Characterization of two distinct phospholipase C enzymes from Burkholderia pseudomallei.

Burkholderia pseudomallei is a serious bacterial pathogen that can cause a lethal infection in humans known as melioidosis. In this study two of its phospholipase C (PLC) enzymes (Plc-1 and Plc-2) were characterized. Starting with a virulent strain, two single mutants were constructed, each with one plc gene inactivated, and one double mutant with both plc genes inactivated. The single plc mutants exhibited decreased extracellular PLC activity in comparison to the wild-type strain, thereby demonstrating that the two genes encoded functional extracellular PLCs. Growth comparisons between the wild-type and PLC mutants in egg-yolk-supplemented medium indicated that both PLCs contributed to egg-yolk phospholipid utilization. Both PLCs hydrolysed phosphatidylcholine and sphingomyelin but neither was haemolytic for human erythrocytes. Experimental infections of eukaryotic cells demonstrated that Plc-1 itself had no effect on plaque-forming efficiency but it had an additive effect on increasing the efficiency of Plc-2 to form plaques. Only Plc-2 had a significant role in host cell cytotoxicity. In contrast, neither Plc-1 nor Plc-2 appeared to play any role in multinucleated giant cell (MNGC) formation or induction of apoptotic death in the cells studied. These data suggested that PLCs contribute, at least in part, to B. pseudomallei virulence and support the view that Plc-1 and Plc-2 are not redundant virulence factors.

Analysis of the role of GADD153 in the control of apoptosis in NS0 myeloma cells.

Apoptosis can limit the maximum production of recombinant protein expression from cultured mammalian cells. This article focuses on the links between nutrient deprivation, ER perturbation, the regulation of (growth arrest and DNA damage inducible gene 153) GADD153 expression and apoptosis. During batch culture, decreases in glucose and glutamine correlated with an increase in apoptotic cells. This event was paralleled by a simultaneous increase in GADD153 expression. The expression of GADD153 in batch culture was suppressed by the addition of nutrients and with fed-batch culture the onset of apoptosis was delayed but not completely prevented. In defined stress conditions, glucose deprivation had the greatest effect on cell death when compared to glutamine deprivation or the addition of tunicamycin (an inhibitor of glycosylation), added to generate endoplasmic reticulum stress. However, the contribution of apoptosis to overall cell death (as judged by morphology) was smaller in conditions of glucose deprivation than in glutamine deprivation or tunicamycin treatment. Transient activation of GADD153 expression was found to occur in response to all stresses and occurred prior to detection of the onset of cell death. These results imply that GADD153 expression is either a trigger for apoptosis or offers a valid indicator of the likelihood of cell death arising from stresses of relevance to the bioreactor environment.