Differential transcriptome response of blood brain barrier spheroids to neuroinvasive Neisseria and Borrelia.

Background: The blood-brain barrier (BBB), a highly regulated interface between the blood and the brain, prevents blood-borne substances and pathogens from entering the CNS. Nevertheless, pathogens like Neisseria meningitidis and Borrelia bavariensis can breach the BBB and infect the brain parenchyma. The self-assembling BBB-spheroids can simulate the cross talk occurring between the cells of the barrier and neuroinvasive pathogens. Methods: BBB spheroids were generated by co-culturing human brain microvascular endothelial cells (hBMECs), pericytes and astrocytes. The BBB attributes of spheroids were confirmed by mapping the localization of cells, observing permeability of angiopep2 and non-permeability of dextran. Fluorescent Neisseria, Borrelia or E. coli (non-neuroinvasive) were incubated with spheroids to observe the adherence, invasion and spheroid integrity. Transcriptome analysis with NGS was employed to investigate the response of BBB cells to infections. Results: hBMECs were localized throughout the spheroids, whereas pericytes and astrocytes were concentrated around the core. Within 1 hr of exposure, Neisseria and Borrelia adhered to spheroids, and their microcolonization increased from 5 to 24 hrs. Integrity of spheroids was compromised by both Neisseria and Borrelia, but not by E. coli infection. Transcriptome analysis revealed a significant change in the expression of 781 genes (467 up and 314 down regulated) in spheroids infected with Neisseria, while Borrelia altered the expression of 621 genes (225 up and 396 down regulated). The differentially expressed genes could be clustered into various biological pathways like cell adhesion, extracellular matrix related, metallothionines, members of TGF beta, WNT signaling, and immune response. Among the differentially expressed genes, 455 (48%) genes were inversely expressed during Neisseria and Borrelia infection. Conclusion: The self-assembling spheroids were used to perceive the BBB response to neuroinvasive pathogens - Neisseria and Borrelia. Compromised integrity of spheroids during Neisseria and Borrelia infection as opposed to its intactness and non-adherence of E. coli (non-neuroinvasive) denotes the pathogen dependent fate of BBB. Genes categorized into various biological functions indicated weakened barrier properties of BBB and heightened innate immune response. Inverse expression of 48% genes commonly identified during Neisseria and Borrelia infection exemplifies unique response of BBB to varying neuropathogens.

Signaling events evoked by domain III of envelop glycoprotein of tick-borne encephalitis virus and West Nile virus in human brain microvascular endothelial cells.

Tick-borne encephalitis virus and West Nile virus can cross the blood-brain barrier via hematogenous route. The attachment of a virion to the cells of a neurovascular unit, which is mediated by domain III of glycoprotein E, initiates a series of events that may aid viral entry. Thus, we sought to uncover the post-attachment biological events elicited in brain microvascular endothelial cells by domain III. RNA sequencing of cells treated with DIII of TBEV and WNV showed significant alteration in the expression of 309 and 1076 genes, respectively. Pathway analysis revealed activation of the TAM receptor pathway. Several genes that regulate tight-junction integrity were also activated, including pro-inflammatory cytokines and chemokines, cell-adhesion molecules, claudins, and matrix metalloprotease (mainly ADAM17). Results also indicate activation of a pro-apoptotic pathway. TLR2 was upregulated in both cases, but MyD88 was not. In the case of TBEV DIII, a MyD88 independent pathway was activated. Furthermore, both cases showed dramatic dysregulation of IFN and IFN-induced genes. Results strongly suggest that the virus contact to the cell surface emanates a series of events namely viral attachment and diffusion, breakdown of tight junctions, induction of virus uptake, apoptosis, reorganization of the extracellular-matrix, and activation of the innate immune system.

Engineering the Single Domain Antibodies Targeting Receptor Binding Motifs Within the Domain III of West Nile Virus Envelope Glycoprotein.

West Nile virus (WNV) is a mosquito-borne neurotrophic flavivirus causing mild febrile illness to severe encephalitis and acute flaccid paralysis with long-term or permanent neurological disorders. Due to the absence of targeted therapy or vaccines, there is a growing need to develop effective anti-WNV therapy. In this study, single-domain antibodies (sdAbs) were developed against the domain III (DIII) of WNV's envelope glycoprotein to interrupt the interaction between DIII and the human brain microvascular endothelial cells (hBMEC). The peripheral blood mononuclear cells of the llama immunized with recombinant DIIIL297-S403 (rDIII) were used to generate a variable heavy chain only (VHH)-Escherichia coli library, and phage display was performed using the M13K07ΔpIII Hyperphages system. Phages displaying sdAbs against rDIII were panned with the synthetic analogs of the DIII receptor binding motifs, DIII-1G299-K307 and DIII-2V371-R388, and the VHH gene from the eluted phages was subcloned into E. coli SHuffle. Soluble sdAbs purified from 96 E. coli SHuffle clones were screened to identify 20 candidates strongly binding to the synthetic analogs of DIII-1G299-K307 and DIII-2V371-R388 on a dot blot assay. Among them, sdAbA1, sdAbA6, sdAbA9, and sdAbA10 blocked the interaction between rDIII and human brain microvascular endothelial cells (hBMECs) on Western blot and cell ELISA. However, optimum stability during the overexpression was noticed only for sdAbA10 and it also neutralized the WNV-like particles (WNV-VLP) in the Luciferase assay with an half maximal effective concentration (EC50) of 1.48 nm. Furthermore, the hemocompatibility and cytotoxicity of sdAbA10 were assessed by a hemolytic assay and XTT-based hBMEC proliferation assay resulting in 0.1% of hemolytic activity and 82% hBMEC viability, respectively. Therefore, the sdAbA10 targeting DIII-2V371-R388 of the WNV envelope glycoprotein is observed to be suitable for in vivo trials as a specific therapy for WNV-induced neuropathogenesis.

Simple and rapid pipeline for the production of cyclic and linear small-sized peptides in E. coli.

Small and medium-sized peptides are gaining popularity in biomedical applications, including therapeutic target development. As an alternative to chemical synthesis, we describe a complete pipeline for the production of linear as well as structurally constrained cyclic peptides in an E. coli expression system in this study. A plasmid vector containing a novel N terminal HOE tag (28 amino acids in length) that fuses with the peptide was created. The HOE tag contains sites for both chemical (CNBr) and enzymatic (enterokinase) cleavage, making it easy to isolate the peptide after production. A total of 21 peptides (17 cyclic and 4 linear) were synthesized, and the HOE tag was successfully removed using either CNBr (9 peptides) or enterokinase (12 peptides). The presence of a disulfide bond was confirmed in six representative cyclic peptides. In this study we have provided detailed instructions on primers design strategy, overexpression and purification of HOE tagged peptides, chemical and enzymatic cleavage, and confirmation of the cyclic form of peptides. We are confident that this pipeline will assist researchers in producing multiple recombinant peptides in a cost-effective and time-efficient manner.

Comprehensive Mapping of the Cell Response to Borrelia bavariensis in the Brain Microvascular Endothelial Cells in vitro Using RNA-Seq.

Borrelia bavariensis can invade the central nervous system (CNS) by crossing the blood-brain barrier (BBB). It is predicted that B. bavariensis evokes numerous signaling cascades in the human brain microvascular endothelial cells (hBMECs) and exploits them to traverse across the BBB. The complete picture of signaling events in hBMECs induced by B. bavariensis remains uncovered. Using RNA sequencing, we mapped 11,398 genes and identified 295 differentially expressed genes (DEGs, 251 upregulated genes and 44 downregulated genes) in B. bavariensis challenged hBMECs. The results obtained from RNA-seq were validated with qPCR. Gene ontology analysis revealed the participation of DEGs in a number of biological processes like cell communication, organization of the extracellular matrix, vesicle-mediated transport, cell response triggered by pattern recognition receptors, antigen processing via MHC class I, cellular stress, metabolism, signal transduction, etc. The expression of several non-protein coding genes was also evoked. In this manuscript, we discuss in detail the correlation between several signaling cascades elicited and the translocation of BBB by B. bavariensis. The data revealed here may contribute to a better understanding of the mechanisms employed by B. bavariensis to cross the BBB.

Development of peptides targeting receptor binding site of the envelope glycoprotein to contain the West Nile virus infection.

West Nile virus (WNV), re-emerging neurotropic flavivirus, can cross the blood-brain barrier (BBB) and cause fatal encephalitis and meningitis. Infection of the human brain microvascular endothelial cells (hBMECs), building blocks of the BBB, represents the pivotal step in neuroinvasion. Domain III (DIII) of the envelope (E) glycoprotein is a key receptor-binding domain, thus, it is an attractive target for anti-flavivirus strategies. Here, two combinatorial phage display peptide libraries, Ph.D.-C7C and Ph.D.-12, were panned against receptor-binding site (RBS) on DIII to isolate peptides that could block DIII. From series of pannings, nine peptides (seven 7-mer cyclic and two 12-mer linear) were selected and overexpressed in E. coli SHuffle T5. Presence of disulfide bond in 7-mer peptides was confirmed with thiol-reactive maleimide labeling. Except for linear peptide 19 (HYSWSWIAYSPG), all peptides proved to be DIII binders. Among all peptides, 4 cyclic peptides (CTKTDVHFC, CIHSSTRAC, CTYENHRTC, and CLAQSHPLC) showed significant blocking of the interaction between DIII and hBMECs, and ability to neutralize infection in cultured cells. None of these peptides showed toxic or hemolytic activity. Peptides identified in this study may serve as potential candidates for the development of novel antiviral therapeutics against WNV.

Transcriptomic analysis of human brain microvascular endothelial cells exposed to laminin binding protein (adhesion lipoprotein) and Streptococcus pneumoniae.

Streptococcus pneumoniae invades the CNS and triggers a strong cellular response. To date, signaling events that occur in the human brain microvascular endothelial cells (hBMECs), in response to pneumococci or its surface adhesins are not mapped comprehensively. We evaluated the response of hBMECs to the adhesion lipoprotein (a laminin binding protein-Lbp) or live pneumococci. Lbp is a surface adhesin recently identified as a potential ligand, which binds to the hBMECs. Transcriptomic analysis was performed by RNA-seq of three independent biological replicates and validated with qRT-PCR using 11 genes. In total 350 differentially expressed genes (DEGs) were identified after infection with S. pneumoniae, whereas 443 DEGs when challenged with Lbp. Total 231 DEGs were common in both treatments. Integrative functional analysis revealed participation of DEGs in cytokine, chemokine, TNF signaling pathways and phagosome formation. Moreover, Lbp induced cell senescence and breakdown, and remodeling of ECM. This is the first report which maps complete picture of cell signaling events in the hBMECs triggered against S. pneumoniae and Lbp. The data obtained here could contribute in a better understanding of the invasion of pneumococci across BBB and underscores role of Lbp adhesin in evoking the gene expression in neurovascular unit.

Identification of the proteins of Borrelia garinii interacting with human brain microvascular endothelial cells.

Lyme borreliosis is one of the major tick-borne diseases in Europe. Events of the translocation of Borrelia across the blood-brain barrier (BBB) involve multiple interactions between borrelial surface proteins and receptors on the brain microvascular endothelial cells (hBMECs). In this study, we aimed to identify proteins of Borrelia that plausibly interact with hBMECs. The surface proteome of live Borrelia (a neuroinvasive strain of B. garinii) was crosslinked with biotin prior to its incubation with hBMECs. The interacting proteins were recovered by affinity purification, followed by SWATH-MS. Twenty-four interacting candidates were grouped into outer membrane proteins (n = 12) and inner membrane proteins (n = 12) based on the subcellular location as per the predictions of LocateP. Other algorithms like TMHMM 2.0 and LipoP, ontology search and literature review were subsequently applied to each of the identified protein candidates to shortlist the most probable interactors. Six proteins namely, LysM domain protein, BESBP-5, Antigen S1, CRASP-1 (Bg071), Erp23 protein and Mlp family Lipoprotein were selected to produce their recombinant forms and experimentally validate their interaction with hBMECs. All the recombinant proteins interacted with hBMECs, in ELISA and immunocytochemistry. We present here a high-throughput approach of generating a dataset of plausible borrelial ligands followed by a systematic bioinformatic pipeline to categorize the proteins for experimental validation.

Comprehensive mapping of the cell response to E. coli infection in porcine intestinal epithelial cells pretreated with exopolysaccharide derived from Lactobacillus reuteri.

Bacterial exopolysaccharides (EPSs) are known to modulate immunity. To date, a plethora of studies have reported the effect of EPSs on intestinal cells; however few works have revealed a complete picture of the signalling events in intestinal epithelial cells induced by bacterial EPSs. Here, using transcriptomics, we comprehensively mapped the biological processes in porcine intestinal epithelial cells challenged with EPS derived from Lactobacillus reuteri alone, enterotoxigenic Escherichia coli (ETEC) or ETEC after pretreatment with EPS. The Gene Ontology analysis of differentially expressed genes (DEGs) showed that ETEC is able to evoke biological processes specifically involved in cell junction reorganization, extracellular matrix degradation, and activation of the innate immune response through the activation of pattern recognition receptors, such as TLRs and CTRs. A total of 495 DEGs were induced in ETEC-challenged cells. On the other hand, EPS pretreatment was able to attenuate overexpression of the genes induced by ETEC infection. The most relevant finding of this study is that EPS has a suppressive effect on the inflammatory response evoked by ETEC infection. On the basis of high-throughput RNA-seq, this report is the first to describe the effects of EPSs derived from L. reuteri used as a pretreatment of global gene expression in porcine epithelial cells.

Single Domain Antibodies Targeting Receptor Binding Pockets of NadA Restrain Adhesion of Neisseria meningitidis to Human Brain Microvascular Endothelial Cells.

Neisseria adhesin A (NadA), one of the surface adhesins of Neisseria meningitides (NM), interacts with several cell types including human brain microvascular endothelial cells (hBMECs) and play important role in the pathogenesis. Receptor binding pockets of NadA are localized on the globular head domain (A33 to K69) and the first coiled-coil domain (L121 to K158). Here, the phage display was used to develop a variable heavy chain domain (VHH) that can block receptor binding sites of recombinant NadA (rec-NadA). A phage library displaying VHH was panned against synthetic peptides (NadA-gdA33-K69 or NadA-ccL121-K158), gene encoding VHH was amplified from bound phages and re-cloned in the expression vector, and the soluble VHHs containing disulfide bonds were overexpressed in the SHuffle E. coli. From the repertoire of 96 clones, two VHHs (VHHF3-binding NadA-gdA33-K69 and VHHG9-binding NadA-ccL121-K158) were finally selected as they abrogated the interaction between rec-NadA and the cell receptor. Preincubation of NM with VHHF3 and VHHG9 significantly reduced the adhesion of NM on hBMECs in situ and hindered the traversal of NM across the in-vitro BBB model. The work presents a phage display pipeline with a single-round of panning to select receptor blocking VHHs. It also demonstrates the production of soluble and functional VHHs, which blocked the interaction between NadA and its receptor, decreased adhesion of NM on hBMECs, and reduced translocation of NM across BBB in-vitro. The selected NadA blocking VHHs could be promising molecules for therapeutic translation.