Poster # B-1062. Type-III Secretion System Determines Pseudomonas aeruginosa Virulence by Inducing. Neutrophil Apoptosis in an Acute Murine Pneumonia ...
Type-III Secretion System Determines Pseudomonas aeruginosa Virulence by Inducing Neutrophil Apoptosis in an Acute Murine Pneumonia Model A.H. Broquet1, C. Jacqueline1, A. Roquilly1,2, M. Davieau1, C. Desessard1,, J. Caillon1, G. Potel1 and K. Asehnoune1,2. UPRES EA 3826 «Thérapeutiques cliniques et expérimentales des infections», Faculté de médecine, 1 rue Veil, 44000 Nantes, France 2 CHU Nantes, Pôle Anesthésie Réanimations, Service d’Anesthésie Réanimation Chirurgicale, Hôtel Dieu, 44093 Nantes, France
1Laboratoire
Poster # B-1062
ABSTRACT
METHODS
Figure 1: T3SS deletion abolished PAO1 virulence in an acute pneumonia model. (A) Mice survival curves and (B) bacteria load in the lungs of mice infected with either PAO1 standard or PAO1 mutants at the indicated times. Survival rates are expressed in percentage (%). Bacteria loads are expressed as Log10 Colony Forming Units (CFU) per gram of lung. (C) Lung histology analysis (upper panel) and P. aeruginosa lung immunohistochemistry (IHC; lower panel) in mice infected with the indicated strains for 6 hours. Bar = 100µm (10x, HPS staining or 200µm (20x, IHC).
C
sham
PAO1
PAO1-3∆
B
AIM OF THE STUDY To assess the ability of the Pseudomonas aeruginosa Type-III Secretion System (T3SS) to induce neutrophil apoptosis in an acute pneumonia mouse model.
•
PAO1 mutant strains used in the study: • PAO1 wild-type: -
exoenzymes (Exo) S, T and Y functional injector
•
PAO1-3∆:
-
no exoenzymes functional injector
•
PAO1-∆pscC:
-
ExoS, T and Y no functional injector
Wild-type strain PAO1
A
B sham
Figure 3. Neutrophil depletion induces mice mortality in mice infected with the PAO1 mutant strains.
PAO1
Intravenous injection of Ly6-G specific antibody (10µg/mouse; clone 1A8) were realized 24 hours prior to bacteria challenge. Mortality rates were monitored up to 6 days. PAO1-3∆
Mutant strains PAO1-3∆ or PAO1-∆pscC
PAO1-∆pscC
Key words: Pseudomonas aeruginosa, virulence factor, host defense, neutrophils, apoptosis
•
CONCLUSIONS
Transtracheal insertion of a 24-G feeding needle for the instillation of 75 µL of the bacterial preparation (7,5 x 107 CFU/mouse)
Fig1: Pneumonia monitoring Survival and bacteria load in lungs Lung histology and P. aeruginosa immunohistochemistry (IHC)
HPS
A
anti-PAO1 PAO1 antibody
Background: Neutrophils are crucial in the defense against bacteria infection. Type-III secretion system (T3SS) is one of the major virulence factor of Pseudomonas aeruginosa (PA). It includes several exoenzymes (Exo) named S, T, Y and U. Exo can interfere with host cellular mechanisms and induce cell apoptosis once injected into targeted cells. The aim of this study was to assess the role of the T3SS in the induction of the neutrophil-apoptosis during a PA-infection model. Methods: PAO1 standard (expressing ExoS, ExoT, ExoY but not ExoU) or its isogenic mutants deleted in either all exo-enzymes (PAO1-∆ExoS ∆ExoT ∆ExoY referred as PAO1-3∆) or deleted in the protein pscC leading to the absence of the injector system (referred as PAO1∆pscC) were used. Female Swiss mice were infected by tracheal instillation at 1.5*107 CFU/mouse and survival curves were tallied up to 7 days. Bacteria load, lung inflammatory status, neutrophil counts and apoptosis status in broncho-alveolar fluid (BALF) and lungs were analyzed by flow cytometry and confocal microscopy. Results: PAO1 pneumonia led to a 50% mortality rate at 48 hours post infection (hpi) in comparison with the 2 mutant strains displaying no mortality. Virulence of the two mutant strains was restored following neutrophils depletion (with an anti-Gr-1 antibody) as 100% mortality rate was observed at 24hpi. Infected mice with PAO1 standard showed a significant decrease of neutrophil accumulation in BALF (62.44±14.35%) compared to infection with the mutant strains PAO1-3∆ and PAO1-∆pscC (90.66±3.95% and 88.27±5.59% respectively (24hpi; P
