| Pseudomonas aeruginosa is one of the most common pathogenic bacteria causing hospital infections. As an opportunistic pathogen, P. aeruginosa mainly infects immunocompromised people and people with severe burns, and can cause wound infections and serious pneumonias. P. aeruginosa can survive in a variety of environments, especially in the moist environment. It also causes serious environmental pollution and instrument equipment pollution. P. aeruginosa is highly resistant to multiple antibiotics, and is hardly to clear away once infected. Therefore, the prevention and treatment of P. aeruginosa infections are gaining more and more attentions.Recent researches have shown that the antibiotic resistance of P. aeruginosa is mainly associated with the formation of biofilms. Biofilm is a group of microorganisms in which cells stick to each other on a surface. These adherent cells are frequently embedded within a self-produced matrix of extracellular polymeric substance (EPS). Most bacteria exist as biofilms in nature. In contrast to planktonic cells, bacteria in biofilms tend to be highly resistant to antimicrobial treatment and host immune responses, and obtain great changes in cell morphology and physiology. EPS is the major component, and occupies 85% of the biofilm volume. EPS provides support for the formation of biofilms, and can promote the communication between biofilm cells. EPS mainly contains extracellular polysaccharides, proteins, nucleic acids, lipids and cell debris. Among them, extracellular polysaccharide is the main component. Due to the widely distribution and strong ability to form biofilms, P. aeruginosa has become a model strain for biofilm research.As an important virulence factor, flagellum plays important roles in bacteria motility and the process of biofilm formation. Expression and regulation of the polar flagellum in P. aeruginosa involves more than 40 different genes, which are clarified into four classes. FleQ is the master regulator, and directly or indirectly regulates the expression of the majority of flagellar genes. Previous studies also found that FleQ is a novel c-di-GMP receptor and can regulate EPS gene transcription. Sequence alignment showed that FleQ belongs to the NtrC phosphorylation two-component family. However, it lacks the conserved phosphorylation site and the corresponding sensor kinase. Besides, FleQ does not contain any conserved c-di-GMP binding motifs. As a consequence, how FleQ binds to c-di-GMP and how it regulates gene transcription has been of great interest to scientists.The first part of our work mainly focused on the structural and functional analysis of FleQ REC domain (FleQR). We purified and crystallized FleQR, and finally got its crystal structure. Through structural comparison with phosphorylation proteins NtrC and StyR, we found that FleQR showed obvious differences with these proteins. According to the specificity of FleQR, we performed experiments to examine whether it plays a role in FleQ function. We constructed the fleQ gene deletion and complementary strains, and subjected them to in vivo functional analysis as well as in vitro biochemical experiments. Experiment results showed that bacteria failed to activate flagellar gene transcription and lost the motility ability due to the lack of REC domain. In contrast, the transcript levels of EPS genes showed obvious increase in these strains. These indicated that REC domain is essential for FleQ function. Through further structural analysis and biochemical experiments, we found that FleQR exists as "transverse dimer" in solution, and F26 residue plays a key role in dimer formation. This has never been reported in other homologous proteins. Our results showed that the dimeric nature is important for FleQ function. Disruption of this interface resulted in a FleQ that tends to form monomer with significantly reduced activity. In addition, through structural modelling and in vivo experiments, we determined that residues R144 and R185 are responsible for c-di-GMP binding, which has been verified by ITC experiments. Our results also indicated that binding of c-di-GMP and ATP can interfere with each other because the binding sites are adjacent. What’s more, the dimeric nature is also essential for FleQ to bind c-di-GMP. Finally, based on all these results, we proposed a simple model for FleQ to regulate flagellar gene transcription.The second part of our work mainly focused on the structural and functional analysis of PslG, a glycosyl hydrolase. As mentioned above, EPS are important for bacterial biofilm formation. P. aeruginosa mainly produces three kinds of polysaccharides:alginate, Pel and Psl. In P. aeruginosa PAO1, Psl is more important than Pel for biofilm microcolony formation and antibiotic resistance. Psl, a repeating pentasaccharide containing D-mannose, D-glucose and L-rhamnose acts as "molecular glue" to promote bacterial cell-cell and cell-surface interactions and it can form a fiber-like web to enmesh bacterial communities.Biosynthesis and secretion of Psl in P. aeruginosa relies on 12 psl genes (PA2231-PA2242). PslG exists in the periplasm and is essential for Psl synthesis. The pslG deletion strain could not synthesize Psl and the biofilm formation was obviously reduced. However, the specific function of PslG is unclear. Interestingly, sequence BLAST against the NCBI database and previous structural forecast suggested that PslG belongs to the CAZy glycosyl hydrolase family 39.Through cooperation with Prof Luyan Z. Ma’s lab from Institute of Microbiology, Chinese Academy of Sciences, we found that PslG was able to inhibit bacteria biofilm formation when over-expressed in vivo or supplied exogenously. It could also disrupt a pre-formed biofilm. Anti-Psl immunoblotting and polysaccharide hydrolysis experiments indicated that PslG could degrade Psl directly. To gain insight into the molecular mechanism, we have solved the crystal structure of PslG and determined its catalytic domain and carbohydrate binding domain (CBM). Glul65 and Glu276 are the key catalytic residues, and there are some other residues which contribute to the formation of the catalytic cleft. Structural analysis indicated that PslG processes typical features of an endoglycosidase. Through molecule docking, we proposed a model that represented the binding of a portion of Psl repeat unit containing 3 mannoses and 1 glucose to the catalytic site. This model suggests that PslG might cleave the bond between β-D-Man and α-L-Rha. In addition, PslG gave an abnormal large elution volume while loaded onto a superdex 200 column. This might suggest that the CBM domain was responsible for carbohydrate binding.Briefly, our studied were mainly focused on the structural and functional analysis of two proteins FleQ and PslG, which are related to P. aeruginosa biofilm formation. The function mechanisms have been preliminarily proposed in this paper. As introduced in the paper, FleQ could regulate flagella and biofilm formation, and PslG was able to inhibit and disrupt bacteria biofilms. Since both flagella and biofilm play important roles in P. aeruginosa pathogenicity, our researches will provide significant clues for the treatment of this kind of bacterial infections. |
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