Transcript From Bedside to Bench and Back again

Acute P. aeruginosa InfectionsFrom 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
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
1
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??
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
2
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
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
3
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
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
4
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??
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
5
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
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
6
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
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
7
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
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
8
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
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
9
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
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
10
Pseudomonas aeruginosa
induced lung injury
• Airspace instillation of live P.aeruginosa
induced alveolar edema and pleural
effusions--airspace instillation of heatkilled 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
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
11
Not all Pseudomonas 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
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
12
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
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
13
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
14
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
15
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
16
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
17
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
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
18
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
19
J774A.1 Macrophage - PA103 pUCPngfp
Staining with Rhodamine-Wheat
germ agglutinin
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
20
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
21
Phagocytosed P. aeruginosa
by Alveolar Macrophages in Mice
V
PA103 pcrV
PA103 with anti-PcrV IgG
P. aeruginosa labeled by pUCPngfp, macrophages
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
- Rhodamine Wheat
Germ
Aggulutinin
22
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
23
Mean Arterial Pressure in Airspace
Instilled Rabbits
120
% of
baseline
100
*
80
No bacteria
Anti-PcrV IgG
60
40 0 1 2 3 4 5 6 7 8 9
Control IgG
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
24
Bacteremia in airspace instilled rabbits
CFU in 100µl/L of blood
Control IgG
103
102
101
100
0
*
Anti-PcrV IgG
No bacteria
0 1 2 3 4 5 6 7 8 9
Time (hours)
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
25
Plasma TNF-a Concentration
(pg/ml)
103
Control IgG
102
Anti-PcrV IgG
*
101
100
0
No bacteria
0 1 2 3 4 5 6 7 8 9
Time (hours)
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
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
27
Conclusions on Pseudomonasinduced 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
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
28