The hemolytic effect of Salmonella typhi Ty 2 porins

Abstract

Two outer membrane proteins of Salmonella typhi Ty 2 were extensively co-purified. According to their migration in dodecylsulfate/polyacrylamide gel electrophoresis and solubility characteristics, these proteins are homologous to the 35-kDa and 36-kDa porins found in Salmonella typhimurium. A porin homologous to the 34-kDa one has not been found in S. typhi Ty 2. A critical step in the purification of porins is heating at 100 "C in 2 sodium dodecyl sulfate before Sephadex gel filtration. The absence of detergent in aqueous suspensions enhances porin aggregation, these aggregations inducing human red cell lysis. Porins obtained by an alternative procedure consisting of heating at 60 'C instead of 1OO'C were also hemolytic. Using nanomolar concentration of porins a strong influence of temperature on the hemolytic effect was observed. Porin-induced hemolysis was inhibited with anti-porin serum, as well as by a treatment with phenylglyoxal, which reacts with thc arginine residues of proteins. The membrane-disrupting ability of porins aggregates might explain some pathogenic Characteristics of gramnegative bacterial infections.Outer-membrane surface components of gram-negative bacteria have been shown to be deeply involved in the interactions with the host cells during infection [l -41. Adherence to epithelial cells followed by penetration and invasion of the host systemic circulation are likely to be mediated by outer-membrane components of some gram-negative bacteria, although periplasmic enzymes might also contribute to the invasiveness of bacteria [5].Fluidity changes and increased hydrophobicity of epithelial cells are induced by bacterial surface components [5] and it is possible that outer-membrane proteins may also alter the permeability of the host cells. It has been shown that Escherichia coli, Salmonellu typhimurium and Proteus mirabilis porins [6-81 are capable of forming transmembrane permeability channels in artificial vesicle membranes and that this capability is dependent upon the formation of porin oligomers [7,9]. This property resembles the behaviour of alamethicin [lo], and other related peptide antibiotics that have channelforming ability [11 -141. As a result of this ability, alamethicin will also induce human red cell lysis [15], leukocyte lysis [16], increased calcium permeability of sarcoplasmic reticulum vesicles [ 171, uncovering of latent adenylate cyclase activity [18] and fusion of lipid vesicles [19].

Clinical isolate of a porinless Salmonella typhi resistant to high levels of chloramphenicol

“…sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (4). Furthermore, the cloning and sequencing of the S. typhi Ty2 ompC gene confirmed the close similarity among OmpC porins from members of the family Enterobacteriaceae (1,33,34).…”

“…SDS-PAGE was performed in 12% polyacrylamide slabs as described by Laemmli (14). All samples were heated at 100°C in the sample buffer before electrophoresis (4 hypothetical chloramphenicol-inactivating activity in strain 1895, as revealed by two bioassays.…”

“…3, lane c), an OmpF-S. typhi Ty2 spontaneous mutant selected by its resistance to carbenicillin (9). In addition, the mutant strain was resistant to chloramphenicol (4 drug per ml. The expression of E. coli OmpF was confirmed by SDS-PAGE (Fig.…”

Molecular cloning and expression in E. coli of a Salmonella typhi porin gene

“…Porin purification from S. typhi Ty2 was carried out as described by Calder6n ¢t al. [13]. This procedure results in the copurification of both porins.…”

“…In S. typhimurium, four porins have been reported: OmpF, OmpC, OmpD [4] and recently, a PhoE-like protein [12]. For S. typhi, the causal agent of typhoid fever, two porins of 35 and 36 kDa have been isolated [13] but no information regarding their gene structure is available. Since it has been demonstrated that S. typhi porins induce high titer antibodies in typhoid patients [14] and that S. typhimurium porins have a protective effect in mice under challenge infection [15,16], it appears very attractive to explore their use as a Correspondence address: A. Venegas, Laboratorio de Bioquimica, Pontificia Universidad Cat61ica de Chile, Casilla 114-D, Santiago, Chile diagnostic tool for typhoid fever and in the development of a vaccine for humans.…”