Global Transcriptome Transcriptome Analysis of Analysis of Global - PowerPoint PPT Presentation

Global Transcriptome Transcriptome Analysis of Analysis of Global Pseudomonas aeruginosa aeruginosa Response Response to to Pseudomonas Ortho-phenylphenol phenylphenol Ortho- 48 th Annual ICAAC/ IDSA 46 th Annual Meeting

Background Background • June 26, 2000 President Clinton met with the • June 26, 2000 President Clinton met with the Director of the Human Genome Program and the Director of the Human Genome Program and the CEO of Celera Genomics announcing the completion CEO of Celera Genomics announcing the completion of the sequencing of the Human Genome of the sequencing of the Human Genome • August 2000, the complete genome sequence of • August 2000, the complete genome sequence of Pseudomonas aeruginosa aeruginosa PA01 published (Cystic PA01 published (Cystic Pseudomonas Fibrosis Foundation) Fibrosis Foundation) • July 2003 the Microarray Research Laboratory • July 2003 the Microarray Research Laboratory (MARL) was established at Fort Meade, MD (MARL) was established at Fort Meade, MD

Pseudomonas aeruginosa aeruginosa: : Nosocomial Nosocomial infections infections Pseudomonas • Nosocomial • Nosocomial infections: infections: – Estimated to occur in 5% of all acute-care hospitalizations. – Estimated to occur in 5% of all acute-care hospitalizations. – More than 2 million cases each year – More than 2 million cases each year – Cost of 4.5 billion dollars – Cost of 4.5 billion dollars but most importantly 90,000 die. but most importantly 90,000 die. • P. aeruginosa – Gram negative rods – Most common opportunistic pathogen http://www.imclive.com/bacteria1.jpg – Cystic fibrosis patients: Chronic lung infections • Increasing prevalence of • Increasing prevalence of nosocomial nosocomial infections: infections: – – Linked to increasing antimicrobial and disinfectant Linked to increasing antimicrobial and disinfectant resistant pathogens. resistant pathogens.

Ortho-phenylphenol phenylphenol and and P. P. aeruginosa aeruginosa Ortho- • Ortho- • Ortho-phenylphenol phenylphenol (OPP): (OPP): – EPA approved chemical – EPA approved chemical – Active ingredient in disinfectants. – Active ingredient in disinfectants. – Mode of action in bacteria has not been elucidated – Mode of action in bacteria has not been elucidated • Use of OPP as a hospital disinfectant necessitates • Use of OPP as a hospital disinfectant necessitates an understanding of the cellular functions that it an understanding of the cellular functions that it affects in different pathogenic bacteria. affects in different pathogenic bacteria. – Facilitate determination of mode of action – Facilitate determination of mode of action – Development of antimicrobials which target specific – Development of antimicrobials which target specific pathogenic bacteria and exert nominal effects on other pathogenic bacteria and exert nominal effects on other species species

GOALS GOALS • What genes, proteins (enzymes), and • What genes, proteins (enzymes), and ultimately metabolic pathways are affected in ultimately metabolic pathways are affected in P. aeruginosa aeruginosa as a result of OPP treatment? as a result of OPP treatment? P. • What are the potential modes of action by • What are the potential modes of action by which OPP inhibits P. P. aeruginosa aeruginosa growth? growth? which OPP inhibits

Methods Methods • • Sublethal concentration of OPP that will produce strong growth concentration of OPP that will produce strong growth Sublethal inhibition: 0.82mM 0.82mM inhibition: • • Early and late transcriptomic transcriptomic response to OPP : RNA extracted response to OPP : RNA extracted Early and late after 20 20 and and 60 60 minutes. minutes. after • • Agilent 2100 Bioanalyzer Bioanalyzer & RNA & RNA LabChip LabChip Agilent 2100 23S 16S

Methods Methods • 4 replicates each: control, 20 min, 60min • 4 replicates each: control, 20 min, 60min • P. • P. aeruginosa aeruginosa GeneChip GeneChip arrays ( arrays (Affymetrix Affymetrix) ) • Real-time PCR: Validation of microarray results • Real-time PCR: Validation of microarray results GeneChip

Analysis and Results Analysis and Results • GeneChip • GeneChip Operating Software ( Operating Software (Affymetrix Affymetrix) ) • GeneSpring • GeneSpring Software(Agilent Software(Agilent Technologies) Technologies) • One-way ANOVA • One-way ANOVA: : 1012 out of 5900 genes ( 1012 out of 5900 genes ( P. P. aeruginosa genome) were statistically significant genome) were statistically significant aeruginosa (p ≤ ≤ 0.05). 0.05). (p • Fold Changes • Fold Changes: : Calculated as the ratios between the Calculated as the ratios between the signal averages of four untreated (control) and four ntreated (control) and four signal averages of four u OPP-treated (experimental) cultures. OPP-treated (experimental) cultures. – 509 genes: – 509 genes: Upregulated Upregulated ( ( ≥ ≥ 2-fold) 2-fold) and and downregulated ( ( ≤ ≤ 2-fold) after 20 and 60 minutes 2-fold) after 20 and 60 minutes downregulated exposure to 0.82mMOPP exposure to 0.82mMOPP

Results and Discussion Results and Discussion • • Functional classes: P. P. aeruginosa aeruginosa Community Annotation Project Community Annotation Project Functional classes: • • Upregulation Upregulation: Filled bars : Filled bars • • Downregulation: Empty bars : Empty bars Downregulation

Results and Discussion Results and Discussion Group I: Up 20, Up 60 Group IV: No change 20, Up 60 Group II: Up 20, No change 60 Group V: No change 20, Down 60 Group III: Down 20, No change 60 Group VI: Down 20, Down 60

Results and Discussion Results and Discussion • Group I : Up 20 min; Up 60 min • Group I : Up 20 min; Up 60 min – Genes encoding 30 and 50s ribosomal proteins, translation – Genes encoding 30 and 50s ribosomal proteins, translation initiation and elongation factors. initiation and elongation factors. – – Membrane transport proteins: Membrane transport proteins: sec sec Y Y, , sec sec E E and and sec sec G G . . – Virulence genes: – Virulence genes: hit hit A A (ferric iron binding (ferric iron binding periplasmic periplasmic protein); hit hit B B ( Iron III transport system ( Iron III transport system permease permease). ). protein); – Type IV – Type IV pilus pilus assembly proteins: assembly proteins: pil pil C, D, G, I, M, N, O and P. C, D, G, I, M, N, O and P. • Upregulation • Upregulation of virulence genes => protective of virulence genes => protective response to OPP treatment. response to OPP treatment.

Results and Discussion Results and Discussion • Group II : Up 20 min; No change 60 min • Group II : Up 20 min; No change 60 min – nor – nor B B- nitric oxide - nitric oxide reductase reductase subunit B: 4-fold subunit B: 4-fold upregulation upregulation. . – – Nitric oxide Nitric oxide reductase reductase enzyme :expressed under anaerobic enzyme :expressed under anaerobic conditions in P. conditions in P. stutzeri stutzeri . . – Possible shift to anaerobic respiration after 20 min : Nitrate used – Possible shift to anaerobic respiration after 20 min : Nitrate used as final electron acceptor – – Denitrification Denitrification. . as final electron acceptor – – Rhamnosyl Rhamnosyl transferase transferase chain A ( chain A ( rhl rhl A A) ) : : 2.4 fold 2.4 fold upregulation upregulation – rhl – rhl A A: critical for the exhibition of swarming motility by : critical for the exhibition of swarming motility by P. P. aeruginosa – –Environmental adaptation. Environmental adaptation. aeruginosa

Results and Discussion Results and Discussion • Group III : Down 20 min; No change 60 min • Group III : Down 20 min; No change 60 min – hcn – hcn A A, , hcn hcn B B, , hcn hcn C C: Approximately 2-fold : Approximately 2-fold downregulation downregulation. . – hcn – hcn ABC ABC encodes a cyanide encodes a cyanide synthase synthase, which forms hydrogen , which forms hydrogen cyanide from glycine glycine. . cyanide from – P. – P. aeruginosa aeruginosa does not produce cyanide under anaerobic does not produce cyanide under anaerobic conditions: nitrate being used as the terminal electron conditions: nitrate being used as the terminal electron acceptor. acceptor. – Supports theory: Possible transient switch to anaerobic – Supports theory: Possible transient switch to anaerobic respiration after 20 minutes of OPP treatment. respiration after 20 minutes of OPP treatment.