Transcription Analysis of Transcription Analysis of Tetracylcine Resistant Genes in Tetracylcine Resistant Genes in

Chlamydia suisChlamydia suis

Presented byPresented by Erika K. VanDenBergErika K. VanDenBerg

MentorMentor

Dr. Dan RockeyDr. Dan Rockey

Department of MicrobiologyDepartment of Microbiology

What is Chlamydia?What is Chlamydia?

• Chlamydia is a bacteriumChlamydia is a bacterium• Obligate intracellular pathogenObligate intracellular pathogen

Chlamydia trachomatisChlamydia trachomatis ((C.C. trachomatistrachomatis))

• #1 Sexually #1 Sexually transmitted disease transmitted disease (STD) in U.S.(STD) in U.S.

• 3 million 3 million Americans become Americans become infected yearlyinfected yearly

• NO SYMPTOMSNO SYMPTOMS

• Causes sterility if untreated and can Causes sterility if untreated and can lead to lead to life-threatening problemslife-threatening problems

C. trachomatisC. trachomatis

• #1 cause of preventable blindness #1 cause of preventable blindness worldwideworldwide

-500 million people suffer from -500 million people suffer from trachomatrachoma

C. pneumoniaeC. pneumoniae• 10-20% of pneumonia worldwide10-20% of pneumonia worldwide• Associated with-Associated with-

1. Coronary 1. Coronary atherosclerosisatherosclerosis

2. Heart disease2. Heart disease

• Chlamydia suisChlamydia suis ( ( C. suisC. suis) found in all ) found in all farmed pigsfarmed pigs

• C. suisC. suis has acquired tetracycline (tet) has acquired tetracycline (tet) resistanceresistance

• TET is a class of antibiotic, TET is a class of antibiotic, inexpensive, and commonly used to inexpensive, and commonly used to treat chlamydial infectionstreat chlamydial infections

• Over 50 yrs TET has been added to Over 50 yrs TET has been added to animal feed in high doses evolving animal feed in high doses evolving microbes to acquire resistance to microbes to acquire resistance to antibioticsantibiotics

Significance of Significance of C. suisC. suis acquiring acquiring tettet-resistance-resistance

1.1. First example of genes recombining First example of genes recombining into Chlamydia or any other obligate into Chlamydia or any other obligate intracellular pathogen.intracellular pathogen.

2.2. Resistance could eventually occur in Resistance could eventually occur in the human pathogens.the human pathogens.

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

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.

E. E. colicoli+pSC101+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

E. E. colicoli+pSC101+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

C. suisC. 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

C. suisC. 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

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.

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.

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.

Acknowledgements to-Acknowledgements to-• Rockey Lab

• Dr. Dan Rockey

• Jae Dugan

• Dr. Kevin Ahern

• Howard Hughes Medical Institute