Regulation of the periplasmic stress responses in E. coli and P. aeruginosa

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2008.Includes bibliographical references.The ability to adapt to changing environments is essential to survival. Bacteria have developed sophisticated means by which they sense and respond to stresses imposed by changes in the environment. I have undertaken the study of elements of the TE stress response pathway in the bacterium Escherichia coli and the orthologous pathway in the bacterium Pseudomonas aeruginosa. These pathways sense stress in the periplasm and relay the signal into the cytoplasm by a series of proteolytic cleavages of a transmembrane regulatory protein. In E. coli, I have undertaken the study of the regulation of the cleavage of transmembrane regulator RseA by the first protease, DegS. I discovered that RseB, an RseA-binding protein, inhibits cleavage of RseA by DegS. The interaction between RseA and RseB is strong and specific, and the inhibition of cleavage is independent of the autoinhibition of DegS by its PDZ domain. In P. aeruginosa, I have demonstrated that AlgW, the homolog of DegS, cleaves the transmembrane regulator MucA. I have shown similar inhibitory effects of the ortholog of RseB on the ortholog of RseA. Interestingly, the PDZ domain of AlgW appears to function differently from that of DegS. In addition, I observed that a regulatory loop in the AlgW protease plays an inhibitory role in the binding of substrate.by Brent Cezairliyan.Ph.D

Distinct Pathogenesis and Host Responses during Infection of C. elegans by P. aeruginosa and S. aureus

The genetically tractable model host Caenorhabditis elegans provides a valuable tool to dissect host-microbe interactions in vivo. Pseudomonas aeruginosa and Staphylococcus aureus utilize virulence factors involved in human disease to infect and kill C. elegans. Despite much progress, virtually nothing is known regarding the cytopathology of infection and the proximate causes of nematode death. Using light and electron microscopy, we found that P. aeruginosa infection entails intestinal distention, accumulation of an unidentified extracellular matrix and P. aeruginosa-synthesized outer membrane vesicles in the gut lumen and on the apical surface of intestinal cells, the appearance of abnormal autophagosomes inside intestinal cells, and P. aeruginosa intracellular invasion of C. elegans. Importantly, heat-killed P. aeruginosa fails to elicit a significant host response, suggesting that the C. elegans response to P. aeruginosa is activated either by heat-labile signals or pathogen-induced damage. In contrast, S. aureus infection causes enterocyte effacement, intestinal epithelium destruction, and complete degradation of internal organs. S. aureus activates a strong transcriptional response in C. elegans intestinal epithelial cells, which aids host survival during infection and shares elements with human innate responses. The C. elegans genes induced in response to S. aureus are mostly distinct from those induced by P. aeruginosa. In contrast to P. aeruginosa, heat-killed S. aureus activates a similar response as live S. aureus, which appears to be independent of the single C. elegans Toll-Like Receptor (TLR) protein. These data suggest that the host response to S. aureus is possibly mediated by pathogen-associated molecular patterns (PAMPs). Because our data suggest that neither the P. aeruginosa nor the S. aureus–triggered response requires canonical TLR signaling, they imply the existence of unidentified mechanisms for pathogen detection in C. elegans, with potentially conserved roles also in mammals

Pseudomonas aeruginosa PA14 Pathogenesis in Caenorhabditis elegans

Pseudomonas aeruginosa is a potentially deadly super bug that is one of the top causes of hospital-acquired infections; it is therefore essential to understand its biology and pathogenesis. This chapter describes methods for using the powerful genetic model organism C. elegans as a host for P. aeruginosa infections so that scientists can safely study the immune response to this fascinating and clinically important bacteria