| Pseudomonas aeruginosa(PA) is a very common opportunistic pathogen. When there is a wound or the immune system is very low, it will infect individuals and cause diseases. What’s more, it is very difficult to remove and leads to chronic infection, which eventually leading to death. It is easy to infect the wound, so it is a major infection source of hospital surgery and some treatments, and often exists in patients with cystic fibrosis to cause pulmonary infection.Quorum sensing is a kind of communications between cells, which can regulate gene expression according to cell concentration. The quorum sensing pathway contains a large number of regulators, which regulate the expression of many virulence factors and help to adapt to various environments and resist various treatments. There are two major quorum sensing systems las and rhl in Pseudomonas aeruginosa, and the las system regulates the rhl system positively to form a hierarchical regulation network.Bacteria are individuals with relatively simple structure, but they usually get together to influence others and survive. Biofilm is one of the common ways of living. After the formation of biofilm, the resistance of colony to antibiotics and other drugs will be significantly enhanced. Cyclic diguanylate (C-di-GMP) is a second messenger molecule in many regulation pathways, and is widely found in bacteria. It can regulate the formation of biofilm. The synthesis and decomposition of C-di-GMP is mainly catalyzed by guanosine cyclic acid enzyme (DGC) and phosphodiesterase (PDE), and EAL domain is a kind of the PDE enzymes which can decompose C-di-GMP into PGPG.Type III secretion system (T3SS) is a major virulence factor that acts on eukaryotic host cells, and is widely found in many pathogens. It is a special needle-like structure, which can transfer the effector toxins from bacteria to the host cytoplasm. The terminal of needle inserts into the host cell membrane to form a transport channel, which is mainly formed by PopB and PopD.There is a las box on the upstream of the QS regulator vqsR, and it can be recognized and bonded by LasR specifically, so the las system can regulate transcription and expression of the vqsR directly. Another quorum sensing regulator QscR is an inhibitor of las and rhl system, and VqsR can inhibit qscR by binding the promoter specifically. It may indicate that VqsR can affect the regulation of QS by QscR, so VqsR is a very important regulator. We did not get the VqsR structure by protein purification, crystal experiments and structure analysis, but we got the structure of N-terminal part of VqsR, which will help us find the small molecules that VqsR may be combined with, and study how VqsR receives signals and regulates expression of downstream genes.Gene arr was found to have an important role in induction by aminoglycoside antibiotic to form biofilm in Pseudomonas aeruginosa. Arr is a membrane phosphodiesterase and its substrate is the second messenger C-di-GMP. Its periplasmic part can accept the stimulation of aminoglycoside antibiotics and regulate the activity of EAL domain, then regulate the intracellular level of C-di-GMP and affect the biofilm formation. We successfully purified the soluble membrane protein of Arr. Although there was no full-length crystal and structure, we tested its activity in vitro and verified the influence of different antibiotics on its activity. We verified aminoglycoside antibiotics could influence activity of EAL domain by the periplasmic domain. In addition, we got the structure of EAL domain.For membrane protein PopB and PopD of translocation pore in type Ⅲ secretion system, we purified the soluble proteins, got no PopB crystal and got PopD crystals which is small and unable to do X-ray diffraction experiments. We know that PopD and PopB can form a ring structure on the membrane, so we will try to co-express PopB and PopD or purify protein separately and react together for crystal screen. |
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