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

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.

Deciphering the Interactome of Neisseria meningitidis With Human Brain Microvascular Endothelial Cells

“…Results presented in this publication together with our recently published study (Jimenez-Munguia et al, 2018 ), validate the experimental and bioinformatic approach designed to uncover potential bacterial ligands interacting with the host cells.…”

“…Proteins of N. meningitidis were extracted exactly as described before (Jimenez-Munguia et al, 2018 ) in non-denaturating lysis buffer containing 20 mM CHAPS, 300 mM NaCl, 0.01% sodium azide and 1 × proteases inhibitors, and biotinylated with EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific) as per manufacturer's instructions. Biotinylation was confirmed with NeutrAvidin (Thermo Fisher Scientific) capture followed by SDS-PAGE as per manufacturer's instructions.…”

“…Human BMECs (D3 cell line), were cultured as previously described (Jimenez-Munguia et al, 2018 ). Cells were either incubated with biotinylated proteins of N. meningitidis or scrapped for protein isolation.…”

“…Bioinformatic analysis applied to a large set of data can help to improve selection of target molecules that are most relevant to the study. We have recently applied a set of bioinformatic tools to select pneumococcal surface proteins that may interact with hBMECs (Jimenez-Munguia et al, 2018 ). Similar bioinformatic pipeline was set in this study using freely available bioinformatic tools (such as CELLO, Psortb, SignalP, LipoP, HMMtop and TMHMM) to identify bacterial proteins that have less chance to bind host cells (e.g., cytoplasmic proteins) and select interacting proteins (plausible interactome).…”

Targeted Proteomics for Studying Pathogenic Bacteria

“…Despite the non‐targeted way of collecting data, the analysis of DIA data can be performed in both a targeted manner as well as non‐targeted (see below). Recently, DIA/SWATH‐MS was employed to assess differential proteome signatures between pathogenic and nonpathogenic Rickettsia inside macrophages and unraveled the ligands of Streptococcus pneumonia interacting with human brain endothelial cells …”