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[A new oxygen-labile hemolysin in Klebsiella pneumoniae]
I Albesa1, L I Barberis, M C Pájaro
1Departamento de Microbiología de Inmunología, Facultad de Ciences Exactas, Universidad Nacional de Río Cuarto, Provincia de Córdoba, Argentina.
Abstract:
The hemolytic activity of sixty K. pneumoniae strains was investigated in tryptic soy agar with rabbit, dog, sheep, human, chicken and mouse blood. All of them were lytic only for rabbit red cells. In liquid medium it was necessary a 2 mercaptoethanol treatment to detect a good degree of hemolysis. Cultures in tryptic soy broth gave 100% hemolysis in assays with rabbit erythrocytes and only when hemolysin was concentrated by purification was it active on dog and sheep but in a lesser degree (7.5% hemolysis). Supernatants of cultures were precipitated at different saline concentrations; the fraction obtained with 30-50% (NH4)2SO4 had hemolytic activity after dialysis and 2 mercaptoethanol treatment. Then this fraction was eluted in a Sephadex G-100 column, but electrophoresis in polyacrylamide gel showed that the hemolytic molecules obtained by gel filtration were contaminated with protein structures which had different electrophoretic migration. Ion-exchange chromatography showed best purification index and the recovery of activity was over 100%, it was possible to explain this good recovery once an inhibitor was detected. Two rabbit red cell lysins were purified, both shared several properties: SH-activation, pH optimum, thermolability, selectivity for rabbit red cells, mechanism of action, inhibition by cholesterol and divalent cations.
Insights
Klebsiella pneumoniae produces hemolysins that specifically lyse rabbit red blood cells. Purification revealed two active lysins, with activity enhanced by 2-mercaptoethanol and inhibited by cholesterol.
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- Klebsiella pneumoniae is an opportunistic pathogen known for its diverse virulence factors.
- Hemolytic activity is a significant virulence factor in many bacterial pathogens.
- Understanding the specific mechanisms of K. pneumoniae hemolysis can inform therapeutic strategies.
Purpose of the Study:
- To investigate the hemolytic activity of Klebsiella pneumoniae strains.
- To characterize the properties of K. pneumoniae hemolysins.
- To purify and identify the specific molecules responsible for hemolysis.
Main Methods:
- Culturing K. pneumoniae strains on various blood agars.
- Assessing hemolytic activity in liquid cultures with and without 2-mercaptoethanol.
- Purification of hemolysins using salting out, gel filtration (Sephadex G-100), and ion-exchange chromatography.
- Analysis of purified hemolysins using polyacrylamide gel electrophoresis.
- Characterization of hemolysin properties including activation, pH optimum, stability, and inhibition.
Main Results:
- Sixty K. pneumoniae strains exhibited hemolytic activity exclusively against rabbit red blood cells.
- Hemolytic activity was significantly enhanced by 2-mercaptoethanol treatment and concentration.
- Purification yielded two distinct rabbit red blood cell lysins.
- Purified lysins demonstrated SH-activation, pH optimum, thermolability, and selectivity for rabbit erythrocytes.
- Inhibition by cholesterol and divalent cations was observed, along with the detection of an inhibitor that explained >100% activity recovery.
Conclusions:
- Klebsiella pneumoniae produces specific hemolysins targeting rabbit erythrocytes.
- The purification process identified key properties of these lysins, including their requirement for SH-activation and susceptibility to inhibition.
- The presence of an inhibitor suggests a complex regulatory mechanism influencing hemolytic activity.