Carboxyl-Terminal Residues N478 and V479 Required for the Cytolytic Activity of Listeriolysin O Play a Critical Role in Listeria monocytogenes Pathogenicity

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.

Listeriolysin O Pore-Forming Activity Is Required for ERK1/2 Phosphorylation During Listeria monocytogenes Infection

“…Recombinant LLO proteins used in this study were expressed as fusion proteins to the N-terminal His-tag using pET30a(+) as the expression vector (4). Briefly, the hly gene from EGDe genome was amplified and inserted into the pET30a(+) vector, and finally transformed into Rosetta competent cells.…”

“…Listeriolysin O (LLO) is a key determinant of L. monocytogenes pathogenesis, mediating vacuole degradation and escape. LLO is a member of the cholesterol-dependent cytolysins (CDCs), which is the largest family of bacterial pore-forming toxins (PFTs) produced by many pathogenic Gram-positive bacteria (4)(5)(6). LLO is a phagosome-specific cytolysin that forms pores in host membranes and is continuously expressed throughout the intracellular lifecycle of L. monocytogenes.…”

Deletion of glutaredoxin promotes oxidative tolerance and intracellular infection in Listeria monocytogenes

“…The sequences were aligned with the MUSCLE method by using CLC Sequence software. The phylogenetic tree was constructed with the Neighbor-Joining (NJ) method using 100 bootstrap replicates [16].…”

“…Intracellular multiplication assay was conducted according to our previous work [16]. The Caco-2 cells were infected with Listeria at MOI of 10 at 37°C for 1.5 h. Extracellular bacteria were then killed with 50 μg/mL gentamycin for 1.5 h, and incubated for an additional 6 h. Cells were washed gently with 10 mM PBS (pH 7.4), fixed with 4% paraformaldehyde and then permeabilized with 0.5% Triton X-100.…”

Asidik Koşullar Altında Listeria monocytogenes’in Glutamat Dekarboksilazlarının Asit Direnç Sistemlerine Katkılarının Değerlendirilmesi ve Farklı Rollerinin Araştırılması

“…GadD1, GadD2 and GadD3 were expressed as fusion proteins with His-tag using the expression vector pET30a (Invitrogen, U.S.A.) as previously shown [18] . The full-length gadD1, gadD2 and gadD3 were amplified with primer pairs listed in Table 3.…”