Delivery of Chicken Egg Ovalbumin to Dendritic Cells by Listeriolysin O-Secreting Vegetative Bacillus Subtilis
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J. Microbiol. Biotechnol. (2018), 28(1), 122–135 https://doi.org/10.4014/jmb.1706.06057 Research Article Review jmb Delivery of Chicken Egg Ovalbumin to Dendritic Cells by Listeriolysin O-Secreting Vegetative Bacillus subtilis Katarzyna Roeske*, Radosław Stachowiak, Tomasz Jagielski, Michał Kamiński, and Jacek Bielecki Department of Applied Microbiology, Faculty of Biology, University of Warsaw, 02-096 Warsaw, Poland Received: June 28, 2017 Revised: October 14, 2017 Listeriolysin O (LLO), one of the most immunogenic proteins of Listeria monocytogenes and its Accepted: October 22, 2017 main virulence factor, mediates bacterial escape from the phagosome of the infected cell. Thus, First published online its expression in a nonpathogenic bacterial host may enable effective delivery of heterologous January 24, 2018 antigens to the host cell cytosol and lead to their processing predominantly through the *Corresponding author cytosolic MHC class I presentation pathway. The aim of this project was to characterize the Phone: +48-22-5541312; delivery of a model antigen, chicken egg ovalbumin (OVA), to the cytosol of dendritic cells by Fax: +48-22-5541404; E-mail: [email protected] recombinant Bacillus subtilis vegetative cells expressing LLO. Our work indicated that LLO produced by non-sporulating vegetative bacteria was able to support OVA epitope presentation by MHC I molecules on the surface of antigen presenting cells and consequently influence OVA-specific cytotoxic T cell activation. Additionally, it was proven that the genetic context of the epitope sequence is of great importance, as only the native full-sequence OVA fused to the N-terminal fragment of LLO was sufficient for effective epitope delivery and activation of CD8+ lymphocytes. These results demonstrate the necessity for further verification of the fusion antigen potency of enhancing the MHC I presentation, and they prove that LLO-producing B. subtilis may represent a novel and attractive candidate for a pISSN 1017-7825, eISSN 1738-8872 vaccine vector. Copyright© 2018 by The Korean Society for Microbiology Keywords: Antigen delivery, Bacillus subtilis, cellular response, listeriolysin O, vaccine design and Biotechnology Introduction Different strategies can be used to deliver antigens to the cytosol of APCs, such as using (i) nanoparticles : liposomes, Cytotoxic CD8+ T cell lymphocytes (CTLs) play an immune-stimulating complexes, or nucleoproteins, (ii) important role in the eradication of many intracellular viruses, or (iii) pathogenic and nonpathogenic bacteria [4]. pathogens, as well as in the destruction of cancer cells [1]. Among them, the use of bacterial carriers constitutes The interaction of the T cell receptor of the lymphocytes probably the most studied and the most promising (TCR) with major histocompatibility complex class I (MHC strategy. Both attenuated and commensal microorganisms class I) molecules located at the surface of the antigen- are used in the next-generation vaccine design; however, presenting cells (APCs), such as dendritic cells (DCs) or owing to the potential risk of conversion to a virulent strain macrophages, provide the specificity of the interaction and causing infection or the loss of immunogenic potency, between those two types of cells. After recognizing the pathogenic vectors are less likely to be used [5]. Bacillus MHC I-peptide complexes of infected cells and receiving subtilis, a gram-positive nonpathogenic soil bacterium, has costimulation from the CD4+ T lymphocytes, CTLs become been widely explored as a host for the expression of foreign activated, leading to removal of the target cells by apoptosis proteins with pharmacological and immunological activities and release of membrane-perforating granzymes and [6-8], which is due to (i) its lack of an outer membrane, perforins [2]. More importantly, a population of memory allowing efficient transport of secretion proteins outside of T cells is generated, leading to resistance to a pathogen the cell, (ii) available knowledge on the genetics and physiology whose antigen was presented [3]. of this species, (iii) its well-established nonpathogenicity, J. Microbiol. Biotechnol. Bacillus subtilis Vegetative Cells and Antigen Delivery 123 (iv) production of spores, enabling survival in adverse adjuvant candidate [22-24]. Another feature of LLO that conditions, and (v) the low costs of culturing. B. subtilis has supports its use in delivering antigens to the cytoplasm of been successfully explored as a host for the expression of host cells is the presence of a PEST-like sequence (P, Pro; E, foreign proteins with immunological activities [9, 10]. Glu; S, Ser; T, Thr) in the structure of the toxin. This N- Genetically modified B. subtilis spores have been also engaged terminal 26-amino-acid sequence was previously thought in a presentation of antigens fused to spore coat proteins, to mediate proteasomal degradation by the host, thereby such as fragments of FliD flagellar cap protein of Clostridium limiting the activity of the cytolysin to the vacuole and tetani [11], UreA protein of Helicobacter acinonychis [12], or protecting the host cell from damage [25]. However, this VP28 protein of white spot syndrome virus [13]. hypothesis has since been retracted by the authors as they Despite the confirmed immunogenicity of such vaccines, proved that the PEST-like region mediates LLO cytotoxicity there are problems that need to be resolved, such as the low by influencing the amount of protein that is synthesized or levels of systemic humoral and cellular response, particularly secreted by the bacteria when they are growing in the host when administering orally [14-16]. It may be caused by a cytosol [26]. The immunogenic potential of this sequence widespread presence of spores in the environment and was examined by Sewell et al. [24], who demonstrated that their structure devoid of the molecular patterns that elicit a the coexpression of the antigen E7 of HPV-16 enhances the proinflammatory response in mammals. These features, immunogenicity of L. monocytogenes and leads to tumor along with a lack of invasiveness, make it necessary to regression in a mouse model. However, studies on the repeat the vaccination and to administer extremely high ability of the fusion antigens carrying the PEST-like sequence doses of bacteria [14]. A more promising perspective in produced by nonpathogenic bacterial vectors to induce bacterial vaccine development is using vegetative non- CTLs still have to be carried out. sporulating strains, as only vegetative recombinant strains In this study, we constructed a set of strains to test are able to induce a strong CD8+ lymphocyte response [17]. whether non-sporulating vegetative B. subtilis could be a The immunogenicity of nonpathogenic B. subtilis-based novel vector for delivery of a model antigen directly to the vectors can be increased by expression of virulence cytosol of APCs. The new constructions were based on determinants of intracellular pathogens. Among the proteins previous knowledge on the immunological potency of both that can lead to bacterial cell invasion and escape from the listeriolysin O and its N-terminal PEST-like sequence [24, 25], vacuole to the cytoplasm of the host cell, there are virulence as well as on the availability of molecular tools allowing determinants of an intracellular pathogen, Listeria detection of the OVA-peptide presentation and OVA- monocytogenes. To facilitate the presentation of the antigen specific T cell activation. Ovalbumin is one of the most in a complex with MHC I molecules and induction of the frequently used model antigens in research to demonstrate response of CD8+ T cells, the antigen should be delivered antigen delivery and study immune response or anticancer directly to the cytoplasm of the host cell, which can be therapies [27-29]. We evaluated whether fusions of the provided by the activity of listeriolysin O (LLO). L. monocytogenes full-length LLO or its N-terminal sequence LLO, a member of the cholesterol-dependent cytolysin could improve the secretion of a model antigen (OVA) or family of bacterial toxins, is a major virulence factor of its epitope SIINFEKL in B. subtilis. We also hypothesized L. monocytogenes and was originally shown to be crucial for that by the pore-forming activity of LLO, the cytoplasmic bacterial escape from the internalization vacuole after entry delivery to APCs would be facilitated, thereby leading to into eukaryotic cells [18]. Bacterial translocation to the effective OVA antigen presentation by MHC I complexes cytosol mediated by LLO results in presentation of the and subsequent activation of OVA-specific cytotoxic T cells. antigen through the MHC I pathway. Both CD4+ and CD8+ lymphocytes are induced, thereby leading to a complex Materials and Methods cell-mediated response to the main listerial antigens [19]. LLO activity provides access of the accompanying antigens Construction of LLO-OVA B. subtilis Mutant Strains to the cytosolic compartment and entry to the presentation All constructions encoding the LLO-OVA fusions of different pathway through transporter associated with antigen construction were prepared on the basis of pUR10, an LLO- presentation (TAP). Such coexpression has been widely encoding integration plasmid and derivative of pAG58-1-ble. The investigated in research on live vaccine vectors [20, 21]. original integrative vector was modified as follows: the nonessential sequence was removed in order to reduce the vector size and enable Interestingly, LLO is