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

Abstract

Neisseria meningitidis is able to translocate the blood-brain barrier and cause meningitis. Bacterial translocation is a crucial step in the onset of neuroinvasion that involves interactions between pathogen surface proteins and host cells receptors. In this study, we applied a systematic workflow to recover and identify proteins of N. meningitidis that may interact with human brain microvascular endothelial cells (hBMECs). Biotinylated proteome of N. meningitidis was incubated with hBMECs, interacting proteins were recovered by affinity purification and identified by SWATH-MS. Interactome of N. meningitidis comprised of 41 potentially surface exposed proteins. These were assigned into groups based on their probability to interact with hBMECs: high priority candidates (21 outer membrane proteins), medium priority candidates (14 inner membrane proteins) and low priority candidates (six secretory proteins). Ontology analysis provided information for 17 out of 41 surface proteins. Based on the series of bioinformatic analyses and literature review, five surface proteins (adhesin MafA1, major outer membrane protein P.IB, putative adhesin/invasion, putative lipoprotein and membrane lipoprotein) were selected and their recombinant forms were produced for experimental validation of interaction with hBMECs by ELISA and immunocytochemistry. All candidates showed interaction with hBMECs. In this study, we present a high-throughput approach to generate a dataset of plausible meningococcal ligands followed by systematic bioinformatic pipeline to categorize the proteins for experimental validation.

Transcriptome analysis of human brain microvascular endothelial cells response to Neisseria meningitidis and its antigen MafA using RNA-seq

“…It was previously proposed that since MafA binds cellular glycolipid such as GgO 3 and GgO 4 21 , it could mediate attachment of Neisseria or OMVs to eukaryotic cells via an as-yet-unknown receptor 26 . The binding ability of MafA to the hBMECs was confirmed recently by us with ELISA and immunocytochemistry 20 .…”

“…However, till to date, scanty reports are available on the members of Maf family (MafA, MafB, etc.). Earlier studies on MafA like adhesin protein (gangliotetraosylceramide binding adhesin) have described this candidate as a surface adhesin that binds to the glycolipid receptors 21 , and have highlighted its interaction with hBMECs 20 . Being a principle components of OMVs 24,25 and encoded by mafA gene located on the maf meningococcal pathogenicity island-like region wherein majority of the genes are related to virulence factors such as toxins, adhesins or invasins 22 , the probability of the involvement of MafA in host-pathogen crosstalk and neuroinvasion is high.…”

“…This isolate was used in the study mainly because of its low passage, neuroinvasiveness, high virulance and known clinical symptoms. N. meningitidis was cultured as described in our previous publication 20 . In brief, very low passaged (P02) isolate was inoculated on Thayer Martin agar (Becton-Dickinson, USA) and a single isolated colony was transferred into 50 mL of Brain Heart Infusion Broth (BHI) enriched with 10 mM MgCl 2 .…”

“…For example type IV pili induce signaling events that initiate transcellular passage 12 , opacity-associated protein c (Opc) interacts with cytoskeletal α-actinin, which has an impact on the modulation of various signaling pathways and cytoskeletal functions enabling meningococci to translocate across endothelial layer 17 , whereas Opa of Neisseria gonorrhoeae binds to the epithelial CD66 receptor and mediates tight contact leading to the transepithelial traversal 18 . In addition to these three surface proteins, meningococcus expresses several adhesins such as NadA 19,20 , MafA 20,21 , MafB 22 , major outer membrane protein P.IB 23 and lipoproteins 20 . Here, members of Maf ( m ultiple a dhesin f amily) are of particular interest.…”

Genetic incorporation of non-canonical amino acid photocrosslinkers in Neisseria meningitidis: New method provides insights into the physiological function of the function-unknown NMB1345 protein

“…Among the factors described above, meningococcal pili, which are mainly composed of the major pilin, PilE, and three minor pilins, PilV, PilX, and ComP, play the most important roles in the meningococcal infection processes involved in interactions with human epithelial and endothelial cells (reviewed in [17,18]). To elucidate the molecular mechanisms underlying meningococcal pathogenesis in more detail, functional genomics by linking genotypes to phenotypes have allowed investigation of the relationships between gene transcript abundance or deficiencies and the capacity to function under various physiological conditions to be investigated, by genome-wide signature-tagged mutagenesis (STM) with an infant rat infection model [19], with human cultured cells [20], and in different media [21], microarrays with infection in human cultured cells [22], comparative genomics [23,24], transcriptomics [25][26][27][28], and proteomics [29][30][31]. However, the mechanisms by which N. meningitidis causes septicemia and meningitis in humans cannot be completely explained by these characterized factors [32].…”