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  • Ribonucleotide reductase (RNR) is an enzyme that controls the cellular concentration of deoxyribonucleotides . Biosynthesis begins with the building up of essential molecules that RNR processes in a catalyzed reaction to make deoxyribonucleotides. RNR assembles deoxyribonucleotides for the synthesis of DNA . The processes are identical in all living organisms. What makes RNR unique from other enzymes is the need for a free radical . Hydroxyurea is a scavenger of free radicals.
  • Thesis ppt

    1. 1. Involvement of SOS response and C-di-GMP signaling in biofilm formation of Pseudomonas aeruginosa via DNA and Protein release. Supervising Professors Dr. Tao Wei, Chair Dr. Neal Guentzel Dr. Jilani Chaudry Nagarjun Kasaraneni, B.Tech
    2. 2. Pseudomonas aeruginosa <ul><li>Pseudomonas aeruginosa is a Gram-negative rod shaped bacteria . It is a common inhabitant of soil, water, and vegetation. </li></ul><ul><li>It is an opportunistic pathogen : The disease process begins with some alteration or circumvention of normal host defenses. </li></ul><ul><li>Nearly 70% of people with Cystic Fibrosis are chronically infected with Pseudomonas aeruginosa . </li></ul><ul><li>It causes a wide range of diseases, which include septicemia, urinary tract infections, pneumonia, chronic lung infections, endocarditis, dermatitis, and osteochondritis. </li></ul>
    3. 3. <ul><li>According to the Centers for Disease Control , </li></ul><ul><li>The overall incidence of P. aeruginosa infections in U.S. hospitals averages about 0.4 percent (4 per 1000 discharges). </li></ul><ul><li>The bacterium is the fourth most commonly-isolated nosocomial pathogen accounting for 10.1 percent of all hospital-acquired infections. </li></ul><ul><li>Pseudomonas accounts for about 25 percent of all hospital acquired Gram-negative bacteria. </li></ul><ul><li>It is a model organism for biofilm formation research. </li></ul><ul><ul><ul><ul><li> </li></ul></ul></ul></ul><ul><li> </li></ul>Significance of the P.aeruginosa infections
    4. 4. Biofilm Importance in Present Day Antimicrobial Therapy <ul><li>Mechanisms to evade antimicrobial therapy by pathogenic bacteria </li></ul><ul><li>An increased ability to degrade antibacterial compounds . </li></ul><ul><li>Decreased permeability . </li></ul><ul><li>Decreased affinity for the antibiotic . </li></ul><ul><li>The ability to either form or live in biofilms </li></ul><ul><li>Estimations from the National Institute of Health (NIH) : 80% of human infections are caused by biofilm-forming bacteria. </li></ul><ul><li>Most of the diseases caused by P.aeruginosa are highly persistent due to ability of the organism to form Biofilms . </li></ul><ul><li>Biofilms are highly resistant against antimicrobial agents and the actions of the immune system. </li></ul>
    5. 5. Biofilm Matrix-enclosed bacterial populations adherent to each other and/or to surfaces Singh et al . (2002) Nature 417: 552
    6. 6. Developmental cycle of a biofilm J.W.Costerton et al., Annu. Rev. Microbiol, 1995
    7. 7. Strategies to prevent biofilm formation Rickard et al. (2003) TIM 11: 94 .
    8. 8. Published Data <ul><li>Hoffman, L. R. et al. Aminoglycoside antibiotics induce bacterial biofilm formation. Nature 436, 1171-1175 (2005). </li></ul><ul><li>Sub inhibitory concentrations of aminoglycoside antibiotics (Tobramycin, MIC= 0.3 ug/ml) induce biofilm formation. </li></ul><ul><li>The biofilm induction is regulated by the Cyclic-di-GMP Signaling via Arr. </li></ul><ul><li>Gotoh, H., Zhang, Y. , Dallo, S.F., Hong, S., Kasaraneni, N., Weitao, T. Pseudomonas aeruginosa under DNA replication inhibition tends to form biofilms via Arr. Research in Microbiology 159, 294-302 (2008). </li></ul><ul><li>Replication inhibitors (Hydroxyurea, MIC= 2mM) induce biofilm formation. </li></ul><ul><li>The cells with replication inhibition tends to from Biofilms. </li></ul><ul><li>The biofilm response requires Arr. </li></ul>
    9. 9. Riboncleiotide reductase & Hydroxyurea Riboncleiotide reductase (RNR) Cysteine thiyl radical attacks OH-C3’ leading to removal of hydroxide ion of C-2 ’ NTPs dNTPs
    10. 10. Hypothesis based on published data <ul><li>Replication inhibition by hydroxyurea </li></ul><ul><li>Global DNA damage </li></ul><ul><li>SOS response </li></ul><ul><li>Cyclic-di-GMP Signaling </li></ul><ul><li>Biofilm formation </li></ul>
    11. 11. Mechanism of SOS response Bianco and Kowalczykowski, 1998
    12. 12. Cyclic-di-GMP signaling <ul><li>Cyclic di-GMP is a novel global messenger in bacteria </li></ul><ul><li>It regulates transition from sessility to motility in numerous bacteria. </li></ul><ul><li>The intracellular levels of C-di-GMP are regulated by proteins that have GGDEF (Diguanuylate cyclases) and EAL (Phosphodiesterases) domains. </li></ul>
    13. 13. Cyclic-di-GMP signaling proteins in different bacteria Simm etal., Mol.Microbiol, 2004
    14. 14. Aminoglycoside response regulator (Arr) Hoffman et al., Nature 436, 1171, 2005
    15. 15. Outline of the work <ul><li>SOS involvement in Biofilm stimulation </li></ul><ul><li>SOS involvement in DNA release </li></ul><ul><li>SOS involvement in Protein release </li></ul><ul><li>Regulation of Protein release by Arr </li></ul><ul><li>Biofilm rehabilitation </li></ul>
    16. 16. Biofilm stimulation is RecA dependent Hydroxyurea –inducible biofilm formation appears RecA-dependent .
    17. 17. SOS involvement in biofilm stimulation
    18. 18. Conclusion <ul><li>The biofilm mass reduced by 4-fold in recA compared to wild type. </li></ul><ul><li>The increase in depth in 24hrs biofilm in presence of hydroxyurea in wild type Pao1. </li></ul><ul><li>Summary </li></ul><ul><li>Hydroxyurea inducible biofilm formation is RecA dependent . </li></ul><ul><li>SOS repressor LexA represses the induction. </li></ul><ul><li>Question </li></ul><ul><li>How the RecA-LexA interplay control the biofilm induction? </li></ul><ul><li>Challenges </li></ul><ul><li>Extracellular Polymeric Substance contains DNA and Proteins. </li></ul><ul><li>RecA-LexA interplay might control biofilm induction by regulating </li></ul><ul><li>DNA and Protein release . </li></ul>
    19. 19. DNA release in response to hydroxyurea Whether the release of DNA is under SOS control? [DNA μ g/ml]/cell
    20. 20. SOS controlled DNA release
    21. 21. Conclusion <ul><li>The increase in levels of extracellular DNA in the time course in treated wild type cultures suggest DNA release in response to hydroxyurea. </li></ul><ul><li>The increase in levels of Extracellular DNA in 24 hr culture in presence of hydroxyurea in wild type. </li></ul><ul><li>Summary </li></ul><ul><li>DNA released in response to hydroxyurea is SOS controlled. </li></ul><ul><li>Challenges </li></ul><ul><li>Is protein release SOS controlled </li></ul>
    22. 22. Protein release in response to hydroxyurea 60 kDa : Chaperonin GroEL 30 kDa : Elastase elsB 47 kDa : Elongation factor-TU 1: Cell lysate proteins HU- 2: Cell lysate proteins HU+ 3: Wt -HU Supernatent proteins 4: Wt+HU Supernatent proteins 5: RecA –HU supernatent proteins 6: RecA +HU Supernatent proteins 7: RecA Comp –HU Supernatent proteins 8: RecA Comp +HU Supernatent proteins.
    23. 23. Conclusion <ul><li>recA protein levels are unaffected in treated cultures. </li></ul><ul><li>SDS gels show low band profile in recA samples. </li></ul><ul><li>Summary </li></ul><ul><li>Hydroxyurea-induced protein yields appear RecA-dependent. </li></ul><ul><li>Question </li></ul><ul><li>Is Protein release SOS-controlled? </li></ul><ul><li>Protein sequencing and literature search provides data for this. </li></ul><ul><li>Challenges </li></ul><ul><li>Correlation of macromolecule release to biofilm stimulation </li></ul>
    24. 24. Correlation of macromolecule release with biofilm stimulation. <ul><li>Dnase 1 treatment reduced 30% of the wild type biofilm culture </li></ul><ul><li>Pronase E treatment reduced 25% of the wild type biofilm culture </li></ul><ul><li>Summary </li></ul><ul><li>The released macromolecules contribute to establishing biofilms . </li></ul>
    25. 25. Correlation of protein release with biofilm stimulation
    26. 26. Conclusion <ul><li>Released macro molecules contribute to biofilm stimulation. </li></ul><ul><li>Protein release is RecA dependent. </li></ul><ul><li>Challenges </li></ul><ul><li>To confirm the correlation we wanted to check the complementation of biofilm in recA mutant </li></ul>
    27. 27. Complementation of the recA mutant biofilm with wild type proteins
    28. 28. Conclusion <ul><li>The recA biofilm increased by 60% in presence of wild type proteins . </li></ul><ul><li>Summary </li></ul><ul><li>The deficiency of the recA mutant to stimulate biofilm may due the less protein release . </li></ul><ul><li>Challenges </li></ul><ul><li>Is the protein release regulated through Arr. </li></ul>
    29. 29. Protein release is regulated by Arr Summary From the Protein concentrations and SDS gel profiles suggests the Protein release in response to hydroxyurea appears to be regulated by Arr Wt arr 1 : Wt –HU Supernatent proteins 2 : Wt+HU Supernatent proteins 3 : arr –HU Supernatent proteins 4 : arr +HU Supernatent proteins Extracted at 8 hr time period.
    30. 30. Hypothesis based on results <ul><li>Replication inhibition by hydroxyurea </li></ul><ul><li>Global DNA damage </li></ul><ul><li>SOS response </li></ul><ul><li>RecA </li></ul><ul><li>LexA </li></ul><ul><li>Arr (SOS regulon) </li></ul><ul><li>Cyclic-di-GMP Signaling </li></ul><ul><li>Biofilm formation </li></ul>
    31. 31. Evidence for biofilm rehabilitation <ul><li>As cells carrying DNA damage tend to form biofilms. </li></ul><ul><li>We hypothesis that biofilms may serve as a safe haven against DNA damage. </li></ul><ul><li>5’-deoxyadenosycobalamin, a class II co-factor ,rescues growth arrest by hydroxyurea </li></ul>1:planktonic cells –HU 8hr 2:Planktonic cells +HU 8hr 3:Biofilm cells –HU 8hr 4:Biofilm cells +HU 8hr 5:Biofilm cells -HU 24hr 6:Biofilm cells +HU 24hr 1:planktonic cells –HU 8hr 2:Planktonic cells +HU 8hr 3:Planktonic cells +AboCbl 8hr 4:Planktonic cells +HU + AboCbl 8hr
    32. 32. Acknowledgements <ul><li>Dr. Tao Wei </li></ul><ul><li>Dr. Jilani Chaudry </li></ul><ul><li>Dr. Neal Guentzel </li></ul><ul><li>Dr. Tao Wei Lab </li></ul><ul><li>Shatha.F.Dallo, Ph.D </li></ul><ul><li>Gotoh.Hideo, Ph.D </li></ul><ul><li>Soonbae Hong, M.S </li></ul><ul><li>Confocal laser Scanning Microscopy </li></ul><ul><li>Dr. Colleen Witt </li></ul><ul><li>Dr. Richard.G.LeBron </li></ul><ul><li>M.M.Navarro </li></ul>
    33. 33. Thank you
    34. 34. Cynthia j.Kenyon. , Biomedical press, 1983 Cartoon of the SOS response