Hyperexpression of listeriolysin in the nonpathogenic species Listeria innocua and high yield purification

doi:10.1016/0168-1656(95)00138-7

Journal of Biotechnology

Volume 43, Issue 3, 15 December 1995, Pages 205-212

https://doi.org/10.1016/0168-1656(95)00138-7 Get rights and content

Abstract

Listeriolysin, the hemolysin of the pathogenic species Listeria monocytogenes, was expressed in the non-pathogenic species Listeria innocua. Coexpression of the positive regulatory factor prfA in the plasmid vector in conjunction with the structural gene hly increased the expression over 500-fold. Purification from supernatant fluids was achieved by two steps of ion exchange chromatography. The procedure resulted in over 60% yield of a hemolytically active, homogenous 58 kDa protein which was used to produce monospecific antibodies. As shown by immunoblot the purified listeriolysin was free of p60, a highly immunogenic protein of similar size also produced by Listeria spp., which otherwise would interfere with immunoassays. Listeriolysin retained full activity for more than 6 months at −70°C.

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Ruminants fed contaminated forage may shed Listeria monocytogenes in their faeces, and prolonged low daily doses of L. monocytogenes could cause listerial infection [Maijala, R., Lyytikainen, O., Autio, T., Aalto, T., Haavisto, L., Honkanen-Buzalski, T., 2001. Exposure of Listeria monocytogenes within an epidemic caused by butter in Finland. Int. J. Food Microbiol. 70, 97–109]. To compare listerial infection following single or repeated doses and the contamination of the environment with the excreted bacteria, ewes were orally inoculated with either 10⁴, 10⁶ or 10¹⁰ cfu L. monocytogenes once, or daily for 10 days. Serological responses were monitored with indirect ELISAs using recombinant listeriolysin O (LLO), internalin A (InlA) and internalin A-related protein (IrpA). The 24 inoculated animals displayed no symptoms, except for a transient hyperthermia in two animals given 10¹⁰ cfu. One ewe died on day 9 after non-listerial mastitis followed by listerial septicaemia. L. monocytogenes was recovered from day 1 post-inoculation until day 17 from the faeces of ewes inoculated with 10⁶ or 10¹⁰ cfu. No antibodies were detected in ewes given 10⁴ or 10⁶ cfu. Anti-LLO and anti-IrpA antibodies were detected from day 15 in animals inoculated with 10¹⁰ cfu, and this strengthened the conclusion that these long-lasting shedders were infected but asymptomatic carriers. An anti-InlA response was detected only at a very low level. These results suggest that repeated daily doses are no more effective than a single dose in causing infection in ewes.
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Regular paperHyperexpression of listeriolysin in the nonpathogenic species Listeria innocua and high yield purification

Abstract

Lancet

J. Biochem. Biophys. Methods

Res. Microbiol.

Cell

J. Chromatogr.

Effects of Listeria monocytogenes and Yersinia enterocolitica on cytokine gene expression and release from human polymorphonuclear granulocytes and epithelial cells (HEp2 cells)

Infect. Immun.

Damage to mammalian cells by proteins that form transmembrane pores

Rev. Physiol. Biochem. Pharmacol.

Bacillus subtilis expressing a haemolysin gene from Listeria monocytogenes can grow in mammalian cells

Nature

Listeriolysin O is a target of the immune response to Listeria monocytogenes

J. Exp. Med.

The homologous and heterologous regions within the iap gene allow genus- and species-specific identification of Listeria spp. by polymerase chain reaction

Appl. Environ. Microbiol.

Listeriolysin O is essential for virulence of Listeria monocytogenes: direct evidence obtained by gene complementation

Infect. Immun.

Detection of a prfAindependent promoter responsible for listeriolysin gene expression in mutant Listeria monocytogenes strains lacking the PrfA regulator

Infect. Immun.

Listeriolysin provides access to the MHC class I presentation pathway for soluble proteins in vitro and in vivo

Eur. J. Immunol.

In vitro model of penetration and intracellular growth of Listeria monocytogenes in the human enterocyte cell line Caco2

Infect. Immun.

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

Infect. Immun.

Purification, characterization, and toxicity of the sulfhydrylactivated hemolysin listeriolysin 0 from Listeria monocytogenes

Infect. Immun.

CD8 + T cells specific for a single nonamer epitope of Listeria monocytogenes are protective in vivo

J. Exp. Med.

Regular paper
Hyperexpression of listeriolysin in the nonpathogenic species Listeria innocua and high yield purification