Proteomic and bioinformatic pipeline to screen the ligands of S. pneumoniae interacting with human brain microvascular endothelial cells

Irene Jiménez-Munguía,Petra Majerova,Katarina Bhide,Lubos Comor,Andrej Kovac,Mangesh Bhide,Ivana Sirochmanova,Zuzana Tomeckova,Evelina Kanova,Lucia Pulzova

doi:10.1038/s41598-018-23485-1

Abstract

The mechanisms by which Streptococcus pneumoniae penetrates the blood-brain barrier (BBB), reach the CNS and causes meningitis are not fully understood. Adhesion of bacterial cells on the brain microvascular endothelial cells (BMECs), mediated through protein-protein interactions, is one of the crucial steps in translocation of bacteria across BBB. In this work, we proposed a systematic workflow for identification of cell wall associated ligands of pneumococcus that might adhere to the human BMECs. The proteome of S. pneumoniae was biotinylated and incubated with BMECs. Interacting proteins were recovered by affinity purification and identified by data independent acquisition (DIA). A total of 44 proteins were identified from which 22 were found to be surface-exposed. Based on the subcellular location, ontology, protein interactive analysis and literature review, five ligands (adhesion lipoprotein, endo-β-N-acetylglucosaminidase, PhtA and two hypothetical proteins, Spr0777 and Spr1730) were selected to validate experimentally (ELISA and immunocytochemistry) the ligand-BMECs interaction. In this study, we proposed a high-throughput approach to generate a dataset of plausible bacterial ligands followed by systematic bioinformatics pipeline to categorize the protein candidates for experimental validation. The approach proposed here could contribute in the fast and reliable screening of ligands that interact with host cells.

Highlights

Streptococcus pneumoniae is one of the longest-known pathogens responsible for sepsis, meningitis and pneumonia
Ligands of S. pneumoniae plausibly interacting with human brain microvascular endothelial cells (BMECs)
When we compared surface-exposed proteins putatively binding to human BMECs with pansurfome of S. pneumoniae R6, we found that 3 out of 11 cell-wall anchored proteins predicted from the R6 genome were identified in our study

Summary

Introduction

Streptococcus pneumoniae is one of the longest-known pathogens responsible for sepsis, meningitis and pneumonia. Classical gel-based proteomic approaches have been applied for analysis of surface-exposed bacterial ligands and their receptors on the host cells[15,16,17]. These techniques have several limitations e.g. it is difficult to resolve complex proteins with highly hydrophobic moieties[18], they are suboptimal for study of membrane-embedded and low-abundant proteins, and techniques are laborious, time consuming and extremely low-throughput. Gel-based techniques become bottle neck in the ligand-receptor interactions experiments especially when biological material is scanty (e.g. primary cells from brain microvasculature). To our knowledge the role surface-exposed proteins of pneumococci in meningeal infection has not been addressed yet with robust proteomic approaches

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Conclusion