another search was performed to find other factors that negatively regulate the hilA gene.

11. hilA Expression Under Different Growth Conditions - Low oxygen/high osmolarity - increased growth from 2.5 to 3.5 fold • High oxygen/low osomolarity - repressed growth - Therefore osmolarity has more of an effect on the expression of the hilA gene

12. What will Decrease the Expression of the hilA Gene • The strain BJ70 was transformed with a plasmid gene bank of the serovar Thyphimurium SL1344 chromsome and plated ampicilin- resistant transformants on MacConkey lactose agar.

13. What is the point? • The purpose was to have a plasmid strain that would definitely repress hilA expression.

14. Repressing hilA Expression • White transformants that were able to repress the expression of the hilA gene were transformed back into strain BJ70. • Of these, one isolate was selected due to the possession of a plasmid that was able to repress hilA expression 75% more effective than growth conditions. • This plasmid is known as pTF120.

15. pTF120 • The cloned gene was sequenced and used to search databases at the National Center for Biotechnology Information. • The intention was to identify the cloned DNA fragments.

16. What is the importance of the sequencing? • The sequencing identified pTF120 to have a similar genetic makeup to that of E. coli. • This enables the scientists to further experiment interchanging different material between the bacteria.

17. Interesting Find • The database identified pTF120 to have a very similar sequencing to the hha gene of E. coli. • The hha gene of E. coli is known to repress hemolysin production. • A functional homolog of the hha gene of Yersinia enterocolitica, known as ymoA, also shows the ability to regulate virulence genes.

18. Can the hha gene of Salmonella function to repress the hemolysin activity of E. coli • Hemolytic activity is detected by inoculating a blood agar with E. coli. • When E. coli was mixed with the Salmonella hha gene, the zone of clearing was completely eliminated. • This is an indication that the Salmonella hha gene functions to stop hemolysis.

19. More Cloning is Conducted to see if hha can Repress hilA Expression • hha from the serovar Typhimurium was amplified and cloned into pGEM-T. • This created pTF137. • The pTF137 was introduced to strain BJ70. • Overexpression of pTF137 reduced hilA expression by more than sevenfold.

20. Rationale for Further Experimenation • Experimentation on overexpression of the hha gene was done to find its effect on other invasive genes. • The following genes were traced: - invF - prgH - sipC

21. Experiment • Plasmid TF137 were fused separately with each of the invasive genes. • Not only did pTF137 decrease repress hilA, but it also decreased the expression of the three invasive genes.

22. Salmonella hha null mutant gene TF79 • TF79 was created by replacing the majority of the hha gene in BJ70 with a kanamycin cassette. • It was found that the hilA gene was still being controlled by environmental signals. • Further experimentation was necessary.

23. The role of hha • Expression of hha affects the expression of hilA.

24. Experimentation of hilA in TF79 • The first expriment involving low oxygen/high osmolarity and vice versa was repeated for the hilA gene in TF79.

25. hha on the expression of hilA • Expression of hilA after mutation of hha • Comparitive analysis between wild type strain and hha mutated

26. Wild type Oxygen limiting- 100mu, low hilA expression High oxygen- 5 mu, very low expression Low osmolarity- 15 mu, very low hilA expression Hha mutated Oxygen limiting- 300 mu, very high hilA expression High oxygen- 15 mu- very low expression Low osmolarity- 100 mu, great variation from wild type Results

27. Effect of hha mutation on invasiveness • Regulatory role of hha on invasiveness of Salmonella enterica

28. Assessment of affecting factors • Three factors had to be assessed prior to experimentation: • The invasiveness of of SL1344 with plasmid GEM-T • The hha mutant TF80 with plasmid GEM-T • TF80 with plasmid TF137

29. Results • Invasive phenotype of TF80 was higher than SL1344 • Lower invasion with the overexpression of Hha from plasmid TF137 in strain TF80 • Repressed invasion of SL1344 with plasmid TF137

30. Comparative analysis of invasion between wild type strain and Hha mutated strain

31. Results • Under repressing conditions -Hha mutated gene- showed increase in invasiveness • High oxygen -invasion was sixfold higher • Low osmolarity -invasion was twelve-fold higher • High oxygen and low osmolarity -invasion was sixfold higher

32. Conclusion • What is Salmonella’s relationship with the hilA gene? • By repressing the hilA gene, the invasive phenotype of Salmonella can also be repressed. • Many things affect the repression of the hilA gene: • Level of oxygen • Level of osmolarity • Genetic engineering