1. Transcription Analysis of Tetracylcine Resistant Genes in Chlamydia suis Presented by Erika K. VanDenBerg Mentor Dr. Dan Rockey Department of Microbiology

2. What is Chlamydia? Chlamydia is a bacterium Chlamydia is a bacterium Obligate intracellular pathogen Obligate intracellular pathogen

3. Chlamydia trachomatis (C. trachomatis) #1 Sexually transmitted disease (STD) in U.S. #1 Sexually transmitted disease (STD) in U.S. 3 million Americans become infected yearly 3 million Americans become infected yearly NO SYMPTOMS NO SYMPTOMS Causes sterility if untreated and can lead to Causes sterility if untreated and can lead to life-threatening problems life-threatening problems

4. C. trachomatis #1 cause of preventable blindness worldwide #1 cause of preventable blindness worldwide -500 million people suffer from trachoma C. pneumoniae 10-20% of pneumonia worldwide 10-20% of pneumonia worldwide Associated with- Associated with- 1. Coronary atherosclerosis 2. Heart disease

5. Chlamydia suis ( C. suis) found in all farmed pigs Chlamydia suis ( C. suis) found in all farmed pigs C. suis has acquired tetracycline (tet) resistance C. suis has acquired tetracycline (tet) resistance TET is a class of antibiotic, inexpensive, and commonly used to treat chlamydial infections TET is a class of antibiotic, inexpensive, and commonly used to treat chlamydial infections Over 50 yrs TET has been added to animal feed in high doses evolving microbes to acquire resistance to antibiotics Over 50 yrs TET has been added to animal feed in high doses evolving microbes to acquire resistance to antibiotics

6. Significance of C. suis acquiring tet-resistance 1. First example of genes recombining into Chlamydia or any other obligate intracellular pathogen. 2. Resistance could eventually occur in the human pathogens.

7. C. suis tet-resistant genes tet R and tet C share its operator sequence. Plasmids pSC101 and pRAS have these genes as well. Mechanism for plasmids pSC101 and pRAS is known. TET is present tet C is being transcribed TET is not present tet C is not transcribed

8. The induction of tet C was analyzed using pSC101 (in E. coli), pRAS (in E. coli), and tetracycline resistant C. suis by performing Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) to analyze transcription in the presence and absence of TET.

9. E. coli+pSC101 1 2 3 4 5 6 7 8 9 10 11 1)100 base pair Ladder 2)tet C expressed in presence of TET 3)tet C not expressed in absence of TET 4)Negative control (DNA) for tet C in presence of TET 5)Negative control (DNA) for tet C in absence of TET 6)Positive control for tet C 7)tet R expressed in presence of TET 8)tet R expressed in absence of TET 9)Negative control (DNA) for tet R in presence of TET 10)Negative control (DNA) for tet R in absence of TET 11)Positive control for tet R E. coli+pRAS had same results

10. E. coli+pSC101 1 2 3 4 5 6 7 8 9 10 11 1)100 base pair Ladder 2)tet C expressed in presence of TET 3)tet C not expressed in absence of TET 4)Negative control (DNA) for tet C in presence of TET 5)Negative control (DNA) for tet C in absence of TET 6)Positive control for tet C 7)tet R expressed in presence of TET 8)tet R expressed in absence of TET 9)Negative control (DNA) for tet R in presence of TET 10)Negative control (DNA) for tet R in absence of TET 11)Positive control for tet R E. coli+pRAS had same results

11. C. suis C. suis R19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1, 2) Positive control for presence of chlamydial RNA 3, 4) Negative control for presence of DNA in chlamydial RNA 5) PCR of R19 gDNA 6) tet R expressed in absence of TET 7) tet R expressed in presence of TET 8) Negative control (DNA) for tet R in absence of TET 9) Negative control (DNA) for tet R in presence of TET 10) Positive control for tet R 11) tet C expressed in absence of TET 12) tet C expressed in presence of TET 13) Negative control (DNA) for tet C in absence of TET 14) Negative control (DNA) for tet C in presence of TET 15) Positive control for tet C

12. C. suis C. suis R19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1, 2) Positive control for presence of chlamydial RNA 3, 4) Negative control for presence of DNA in chlamydial RNA 5) PCR of R19 gDNA 6) tet R expressed in absence of TET 7) tet R expressed in presence of TET 8) Negative control (DNA) for tet R in absence of TET 9) Negative control (DNA) for tet R in presence of TET 10) Positive control for tet R 11) tet C expressed in absence of TET 12) tet C expressed in presence of TET 13) Negative control (DNA) for tet C in absence of TET 14) Negative control (DNA) for tet C in presence of TET 15) Positive control for tet C

13. Results- In E. coli+pSC101 and E. coli+pRAS tet C is only expressed in the presence of TET, where as, in C. suis tet C is constitutive.

14. The two sequenced chlamydial strains of C. suis showed that tet C and tet R had a 6 base pair deletion in its operator region in comparison to plasmids pSC101 and pRAS.

15. The two sequenced chlamydial strains of C. suis showed that tet R had a truncation. The Rockey Lab is currently investigating whether or not these two factors are the reason tet C is constitutive.

16. Acknowledgements to- Rockey Lab Dr. Dan Rockey Jae Dugan Dr. Kevin Ahern Howard Hughes Medical Institute