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Aleksandra Snyder, Ph.D.

Vasil Lab


 

THESIS


Complete Title of Thesis:
"Analysis of the Twin Arginine Tranport System in Secretion of the Pseudomonas aeruginosa Hemolytic Phospholipase C and in Bacterial Pathogenesis"


Prepared under the direction of:   Michael L. Vasil, Ph.D.


SUMMARY

The hemolytic phospholipase C (PlcH), an extracellular virulence factor of Pseudomonas aeruginosa, was recently shown to depend on the Twin Arginine Transport (Tat) for secretion. PlcH, like all Tat secreted substrates, is synthesized with a Tat secretion signal which contains two conserved arginines (R9 and R10) within a consensus motif as well as additional arginines (R8, R34, R35) not typically found in E.coli Tat signal sequences. In these studies, I examined the role of the signal sequence arginines as well as the entire signal sequence on PlcH secretion. Mutagenesis of R9 and R10 caused a mislocalization of PlcH to the inner membrane, indicating a critical role for these residues in secretion. Mutagenesis of R8, R34 and R35 did not affect PlcH secretion, but did affect protein stability. Deletion of the entire signal sequence resulted in plcH transcript degradation, suggesting a role for the region encoding the signal sequence in transcript stability.

Previous studies have shown that the Tat translocation machinery seems to be species-specific. I examined whether the PlcH signal sequence was compatible with the E.coli Tat machinery by localizing PlcH expressed in an E.coli host and a P. aeruginosa Δtat host complemented with the E.coli Tat machinery. PlcH was transported across the inner membrane only in the P.aeruginosa complemented host, indicating that although the E.coli Tat machinery is compatible with the PlcH signal sequence, additional accessory factors may be required for PlcH secretion which are absent in the E.coli host.

The role of Tat in P. aeruginosa pathogenesis was also examined in a chronic pulmonary rat infection model. A P. aeruginosa ΔtatC mutant was able to survive in rat lungs, but was unable to cause the pathology observed upon wild type infection. Genechip analysis of global gene expression indicated upregulation of alternative pathways to compensate for loss of function. Additionally, genes involved in Type III secretion were downregulated in the ΔtatC mutant, suggesting a link between Tat and Type III secretion. Finally, a P. aeruginosa ΔtatC mutant was unaffected in outer membrane integrity, contrary to results shown for an E.coli ΔtatC mutant.