Low-dosage ultraviolet confocal fluorescence microscopy and filipin staining showed that cholesterol accumulated at <i>Salmonella</i> entry foci, and was retained by <i>Salmonella-</i>containing vacuoles (SCVs) following bacterial uptake. This was particularly evident in the previously uncharacterised membrane ruffles induced on cultured fibroblasts, when viewed by scanning electron microscopy and three-dimensional fluorescence rendering. Cellular cholesterol redistribution required bacterial effector delivery directed by the SPI1 encoded TTSS. These results showed that host cholesterol is essential for <i>Salmonella </i>uptake, and indicated the involvement of host cell rafts in the entry process. Purified SipB was identified as a cholesterol binding protein in novel gel filtration assays <i>in vitro</i>. Upon cholesterol depletion of cultured eukaryotic cells, addition of purified SipB was sufficient to restore correct localisation of the raft marker, caveolin-1. Fluorescence microscopy, immunogold electron microscopy and biochemical assays demonstrated that cellular binding of purified SipB was reduced in cholesterol depleted cells compared with untreated cells. Localisation of SipB to rafts was observed in <i>Salmonella </i>infected cells, or cells treated with purified SipB. Enhanced localisation for purified SipB was observed when added to cells in complex with SipC, or following treatment of cells with cytoskeleton-disrupting drugs. In combination, these data indicate that SipB specifically targets hosts rafts during entry. New insights into the uptake of <i>Salmonella</i> and their attached flagella were gained by immunoflourescence microscopy. Flagella are lengthy, rigid, helical structures and their fate, following cell entry, was previously unknown. The flagella filament was observed to adopt novel, uncharacterised conformations within the host cell and whilst further flagella assembly was not apparent, pre-existing flagella exhibited long term intracellular stability. This study addresses the issue of the fate <i>Salmonella</i> flagella after cell entry and the nature of the SCV microenvironment.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:599320 |
| Date | January 2004 |
| Creators | Garner, M. J. |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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