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Binding to The Target Cell Surface Is The Crucial Step in Pore Formation of Hemolysin BL from Bacillus cereus.

ABSTRACT: A major virulence factor involved in Bacillus cereus food poisoning is the three-component enterotoxin hemolysin BL. It consists of the binding component B and the two lytic components L₁ and L₂. Studying its mode of action has been challenging, as natural culture supernatants additionally contain Nhe, the second three-component enterotoxin, and purification of recombinant (r) Hbl components has been difficult. In this study, we report on pore-forming, cytotoxic, cell binding and hemolytic activity of recently generated rHbl components expressed in E. coli. It is known that all three Hbl components are necessary for cytotoxicity and pore formation. Here we show that an excess of rHbl B enhances, while an excess of rHbl L₁ hinders, the velocity of pore formation. Most rapid pore formation was observed with ratios L₂:L₁:B = 1:1:10 and 10:1:10. It was further verified that Hbl activity is due to sequential binding of the components B - L₁ - L₂. Accordingly, all bioassays proved that binding of Hbl B to the cell surface is the crucial step for pore formation and cytotoxic activity. Binding of Hbl B took place within minutes, while apposition of the following L₁ and L₂ occurred immediately. Further on, applying toxin components simultaneously, it seemed that Hbl L₁ enhanced binding of B to the target cell surface. Overall, these data contribute significantly to the elucidation of the mode of action of Hbl, and suggest that its mechanism of pore formation differs substantially from that of Nhe, although both enterotoxin complexes are sequentially highly related.

SUBMITTER: Jessberger N

PROVIDER: S-EPMC6563250 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Binding to The Target Cell Surface Is The Crucial Step in Pore Formation of Hemolysin BL from Bacillus cereus.

Jessberger Nadja N Dietrich Richard R Schwemmer Stefanie S Tausch Franziska F Schwenk Valerie V Didier Andrea A Märtlbauer Erwin E

Toxins 20190520 5

A major virulence factor involved in Bacillus cereus food poisoning is the three-component enterotoxin hemolysin BL. It consists of the binding component B and the two lytic components L1 and L2. Studying its mode of action has been challenging, as natural culture supernatants additionally contain Nhe, the second three-component enterotoxin, and purification of recombinant (r) Hbl components has been difficult. In this study, we report on pore-forming, cytotoxic, cel ...[more]

PMID: 31137585

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Project description:BackgroundBacillus cereus group isolates that produce diarrheal or emetic toxins are frequently isolated from raw milk and, in spore form, can survive pasteurization. Several species within the B. cereus group are closely related and cannot be reliably differentiated by established taxonomical criteria. While B. cereus is traditionally recognized as the principal causative agent of foodborne disease in this group, there is a need to better understand the distribution and expression of different toxin and virulence genes among B. cereus group food isolates to facilitate reliable characterization that allows for assessment of the likelihood of a given isolate to cause a foodborne disease.ResultsWe performed whole genome sequencing of 22 B. cereus group dairy isolates, which represented considerable genetic diversity not covered by other isolates characterized to date. Maximum likelihood analysis of these genomes along with 47 reference genomes representing eight validly published species revealed nine phylogenetic clades. Three of these clades were represented by a single species (B. toyonensis -clade V, B. weihenstephanensis - clade VI, B. cytotoxicus - VII), one by two dairy-associated isolates (clade II; representing a putative new species), one by two species (B. mycoides, B. pseudomycoides - clade I) and four by three species (B. cereus, B. thuringiensis, B. anthracis - clades III-a, b, c and IV). Homologues of genes encoding a principal diarrheal enterotoxin (hemolysin BL) were distributed across all, except the B. cytotoxicus clade. Using a lateral flow immunoassay, hemolysin BL was detected in 13 out of 18 isolates that carried hblACD genes. Isolates from clade III-c (which included B. cereus and B. thuringiensis) consistently did not carry hblACD and did not produce hemolysin BL. Isolates from clade IV (B. cereus, B. thuringiensis) consistently carried hblACD and produced hemolysin BL. Compared to others, clade IV was significantly (p = 0.0001) more likely to produce this toxin. Isolates from clade VI (B. weihenstephanensis) carried hblACD homologues, but did not produce hemolysin BL, possibly due to amino acid substitutions in different toxin-encoding genes.ConclusionsOur results demonstrate that production of diarrheal enterotoxin hemolysin BL is neither inclusive nor exclusive to B. cereus sensu stricto, and that phylogenetic classification of isolates may be better than taxonomic identification for assessment of B. cereus group isolates risk for causing a diarrheal foodborne disease.

Dataset Information

Binding to The Target Cell Surface Is The Crucial Step in Pore Formation of Hemolysin BL from Bacillus cereus.

Publications

Binding to The Target Cell Surface Is The Crucial Step in Pore Formation of Hemolysin BL from <i>Bacillus cereus</i>.

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