Examination of the regulation of the leukotoxin operon in Mannheimia haemolytica.

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Mannheimia haemolytica is a Gram negative bacterium and a commensal microorganism in the nasal pharyngeal passage of cattle. When the animals are stressed or suffer from concurrent viral infections, their immune systems are compromised and can not clear the bacterium inhaled into the lungs. The bacterium is able to adhere/colonize the lungs, evade the immune clearance and cause pneumonia due to destruction of the lung tissue. One of the major virulence factor produced by the bacterium is a leukotoxin which targets the bovine leukocytes. The leukotoxin lyses the bovine macrophages to prevent them from attacking the invading bacterium. Lysis of the macrophages release cellular lytic components that leads to destruction of the lung tissue and the loss of lung function. The leukotoxin is a member of the RTX family of cytolysins found in many bacterial pathogens. Even though the genes that code for the leukotoxin have been characterized for over 25 years, very little is known about their regulation of expression. To gain a better understanding on how and when the bacterium decides to produce the leukotoxin, I proposed to investigate the genetic mechanism that regulate expression of the leukotoxin genes. This will be accomplished by mutagenesis experiments and the isolation of non-haemolytic mutants. This genetic screen utilized the haemolytic activity of the leukotoxin which produces a zone of clearance around the bacterial colony on sheep's blood agar plates. The non-haemolytic mutant could be defective in the actual leukotoxin genes, or in another 'factor' required for expression of the leukotoxin genes. After sorting out the mutant into the above two groups, those with mutations not in the leukotoxin genes will be further characterized by complementation with a genomic library to screen for restoration of the haemolytic phenotype. Any recombinant plasmid that can restore the haemolytic phenotype should contain the missing or mutated function. This should identify any novel factor required for expression of the leukotoxin genes. The results from this study could be extended to the regulation of expression of RTX genes in other bacterial pathogens.