Lucia Pulzova

University of Veterinary Medicine and Pharmacy in Košice, Kassa, Košický, Slovakia

Are you Lucia Pulzova?

Claim your profile

Publications (22)30.52 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Single Domain Antibody (VHH) is promising building block for various antibody-based applications. Ribosome display can be used successfully in the production of VHH. However, construction of the expression cassette, confirmation of the translation and proper folding of nascent chain and purification of the ribosome complexes still remain cumbersome tasks. Additionally, selection of the most suitable expression system is challenging. We designed primers that will amplify virtually all Camelidae VHH. With the help of double-overlap extension (OE) PCR we fused VHH with F1 fragment (T7 promoter and species independent translation sequence) and F2 fragment (mCherry, Myc-tag, tether, SecM arrest sequence and 3’ stem loop) to generate full-length DNA cassette. OE-PCR generated fragments were incubated directly with cell-free lysates (Leishmania torentolae, rabbit reticulocyte or E. coli) for 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. Results showed that the same expression cassette could be used to synthesize ribosome complexes with different expression systems. mCherry reporter served to confirm synthesis and proper folding of the nascent chain, Myc-tag was useful in rapid purification of ribosome complexes, and combination of 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 be used effectively in antibody ribosome display and VHH production.
    Molecular BioSystems 04/2015; DOI:10.1039/C5MB00026B · 3.18 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: 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. Copyright © 2015. Published by Elsevier Ltd.
    Microbial Pathogenesis 03/2015; 81. DOI:10.1016/j.micpath.2015.03.007 · 2.00 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: 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-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.
    Microbiological Research 09/2014; DOI:10.1016/j.micres.2014.09.003 · 1.94 Impact Factor
  • Source
    ICBMB 2014 International conference in biochemistry and molecular biology 17-19th April 2014 Vienna, Austria; 04/2014
  • Source
    ICBMB 2014 International conference on biochemistry and molecular biology, 17-19 April 2014, Vienna, Austria; 04/2014
  • Parasites & Vectors 04/2014; 7 (Suppl 1) P10(Suppl 1). DOI:10.1186/1756-3305-7-S1-P10 · 3.25 Impact Factor
  • Lucia Pulzova, Mangesh Bhide
    [Show abstract] [Hide abstract]
    ABSTRACT: Lyme borreliosis (LB), caused by Borrelia burgdorferi(B.b.), is the most frequently diagnosed tick-borne zoonosis in temperate zones of the Northern hemisphere. Borreliais 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 Borreliain different niches and multifunctionality of proteins are the major strategies of Borreliato 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.
    Current Protein and Peptide Science 02/2014; DOI:10.2174/1389203715666140221124213 · 2.33 Impact Factor
  • Source
    VIII ItPA National Congress Padua, June 18th-21st, 2013; 06/2013
  • [Show abstract] [Hide abstract]
    ABSTRACT: The uniflagellate protozoan parasite Trypanosoma brucei multiplies extracellularly in blood and escapes elimination by the complement system. Activated complement elicits potent biological activities including direct lysis of pathogens (Mollnes et al., 2002; Song et al., 2000; Walport, 2001). One strategy adopted by pathogens to avoid clearance and destruction by complement is to acquire host fluid-phase regulators, like C4b-binding protein or factor H. The acquisition of fluid-phase regulators on the surface of a given pathogen normally results in downregulation of complement activation.
    Part III edited by Andre de Almeida, David Eckersall, Elena, Bencurova, Saskia Dolinska, Patrik Mlynarcik, Miroslava Vincova, Mangesh Bhide, 04/2013; Wageningen Academic Publishers., ISBN: 978-90-8686-776-9
  • [Show abstract] [Hide abstract]
    ABSTRACT: In a previous study we have shown, that the OspA of Borrelia garinii (neuroinvasive strain SKT-7.1) is crucial for its transient tethering to the rat brain microvascular endothelial cells (RBMEC) via 31.8 kDa receptor CD40. Transient tethering is then followed by stationary adhesion in which ICAM-1 or VCAM-1 might be the potential molecules. Both transient and stationary adhesions are necessary steps in the traversal of Borrelia across the blood-brain barrier (BBB). Understanding the basic mechanisms involved in crossing the ...
    Part III edited by Andre de Almeida, David Eckersall, Elena, Bencurova, Saskia Dolinska, Patrik Mlynarcik, Miroslava Vincova, Mangesh Bhide, 04/2013; Wageningen Academic Publishers., ISBN: 978-90-8686-776-9
  • [Show abstract] [Hide abstract]
    ABSTRACT: Neural Trypanosomiasis is fatal disease caused by Trypanosoma brucei. T. brucei complex consists of three major subspecies namely T. brucei brucei, T. brucei gambiense, T. brucei rhodesiense. They are transmitted by the saliva of the infected tsetse flies of genus Glossina during blood meal. Trypanosomas are extracellular, spread through the blood stream to various niches, and consequently invade the central nervous system (Masocha et al., 2007). It is necessary to know how T. brucei interacts with brain microvascular endothelial cells (BMECs) to cross the blood brain barrier. Previous studies have shown that majority of extracellular pathogens have to adhere to BMEC first (transient adhesion), which then induces series of cell events causing more firm adhesion of pathogen to BMECs (stationary adhesion), followed by alteration in the extracellular matrix near tight junctions allowing more space for pathogen to cross BBB (reviewed in Bencurova et al. 2011). To this background, the study was aimed to identify putative surface adhesion receptors on BMECs that may take part in the Trypanosoma:BMEC interface during transient adhesion. Unfolding the underlying principles of ligand:receptor interactions, which take place during passage of Trypanosoma through BBB is important to understand its neuroinvasive mechanisms.
    Part III edited by Andre de Almeida, David Eckersall, Elena, Bencurova, Saskia Dolinska, Patrik Mlynarcik, Miroslava Vincova, Mangesh Bhide, 04/2013; Wageningen Academic Publishers., ISBN: 978-90-8686-776-9
  • Mucha R., Pulzova L., Mlynarcik P.
    New trends in bioinformatics analysis focused of data from genomics and proteomics, Edited by Mangesh R. Bhide& Saskia Dolinska, 01/2013: chapter Chapter 1; The University of veterinary medicine and pharmacy in Kosice., ISBN: ISBN 978-80-8077-321-2
  • Edited by André de Almeida, David Eckersall, Elena Bencurova, Saskia Dolinska, Patrik Mlynarcik, Miroslava Vincova and Mangesh Bhide, 01/2013: pages 151-154; Farm animal proteomics.
  • [Show abstract] [Hide abstract]
    ABSTRACT: Objectives Francisella tularensis (Ft), a small Gram-negative facultative intracellular bacterium, is the causative agent of tularemia. Disease is transmitted to human and animals mostly by vectors such as ticks, flies and mosquitoes. Tularemia is endemic in many parts of the northern hemisphere and has been detected in over 250 animals. Ft can invade many organs in the host body, such a liver, eyes, lung and central nervous system. Neural form of tularemia is rare, but often ends with fatal consequences (Gangat, 2007; van de Beek et al., 2007). One of the crucial steps in CNS invasion is the crossing of blood-brain barrier (BBB), while, bacterial translocation initiates with their transient adhesion on brain microvascular endothelial cells (BMECs). Present study is aimed at investigation of the molecules responsible for adhesion of Ft to BMECs. These molecules could be the important candidates in the development of prophylactic drugs against meningitides caused by Francisella. Material and methods Cultivation of Francisella and BMECs Francisella strain LVS was cultivated for 4 days at 37 °C on chocolate agar enriched with 1% glucose and 0.1% L-cystein. Bacterial cells were harvested and whole cell lysate was prepared by sonication. BMECs were prepared from Wistar rats as described previously (Pulzova et al., 2009; Veszelka et al., 2007). Forebrains were cleaned of meninges, minced into small pieces and digested with collagenase type II and DNase. Fragments were separated from myelin layer by centrifugation and microvessels were digested with collagenase-dispase and DNase. Microvessel endothelial cell clusters were separated on a 33% continuous Percoll and endothelial cell clusters were then directly plated on collagen type IV coated chamber slide. Cells were cultivated in DMEM supplemented with 20% plasma derived, gentamicine, L-glutamine, heparin, 1 basic fibroblast growth factor and puromycin. Cells were harvested and whole cell lysate was prepared. Ligand capture assay to search molecules in BMEC:Francisella interface
    01/2013: pages 99-102; Farm animal proteomics.
  • International Journal of Infectious Diseases 06/2012; 16:e466. DOI:10.1016/j.ijid.2012.05.676 · 2.33 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    Journal of proteomics 04/2012; 75(14):4520-8. DOI:10.1016/j.jprot.2012.04.013 · 3.93 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    Journal of proteomics 03/2012; 75(14):4529-35. DOI:10.1016/j.jprot.2012.03.001 · 3.93 Impact Factor
  • 75-79, 01/2012; Farm animal proteomics.
  • 01/2012: pages 94-97; Farm animal proteomics.
  • [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    Scientific Reports 09/2011; 1:86. DOI:10.1038/srep00086 · 5.08 Impact Factor

Publication Stats

20 Citations
30.52 Total Impact Points


  • 2012–2015
    • University of Veterinary Medicine and Pharmacy in Košice
      Kassa, Košický, Slovakia
  • 2009–2012
    • University of Veterinary Medicine in Kosice - Univerzita veterinarneho lekarstva v Kosiciach
      • Laboratory of Biomedical Microbiology and Immunology
      Kassa, Košický, Slovakia
  • 2011
    • Slovak Academy of Sciences
      • Institute of Neuroimmunology
      Presburg, Bratislavský, Slovakia