| In recent years, the antibiotic resistance of clinical pathogenic bacteria has become an increasingly severe problem as a result of the wide use of antibiotics. As a gram-negative bacteria and opportunistic pathogen, Pseudomonas aeruginosa is the third source of nosocomial infections and can cause serious infections during medical treatment. Additionally, the treatment of such infections is fairly difficult due to the high innate antibiotic and acquired resistance of P.aeruginosa. Thus, it is of significant meaning to carry out research into both the infection and innate antibiotic resistance mechanisms of P.aeruginosa, which will provide new measures for prevention and treatment of infections caused by P.aeruginosa.The pathogenesis of P. aeruginosa is modulated by signaling pathways. This study focuses on the following signaling pathways:quorum-sensing systems in P. aeruginosa, signal molecule of sub-inhibitory concentration antibiotics and inter-species signal autoinducer-2. All these three pathways are related to the modulation off P.aeruginosa pathogenesis. The virulence factors are modulated by species-specific acyl-homoserine lactone (AHL) mediated quorum sensing as well as quinolone mediated quorum sensing systems. There are two quorum-sensing systems based on the AHL signal, namely Las system and Rhl system. In addition, there is a third quorum sensing system based on the Pseudomonas quinolone signal (PQS) and2-heptyl-4-quinolone (HHQ). Besides the ability to inhibit the growth of and kill bacteria, antibiotics of sub-inhibitory concentrations can act as the signal between bacteria interaction and participate in gene regulation. Moreover, research also shows that signal AI-2between universal quorum-sensing systems can modulate the P. aeruginosa pathogenesis. Furthermore, these signaling pathways also interact with one another. Research of interaction between cells is of significance for understanding the pathogenesis mechanisms of pathogen, and it also provides a basis for the development of novel drugs.This thesis mainly focuses on the modulation mechanisms of signaling pathway on P. aeruginosa pathogenesis, as well as the interactions between these signaling pathways. The major work and contributions of this thesis are as follows:(1) This study investigates the phenazine regulation by sub-inhibitory concentration of antibiotics in P. aeruginosa. Based on existing research, this work reveals that phenazine is regulated by sub-inhibitory concentration of tetracycline. At the genome level, the transposon mutation method is used to screen out the genes and the regulation pathway, involved in regulation of operons phzA1B1C1D1E1F1G1(phzA1) and phzA2B2C2D2E2F2G2(phzA2), which are responsible for phenazine production, especially, the genes and pathways involved in regulation of phzAl and phzA2by spectinomycin. Meanwhile, the relationship between quorum-sensing system and sub-inhibitory antibiotic modulation is investigated based on operon phzA1/phzA2expression.In preliminary work, we have discovered that sub-inhibitory concentrations of spectinomycin can activate the expression of phzAl and phzA2. By adding spectinomycin, the expression of phzAl is30-fold higher and phzA2expression increases by4-fold. With the employment of the transposon mutation method, we screen the whole P.aeruginosa genome database for the genes involved in Spc mediated alternation of phzA1/phzA2expression. Among them, genes lasl, mvfR, vqsM,pqsE can positively regulate the expression of phzA1and phzA2, which well agrees with existing results. Besides, we have screened out eight genes that are identified to be related to the activation of spectinomycin for phzAllphzA2, indicating that these genes may be involved in this regulation. With the disruption of genes ppk and pcnB, the expression of phzA1increases after incubating with Spc. Ppk is a polyphosphate kinase while PcnB is a poly (A) polymerase. With the disruption of pilH, pilY and pilX, the expression of phzA2increases when exposed to Spc, indicating these three genes are related to the type4fimbrial biogensis protein production. In the algC deletion strain, activation of phzA2mediated by Spc was decreased, and algC encodes the phosphomannomutase. With the disruption of truA and rsaL, the alternation made by Spc disappeares, which indicates that Spc has no effect on phzA2expression in these two mutants. Thus, gene truA encodes the tRNA-pseudouridine synthase I while rsaL encodes the regulator protein related to quorum sensing in P.aeruginosa.(2) This work investigates the modulation mechanism on phenazine operons by quorum sensing systems in P. aeruginosa. Research shows that operons phzA1and phzA2in P. aeruginosa PAO1, which is opposite in PA14, most phenazine production is controlled by phzA1operon. In order to investigate the function of phzA1and phzA2, as well as the different regulation of these two genes in P. aeruginosa, we constructe and collecte several genes deletion strains which are the essential genes in regulatory systems in P. aeruginosa, and the promoter-reporter system of phzA1and phzA2are tested in the mutants. The study shows that, in the mutantâ–³lasI/â–³rhll/â–³pqsR, the expression of phzA1and phzA2are inhibited. However, in the gacA deletion strain, the essential regulator in two-component regulatory system, the expression of phzAl was inhibited while the expression of phzA2was activated. The result above means gacA can positively regulate phzA1while negatively regulate phzA2. Additionally, in the mutants of vqsR, PA0011and PA1243, experiments demonstrate that the expression of phzA2mediated by Spc is activated more than control, while the activation of phzAl by Spc remaines unchanged. These phenomenons reveal the difference in phzAl and phzA2expression, represented by the distinct modulation of phzA1and phzA2, may be related to these genes. Meanwhile, in studying the regulation of LasR, RhlR and PqsR on phzA1, experimental results show that LasR and RhlR indirectly modulated phzA1instead of directly binding to phzA1promoter region.(3) While investigating the relationship between quorum-sensing system and antibiotics regulation at sub-inhibitory concentration, the element las-box, the modulation responding element, is found in the upstream region of operon phzA1promoter. Previous study has revealed that alternation of phzA1expression by sub-inhibitory concentration of tetracycline is dependent on las-box. The expression of phzA1is higher under the existence of las-box. Research work in this thesis directly confirms the interaction between las-box and tetracycline. Specifically, las-box is not the only activate element for phzA1expression, but also the element for tetracycline modulation of phzA1.(4) This thesis further studies the signal AI-2effection on pathogenesis of P. aeruginosa and its mechanisms. Study has found that exogenous AI-2can affect pathogenesis of P. aeruginosa. The rat lung model experiment demonstrates that the existing of AI-2can aggravate lung infection by P. aeruginosa. Compared with pure P.aeruginosa infected condition, the lung infection is heavier under the existence of AI-2. While investigating the mechanism of AI-2function, we find that AI-2can regulate the Rhl system by activating the expression of gene rhll, rhlR and rhlAB in P.aeruginosa. Furthermore, the swarming ability of P.aeruginosa can be activated by AI-2as well. Although, AI-2can activate the major genes in Rhl system, the study shows that when AI-2exists, the activity of signaling molecular C4-HSL decreases, which leads to the speculation that AI-2competes with C4-HSL in regulation. These results reveal that the modulation of P. aeruginosa pathogenesis by AI-2may be achieved through the regulation of Rhl quorum sensing system. |
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