Abstract
Listeria monocytogenes is a facultative intracellular pathogen that secretes the cytolysin listeriolysin O (LLO), which enables the bacteria to cross the phagosomal membrane. L. monocytogenes regulates LLO activity in the phagosome and minimizes its activity in the host cytosol. Mutants that fail to compartmentalize LLO activity are cytotoxic and have attenuated virulence. Here, we showed that residues N478 and V479 of LLO are required for LLO hemolytic activity and bacterial virulence. A single N478A mutation (LLON478A) significantly increased the hemolytic activity of LLO at a neutral pH, while no difference was observed at the optimum acidic pH, compared with wild-type LLO. Conversely, the mutant LLOV479A exhibited lower hemolytic activity at the acidic pH, but not at the neutral pH. The double mutant LLON478AV479A showed a greater decrease in hemolytic activity at both the acidic and neutral pHs. Interestingly, strains producing LLON478A or LLOV479A lysed erythrocytes similarly to the wild-type strain. Surprisingly, bacteria-secreting LLON478AV479A had barely detectable hemolytic activity, but exhibited host cell cytotoxicity, escaped from the phagosome, grew intracellularly, and spread cell-to-cell with the same efficiency as the wild-type strain, but were highly attenuated in virulence in mice. These data demonstrate that these two residues are required for LLO hemolytic activity and pathogenicity in mice, but not for escape from the phagosome and cell-to-cell spreading. The finding that the nearly non-hemolytic LLON478AV479A mutant grew intracellularly indicates that mutagenesis of a virulence determinant is a novel approach for the development of live vaccine strains.
Highlights
The Gram-positive, facultative intracellular bacterium Listeria monocytogenes is the causative agent of listeriosis, a severe foodborne infection with a high mortality rate [1]
Its ability to lyse erythrocytes was completely impaired, suggesting that the deleted 12 residues might be required for listeriolysin O (LLO) cytolytic activity, which prompted us to further investigate which single amino acid among these 12 residues plays a decisive role in modulating the LLO activity
We found that among all the LLO mutants, only the mutant proteins LLON478AV479AY480A and LLON478AV479A completely lost their hemolytic activity, while the others lysed erythrocytes as efficiently as wild-type LLO (Figure 1C), suggesting that residues N478 and V479 are critical for LLO activity
Summary
The Gram-positive, facultative intracellular bacterium Listeria monocytogenes is the causative agent of listeriosis, a severe foodborne infection with a high mortality rate [1]. This pathogen is ubiquitous, and it has been isolated from humans and animals, as well as from raw and ready-to-eat foods [2, 3], and it is capable of invading a wide variety of eukaryotic cells, including endothelial cells and macrophages [4, 5]. LLO belongs to the family of cholesterol-dependent cytolysins (CDCs) that are secreted by many pathogenic Gram-positive bacteria [9], and CDCs are the largest family among the bacterial pore-forming toxins [10, 11]. All CDCs are secreted as soluble monomers by their cognate bacteria and are characterized by their ability to bind to the cholesterol of host membranes and form large pores [8]
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