Type III secretion system | EPFL Graph Search

The type III secretion system (T3SS or TTSS), also called the injectisome, is one of the bacterial secretion systems used by bacteria to secrete their effector proteins into the host's cells to promote virulence and colonisation. The T3SS is a needle-like protein complex found in several species of pathogenic gram-negative bacteria. The term Type III secretion system was coined in 1993. This secretion system is distinguished from at least five other secretion systems found in gram-negative bacteria. Many animal and plant associated bacteria possess similar T3SSs. These T3SSs are similar as a result of convergent evolution and phylogenetic analysis supports a model in which gram-negative bacteria can transfer the T3SS gene cassette horizontally to other species. The most researched T3SSs are from species of Shigella (causes bacillary dysentery), Salmonella (typhoid fever), Escherichia coli (Gut flora, some strains cause food poisoning), Vibrio (gastroenteritis and diarrhea), Burkholderia (glanders), Yersinia (plague), Chlamydia (sexually transmitted disease), Pseudomonas (infects humans, animals and plants) and the plant pathogens Erwinia, Ralstonia and Xanthomonas, and the plant symbiont Rhizobium. The T3SS is composed of approximately 30 different proteins, making it one of the most complex secretion systems. Its structure shows many similarities with bacterial flagella (long, rigid, extracellular structures used for motility). Some of the proteins participating in T3SS share amino-acid sequence homology to flagellar proteins. Some of the bacteria possessing a T3SS have flagella as well and are motile (Salmonella, for instance), and some do not (Shigella, for instance). Technically speaking, type III secretion is used both for secreting infection-related proteins and flagellar components. However, the term "type III secretion" is used mainly in relation to the infection apparatus. The bacterial flagellum shares a common ancestor with the type III secretion system. T3SSs are essential for the pathogenicity (the ability to infect) of many pathogenic bacteria.

Two strategies to improve transient gene expression in HEK293E and/or CHO-DG44 cells

Guillaume Lüthi

Transient gene expression (TGE) is based on episomal DNA expression. This technique is used for the production of recombinant proteins that can be used as therapeutics. TGE is for example extremely useful for shortening the time between candidate proteins screening and their production. Understanding and optimizing TGE is a major concern in the field of recombinant proteins. Thus, we selected here two different approaches to further improve TGE. First, we studied effect of signal peptide (SP) modification on TNFR-Fc protein in transiently transfected HEK293E and CHO-DG44 cells. Secondly, we studied effect of Valproic acid on the production of TNFR-Fc protein in HEK293E cells and on its influence on plasmid methylation. Modification of TNFR-Fc was done with eighteen others SPs. Artificial SPs exhibiting different scores using a prediction method and SPs coming from naturally secreted proteins were tested. None of them could induce a better secretion than the native SP. Nevertheless, they all showed comparable secretion abilities, except one that could not induce protein secretion. We showed here that SP modification could not increase protein secretion in HEK293E or in CHO-DG44. But also that without a valid SP, protein secretion is inhibited. Addition of VPA in HEK293E cells transfected with pXLG-A3 plasmid increased IgG productivity by 11 fold. Nevertheless, we showed here that VPA did not increase TNFR-Fc productivity to the same extent (1.2 fold) even by optimization of its concentration. Furthermore, we investigated the effect of plasmid methylation (TNFRFc and IgG) on TGE using VPA. We found that IgG expression was less influenced by this modification than TNFR-Fc. In fact, TNFR-Fc plasmid methylation drastically decreased specific productivity (0.2 fold). And VPA could almost entirely compensate this effect (0.8 fold). We therefore hypothesized that VPA has an impact on pDNA methylation in the context of TGE.

2011

Two strategies to improve transient gene expression in HEK293E and/or CHO-DG44 cells

Guillaume Lüthi

2011