Truncated Hemolysin II and Cytotoxin K2 Forms of Bacillus cereus

Objective: The β-pore-forming toxins hemolysin II (HlyII) and cytotoxin K2 (CytK2) are important pathogenic factors of the opportunistic bacterium Bacillus cereus and are secreted as monomers that can oligomerize in the presence of a target cell to form transmembrane channels. Analysis of the nucleotide sequence of HlyII and CytK2 suggests the possibility of truncated forms of these proteins that was confirmed by immunochemical, and chromatography-mass spectroscopic analyzes. Methods: HlyII and CytK2 were expressed in recombinant strains of E. coli BL21(DE3) carrying plasmids pET29 which produced the intracellular proteins HlyII and CytK2 from B. cereus ATCC14579^T. In the case of HlyII, fractions containing soluble intracellular proteins, periplasmic proteins, and cellular debris were obtained and analyzed using monoclonal antibodies. The truncated protein forms HlyII and CytK2 were analyzed by chromatography-mass spectrometry analysis. Results and Discussion: Analysis of the primary amino acid sequences of HlyII and CytK2 revealed the presence of potential internal translation initiation sites within the sequence. The presence of the truncated forms of HlyII was confirmed by immunochemical analysis using monoclonal antibodies and by chromatography-mass spectrometry. During expression of the CytK2 protein in E. coli cells, a protein fragment was identified and excised from a polyacrylamide gel, the identity of which with the CytK2 protein was confirmed by chromatography-mass spectrometry analysis. Conclusions: The results obtained confirm the general concept that the bacterial genome is capable of encoding a wider range of proteins than the number of genes it contains. The opportunistic bacterium B. cereus is often the cause of nosocomial infections, one of its key virulent factors are hemolysin II and cytotoxin K2. Using monoclonal antibodies and mass spectrometric analysis it has been shown that these hemolytic enzymes are capable of forming shortened forms.