A Monoclonal Antibody against the C-Terminal Domain of Bacillus cereus Hemolysin II Inhibits HlyII Cytolytic Activity.

Natalia Rudenko,Zhanna Andreeva-Kovalevskaya,Alexander Solonin,Alexander Siunov,Alexey Nagel,Bogdan Melnik,Anna Zamyatina,Vadim Salyamov,Khanafiy Boziev,Anna Karatovskaya,Fedor Brovko,Alexander Kolesnikov,Anna Shepelyakovskaya

doi:10.3390/toxins12120806

Abstract

Bacillus cereus is the fourth most common cause of foodborne illnesses that produces a variety of pore-forming proteins as the main pathogenic factors. B. cereus hemolysin II (HlyII), belonging to pore-forming β-barrel toxins, has a C-terminal extension of 94 amino acid residues designated as HlyIICTD. An analysis of a panel of monoclonal antibodies to the recombinant HlyIICTD protein revealed the ability of the antibody HlyIIC-20 to inhibit HlyII hemolysis. A conformational epitope recognized by HlyIIC-20 was found. by the method of peptide phage display and found that it is localized in the N-terminal part of HlyIICTD. The HlyIIC-20 interacted with a monomeric form of HlyII, thus suppressing maturation of the HlyII toxin. Protection efficiencies of various B. cereus strains against HlyII were different and depended on the epitope amino acid composition, as well as, insignificantly, on downstream amino acids. Substitution of L324P and P324L in the hemolysins ATCC14579T and B771, respectively, determined the role of leucine localized to the epitope in suppressing the hemolysis by the antibody. Pre-incubation of HlyIIC-20 with HlyII prevented the death of mice up to an equimolar ratio. A strategy of detecting and neutralizing the toxic activity of HlyII could provide a tool for monitoring and reducing B. cereus pathogenicity.

Highlights

Bacillus cereus is the fourth most common cause of foodborne illnesses [1]
B. cereus hemolysin II (HlyII), using to which the HlyIICTD is shown to be capable of independently binding erythrocyte membranes
The affinity constants characterizing the interaction of this antibody with HlyIICTD, intact HlyII14579 and HlyII771 were determined by indirect ELISA

Summary

Introduction

Bacillus cereus is the fourth most common cause of foodborne illnesses [1]. Modern phylogeny singles out nine species in seven phylogenetic clades in the B. cereus group [2]. B. cereus hemolysin II (HlyII) belongs to pore-forming β-barrel toxins [7,8,9,10]. The hemolysin II gene is found in all clades of B. cereus sensu lato [11], but is more common among various natural isolates of Bacillus thuringiensis [12]. This places HlyII in the focus of the study, since B. thuringiensis is widely used as a biological insecticide for plant protection [6]. The HlyII protein possesses a C-terminal extension of 94 amino acid residues designated as HlyIICTD (C-terminal domain) [7,14] previously undescribed for this class of toxins. HlyII∆C shows an 8-fold lower hemolytic activity in affecting rabbit erythrocytes [14]; monoclonal antibodies (mAbs) against HlyIICTD proved useful in revealing the CTD role at various stages of pore formation

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Conclusion