Tn916-induced mutations in the hemolysin determinant affecting virulence of Listeria monocytogenes

Abstract

A genetic determinant essential for hemolysin production by Listeria monocytogenes has been inactivated by insertion of transposon Tn916 into L. monocytogenes DNA. The transposon was transferred by means of conjugation of a streptomycin-resistant L. monocytogenes recipient strain with Streptococcus faecalis CG110 on membrane filters. Among the tetracycline-resistant transconjugants, mutants were detected which had lost hemolytic activity. When tested in a mouse model, these mutants appeared to have lost the virulence that characterizes the parental strain. An extracellular protein of 58,000 apparent molecular weight was eliminated in the nonhemolytic mutants. In some of the mutants, the decrease in the production of the 58,000-dalton protein was accompanied by the production of a new protein of 49,000 apparent molecular weight. Hemolytic revertants regained the hemolytic phenotype and virulence and produced the extracellular protein that characterizes the recipient strain. Hybridization studies with Tn916 DNA indicated that the transposon is present in EcoRI and HindIII fragments of the nonhemolytic mutants. Single copies of Tn916 were detected in the chromosomal DNA of two of the three nonhemolytic mutants that were studied in detail. In hemolytic, tetracycline-sensitive revertants Tn916 appeared to be completely excised from the chromosome.

Eukaryotic expression plasmid transfer from the intracellular bacteriumListeria monocytogenesto host cells

“…In addition, the bacterium secretes listeriolysin, a poreforming toxin, that allows its efficient escape from the phagolysosome into the cytosol of the infected cell (Kathariou et al, 1987;Cossart et al, 1989;Jacobs et al, 1998;. Within this compartment, L. monocytogenes harnesses components of the host cell cytoskeleton for intracellular motility and cell-to-cell spread (Chakraborty and Wehland 1997).…”

The arsenal of virulence factors deployed byListeria monocytogenesto promote its cell infection cycle

“…116 L. monocytogenes mutants deficient in LLO have difficulty to reach the host cell cytoplasm and are less virulent. 113,[117][118][119][120] The activity of LLO is optimal at the acidic pH of the phagocytic vacuole and decreases at the neutral pH of the cytoplasm, preventing excessive host cell damage. 121 LLO binds to membranes as monomers that oligomerize into pore-forming complexes.…”

Sterol and pH Interdependence in the Binding, Oligomerization, and Pore Formation of Listeriolysin O

“…As an important control, the binding of 25 nM LLO to the L1 chip alone was checked and found to be negligible. 2 That was an important control to check, because when the dextran layer with hydrophobic anchors is partially exposed the analyte may bind to them, and this can complicate data analysis.…”

“…After the entry into the cell by phagocytosis, it needs to escape from the vacuole into the cytosol for efficient growth and spread to other cells. This is achieved by secretion of the pore-forming toxin (PFT) 1 listeriolysin O (LLO), which destroys the phagosomal membrane and is thus the most critical factor for the escape from phagocytic vacuoles (2)(3)(4)(5)(6).…”