Attenuated mutants of the intracellular bacterium Listeria monocytogenes obtained by single amino acid substitutions in listeriolysin O

Abstract

Listeriolysin O (LLO), a major virulence factor of the intracellular bacterium Listeria monocytogenes, shares with other known 'thiol-activated toxins' a conserved undecapeptide, ECTGLAWEWWR, located in the C-terminal region of the protein and containing the unique cysteine of the molecule. Single amino acid substitutions were created in this region to study the role of cysteine and tryptophan residues in the lytic activity of LLO as well as in the virulence of the bacterium. Transformation of a transposon-induced non-haemolytic mutant with plasmids carrying the mutated genes allowed allele exchange and transfer of mutations on to the chromosome by in vivo recombination. The mutant strains secreted a full-length 59 kilodalton LLO. A decrease of 25% in the haemolytic activity in culture supernatants was observed in the case of mutation Cys-484 to Ala and of 80% for mutation Cys-484 to Ser. Mutations Trp-491 and Trp-492 to Ala decreased activity by, respectively, 95% and 99.9%. LLOs produced by the mutants, as the wild type, were active at low pH, inhibited by cholesterol, and able to bind to cell membranes. A close relationship was found between virulence of mutants in the mouse model and haemolytic activity in their culture supernatants. These results demonstrate that the thiol group of Cys-484 is not essential for either haemolytic activity in vitro or virulence in vivo. In contrast, Trp-492 appears to be required for both haemolytic activity and virulence. The finding that the nearly non-haemolytic mutant Trp-492-Ala persisted in the spleen for several days after inoculation indicates that mutagenesis of a virulence determinant can attenuate virulence and provides a novel approach to the development of live vaccine strains.

Pneumococcal proteins and the pathogenesis of disease caused by Streptococcus pneumoniae

“…The role of the unique cysteine in pneumolysin was probed using sitedirected mutagenesis to change the single cysteine to alanine and, surprisingly, the modified toxin retained full cytolytic and cytotoxic activity . Similar observations have been made with SLO (Pinkney et al, 1989) and listeriolysin (Michel et al, 1990). Thus there is no obvious role for the thiol group in the in vitro activity of pneumolysin, although it remains possible that the thiol group is important in vivo.…”

“…A similar result was obtained when tryptophans at positions 433 and 435 in pneumolysin were changed independently to phenylalanine . Similar findings were made with SLO (Pinkney et al, 1989) and listeriolysin (Michel et al, 1990). Thus the cysteine-containing motif appears to participate in the generation of the functional lesion in toxin-treated membranes after binding and oligomerization.…”

Listeriolysin genes: complete sequence of ilo from Listeria ivanovii and of lso from Listeria seeligeri

“…molysin; Ul streptolysin 0 [15] and also Iisteriolysin 0 (LLOl from L. monocytogenes [14]. Our results show that lLO contains two cysteine residues, one in the conserved region and another one 26 amino acids distal from there (position 509 in Figs.…”

“…monocytogenes [14]. Although the normally conserved alanine is replaced in LSO by another hydrophobic, nonpolar, uncharged amino acid, the bulky aromatic ring of phenylalanine might have some detrimental effect on the hemolytic activity of LSO, compared to LLO.…”

Listeria monocytogenes transiently alters mitochondrial dynamics during infection

“…3A), indicating that LLO is required for fragmentation of host-cell mitochondria. Bacteria whose hemolysin (hly) gene carries a point mutation disrupting pore formation (38) did not affect mitochondrial morphology ( Fig. 3B), revealing that fragmentation depends on the pore-forming ability of LLO.…”