Active membrane transport and receptor proteins from bacteria

A general strategy for the expression of bacterial membrane transport and receptor genes in Escherichia coli is described. Expression is amplified so that the encoded proteins comprise 5-35% of E. coli inner membrane protein. Depending upon their topology, proteins are produced with RGSH6 or a Strep tag at the C-terminus. These enable purification in mg quantities for crystallization and NMR studies. Examples of one nutrient uptake and one multidrug extrusion protein from Helicobacter pylori are described. This strategy is successful for membrane proteins from H. pylori, E. coli, Enterococcus faecalis, Bacillus subtilis, Staphylococcus aureus, Microbacterium liquefaciens, Brucella abortus, Brucella melitensis, Campylobacter jejuni, Neisseria meningitides, Streptomyces coelicolor and Rhodobacter sphaeroides. ©2005 Biochemical Society.

Macrophages and lymphocyte responiveness to mitogen

Purified B-cells fail to proliferate in response to the strong thymus-independent (TI) antigen Lipopolysaccharide (LPS) in the absence of macrophages (Corbel and Melchers, 1983). The fact that macrophages, or factors derived from them are required is supported by the inability of marginal zone B-cells in infants to respond to highly virulent strains of bacteria such as Neisseria meningitidis and Streptococcus pneumoniae (Timens, 1989). This may be due to the lack of CD21 expression on B-cells in infants which could associate with its co-receptor (C3d) on adjacent macrophages. It is not clear whether cell surface contacts and/or soluble products are involved in lymphocyte-macrophage interactions in response to certain antigens. This thesis describes the importance of the macrophage in lymphocyte responses to T-dependent (TD) and TI antigens. The major findings of this thesis were as follows: (1). Macrophages were essential for a full proliferative response to a range of T - and B-cell mitogens and TI-1 and TI-2 antigens, including Concanavalin A, LPS, Pokeweed mitogen (PWM), Dextran sulphate, Phytohaemagglutinin-P (PHA-P) and Poly[I][C]. (2). A ratio of 1 macrophage to 1000 lymphocytes was sufficient for the mitogens to exert their effects. (3). The optimal conditions were established for the activation of an oxidative burst in cells of the monocyte/macrophage lineage as measured by luminometry. The order of ability was OpZ >PMA/lonomycin >f-MLP >Con A >DS >PHA >Poly[I][C] >LPS >PWM. Responses were only substantial and protracted with OpZ and PMA. Peritoneal macrophages were the most responsive cells, whereas splenic and alveolar macrophages were significantly less active and no response could be elicited with Kupffer cells, thus demonstrating heterogeneity between macrophages. (4). Activated macrophages that were then fixed with paraformaldehyde were unable to restore mitogenic responsiveness, even with a ratio of 1 macrophage to 5 lymphocytes. (5). Although highly purified T- and B-cells could respond to mitogen provided live macrophages were present, maximum activation was only observed when all 3 cell types were present. (6). Supernatants from purified macrophage cultures treated with a range of activators were able to partially restore lymphocyte responses to mitogen in macrophage-depleted splenocyte cultures, and purified T - and B-cell cultures. In fact supernatants from macrophages treated with LPS for only 30 minutes could restore responsiveness. Supernatants from OpZ treated macrophages were without effect. (7). Macrophage supernatants could not induce proliferation in the absence of mitogen. They therefore provide a co-mitogenic signal required by lymphocytes in order to respond to mitogen. (8). Macrophage product profiles revealed that LPS and Con A-treated macrophage supernatants showed elevated levels of IL-1β, TNF -α L TB4 and TXB2. These products were therefore good candidates as the co-mitogenic factor. The possible inhibitory factors secreted by OpZ-treated macrophages were PGE2, IL-10 and NO. (9). The removal of cytokines, eicosanoids and TNF-α from LPS-treated macrophage supernatants using Cycloheximide, Dexamethasone and an MMPI respectively, resulted in the inability of these supernatants to restore macrophage-depleted lymphocyte responses to mitogen. (10). rIL-1β and rTNF-α are co-mitogenic factors, as macrophage-depleted lymphocytes incubated with rIL-1β and rTNF-α can respond to mitogen.