Mapping of Recognition Sites of Monoclonal Antibodies Responsible for the Inhibition of Pneumolysin Functional Activity

Indre Kucinskaite-Kodze,Justas Dapkunas,Aurelija Zvirbliene,Milda Pleckaityte,Martynas Simanavicius

doi:10.3390/biom10071009

Abstract

The pathogenicity of many bacteria, including Streptococcus pneumoniae, depends on pore-forming toxins (PFTs) that cause host cell lysis by forming large pores in cholesterol-containing cell membranes. Therefore, PFTs-neutralising antibodies may provide useful tools for reducing S. pneumoniae pathogenic effects. This study aimed at the development and characterisation of monoclonal antibodies (MAbs) with neutralising activity to S. pneumoniae PFT pneumolysin (PLY). Five out of 10 produced MAbs were able to neutralise the cytolytic activity of PLY on a lung epithelial cell line. Epitope mapping with a series of recombinant overlapping PLY fragments revealed that neutralising MAbs are directed against PLY loops L1 and L3 within domain 4. The epitopes of MAbs 3A9, 6E5 and 12F11 located at L1 loop (aa 454–471) were crucial for PLY binding to the immobilised cholesterol. In contrast, the MAb 12D10 recognising L3 (aa 403–423) and the MAb 3F3 against the conformational epitope did not interfere with PLY-cholesterol interaction. Due to conformation-dependent binding, the approach to use overlapping peptides for fine epitope mapping of the neutralising MAbs was unsuccessful. Therefore, the epitopes recognised by the MAbs were analysed using computational methods. This study provides new data on PLY sites involved in functional activity.

Highlights

Streptococcus pneumoniae is the most common cause of bacterial otitis media, pneumonia, meningitis, sepsis and other severe illnesses [1]
The specificity of the antibodies against PLY was confirmed by enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) (Table 5, Figure 1)
The monoclonal antibodies (MAbs) did not react with irrelevant bacterial proteins extract from E. coli in WB used as a negative control

Summary

Introduction

Streptococcus pneumoniae (pneumococcus) is the most common cause of bacterial otitis media, pneumonia, meningitis, sepsis and other severe illnesses [1]. The currently available pneumococcal vaccines based on polysaccharide capsules can protect from about a quarter of known S. pneumoniae serotypes [3] They do not protect from colonisation or infection by nonencapsulated pathogenic pneumococci [3,4]. Pneumolysin (PLY), a pore-forming toxin (PFT) produced by pneumococcus, is a major protein virulence factor and a potential candidate for developing protein-based vaccines [5]. It is well-recognised that PLY plays a significant role in severe outcomes of pneumococcal disease, in particular in the pathogenesis of lung and myocardial dysfunction [6]. Strategies for neutralisation of the toxic activity of PLY might provide a tool for reducing S. pneumoniae pathogenicity

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