Lucia Pulzova

Pathogen translocation across the blood-brain barrier

Neurological manifestations caused by neuroinvading pathogens are typically attributed to penetration of the blood-brain barrier (BBB) and invasion of the central nervous system. However, the mechanisms used by many pathogens (such as Borrelia) to traverse the BBB are still unclear. Recent studies revealed that microbial translocation across the BBB must involve a repertoire of microbial-host interactions (receptor-ligand interactions). However, the array of interacting molecules responsible for the borrelial translocation is not yet clearly known. Pathogens bind several host molecules (plasminogen, glycosaminoglycans, factor H, etc.) that might mediate endothelial interactions in vivo. This review summarizes our current understanding of the pathogenic mechanisms involved in the translocation of the BBB by neuroinvasive pathogens.

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.

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.

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-1β 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-1β and IL-6 (P<0.01).

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 hosts 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.

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.

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

BackgroundCamelids 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).ResultsIn 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.ConclusionA 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.

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.

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.