The overall goal of the project is to delineate the molecular processes that lead to P. aeruginosa exoenzyme S expression. Exoenzyme S is a part of the ADP-ribosyltransferase family of bacterial toxins, but little is known about intoxication with this molecule. Previous work has focused on the characterization of a trans-regulatory locus that controls the synthesis of exoenzyme S. The results indicate that three potential protein products are required for exoenzyme S synthesis in transposon mutants. Determination of the nucleotide sequence of this region has resulted in the identification of homologous regions related to the response regulator and histidine protein kinase classes of bacterial regulatory proteins. Preliminary studies on the transcriptional control of the trans-regulatory locus suggest that ORF1 and ORF3 are transcribed from separate promoter regions, while ORF2 has not been tested. Two additional promoter regions were found that flank the trans-regulatory locus. ORF1 and the flanking promoter regions are coordinately regulated with exoenzyme S production; ORF3 is regulated differently.
The aim of this proposal is to extend these preliminary studies to express and purify the ORF1, ORF2, and ORF3 regulatory proteins and to determine their function in DNA binding and in vitro transcriptional systems.
The specific aims of this project are to: 1, Express and localize the transcriptional activator for exoenzyme S; 2, Clone the structural gene for exoenzyme S; 3, Identify the mechanisms that mediate exoenzyme S transcription; 4, Analyze the function of other genes encoded within the trans-regulatory locus; and 5, Analyze the contribution of genes downstream of the trans-regulatory region. The importance of exoenzyme S production has been demonstrated in burn and chronic lung infections through the use of isogenic mutants in animal models. The selective advantage exoenzyme S production confers on the bacterium during infection appears to be related to the ability of P. aeruginosa to spread from an initial site of colonization to distant organs via the bloodstream. This property is particularly devastating in burn patients and is responsible for the high rate of fatal septicemias associated with this group of immunocompromised individuals. In chronic lung infections of cystic fibrosis patients, P. aeruginosa does not disseminate within the host, but low levels of exoenzyme S production appear to be responsible for tissue damage and necrosis. The coordinated effort to clone and characterize the structural and regulatory elements for exoenzyme S synthesis will be necessary to understand the events that lead to exoenzyme S production in the infected host. Learning how molecular processes regulate P. aeruginosa virulence and pathogenesis can lead to strategies of treatment that down-regulate toxin expression and reduce the high fatality associated with disseminated Pseudomonas infection.
