SlyA, a regulatory protein from Salmonella typhimurium, induces a haemolytic and pore-forming protein in Escherichia coli

Abstract

A chromosomal fragment from Salmonella typhimurium, when cloned in Escherichia coli, generates a haemolytic phenotype. This fragment carries two genes, termed slyA and slyB. The expression of slyA is sufficient for the haemolytic phenotype. The haemolytic activity of E. coli carrying multiple copies of slyA is found mainly in the cytoplasm, with some in the periplasm of cells grown to stationary phase, but overexpression of SlyB, a 15 kDa lipoprotein probably located in the outer membrane, may lead to enhanced, albeit unspecific, release of the haemolytic activity into the medium. Polyclonal antibodies raised against a purified SlyA-HlyA fusion protein identified the overexpressed monomeric 17 kDa SlyA protein mainly in the cytoplasm of E. coli grown to stationary phase, although smaller amounts were also found in the periplasm and even in the culture supernatant. However, the anti-SlyA antibodies reacted with the SlyA protein in a periplasmic fraction that did not contain the haemolytic activity. Conversely, the periplasmic fraction exhibiting haemolytic activity did not contain the 17 kDa SlyA protein. Furthermore, S. typhimurium transformed with multiple copies of the slyA gene did not show a haemolytic phenotype when grown in rich culture media, although the SlyA protein was expressed in amounts similar to those in the recombinant E. coli strain. These results indicate that SlyA is not itself a cytolysin but rather induces in E. coli (but not in S. typhimurium) the synthesis of an uncharacterised, haemolytically active protein which forms pores with a diameter of about 2.6 nm in an artificial lipid bilayer. The SlyA protein thus seems to represent a regulation factor in Salmonella, as is also suggested by the similarity of the SlyA protein to some other bacterial regulatory proteins. slyA- and slyB-related genes were also obtained by PCR from E. coli, Shigella sp. and Citrobacter diversus but not from several other gram-negative bacteria tested.

The Rap and Hor proteins of Erwinia, Serratia and Yersinia: a novel subgroup in a growing superfamily of proteins regulating diverse physiological processes in bacterial pathogens

“…The first 17 amino acids of this protein were characteristic of an N-terminal sec-dependent signal sequence, with a recognition sequence (LVGC) for lipid modification and processing by signal peptidase II (Wu et al, 1983;Yamaguchi et al, 1988). Consistent with this, the predicted protein product shared a high level of sequence identity with Pcp Ye (71%), Pcp Hi (47%) and SlyB (71%), all of which have been classified previously as outer membrane lipoproteins from Yersinia enterocolitica, Haemophilus influenzae and Salmonella typhimurium respectively (Deich et al, 1988;Baumler and Hantke, 1992;Ludwig et al, 1995;Fig. 3).…”

“…2) with SlyA, from Salmonella typhimurium, originally classified as a haemolytic virulence factor (salmolysin) required for survival in murine macrophage (Libby et al, 1994). Recently, SlyA has been reclassified as a positive-acting regulatory protein able to induce the production of a cryptic haemolysin in various Escherichia coli strains (Ludwig et al, 1995;Oscarsson et al, 1996). The second of the complete ORFs, orf3 (468 bp) was predicted to encode a 15 540 Da protein of 155 amino acids.…”

Identification of amino acid residues of Salmonella SlyA that are critical for transcriptional regulation

“…SlyA of S. enterica has been identified as a member of the MarR/SlyA family, based on the similarity of its amino acid sequence to that of other members of this family (Ludwig et al, 1995;Thomson et al, 1997). Although the amino acid sequence similarity of this SlyA is relatively low, we demonstrated that the structure of this SlyA is very similar to the structures of the other MarR family proteins.…”

Identification and Characterization of an Amphipathic Leucine Zipper-like Motif in Escherichia coli Toxin Hemolysin E

“…It is interesting to mention that hemolysin E gene although found in non-pathogenic strains of E. coli, it remains silent under laboratory conditions. However, it is positively regulated by several regulatory proteins, which include SlyA and MprA (1,3,(5)(6)(7)(8)). This probably suggests that hemolysin E is expressed in response to appropriate environmental signals.…”

“…All these data indicate that HlyE similar to other hemolysins (13,14) is likely to be a significant virulence factor responsible for pathogenic infection in human. Experiments in lipid bilayer indicated that hemolysin E forms pores in membrane (4,7). This pore-forming activity of the toxin is considered to be associated with the infection of the pathogenic organism in the target cell.…”