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Drobnak I.,University of Notre Dame | Drobnak I.,Acies Bio d.o.o. | Braselmann E.,University of Notre Dame | Braselmann E.,University of Colorado at Boulder | And 8 more authors.
Molecular Microbiology | Year: 2015

Autotransporter (AT) proteins provide a diverse array of important virulence functions to Gram-negative bacterial pathogens, and have also been adapted for protein surface display applications. The 'autotransporter' moniker refers to early models that depicted these proteins facilitating their own translocation across the bacterial outer membrane. Although translocation is less autonomous than originally proposed, AT protein segments upstream of the C-terminal transmembrane β-barrel have nevertheless consistently been found to contribute to efficient translocation and/or folding of the N-terminal virulence region (the 'passenger'). However, defining the precise secretion functions of these AT regions has been complicated by the use of multiple overlapping and ambiguous terms to define AT sequence, structural, and functional features, including 'autochaperone', 'linker' and 'junction'. Moreover, the precise definitions and boundaries of these features vary among ATs and even among research groups, leading to an overall murky picture of the contributions of specific features to translocation. Here we propose a unified, unambiguous nomenclature for AT structural, functional and conserved sequence features, based on explicit criteria. Applied to 16 well-studied AT proteins, this nomenclature reveals new commonalities for translocation but also highlights that the autochaperone function is less closely associated with a conserved sequence element than previously believed. © 2014 John Wiley & Sons Ltd.

Hjelm A.,University of Stockholm | Soderstrom B.,University of Stockholm | Soderstrom B.,Okinawa Institute of Science and Technology | Vikstrom D.,Xbrane Bioscience AB | And 5 more authors.
Applied and Environmental Microbiology | Year: 2015

Bacterial ghosts are empty cell envelopes of Gram-negative bacteria that can be used as vehicles for antigen delivery. Ghosts aregenerated by releasing the bacterial cytoplasmic contents through a channel in the cell envelope that is created by the controlledproduction of the bacteriophage ΦX174 lysis protein E. While ghosts possess all the immunostimulatory surface properties ofthe original host strain, they do not pose any of the infectious threats associated with live vaccines. Recently, we have engineeredthe Escherichia coli autotransporter hemoglobin protease (Hbp) into a platform for the efficient surface display of heterologousproteins in Gram-negative bacteria, HbpD. Using the Mycobacterium tuberculosis vaccine target ESAT6 (early secreted antigenictarget of 6 kDa), we have explored the application of HbpD to decorate E. coli and Salmonella ghosts with antigens. The use ofdifferent promoter systems enabled the concerted production of HbpD-ESAT6 and lysis protein E. Ghost formation was monitoredby determining lysis efficiency based on CFU, the localization of a set of cellular markers, fluorescence microscopy, flowcytometry, and electron microscopy. Hbp-mediated surface display of ESAT6 was monitored using a combination of a proteaseaccessibility assay, fluorescence microscopy, flow cytometry and (immuno-)electron microscopy. Here, we show that the concertedproduction of HbpD and lysis protein E in E. coli and Salmonella can be used to produce ghosts that efficiently displayantigens on their surface. This system holds promise for the development of safe and cost-effective vaccines with optimal intrinsicadjuvant activity and exposure of heterologous antigens to the immune system. © 2015, American Society for Microbiology.

Daleke-Schermerhorn M.H.,VU University Amsterdam | Daleke-Schermerhorn M.H.,Abera Bioscience AB | Felix T.,Institute Pasteur Paris | Felix T.,French Institute of Health and Medical Research | And 25 more authors.
Applied and Environmental Microbiology | Year: 2014

Outer membrane vesicles (OMVs) are spherical nanoparticles that naturally shed from Gram-negative bacteria. They are rich in immunostimulatory proteins and lipopolysaccharide but do not replicate, which increases their safety profile and renders them attractive vaccine vectors. By packaging foreign polypeptides in OMVs, specific immune responses can be raised toward heterologous antigens in the context of an intrinsic adjuvant. Antigens exposed at the vesicle surface have been suggested to elicit protection superior to that from antigens concealed inside OMVs, but hitherto robust methods for targeting heterologous proteins to the OMV surface have been lacking. We have exploited our previously developed hemoglobin protease (Hbp) autotransporter platform for display of heterologous polypeptides at the OMV surface. One, two, or three of the Mycobacterium tuberculosis antigens ESAT6, Ag85B, and Rv2660c were targeted to the surface of Escherichia coli OMVs upon fusion to Hbp. Furthermore, a hypervesiculating ΔtolR ΔtolA derivative of attenuated Salmonella enterica serovar Typhimurium SL3261 was generated, enabling efficient release and purification of OMVs decorated with multiple heterologous antigens, exemplified by the M. tuberculosis antigens and epitopes from Chlamydia trachomatis major outer membrane protein (MOMP). Also, we showed that delivery of Salmonella OMVs displaying Ag85B to antigen-presenting cells in vitro results in processing and presentation of an epitope that is functionally recognized by Ag85B-specific T cell hybridomas. In conclusion, the Hbp platform mediates efficient display of (multiple) heterologous antigens, individually or combined within one molecule, at the surface of OMVs. Detection of antigen- specific immune responses upon vesicle-mediated delivery demonstrated the potential of our system for vaccine development. © 2014, American Society for Microbiology.

Van Ulsen P.,VU University Amsterdam | Rahman S.U.,VU University Amsterdam | Jong W.S.P.,VU University Amsterdam | Jong W.S.P.,Abera Bioscience AB | And 4 more authors.
Biochimica et Biophysica Acta - Molecular Cell Research | Year: 2014

The two membranes of Gram-negative bacteria contain protein machines that have a general function in their assembly. To interact with the extra-cellular milieu, Gram-negatives target proteins to their cell surface and beyond. Many specialized secretion systems have evolved with dedicated translocation machines that either span the entire cell envelope or localize to the outer membrane. The latter act in concert with inner-membrane transport systems (i.e. Sec or Tat). Secretion via the Type V secretion system follows a two-step mechanism that appears relatively simple. Proteins secreted via this pathway are important for the Gram-negative life-style, either as virulence factors for pathogens or by contributing to the survival of non-invasive environmental species. Furthermore, this system appears well suited for the secretion of biotechnologically relevant proteins. In this review we focus on the biogenesis and application of two Type V subtypes, the autotransporters and two-partner secretion (TPS) systems. For translocation across the outer membrane the autotransporters require the assistance of the Bam complex that also plays a generic role in the assembly of outer membrane proteins. The TPS systems do use a dedicated translocator, but this protein shows resemblance to BamA, the major component of the Bam complex. Interestingly, both the mechanistic and more applied studies on these systems have provided a better understanding of the secretion mechanism and the biogenesis of outer membrane proteins. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey. © 2013 Elsevier B.V.

Kuipers K.,Radboud University Nijmegen | Daleke-Schermerhorn M.H.,VU University Amsterdam | Daleke-Schermerhorn M.H.,Abera Bioscience AB | Jong W.S.P.,VU University Amsterdam | And 8 more authors.
Vaccine | Year: 2015

Bacterial outer membrane vesicles (OMVs) are attractive vaccine formulations because they have intrinsic immunostimulatory properties. In principle, heterologous antigens incorporated into OMVs will elicit specific immune responses, especially if presented at the vesicle surface and thus optimally exposed to the immune system. In this study, we explored the feasibility of our recently developed autotransporter Hbp platform, designed to efficiently and simultaneously display multiple antigens at the surface of bacterial OMVs, for vaccine development. Using two Streptococcus pneumoniae proteins as model antigens, we showed that intranasally administered Salmonella OMVs displaying high levels of antigens at the surface induced strong protection in a murine model of pneumococcal colonization, without the need for a mucosal adjuvant. Importantly, reduction in bacterial recovery from the nasal cavity was correlated with local production of antigen-specific IL-17A. Furthermore, the protective efficacy and the production of antigen-specific IL-17A, and local and systemic IgGs, were all improved at increased concentrations of the displayed antigen. This discovery highlights the importance of an adequate antigen expression system for development of recombinant OMV vaccines. In conclusion, our findings demonstrate the suitability of the Hbp platform for development of a new generation of OMV vaccines, and illustrate the potential of using this approach to develop a broadly protective mucosal pneumococcal vaccine. © 2015 Elsevier Ltd.

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