Transcription Analysis of Tetracylcine Resistant Genes in Chlamydia suis

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Transcription Analysis of Tetracylcine Resistant
Genes in Chlamydia suis

• Presented by
• Erika K. VanDenBerg
• Mentor
• Dr. Dan Rockey
• Department of Microbiology

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What is Chlamydia?

• Chlamydia is a bacterium
• Obligate intracellular pathogen

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Chlamydia trachomatis (C. trachomatis)

• 1 Sexually transmitted disease (STD) in U.S.
• 3 million Americans become infected yearly
• NO SYMPTOMS

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

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C. trachomatis

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

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• Chlamydia suis ( C. suis) found in all farmed
  pigs
• C. suis has acquired tetracycline (tet)
  resistance
• 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

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Significance of C. suis acquiring tet-resistance

• First example of genes recombining into Chlamydia
  or any other obligate intracellular pathogen.
• Resistance could eventually occur in the human
  pathogens.

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• 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

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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.
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E. colipSC101

• 100 base pair Ladder
• tet C expressed in presence of TET
• tet C not expressed in absence of TET
• Negative control (DNA) for tet C in presence of
  TET
• Negative control (DNA) for tet C in absence of
  TET
• Positive control for tet C
• tet R expressed in presence of TET
• tet R expressed in absence of TET
• Negative control (DNA) for tet R in presence of
  TET
• Negative control (DNA) for tet R in absence of
  TET
• Positive control for tet R

1 2 3 4 5 6 7 8 9 10 11
E. colipRAS had same results
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E. colipSC101

• 100 base pair Ladder
• tet C expressed in presence of TET
• tet C not expressed in absence of TET
• Negative control (DNA) for tet C in presence of
  TET
• Negative control (DNA) for tet C in absence of
  TET
• Positive control for tet C
• tet R expressed in presence of TET
• tet R expressed in absence of TET
• Negative control (DNA) for tet R in presence of
  TET
• Negative control (DNA) for tet R in absence of
  TET
• Positive control for tet R

1 2 3 4 5 6 7 8 9 10 11
E. colipRAS had same results
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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. suis R19
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
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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. suis R19
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
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Results- In E. colipSC101 and E. colipRAS tet
C is only expressed in the presence of TET, where
as, in C. suis tet C is constitutive.
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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.
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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.
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• Acknowledgements to-
• Rockey Lab
• Dr. Dan Rockey
• Jae Dugan
• Dr. Kevin Ahern
• Howard Hughes Medical Institute