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Title: Identification of a novel substrate of the Salmonella protein tyrosine phosphatase SptP
Author: Humphreys, D.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2007
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A novel cellular target was captured when purified SptP substrate-trapping derivatives and cell extracts were combined, which was isolated and identified by MALDI-TOF mass spectrometry as VCP (valosin-containing protein). VCP is a AAA family ATPase that regulates multiple ubiquitin-dependent cellular processes, including proteasome-dependant protein degradation, protein-membrane association and organelle biogenesis. Next, interaction between native SptP and VCP was reconstituted in vitro. Further biochemical analyses revealed that binding was blocked by phosphatase inhibitors and required both SptP domains (GAP and PTP). Furthermore, SptP exhibited potent phosphatase activity towards VCP in vitro, and TTSS-delivered SptP associated with and specifically dephosphorylated VCP during infection of cultured cells with S. typhimurium. Two additional entry effectors, the Salmonella GTP exchange factor mimics SopE/2 and inositol phosphatase SopB are ubiquitinated following their delivery into target cells. Immunoprecipitation of SopE2 and SopB from cells following infection with a S. typhimurium ΔsptP null mutant localisation occurred independently of SptP and therefore VCP dephosphorylation. Bacterial entry efficiency was unaffected by VCP gene silencing by RNA interference (RNAi), suggesting that SptP-dependent VCP dephosphorylation was relevant later during infection. Such a role was further supported by immunofluorescence and immunoprecipitation analyses demonstrating that SptP persists within infected cells. Immunofluorescence microscopy  showed that both SptP and VCP transiently associate with internalised S. typhimurium  and that this SptP targeting was dependent upon it s GAP activity. Infection with S. typhimurium engineered to delivery augmented levels of SptP resulted in a 2.1-fold increase in bacterial replication 8-hours post-infection and reciprocally, a 1.7-fold reduction was observed at equivalent time-points after cells were infected with the S. typhimurium ΔsptP  mutant. VCP knockdown resulted in increased bacterial replication and parallel immunofluorescence microscopy revealed elevated numbers of Salmonella localised in the infected cell cytosol. Examination of wild-type SCV morphology after VCP knockdown demonstrated that Sif formation was abolished. This thesis reports that the Salmonella entry effector SptP persists after invasion and regulates S. typhimurium replication at late stages of infection through alteration of the phosphorylation status of VCP, a novel cellular target of the SptP PTP domain located on the SCV.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available