The role of pneumolysin in mediating lung damage in a lethal pneumococcal pneumonia murine model

María Del Mar García-Suárez,Aurora Astudillo,Noelia Flórez,Kevin Fabrizio,Liise-Anne Pirofski,Fernando Vázquez,Francisco J Méndez,Roberto Villaverde

doi:10.1186/1465-9921-8-3

María Del Mar García-Suárez, Aurora Astudillo + Show 6 more

Open Access

https://doi.org/10.1186/1465-9921-8-3

Copy DOI

Abstract

BackgroundIntranasal inoculation of Streptococcus pneumoniae D39 serotype 2 causes fatal pneumonia in mice. The cytotoxic and inflammatory properties of pneumolysin (PLY) have been implicated in the pathogenesis of pneumococcal pneumonia.MethodsTo examine the role of PLY in this experimental model we performed ELISA assays for PLY quantification. The distribution patterns of PLY and apoptosis were established by immunohistochemical detection of PLY, caspase-9 activity and TUNEL assay on tissue sections from mice lungs at various times, and the results were quantified with image analysis. Inflammatory and apoptotic cells were also quantified on lung tissue sections from antibody treated mice.ResultsIn bronchoalveolar lavages (BAL), total PLY was found at sublytic concentrations which were located in alveolar macrophages and leukocytes. The bronchoalveolar epithelium was PLY-positive, while the vascular endothelium was not PLY reactive. The pattern and extension of cellular apoptosis was similar. Anti-PLY antibody treatment decreased the lung damage and the number of apoptotic and inflammatory cells in lung tissues.ConclusionThe data strongly suggest that in vivo lung injury could be due to the pro-apoptotic and pro-inflammatory activity of PLY, rather than its cytotoxic activity. PLY at sublytic concentrations induces lethal inflammation in lung tissues and is involved in host cell apoptosis, whose effects are important to pathogen survival.

Highlights

Intranasal inoculation of Streptococcus pneumoniae D39 serotype 2 causes fatal pneumonia in mice
Outbred MF-1 mice (Oxon, Harland Olac Ltd., Bicester, England) weighing 30 ± 3 g were lightly anaesthetized with 3% (v/v) halothane over oxygen (3–4 l/min) using a methacrylate box connected to Fluovac 240 (Anaesthetizing system, Cheshire, England) and intranasally infected with a lethal dose of 5 × 106 colony-forming units (CFU) of S. pneumoniae D39 serotype 2 NCTC 7466 (Spanish Type Culture Collection, Valencia, Spain) in 50 μl of phosphate-buffered saline (PBS), applied atraumatically to the tip of the nose and involuntarily inhaled
PLY quantification and pneumococci localization Quantification of PLY was performed in bronchoalveolar lavages (BAL) obtained at different time points during pneumococcal pneumonia from mice infected intranasally with S. pneumoniae D39 serotype 2 (Figure 1A)

Summary

Introduction

Intranasal inoculation of Streptococcus pneumoniae D39 serotype 2 causes fatal pneumonia in mice. The cytotoxic and inflammatory properties of pneumolysin (PLY) have been implicated in the pathogenesis of pneumococcal pneumonia. Pneumococcus usually colonizes the nasopharynx of humans asymptomatically, on occasions it passes from this niche to the lungs, brain, and blood [1,2]. This can lead to diseases associated with high morbidity and mortality such as pneumonia, septicemia, and meningitis. Pneumolysin (PLY) is a 53-kDa toxic protein that belongs to the family of antigenically related thiolactivated, cholesterol-binding cytolysins [3]. PLY is considered to be an excellent candidate to include in a pneumococcal vaccine [1,12]

Methods

Results

Discussion

Conclusion