Screening for the Key Proteins Associated with Rete Testis Invasion in Clinical Stage I Seminoma via Label-Free Quantitative Mass Spectrometry.

Rete testis invasion (RTI) is an unfavourable prognostic factor for the risk of relapse in clinical stage I (CS I) seminoma patients. Notably, no evidence of difference in the proteome of RTI-positive vs. -negative CS I seminomas has been reported yet. Here, a quantitative proteomic approach was used to investigate RTI-associated proteins. 64 proteins were differentially expressed in RTI-positive compared to -negative CS I seminomas. Of them, 14-3-3γ, ezrin, filamin A, Parkinsonism-associated deglycase 7 (PARK7), vimentin and vinculin, were validated in CS I seminoma patient cohort. As shown by multivariate analysis controlling for clinical confounders, PARK7 and filamin A expression lowered the risk of RTI, while 14-3-3γ expression increased it. Therefore, we suggest that in real clinical biopsy specimens, the expression level of these proteins may reflect prognosis in CS I seminoma patients.

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.

XPA: DNA Repair Protein of Significant Clinical Importance.

The nucleotide excision repair (NER) pathway is activated in response to a broad spectrum of DNA lesions, including bulky lesions induced by platinum-based chemotherapeutic agents. Expression levels of NER factors and resistance to chemotherapy has been examined with some suggestion that NER plays a role in tumour resistance; however, there is a great degree of variability in these studies. Nevertheless, recent clinical studies have suggested Xeroderma Pigmentosum group A (XPA) protein, a key regulator of the NER pathway that is essential for the repair of DNA damage induced by platinum-based chemotherapeutics, as a potential prognostic and predictive biomarker for response to treatment. XPA functions in damage verification step in NER, as well as a molecular scaffold to assemble other NER core factors around the DNA damage site, mediated by protein-protein interactions. In this review, we focus on the interacting partners and mechanisms of regulation of the XPA protein. We summarize clinical oncology data related to this DNA repair factor, particularly its relationship with treatment outcome, and examine the potential of XPA as a target for small molecule inhibitors.

Increased levels of XPA might be the basis of cisplatin resistance in germ cell tumours.

BACKGROUND: Germ cell tumours (GCTs) represent a highly curable malignity as they respond well to cisplatin (CDDP)-based chemotherapy. Nevertheless, a small proportion of GCT patients relapse or do not respond to therapy. As this might be caused by an increased capacity to repair CDDP-induced DNA damage, identification of DNA repair biomarkers predicting inadequate or aberrant response to CDDP, and thus poor prognosis for GCT patients, poses a challenge. The objective of this study is to examine the expression levels of the key nucleotide excision repair (NER) factors, XPA, ERCC1 and XPF, in GCT patients and cell lines. METHODS: Two hundred seven GCT patients' specimens with sufficient follow-up clinical-pathological data and pairwise combinations of CDDP-resistant and -sensitive GCT cell lines were included. Immunohistochemistry was used to detect the ERCC1, XPF and XPA protein expression levels in GCT patients' specimen and Western blot and qRT-PCR examined the protein and mRNA expression levels in GCT cell lines. RESULTS: GCT patients with low XPA expression had significantly better overall survival than patients with high expression (hazard ratio = 0.38, 95% confidence interval: 0.12-1.23, p = 0.0228). In addition, XPA expression was increased in the non-seminomatous histological subtype, IGCCCG poor prognosis group, increasing S stage, as well as the presence of lung, liver and non-pulmonary visceral metastases. Importantly, a correlation between inadequate or aberrant CDDP response and XPA expression found in GCT patients was also seen in GCT cell lines. CONCLUSIONS: XPA expression is an additional independent prognostic biomarker for stratifying GCT patients, allowing for improvements in decision-making on treatment for those at high risk of refractoriness or relapse. In addition, it could represent a novel therapeutic target in GCTs.

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

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.

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

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-ß-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.

Design, construction and evaluation of multi-epitope antigens for diagnosis of Lyme disease.

INTRODUCTION: Introduction and objective. Lyme disease (LD) is the most common vector-borne disease in the temperate zone of the Northern Hemisphere. Diagnosis of LD is mainly based on clinical symptoms supported with serology (detection of anti-Borrelia antibodies) and is often misdiagnosed in areas of endemicity. MATERIAL AND METHODS: In this study, the chimeric proteins (A/C-2, A/C-4 and A/C-7.1) consisting of B-cell epitopes of outer surface proteins OspA and OspC from Borrelia genospecies prevalent in Eastern Slovakia, were designed, over-expressed in E. coli, and used to detect specific anti-Borrelia antibodies in serologically characterized sera from patients with Lyme-like symptoms to evaluate their diagnostic potential. RESULTS: Results showed that chimeras vary in their immuno-reactivity when tested with human sera. Compared with the results obtained from a two-tier test, the application of recombinant multi-epitope chimeric proteins as diagnosis antigens, produced fair agreement in the case of A/C-2 (0.20<κ<0.40) and good agreement (0.60<κ<0.80) when A/C-7.1 was used as capture antigen. Chimera A/C-4 were excluded from further study due to loss of reactivity with OspA-specific antibodies. CONCLUSIONS: The combination of specific B-cell epitopes from OspA and OspC proteins may improve the diagnostic accuracy of serologic assays, but further studies are required to address this hypothesis.

Joining the in vitro immunization of alpaca lymphocytes and phage display: rapid and cost effective pipeline for sdAb synthesis.

BACKGROUND: Camelids possess unique functional heavy chain antibodies, which can be produced and modified in vitro as a single domain antibody (sdAb or nanobody) with full antigen binding ability. Production of sdAb in conventional manner requires active immunization of Camelidae animal, which is laborious, time consuming, costly and in many cases not feasible (e.g. in case of highly toxic or infectious antigens). RESULTS: In this study, we describe an alternative pipeline that includes in vitro stimulation of naïve alpaca B-lymphocytes by antigen of interest (in this case endothelial cell binding domain of OspA of Borrelia) in the presence of recombinant alpaca interleukins 2 and 4, construction of sdAb phage library, selection of antigen specific sdAb expressed on phages (biopanning) and confirmation of binding ability of sdAb to the antigen. By joining the in vitro immunization and the phage display ten unique phage clones carrying sdAb were selected. Out of ten, seven sdAb showed strong antigen binding ability in phage ELISA. Furthermore, two soluble forms of sdAb were produced and their differential antigen binding affinity was measured with bio-layer interferometry. CONCLUSION: A proposed pipeline has potential to reduce the cost substantially required for maintenance of camelid herd for active immunization. Furthermore, in vitro immunization can be achieved within a week to enrich mRNA copies encoding antigen-specific sdAbs in B cell. This rapid and cost effective pipeline can help researchers to develop efficiently sdAb for diagnostic and therapeutic purposes.

Alterations in Outer Membrane Permeability Favor Drug-Resistant Phenotype of Klebsiella pneumoniae.

The increasing number of infections caused by multidrug-resistant and pandrug-resistant bacteria represents a serious worlwide problem. Drug resistance limits available antimicrobials and can lead to suboptimal treatment of bacterial infections. It can be predicted that resistance to more antimicrobial drugs will be acquired by even more bacteria in the future. Among the distinct resistance strategies, preventing drug entrance to intracellular compartment through modification of membrane permeability (bacterial influx) and active export of compounds to the external environment (bacterial efflux) are of particular importance as they limit the interaction of the drug with its intracellular targets and, consequently, its deleterious effects on the cell. Several current studies have extended our understanding of drug resistance mechanisms associated with altered membrane permeability in gram-negative bacteria. In this study, we propose a summary of resistance mechanisms associated with transport of drugs across bacterial cell envelope exploited by Klebsiella pneumoniae, one of the most common nosocomial infection-causing pathogens. The better understanding of molecular bases of drug transport in/out of the single cell may have consequence for success in antimicrobial therapy of infection caused by drug-resistant Klebsiella.

Identification of B-cell epitopes of Borrelia burgdorferi outer surface protein C by screening a phage-displayed gene fragment library.

Outer surface protein C (OspC) of Borrelia stimulates remarkable immune responses during early infection and is therefore currently considered a leading diagnostic and vaccine candidate. The sensitivity and specificity of serological tests based on whole protein OspC for diagnosis of Lyme disease are still unsatisfactory. Minimal B-cell epitopes are key in the development of reliable immunodiagnostic tools. Using OspC fragments displayed on phage particles (phage library) and anti-OspC antibodies isolated from sera of naturally infected patients, six OspC epitopes capable of distinguishing between LD patient and healthy control sera were identified. Three of these epitopes are located at the N-terminus (OspC E1 aa19-27, OspC E2 aa38-53, OspC E3 aa62-66) and three at the C-terminal end (OspC E4 aa155-163, OspC E5 aa184-190 and OspC E6 aa201-207). OspC E1, E4 and E6 were highly conserved among LD related Borreliae. To our knowledge, epitopes OspC E2, E3 and E5 were identified for the first time in this study. Minimal B-cell epitopes may provide fundamental data for the development of multi-epitope-based diagnostic tools for Lyme disease.

Exopolysaccharides of Lactobacillus reuteri: Their influence on adherence of E. coli to epithelial cells and inflammatory response.

The aim of the study was to characterize exopolysaccharides (EPS) originated from Lactobacillus reuteri strain DSM 17938 (EPS-DSM17938) and L. reuteri strain L26 Biocenol™ (EPS-L26) and evaluate their influence on adherence of enterotoxigenic Escherichia coli (ETEC) to IPEC-1 cells and proinflammatory gene expression. Both EPS were d-glucan polysaccharides with higher molecular weight (Mw), but differing in spatial conformation and elicited variable cytokine profile. EPS-DSM17938, relatively linear polysaccharide with (1→4) and (1→6) glycosidic linkages, increased IL-1ß gene expression (0.1mg/mL; P<0.05), while EPS-L26, more branched polysaccharide with (1→3) and (1→6) glycosidic linkages, exerted slight but statistically significant up-regulation of NF-κB, TNF-α and IL-6 mRNA (P<0.05). The most significant finding is that preincubation of IPEC-1 cells with both EPS followed by ETEC infection inhibit ETEC adhesion on IPEC-1 cells (P<0.01) and ETEC-induced gene expression of proinflammatory cytokine IL-1ß and IL-6 (P<0.01).

An Introduction to B-Cell Epitope Mapping and In Silico Epitope Prediction.

Identification of B-cell epitopes is a fundamental step for development of epitope-based vaccines, therapeutic antibodies, and diagnostic tools. Epitope-based antibodies are currently the most promising class of biopharmaceuticals. In the last decade, in-depth in silico analysis and categorization of the experimentally identified epitopes stimulated development of algorithms for epitope prediction. Recently, various in silico tools are employed in attempts to predict B-cell epitopes based on sequence and/or structural data. The main objective of epitope identification is to replace an antigen in the immunization, antibody production, and serodiagnosis. The accurate identification of B-cell epitopes still presents major challenges for immunologists. Advances in B-cell epitope mapping and computational prediction have yielded molecular insights into the process of biorecognition and formation of antigen-antibody complex, which may help to localize B-cell epitopes more precisely. In this paper, we have comprehensively reviewed state-of-the-art experimental methods for B-cell epitope identification, existing databases for epitopes, and novel in silico resources and prediction tools available online. We have also elaborated new trends in the antibody-based epitope prediction. The aim of this review is to assist researchers in identification of B-cell epitopes.

A rapid and simple pipeline for synthesis of mRNA-ribosome-V(H)H complexes used in single-domain antibody ribosome display.

The single-domain antibody (VHH) is a promising building block for a number of antibody-based applications. Ribosome display can successfully be used in the production of VHH. However, the construction of the expression cassette, confirmation of the translation and proper folding of the nascent chain, and the purification of the ribosome complexes, remain cumbersome tasks. Additionally, selection of the most suitable expression system can be challenging. We have designed primers that will amplify virtually all Camelidae VHH. With the help of a double-overlap extension (OE) polymerase chain reaction (PCR) we have fused VHH with the F1 fragment (T7 promoter and species-independent translation sequence) and the F2 fragment (mCherry, Myc-tag, tether, SecM arrest sequence and 3' stem loop) to generate a full-length DNA cassette. OE-PCR generated fragments were incubated directly with cell-free lysates (Leishmania torentolae, rabbit reticulocyte or E. coli) for the synthesis of mRNA-VHH-mCherry-ribosome complexes in vitro. Alternatively, the cassette was ligated in pQE-30 vector and transformed into E. coli to produce ribosome complexes in vivo. The results showed that the same expression cassette could be used to synthesize ribosome complexes with different expression systems. mCherry reporter served to confirm the synthesis and proper folding of the nascent chain, Myc-tag was useful in the rapid purification of ribosome complexes, and combination of the SecM sequence and 3' stem loop made the cassette universal, both for cells-free and E. coli in vivo. This rapid and universal pipeline can effectively be used in antibody ribosome display and VHH production.

Exploitation of complement regulatory proteins by Borrelia and Francisella.

Pathogens have developed sophisticated mechanisms of complement evasion such as binding to the host complement regulatory proteins (CRPs) on their surface or expression of CRP mimicking molecules. The ability of pathogens to evade the complement system has been correlated with pathogenesis and host selectivity. Hitherto, little work has been undertaken to determine whether Borrelia and Francisella exploit various CRPs to block complement attack. Seventeen Borrelia (twelve species) and six Francisella (three subspecies) strains were used to assess their ability to bind human, sheep and cattle CRPs or mimic membrane associated complement regulators. A series of experiments including affinity ligand binding experiments, pull-down assays and mass spectrometry based protein identification, revealed an array of CRP binding proteins of Borrelia and Francisella. Unlike Francisella, Borrelia strains were able to bind multiple human CRPs. Three strains of Borrelia (SKT-4, SKT-2 and HO14) showed the presence of a human CD46-homologous motif, indicating their ability to possess putative human CD46 mimicking molecules. Similarly, five strains of Borrelia and two strains of Francisella may have surface proteins with human CD59-homologous motifs. Among ovine and bovine CRPs, the only CRP bound by Francisella (LVS, Tul4 strain) was vitronectin, while ovine C4BP, ovine factor H and bovine factor H were bound to Borrelia strains SKT-2, DN127 and Co53. This study presents an array of proteins of Borrelia and Francisella that bind CRPs or may mimic membrane-CRPs, thus enabling multiphasic complement evasion strategies of these pathogens.

Deciphering the protein interaction in adhesion of Francisella tularensis subsp. holarctica to the endothelial cells.

Extracellular form of Francisella is able to cross various cell barriers and invade multiple organs, such as skin, liver, lung and central nervous system. Transient adhesion of Francisella to endothelial cells may trigger the process of translocation. In this report, we showed that Francisella tularensis subsp. holarctica (Fth) is able to adhere to the endothelial cells, while ICAM-1 may serve as an adhesion molecule for Fth. Pull down and affinity ligand binding assays indicated that the PilE4 could be the probable ligand for ICAM-1. Further deciphering of this ligand:receptor interaction revealed that PilE4 interacts with Ig-like C2-type 1 domain of ICAM-1. To corroborate the role of PilE4 and ICAM-1 interaction in adhesion of extracellular form of Fth to endothelial cells, ICAM-1 was blocked with monoclonal anti-ICAM-1 antibody prior to the incubation with Fth and numbers of adherent bacteria were counted. Blocking of the ICAM-1 significantly reduced (500-fold, P < 0.05) number of adherent Fth compared to unblocked cells. PilE4:ICAM-1 interaction unfolded here may provide a new perspective on molecules involved in the adhesion of extracellular form of Francisella to endothelial cells and probably its translocation across endothelial barriers.

Deciphering the interface between a CD40 receptor and borrelial ligand OspA.

Neuroborreliosis is serious sequelae of Lyme borreliosis. Neuroinvasion is largely relied on successful translocation of Borrelia across the blood-brain barrier. Adherence of Borrelia to brain microvascular endothelial cell (BMEC) seems to be critical for translocation. Here we unfold the interface between OspA and CD40 molecules, major ligand and receptor, that are involved in adhesion of Borrelia to BMECs. We found that a region between Asn127 and Asp205 of OspA forms the CD40-receptor binding site. This region encompasses human umbilical vein endothelial cell (HUVEC) binding domain and contains a potential ligand-binding pocket lined by three amino acid residues: Arg139, Glu160 and Lys189. Disruption of this pocket (by truncation of the HUVEC binding domain) caused complete abrogation of its ability to bind CD40. To identify the amino acid residues within the HUVEC binding domain involved in the CD40 binding, site-directed mutagenesis and binding assays were performed. Results showed that Asp149, Phe165, Ala172, Val186 and Leu192 might form interface with CD40 molecule. Other side of the interface was also identified with the help of a ligand-binding assay with OspA and truncated CD40 fragments. Results exposed that cysteine rich domain 2 (CRD2) of CD40 might be the site for OspA binding. Precise knowledge of the molecular basis of the ligand-receptor interactions is essential in order to understand mechanisms of pathogenesis and could help in the development of novel therapeutics and vaccines.

Outer surface proteins of Borrelia: peerless immune evasion tools.

Lyme borreliosis (LB), caused by Borrelia burgdorferi (B.b.), is the most frequently diagnosed tick-borne zoonosis in temperate zones of the Northern hemisphere. Borrelia is unique among bacteria in its ability to express a wide variety of lipoproteins on its surface, which play an essential role in pathogenesis. Surface proteins of spirochetes are important virulence determinants, immune evasion molecules and adaptation factors in the transmission and interaction with host tissues. Vast diversity in the expressed surface proteome of Borrelia in different niches and multifunctionality of proteins are the major strategies of Borrelia to avoid the destructive effect of immune system. In this review we provide deep insight into the protein:protein interactions that take place between different stages of life of Borrelia. Precise knowledge of surface proteins may help in improvement of the vaccines as well as for therapeutic agents against borreliosis.

Variable regions in the sushi domains 6-7 and 19-20 of factor H in animals and human lead to change in the affinity to factor H binding protein of Borrelia.

Borrelia binds host's complement regulatory factor H (fH) to evade complement attack. However, binding affinities between fH-binding-proteins (FHBPs) of Borrelia and fH from various hosts are disparate. Experiments performed to unfold the underlying molecular basis of this disparity revealed that recombinant BbCRASP-1 (major FHBP of Borrelia burgdorferi) neither interacted with sushi 6-7, nor with sushi 19-20 domains of fH in cattle and pig, however, showed binding affinity to both sushi domains of human fH, sushi 6-7 of mouse and sushi 19-20 of sheep. Further, peptide-spot assay revealed three major binding sites (sushi 6:(335-346), sushi 7:(399-410) and sushi 20:(1205-1227)) in human fH that can form BbCRASP-1:fH interface, while (337)HENMR(341) residues in sushi 6 are crucial for rigid BbCRASP-1:fH complex formation. Amino acid stretches DTIEFTCRYGYRPRTALHTFRTT in ovine sushi 19-20 and SAYWEKVYVQGQ in mouse sushi 7 were important sites for fH:BbCRASP-1 interaction. Comparative analysis of the amino acid sequences of sushi 6 of cattle, pig and human revealed that bovine and porcine fH lack methionine and arginine in HENMR pocket, that may impede formation of fH:BbCRASP-1 interface. Increasing numbers of FHBPs from animal and human pathogens are being discovered, thus results presented here can be important benchmark for study of other FHBPs:FH interactions.

Development of simple and rapid elution methods for proteins from various affinity beads for their direct MALDI-TOF downstream application.

Commercially available desalting techniques, necessary for downstream MALDI-TOF analysis of proteins, are often costly or time consuming for large-scale analysis. Here, we present techniques to elute proteins from various affinity resins, free from salt and ready for MALDI mass spectrometry. We showed that 0.1% TFA in 50% acetonitrile or 40% ethanol can be used as salt-free eluents for His-tagged proteins from variety of polyhistidine-affinity resins, while washing of resin beads twice with double-distilled water prior to the elution effectively desalted and recovered wide-range-molecular size proteins than commercially available desalting devices. Modified desalting and elution techniques were also applied for Flag- and Myc-tag affinity resins. The technique was further applied in co-precipitation assay, where the maximum recovery of wide-range molecular size proteins is crucial. Further, results showed that simple washing of the beads with double distilled water followed by elution with acetonitrile effectively desalted and recovered 150 kDa factor H protein of the sheep and its binding partner ~30 kDa BbCRASP-1 in co-precipitation assay. In summary, simple modifications in the desalting and elution strategy save time, labor and cost of the protein preparation for MALDI mass spectrometry; and large-scale protein purifications or co-precipitations can be performed with ease.

OspA-CD40 dyad: ligand-receptor interaction in the translocation of neuroinvasive Borrelia across the blood-brain barrier.

Lyme borreliosis is the most widespread vector-borne disease in temperate zones of Europe and North America. Although the infection is treatable, the symptoms are often overlooked resulting in infection of the neuronal system. In this work we uncover the underlying molecular mechanism of borrelial translocation across the blood-brain barrier (BBB). We demonstrate that neuroinvasive strain of Borrelia readily crosses monolayer of brain-microvascular endothelial cells (BMECs) in vitro and BBB in vivo. Using protein-protein interaction assays we found that CD40 of BMECs and OspA of Borrelia are the primary molecules in transient tethering of Borrelia to endothelium. OspA of neuroinvasive Borrelia, but not of non-neuroinvasive strain, binds CD40. Furthermore, only the neuroinvasive Borrelia and its recombinant OspA activated CD40-dependent pathway in BMECs and induced expression of integrins essential for stationary adhesion. Demonstration of the CD40-ligand interactions may provide a new possible perspective on molecular mechanisms of borrelial BBB translocation process.