We demonstrate the presence of a glycosylphosphatidylinositol (GPI) anchor-specific endosomal pathway in the protozoan pathogen Trypanosoma brucei. In higher eukaryotes evidence indicates that GPI-anchored proteins are transported in both the endocytic and exocytic systems by mechanisms involving sequestration into specific membrane microdomains and consequently sorting into distinct compartments. This is potentially extremely important in trypanosomatids as the GPI anchor is the predominant mechanism for membrane attachment of surface macromolecules, including the variant surface glycoprotein (VSG). A highly complex developmentally regulated endocytic network, vital for nutrient uptake and evasion of the immune response, exists in T. brucei. In common with mammalian cells an early endosomal compartment is defined by Rab5 small GTPases, which control transport processes through the endosomal system. We investigate the function of two trypanosome Rab5 homologues. TbRAB5A and TbRAB5B, which colocalize in the procyclic stage, are distinct in the bloodstream form of the parasite. TbRAB5A endosomes contain VSG and transferrin, endocytosed by the T. brucei GPI-anchored transferrin receptor, whereas TbRAB5B endosomes contain the transmembrane protein ISG 100 but neither VSG nor transferrin. These findings indicate the presence of trypanosome endosomal pathways trafficking proteins through specific routes depending on the mode of membrane attachment. Ectopic expression of mutant TbRAB5A or -5B indicates that TbRAB5A plays a role in LDL endocytosis, whereas TbRAB5B does not, but both have a role in fluid phase endocytosis. Hence TbRAB5A and TbRAB5B have distinct functions in the endosomal system of T. brucei. A developmentally regulated GPIspecific endosomal pathway in the bloodstream form suggests that specialized transport of GPI-anchored proteins is required for survival in the mammalian host.
Objective Salmonella enterica infections can lead to Reactive Arthritis (ReA), which can exhibit an association with human leucocyte antigen (HLA)-B*27:05, a molecule prone to misfolding and initiation of the unfolded protein response (UPR). This study examined how HLA-B*27:05 expression and the UPR affect the Salmonella life-cycle within epithelial cells.MethodsIsogenic epithelial cell lines expressing two copies of either HLA-B*27:05 and a control HLA-B*35:01 heavy chain (HC) were generated to determine the effect on the Salmonella infection life-cycle. A cell line expressing HLA-B*27:05.HC physically linked to the light chain beta-2-microglobulin and a specific peptide (referred to as a single chain trimer, SCT) was also generated to determine the effects of HLA-B27 folding status on S. enterica life-cycle. XBP-1 venus and AMP dependent Transcription Factor (ATF6)-FLAG reporters were used to monitor UPR activation in infected cells. Triacin C was used to inhibit de novo lipid synthesis during UPR, and confocal imaging of ER tracker stained membrane allowed quantification of glibenclamide-associated membrane.Results S. enterica demonstrated enhanced replication with an altered cellular localisation in the presence of HLA-B*27:05.HC but not in the presence of HLA-B*27:05.SCT or HLA-B*35:01. HLA-B*27:05.HC altered the threshold for UPR induction. Salmonella activated the UPR and required XBP-1 for replication, which was associated with endoreticular membrane expansion and lipid metabolism.ConclusionsHLA-B27 misfolding and a UPR cellular environment are associated with enhanced Salmonella replication, while Salmonella itself can activate XBP-1 and ATF6. These data provide a potential mechanism linking the life-cycle of Salmonella with the physicochemical properties of HLA-B27 and cellular events that may contribute to ReA pathogenesis. Our observations suggest that the UPR pathway maybe targeted for future therapeutic intervention.
Cell engineering to enable step change improvements in bioprocessing can be directed at targets other than increasing product titer. The physical properties of the process suspension such as viscosity, for example, have a major impact on various downstream processing unit operations. The release of chromosomal DNA during homogenization of Escherichia coli and its influence on viscosity is well-recognized. In this current article we demonstrate co-expression of Staphylococcus aureus nuclease in E. coli to reduce viscosity through auto-hydrolysis of nucleic acids. Viscosity reduction of up to 75% was achieved while the particle size distribution of cell debris was maintained approximately constant (d(50) = 0.5-0.6 microm). Critically, resultant step change improvements to the clarification performance under disc-stack centrifugation conditions are shown. The cell-engineered nuclease matched or exceeded the viscosity reduction performance seen with the addition of exogenous nuclease removing the expense and validation issues associated with such additions to a bioprocess. The resultant material dramatically altered performance in scale-down mimics of continuous disc-stack centrifugation. Laboratory scale data indicated that a fourfold reduction in the settling area of a disc-stack centrifuge can be expected due to a less viscous process stream achieved through nuclease co-expression with a recombinant protein.
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