Hemolytic activity of Listeria monocytogenes at different pH and temperature.

VACA L; DI GENARO S; LACIAR A

Merlo, San Luis

Congreso; XXII Reunión anual de la Sociedad de Biología de Cuyo; 2004

Sociedad de Biología de Cuyo

Listeriolysin (LLO), produced by L. monocytogenes (Lm) strains is a 58 kDa hemolysin. LLO is an essential virulence factor, necessary for the intracellular survival of these bacteria. The intracellular environment might impose stress conditions similar to heat shock and pH on LLO hemolytic activity (HA). Lm CLIP 74902 strain was grown in CASO (casein peptone-soymeal peptone) broth at pH 5.5 and at pH 7.2, and incubated at 37 ºC for 6 h with shacking. Cultures were centrifuged and pellets resuspended in 100 mL CASO broth supplemented with 0.5 % yeast extract at pH 5.5 and 7.2, respectively. Both were preheated at 48 ºC and 37 ºC, and held for 1 h. Cells were centrifuged, resuspended in 5 mL EDTA, and sonicated for releasing total intracellular proteins (IP). The supernatant obtained was mixed with chilled acetone to precipite extracellular proteins (EP). A quantitative plate hemolysis assay was performed with 50 µL of protein sample and 100 µL of freshly prepared 3% HRBC. Plates were incubated for 6 h. The HA was determined by reading absorbencies at 545 nm. Assays were triplicated. IP-HA a 48 ºC and pH 7.2 was higher than at 5.5, while at 37 ºC they were similar at both pH at 37 ºC. Previous reports have described that pH 7.2 at 48 ºC had higher intracellular HA. Most likely, excreted LLO has accumulated in the cells during heat-shock.