Control of Autolysis in Pneumococci

Regulation of bacterial autolytic enzymes (cell wall hydrolases) is a highly spohisticated physiological task. Antibiotics such as pencicillin induce bacteriolysis by interfering with the control of the endogenous autolytic enzymes, indicating the major chemotherapeutic relevance of autolysins. Although the binding of antibiotics to cell wall synthetic enzymes has been very well characterized, it is unknown how this event leads to deregulation of autolytic enzymes. It is this aspect of antibiotic activity, revealed as the tolerant phenotype, that is the focus of this proposal. Bacteria which stop growing in response to penicillin but fail to lyse and die are termed tolerant. This property ensures bacterial survival and is the first step for most strains on the way to development of antibiotic resistance. Mechanistically, tolerance arises beyond the level of the drug binding to the vacteria (the site where resistance arises). Classically, tolerance has been induced in the lab by knock out of the lethal autolysin.
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Much of this physiology has been studied in pneumococci becuase it contains only one major autolytic enzyme, this providing a simple, model system from which to learn of elements in the autolytic cascade. Tolerance has also been described in clinical isolates of pneumococci, but, the mechanism of tolerance is unknown since the autolytic enzyme is present and functional but is apparently, not triggered to act in the presence of penicillin bound to the bacterium.</p>
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This proposal seeks to apply a newly developed genetic strategy to identify genetic elements important in control of autolytic activity. A library of insertionally inactivated mutants has been screened for mutants in which loss of function of a gene renders the bacteria tolerant to penicillin despite the presence of normal autolytic enzyme. The genes involved in generation of the tolerant phenotype will be characterized.</p>
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One mutant of particular interest harbors an inactivation in a histidine kinase, suggesting a possible signalling pathway important for the triggering of lytic activity. Very recent analysis of this mutant indicates it is also defective for natural transformation of DNA suggesting a programed link between autolysis and transformation. This will provide information important to the development of new potential antibacterial agents and perhaps suggest why bacteria in the clinical encironment choose to reggulate autolytic activity rather than dispense with sucidal autolysins in the face aof antibiotic pressure.</p>