Insight into the Cellular Alterations Required for Establishing Opportunistic Pseudomonas aeruginosa Infections

The Gram-negative, opportunistic pathogen Pseudomonas aeruginosa (Pa) is responsible for causing disease of high morbidity and mortality in individuals who are immunocompromised, suffer from damaged epithelial barriers, and who have cystic fibrosis. However, the underlying host cell compromise that allows the establishment of Pa infections remains unknown and is of importance to the development of novel strategies that interrupt Pa infections. The type III secretion system (T3S) allows the direct translocation of bacterial effectors into the host cell cytosol, and in this study was used first, to identify host cell properties involved in the initiation of Pa infections, and then second, to dissect the Pa infectious process. Using the T3S effector ExoS to monitor T3S translocation and the initiation of Pa infections in HT-29 epithelial cells, we identified a relationship between leading edge focal complex adhesion properties involved in cell migration and sensitivity to Pa-T3S. Highly migratory T24 epithelial cells were used to further explore the role of host cell migration in Pa infections, using manipulations of ExoS GTPase activating (GAP) and ADP-ribosyltransferase (ADPRT) activities to manipulate T3S. These studies allowed dissection of the Pa infectious process, and found that Pa expressing wild type ExoS preferentially bound to the leading edge of T24 cells, where ExoS GAP activity interfered with Pa internalization, and ExoS ADPRT activity interrupted actin-plasma membrane associations required for T3S translocation. Interestingly, both toxic events limited the Pa infectious process. Further studies of MTC, MTLn3, and MDCK epithelial cells identified a reciprocal relationship between Rac1 and Rho activation at the leading edge and Pa internalization and T3S translocation efficiency. Together, the studies in this dissertation highlight the role of actin-plasma membrane associations and Rho-GTPases in directing T3S translocation and the Pa infectious process, and the ability of Pa to hijack these factors during cell migration. Our studies in turn are consistent with cell migration properties induced in response to tissue damage being the cellular compromise that leads to the initiation of Pa infections, and that effectors such as ExoS are able to interrupt these same properties to limit the infectious process and maintain the opportunistic nature of Pa infections

Metastatic MTLn3 and non-metastatic MTC adenocarcinoma cells can be differentiated by Pseudomonas aeruginosa

Cancer patients are known to be highly susceptible to Pseudomonas aeruginosa (Pa) infection, but it remains unknown whether alterations at the tumor cell level can contribute to infection. This study explored how cellular changes associated with tumor metastasis influence Pa infection using highly metastatic MTLn3 cells and non-metastatic MTC cells as cell culture models. MTLn3 cells were found to be more sensitive to Pa infection than MTC cells based on increased translocation of the type III secretion effector, ExoS, into MTLn3 cells. Subsequent studies found that higher levels of ExoS translocation into MTLn3 cells related to Pa entry and secretion of ExoS within MTLn3 cells, rather than conventional ExoS translocation by external Pa. ExoS includes both Rho GTPase activating protein (GAP) and ADP-ribosyltransferase (ADPRT) enzyme activities, and differences in MTLn3 and MTC cell responsiveness to ExoS were found to relate to the targeting of ExoS-GAP activity to Rho GTPases. MTLn3 cell migration is mediated by RhoA activation at the leading edge, and inhibition of RhoA activity decreased ExoS translocation into MTLn3 cells to levels similar to those of MTC cells. The ability of Pa to be internalized and transfer ExoS more efficiently in association with Rho activation during tumor metastasis confirms that alterations in cell migration that occur in conjunction with tumor metastasis contribute to Pa infection in cancer patients. This study also raises the possibility that Pa might serve as a biological tool for dissecting or detecting cellular alterations associated with tumor metastasis

Role of host cell polarity and leading edge properties in Pseudomonas type III secretion

Type III secretion (T3S) functions in establishing infections in a large number of Gram-negative bacteria, yet little is known about how host cell properties might function in this process. We used the opportunistic pathogen Pseudomonas aeruginosa and the ability to alter host cell sensitivity to Pseudomonas T3S to explore this problem. HT-29 epithelial cells were used to study cellular changes associated with loss of T3S sensitivity, which could be induced by treatment with methyl-beta-cyclodextrin or perfringolysin O. HL-60 promyelocytic cells are innately resistant to Pseudomonas T3S and were used to study cellular changes occurring in response to induction of T3S sensitivity, which occurred following treatment with phorbol esters. Using both cell models, a positive correlation was observed between eukaryotic cell adherence to tissue culture wells and T3S sensitivity. In examining the type of adhesion process linked to T3S sensitivity in HT-29 cells, a hierarchical order of protein involvement was identified that paralleled the architecture of leading edge (LE) focal complexes. Conversely, in HL-60 cells, induction of T3S sensitivity coincided with the onset of LE properties and the development of actin-rich projections associated with polarized cell migration. When LE architecture was examined by immunofluorescent staining for actin, Rac1, IQ-motif-containing GTPase-activating protein 1 (IQGAP1) and phosphatidylinositol 3 kinase (PI3 kinase), intact LE structure was found to closely correlate with host cell sensitivity to P. aeruginosa T3S. Our model for host cell involvement in Pseudomonas T3S proposes that cortical actin polymerization at the LE alters membrane properties to favour T3S translocon function and the establishment of infections, which is consistent with Pseudomonas infections targeting wounded epithelial barriers undergoing cell migration