SummaryBurkholderia pseudomallei is a Gram-negative facultative intracellular pathogen that enters and escapes from eukaryotic cells using the power of actin polymerization. We have identified a bacterial protein (BimA) that is required for the ability of B. pseudomallei to induce the formation of actin tails. BimA contains proline-rich motifs and WH2-like domains and shares limited homology at the C-terminus with the Yersinia autosecreted adhesin YadA. BimA is located at the pole of the bacterial cell at which actin polymerization occurs and mutation of bimA abolished actinbased motility of the pathogen in J774.2 cells. Transient expression of BimA in HeLa cells resulted in F-actin clustering reminiscent of that seen on WASP overexpression. Antibody-mediated clustering of a CD32 chimera in which the cytoplasmic domain was replaced with BimA resulted in localization of the chimera to the tips of F-actin enriched membrane protrusions. We report that purified truncated BimA protein binds monomeric actin in a concentrationdependent manner in cosedimentation assays and that BimA stimulates actin polymerization in vitro in a manner independent of the cellular Arp2/3 complex.
Formation and rearrangement of disulfide bonds during the correct folding of nascent proteins is modulated by a family of enzymes known as thiol isomerases, which include protein disulfide isomerase (PDI), endoplasmic reticulum protein 5 (ERP5), and ERP57. Recent evidence supports an alternative role for this family of proteins on the surface of cells, where they are involved in receptor remodeling and recognition. In platelets, blocking PDI with inhibitory antibodies inhibits a number of platelet activation pathways, including aggregation, secretion, and fibrinogen binding. Analysis of human platelet membrane fractions identified the presence of the thiol isomerase protein ERP5. Further study showed that ERP5 is resident mainly on platelet intracellular membranes, although it is rapidly recruited to the cell surface in response to a range of platelet agonists. Blocking cell-surface ERP5 using inhibitory antibodies leads to a decrease in platelet aggregation in response to agonists, and a decrease in fibrinogen binding and P-selectin exposure. It is possible that this is based on the disruption of integrin function, as we observed that ERP5 becomes physically associated with the integrin  3 subunit during platelet stimulation. These results provide new insights into the involvement of thiol isomerases and regulation of platelet activation. IntroductionIn classical terms, reduction/oxidation systems within a cell have been represented very simply. The cytoplasmic environment is hypoxic and reducing, whereas the extracellular environment is normoxic and oxidizing. The generation of a disulfide bond from 2 cysteine residues is an oxidation reaction. To correctly generate these bonds inside the cell, there are, therefore, a group of enzymes known as the thiol isomerases. These are capable of the formation, reduction, and rearrangement of the disulfide-bonding patterns of proteins, often as part of folding of nascent proteins. The thiol isomerase enzymes are anchored to the endoplasmic reticulum via KDEL-receptor proteins. [1][2][3] Recent studies have suggested additional functions for thiol isomerase enzymes: on the surface of cells, where they participate in receptor activation and remodeling, and substrate processing. [4][5][6] Protein disulfide isomerase (PDI) is the best-characterized thiol isomerase to demonstrate this dual functionality. A number of cell types, including bovine aortic endothelial cells, 7 rat hepatocytes, 8,9 and human B cells, 5,10 have been shown to secrete PDI, which associates with the cell surface. Cell-surface PDI has been implicated in the reduction of the disulfide-linked diptheria toxin heterodimer 11 and events triggering the entry of HIV into lymphoid cells. 6,12 On the basis of a series of investigations, initially by Detweiller and coworkers, a role for PDI in platelet physiology is now established. 4,[13][14][15][16] Early studies demonstrated PDI was present on the external membrane of activated and resting platelets, and proteins with thiol isomerase activity were secreted f...
Recently we identified a bacterial factor (BimA) required for actin-based motility of Burkholderia pseudomallei. Here we report that Burkholderia mallei and Burkholderia thailandensis are capable of actin-based motility in J774.2 cells and that BimA homologs of these bacteria can restore the actin-based motility defect of a B. pseudomallei bimA mutant. While the BimA homologs differ in their amino-terminal sequence, they interact directly with actin in vitro and vary in their ability to bind Arp3.
Listeria, Rickettsia, Burkholderia, Shigella and Mycobacterium species subvert cellular actin dynamics to facilitate their movement within the host cytosol and to infect neighbouring cells while evading host immune surveillance and promoting their intracellular survival. 'Attaching and effacing' Escherichia coli do not enter host cells but attach intimately to the cell surface, inducing motile actin-rich pedestals, the function of which is currently unclear. The molecular basis of actin-based motility of these bacterial pathogens reveals novel insights about bacterial pathogenesis and fundamental host-cell pathways.
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