Roles of the PEST-like sequence of Listeriolysin O from Listeria monocytogenes in activating ERK1/2 phosphorylation

Abstract:

[Objective] The determinant virulence factor Listeriolysin O (LLO) of Listeria monocytogenes, a foodborne pathogen, contains a unique N-terminal amino acid sequence that is absent in other cytolysins and was previously referred as the PEST-like sequence (containing three putative phosphorylation sites, S44, S48, and T51). We here, therefore, aimed to explore the biological roles of the PEST-like sequence in LLO-induced ERK1/2 kinases phosphorylation in human epithelial cells (Caco-2). [Methods] The plasmid for expressing the recombinant LLO was constructed and transformed into E. coli Rosetta, and the his-tagged soluble protein was purified using the nickel-chelated affinity column chromatography. The LLO variants (LLO_ΔPEST, LLO_S44A, LLO_S48A, and LLO_T51A) were then obtained by using the site-directed mutagenesis strategy and expressed as above for LLO. The hemolytic activity for these recombinant proteins was assessed by lysis the erythrocytes, and moreover, effects of LLO or its variants on ERK1/2 kinases phosphorylation in Caco-2 cells was detected by using the Western blotting method. [Results] The results in the present study showed that the recombinant LLO, as well as the four LLO variants were able to lysis the erythrocytes at pH 5.5 and pH 7.4, suggesting that the PEST-like sequence was not required for the pore-forming activity of LLO. Besides, treatment of the LLO or its variants at the cytolytic concentration of 5 nmol/L could significantly induce ERK1/2 kinases phosphorylation in Caco-2 cells. [Conclusion] Our data collectively showed that the PEST-like sequence was not necessary for the LLO-mediated perforation ability on host membranes and not required for the LLO-triggered ERK1/2 signaling, which laid the foundation for further exploration of the potential roles of this motif during L. monocytogenes infection.