Development of antigen-delivery systems, based on the Escherichia coli hemolysin secretion pathway

doi:10.1016/S0378-1119(96)00424-6

Gene

Volume 179, Issue 1, 1996, Pages 133-140

https://doi.org/10.1016/S0378-1119(96)00424-6 Get rights and content

Abstract

We describe the development of plasmid vectors carrying the expression sites, an hlyA cassette and the secretion genes of Escherichia coli hemolysin. These allow the synthesis and secretion of heterologous microbial antigens in E. coli and attenuated Salmonella aroA strains. Genes or gene fragments encoding microbial antigens are inserted in-frame into a residual part of the hlyA gene which essentially encodes the HlyA secretion signal (HlyA₈). In general, the fused genes, carrying the hlyA_s sequence at the 3' terminus, are efficiently expressed, and the synthesized antigens are secreted into the culture supernatant of the producing strain. Attenuated Salmonella strains synthesizing either HlyA_s-fused listeriolysin or p60 of Listeria monocytogenes were constructed by this procedure and shown to provide protective immunity against L. monocytogenes in mice. The most effective protection was obtained when these microbial antigens were secreted by the attenuated Salmonella strains. We further present new approaches which may allow the application of this antigen-delivery system to any microbial antigen.

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The early successful examples of in vitro functionality of Hly secretion system in different Gram-negative vaccine microorganisms include Salmonella serotypes, Shigella spp, V. cholerae, enterohaemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC), S. marcescens, and P. aeruginosa (Spreng et al., 1999; Hahn and von Specht, 2003). Various studies described the successful application of Hly secretion system in secretion of, delivery of, and immunization against different pathogens including bacteria (Gentschev et al., 1996; Spreng et al., 2000a), viruses (Spreng et al., 2000b) and parasites (Gómez-Duarte et al., 2001) using a carrier strain, mostly attenuated Salmonella strains in several model animals including mice, rabbit, and cattle. One of the most encouraging examples of vaccine development using T1SS was reported by Gentschev et al. in 2004.
Bacteria have evolved a diverse range of secretion systems to export different substrates across their cell envelope. Although secretion of proteins into the extracellular space could offer advantages for recombinant protein production, the low secretion titers of the secretion systems for some heterologous proteins remain a clear drawback of their utility at commercial scales. Therefore, a potential use of most of secretion systems as production platforms at large scales are still limited. To overcome this limitation, remarkable efforts have been made toward improving the secretion efficiency of different bacterial secretion systems in recent years.
Here, we review the progress with respect to biotechnological applications of type I secretion system (T1SS) of Gram-negative bacteria. We will also focus on the applicability of T1SS for the secretion of heterologous proteins as well as vaccine development. Last but not least, we explore the employed engineering strategies that have enhanced the secretion efficiencies of T1SS. Attention is also paid to directed evolution approaches that may offer a more versatile approach to optimize secretion efficiency of T1SS.
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Repeat-in-ToXin (RTX) toxins are pathogenic factors produced by a wide variety of Gram-negative bacteria. These toxins are commonly known as hemolysins or leukotoxins, as they are able to form pores in the membrane of their eukaryotic target cells. The most salient structural characteristic of RTX toxins is that they have tandem repetitions of nonapeptidic motifs that constitute specific calcium-binding sites. The RTX toxins are secreted by a type I secretion system (T1SS) and fold in the presence of calcium into their active cytolytic forms. RTX toxins mainly target the immune cells and contribute to the virulence of their bacterial host.
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Many virulence factors of Escherichia coli need to traverse the inner membrane, periplasm, and outer membrane to be effective. The bacterium has a variety of secretion systems to achieve this. Two of these, the type I secretion system (T1SS) and the type V secretion system (T5SS), are explored in this chapter. In E. coli the best-known T1SS is that used to secrete hemolysin, a pore-forming toxin found in both uropathogenic and enterohemorrhagic strains of E. coli. Many proteins are secreted via the T5SS in E. coli, with the proteins having a wide range of virulence functions. This chapter will focus on these two systems; the mechanism of biogenesis, the function of the secreted proteins and current attempts to use these systems in biotechnological applications.
The rate of folding dictates substrate secretion by the Escherichia coli hemolysin type 1 secretion system
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Citation Excerpt :
The export of the 107-kDa toxin HlyA by E. coli into the medium is impressive in terms of its size and secreted amounts (12). Consequently, the Hly system has attracted great interest as an alternative secretion system for biotechnological applications (13). Bypassing the periplasm by targeting the synthesized proteins directly to the external medium has major advantages, such as circumventing cellular toxicity, less proteolysis, and the ease of purification of the exported protein (14, 15).
Secretion of the Escherichia coli toxin hemolysin A (HlyA) is catalyzed by the membrane protein complex HlyB-HlyD-TolC and requires a secretion sequence located within the last 60 amino acids of HlyA. The Hly translocator complex exports a variety of passenger proteins when fused N-terminal to this secretion sequence. However, not all fusions are secreted efficiently. Here, we demonstrate that the maltose binding protein (MalE) lacking its natural export signal and fused to the HlyA secretion signal is poorly secreted by the Hly system. We anticipated that folding kinetics might be limiting secretion, and we therefore introduced the “folding” mutation Y283D. Indeed this mutant fusion protein was secreted at a much higher level. This level was further enhanced by the introduction of a second MalE folding mutation (V8G or A276G). Secretion did not require the molecular chaperone SecB. Folding analysis revealed that all mutations reduced the refolding rate of the substrate, whereas the unfolding rate was unaffected. Thus, the efficiency of secretion by the Hly system is dictated by the folding rate of the substrate. Moreover, we demonstrate that fusion proteins defective in export can be engineered for secretion while still retaining function.
A novel multivalent vaccine based on secretary antigen-delivery induces protective immunity against Vibrio anguillarum and Aeromonas hydrophila
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In our previous work, four secretory antigen-delivery systems based on different signal peptides (SPs) (SP_hlyA, SP_rtxA, SP_vah3 and SP_empA) have been successfully established in attenuated Vibrio anguillarum. To investigate the potential application of the antigen-delivery systems in V. anguillarum multivalent vector vaccine, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from pathogenic Aeromonas hydrophila, as a putative protective antigen, was fused to the four delivery systems, respectively, and introduced into attenuated V. anguillarum strain MVAV6203 to get four GAPDH-delivery strains in this work. Immunodetection of GAPDH indicated that among the four constructs, the SP_empA-mediated GAPDH-delivery strain AV(pGap-empA) showed the highest level of GAPDH expression and secretion, and was determined as the multivalent vaccine candidate. Further immune protection evaluation of AV(pGap-empA) in turbot (Scophtalmus maximus) demonstrated that the vaccine candidate could induce efficient protection against V. anguillarum and A. hydrophila, suggesting that this vector vaccine had great potential in serving as a valuable multivalent live vaccine.
Improvement of the live vaccine strain Salmonella enterica serovar Typhi Ty21a for antigen delivery via the hemolysin secretion system of Escherichia coli
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The attenuated Salmonella enterica serovar Typhi strain Ty21a (Ty21a) is the only attenuated live oral vaccine against typhoid fever. Ty21a is also an attractive carrier for the delivery of heterologous antigens. We have used Ty21a for antigen delivery via the hemolysin (HlyA) secretion system of Escherichia coli, the prototype of the type I secretion system (T1SS). In this study, we identified by genetic complementation that the specific mutation of rpoS correlated with the hemolysin production of strain Ty21a. We furthermore showed that complementation with a plasmid encoding rfaH, which is described to be a downstream target of rpoS, led to increased expression and secretion of hemolysin. Finally, we demonstrated a significant enhancement of antibody responses against the heterologous HlyA antigen of Ty21a after immunization of mice with rfaH complemented S. typhi strain secreting HlyA compared with the same strain without rfaH plasmid.

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^☆: Presented at the Chulabhorn Research Institute International Conference on ‘Biotechnology Research and Applications for Sustainable Development (BRASD)’, Central Plaza Hotel, Bangkok, Thailand, 7–10 August 1995.

²: I.G. and H.M. contributed equally to this publication.

View full text

Development of antigen-delivery systems, based on the Escherichia coli hemolysin secretion pathway☆

Abstract

Trends Biotechnol.

Curr. Opin. Cell Biol.

Cell

Immunol. Today

Curr. Opin. Immunol.

Oral immunisation using live attenuated Salmonella spp. as carrier of foreign antigens

Clin. Microbiol. Rev.

Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in Gramnegative eubacteria

J. Bacteriol.

Internalin-mediated invasion of epithelial cells by Listeria monocytogenes is regulated by the bacterial growth state, temperature and the pleiotropic activator PrfA

Mol. Microbiol.

Topological and functional studies on HlyB of Escherichia coli

Mol. Gen. Genet.

Change in the cellular localization of alkaline phosphatase by alteration of its carboxyterminal

Mol. Gen. Genet.

Identification of p60 antibodies in human sera and presentation of this listerial antigen on the surface of attenuated Salmonellae by HlyB-HlyD secretion system

Infect. Immun.

Synthesis and secretion of bacterial antigens by attenuated Salmonella via the Escherichia coli hemolysin secretion system

Behring Inst. Mitt.

Salmonella secreting active listeriolysin changes its intracellular life style

Infect. Immun.

Mini-TnhlyAs: a new tool for the construction of secreted fusion proteins

Mol. Gene. Genet.

Processing by OmpT of fusion proteins carrying the HlyA transport signal during secretion by Escherichia coli hemolysin transport system

Mol. Gen. Genet.

Vaccination strategies against intracellular microbes

FEMS Immunol. Med. Microbiol.

Analysis of the haemolysin secretion system by PhoA-HlyA fusion proteins

Mol. Gen. Genet.

Listeria monocytogenes p60 supports host cell invasion by and in vivo survival of attenuated Salmonella typhimurium

Infect. Immun.

Superior efficacy of secreted over somatic antigen display in recombinant Salmonella vaccine induced protection against listeriosis

Selection for transport competence of C-terminal polypeptides derived from Escherichia coli hemolysin: the shortest peptide capable of autonomous HlyB-HlyD-dependent secretion comprises the C-terminal 62 amino acids of HlyA

Mol. Gen. Genet.

Salmonella typhimurium initiates murine infection by penetrating and destroying the specialized epithelial M cells of the Peyer's patches

J. Exp. Med.

Immunity to intracellular bacteria

Annu. Rev. Immunol.

Identification of individual amino acids required for secretion within the haemolysin (HlyA) C-terminal targeting region

Mol. Microbiol.

The gene for protein p60 of Listeria monocytogenes and its use as a specific probe for L. monocytogenes

Infect. Immun.

Isolation and analysis of the C-terminal signal directing export of Escherichia coli hemolysin across both bacterial membranes

EMBO J.

Mini-TnhlyA_s: a new tool for the construction of secreted fusion proteins