Acute P. aeruginosa Infections-From Genes to the Bedside The relationship between research and clinical care

1. Acute P. aeruginosa Infections-From Genes to the Bedside The relationship between research and clinical care Jeanine Wiener-Kronish, M.D. Professor of Anesthesia and Medicine Vice Chair, Department of Anesthesia and Perioperative Care

2. OBJECTIVES • Review Pseudomonas aeruginosa infections and importance of virulence factors in infections • Review research into mechanisms of Pseudomonas aeruginosa induction of lung injury • Translation of basic research to patients-are the experimental data relevant to lung injury in patients??

3. Pseudomonas aeruginosa infections • Leading cause of gram negative nosocomial pneumonias and bacteremia in adults and children • **S. aureus more commonly associated with bacteremia, but P. aeruginosa bacteremia associated with increased mortality and increased organ dysfunction • Ped Infect Dis J 2003: 22:686-91 • Chest 2004;125:607-616 • J Hosp Infect 2003;53:18-24

4. Other P.aeruginosa infections • Bacteremia in AIDS • External otitis in diabetics and patients obtaining posts through ear cartilage • Contact lens keratitis • Long term pulmonary infections in CF • Urinary infections • CAP- 3rd most common agent in Spain JAMA 2004;291:981-5; Curr Opin Infect Dis 2003;16: 135-143

5. Why is P.aeruginosa successful? • Infections with P.aeruginosa are severe, unrelenting and are associated with high mortalities--is it because the hosts are so sick or is it because the bacteria is so virulent?? • P.aeruginosa also “colonizes”--are the same strains of P.aeruginosa causing colonization and infections??

6. P. aeruginosa Virulence • List of cell associated virulence factors : Type IV fimbriae, LPS, non-fimbrial adhesins, alginate, flagellum • Secreted toxins, enzymes: Proteases (elastase, alkaline protease); Haemolysins (Phosphlipase C, Rhamnolipid); Lipase Alkaline phosphatase Exotoxin A Iron siderophores (Pyoverdine, Pyochelin) HCN; Pyocyanin ExoS, ExoT, ExoU, ExoY

7. Quorum Sensing-1 example of transcriptional regulation • Cell-density-dependent bacterial intercellular signaling system that enables bacteria to coordinately regulate gene expression in response to cell density--example Las system regulates production of elastase alkaline protease and exotoxin A • Autoinducers (homoserine lactones and quoinolones) binds and activates transcriptional regulators resulting in coordinate transcription of a group of genes

8. Which virulence products are causing disease?? • Dependent on experimental conditions and strains of bacteria Example: Non-piliated mutants of PAK show reduced virulence in mouse model of acute pneumonia vs PA103 non-piliated mutants are fully virulent • Bacteria are able to change virulence gene expression with changing environments Example: Cholera only expresses virulence factors while in the gut of host

9. Conclusions from Backround • P.aeruginosa is a clinically important pathogen that produces a myriad of virulence products • The importance of a virulence product in disease requires documentation of the presence of disease when that virulence product is produced and the lack of disease when the product is missing

10. My goal in the laboratory was to investigate the pathogenesis of acute lung injury….. • Acute lung injury experiments in anesthetized sheep • Airspace instillation of E. coli endotoxin did not cause alveolar edema • Obtained a Pseudomonas aeruginosa strain from a patient who was bacteremic with acute lung injury

11. Pseudomonas aeruginosa induced lung injury • Airspace instillation of live P.aeruginosa induced alveolar edema and pleural effusions--airspace instillation of heat-killed P.aeruginosa (endotoxin)did not J Clin Investigation 88:864, 1991 • Attempts to block alveolar edema with circulatory IgG were not as protective as with the administration of airspace IgG J Clin Investigation 92: 1221, 1993

12. Not allPseudomonas strains seemed to hurt patients… • One strain, PA103 (a clinical isolate), was significantly more injurious when instilled into animals. The instillation of this bacteria consistently created alveolar edema, bacteremia and septic shock Am J Physiology 267: L551, 1994 Infect Immunity 66: 3242, 1998

13. What makes PA103 so injurious?? • Exo U is a novel toxin, with lipase activity, in PA103 is injected into eukaryotic cells and kills them Nature Medicine 5:392, 1999 EMBO J 22: 2959, 2003

18. PcrV- accessible antigen • A 32-kDa type III secreted protein • A homolog of Yersinia LcrV (V-antigen) • Essential to the P. aeruginosa type III secretion • Antibody against PcrV blocks the translocation of type III secreted toxins

20. J774A.1 Macrophage - PA103 pUCPngfp Staining with Rhodamine-Wheat germ agglutinin

22. Phagocytosed P. aeruginosa by Alveolar Macrophages in Mice V PA103pcrV PA103 with anti-PcrV IgG P. aeruginosa labeled by pUCPngfp, macrophages - Rhodamine Wheat Germ Aggulutinin

24. Mean Arterial Pressure in Airspace Instilled Rabbits % of baseline No bacteria Anti-PcrV IgG Control IgG

25. Bacteremia in airspace instilled rabbits CFU in 100µl/L of blood Control IgG Anti-PcrV IgG No bacteria

26. Plasma TNF-a Concentration (pg/ml) Control IgG Anti-PcrV IgG No bacteria

27. Understanding the mechanism of type III secretion led to the development of therapies • Antibodies against PcrV, a bacterial protein involved in the type III secretion, blocked bacteremia, septic shock and death in animals J Clinical Investigation 104:743, 1999 J Immunity 167:5880, 2001

28. Conclusions on Pseudomonas-induced lung toxicity • Pseudomonas toxins, not endotoxin, are involved in the creation of acute lung injury • Pseudomonas strains are more virulent and cause more lung injury if they secrete type III toxins, particularly ExoU • Blockade of the type III secretion system appears feasible using antibodies; antibodies have potential as therapies, perhaps delivered to the airspaces