Cell line differences in bacterially translocated ExoS ADP-ribosyltransferase substrate specificity

Abstract

Exoenzyme S (ExoS) is an ADP-ribosyltransferase (ADPRT) directly translocated into eukaryotic cells by the type III secretory (TTS) process of Pseudomonas aeruginosa. Comparisons of the functional effects of ExoS on human epithelial and murine fibroblastic cells showed that human epithelial cells exhibited an overall increased sensitivity to the effects of bacterially translocated ExoS on cell proliferation, morphology and re-adherence. ExoS was also found to ADP-ribosylate a greater number of low-molecular-mass G (LMMG) proteins in human epithelial cells, as compared to murine fibroblasts. Examination of the cellular mechanism for differences in ExoS ADPRT substrate modification found that the more restricted pattern of substrate modification in murine fibroblasts was not linked to the efficiency of bacterial adherence nor to the efficiency of ExoS internalization by the TTS process. In exploring the cellular nature of patterns of substrate modification, more extensive substrate modification was detected in human and simian cell lines, while rodent cell lines, including rat, mouse and hamster lines, consistently exhibited the more limited pattern of LMMG protein ADP-ribosylation. Patterns of substrate modification were not altered by cellular transformation and occurred independently of cell type. These studies suggest that eukaryotic cell properties, as recognized through studies of cells of different animal origins, affect the substrate targeting of ExoS ADPRT activity, and that this in turn can influence the severity of effects of ExoS on host-cell function.

Ezrin/Radixin/Moesin Proteins Are High Affinity Targets for ADP-ribosylation by Pseudomonas aeruginosa ExoS

“…Recently, the ADP-ribosylation activity of ExoS has been linked to cellular adherence, the maintenance of filopodia, and enhanced lamellipodia and cellular ruffling (32). Different cell lines demonstrate different sensitivities to intoxication, with epithelial cells being the most affected by the action of ExoS (33). These data suggest that ExoS ADP-ribosylates an unidentified host protein(s) that is responsible for cytoskeletal changes and/or cell death.…”

“…Rounding-Recent studies demonstrated that the ADP-r activity of ExoS leads to a loss of cellular adherence upon infection with P. aeruginosa (33). To quantify the effects of the ADP-r activity on cell morphology, HeLa cells were infected with P. aeruginosa expressing either the wild-type ExoS, ExoS deficient in GTPase-activating protein activity (R146K), ExoS deficient for ADP-r and GTPase-activating protein activity (R146K/E381D), or a vector (pUCP) control.…”

Examining the Role of Actin-Plasma Membrane Association in Pseudomonas aeruginosa Infection and Type III Secretion Translocation in Migratory T24 Epithelial Cells

“…To summarize, infections by the opportunistic pathogen P. aeruginosa are influenced by host cell properties (6,41,51,52). Through visualization of ExoS translocation in relation to T24 cell migration, we were able to characterize the mechanisms underlying eukaryotic cell differences in T3S translocation caused by mutations that inactivate ExoS GAP or ADPRT activities.…”

“…The T24 cell model was chosen in this study to further investigate the role of the leading edge and cell migration in P. aeruginosa infections due to its highly migratory mesenchymal phenotype. Eukaryotic cell properties are known to influence ExoS GAP and ADPRT toxicity (51), which leads to predictions that alterations in cell migration properties might affect ExoS GAP and ADPRT toxicity. In this regard, recent studies using real-time phase-contrast imaging to examine the effects of ExoS on immortalized corneal epithelial cells found that infection with P. aeruginosa expressing ADPRT-active ExoS allowed P. aeruginosa to be internalized and replicate within membrane blebs (2).…”

Regulation of Rab5 GTPase activity during Pseudomonas aeruginosa-macrophage interaction

“…Observations from this study showed that ExoS anti-internalization activity was mostly dependent on its ADPr activity which differs from previous studies in HeLa cells where the ExoS antiinternalization function was attributed to RhoGAP activity . One explanation for this discrepancy could be due to the use of a different cell line, and cell line properties are known to influence the substrate specificity of ExoS (Rucks, Fraylick et al 2003). The ability of ExoS ADPr, but not its RhoGAP activity, to inactivate Rab5 ( Figure 10A) provides evidence for cell-type dependent mechanisms of phagocytosis, which can be differentiated by ExoS.…”