| Pseudomonas aeruginosa, one of the three major opportunistic pathogens of human infection, is widely distributed in the the natural world. Pseudomonas aeruginosa is the common cause of nosocomial infection, the treament to the infection of PA seems relatively difficult because of its internal tolerance to the antibiotics and disinfectants.The genome of PA has been sequenced recently. A whole length of 6.3 Mbp, the enormous genome information means much more physiologic adjustment proteins which extend the adaptation ability of PA to various environments.Recent researches showed that the production of the ectotoxic factor of PA is the behavior of a cluster of dense bacteria rather than a single bacterium. The ectotoxic factor excreted from small amount of bacteria activates the immunological response of the host to remove them, but if in large microbial population, the toxic genes will be express coordinately, which leads to a higher level of ectotoxic factor to overcome the host's defense system. Therefore, the process should be blocked before the toxic gene expressed coordinately. The phenomenon of density dependent specific gene expressing coordinately was called quorum sensing(QS) .Using the density response system, PA transfers information and supervises bacteria density through acyl-homoserine lactone(acyl-HSL) to control the production of its ectotoxic factor. It demonstrates that the QS signal system is necessary for the differentiation from a single PA cell to the complicated multi-cellular structure. At the same time, poly phosphate kinase (PPK) was found playing an important role in the formation of biofilm, generation of toxic factor and the motion mode of PA, which implies that the formation of biofilm and generation of toxic factor are regulated by several different genes.The biofilm play an important role to the tolerance of bacteria. The bacteria with the biofilm get the ability against antibiotics 1000 times than free ones. The mechanism of biofilm tolerance is so complex, just as following. ①. Restricting the permeation of antibiotics. Under the BF status, bacteria secret plenty of EPS, EPScould bond single bacterium to form microcolony. Both purify strain and multi-microorganisms can develop to microcolony. In these microcolony, bacteria growth up to the organized group with heterogeneity function. Bulks of microcolony intensify the BF, and the generation of EPS is very important to the development of BF. It has been confirmed that EPS has an apparent resistance effect on some antibiotics and insecticide. Now it is generalized accepted that the BF of PA prevent the permeation of aminoside I antibiotics. (2) Alteration of growth pace and metabolic activity. Some bacteria in the BF stay in a hypometabolism and limited oxygen status, which result in the insensitivity to the antibiotics. ?Transformation of phenotype. The BF bacteria are induced to express the different gene from the free status, thus the BF specific phenotype is generated.According to the genome analysis, there are 12 (resistance-nodulation division family ) RND excretion pump in PA. The P — lactamase produced by PA is its main mechanism of resistance to the P —lactamase antibiotics.The relationship between the QS system and drug tolerance(D QS system and the BF tolerance. QS system play a very important role in the formation of BF(2) QS system and the multi-drug excretion pump. One of the important excretion pump MexAB-OprM takes part in the signal transport of QS signal system.(3) QS system and P —lactamase. Whether the QS system join the regulation of expressing of P —lactamase is still a question.Therefore, interference of QS system is a hopeful path to lower the generation of ectotoxic factor and improve the drug sensitivity though dealing with biofilm. By searching the antagonist of key intermediate metabolite, a fresh way to new drug development and disease therapy will be established. But there are several problems we should resolve first, which kinds of genes relate to the QS system existing, which is necessary to the formation of biofilm and production of toxic factors, and whichkinds of genes cause the delayed reaction of the signal of the QS system.The technique of biochip emerged in the 1980s, and the gene chip is one of the most fully developed ones. Based on the hydrogen bond between base pairs in the nucleic acid double-strands, researchers could check the samples through detecting the fluorescence or isotope signals from the purpose single-strand. Because of its features of rapid * high performance and high-flux , gene chip has been the hot spot in the medical research field . Now the gene chip mainly applicated in finding the new gene, disease diagnosis, drug screening, toxicological genome, optimization of crops, environment detection, food sanitation surveillance and judicial identification. Wherein the chips which detect the gene mutation and level of gene expression are well developed.This research is designed as follow: based on the genome sequences of Pseudomonas aeruginosa , we could amplification , cloning , sequencing the anticipated 5,570 open reading frames. Then optimizating and cloning the key genes which are related to the QS signal system to prepare for the gene chips which can be used to screen the effective component of the drugs. Accordingly we may find several kinds of drugs, which could blockade the signal pass way , inhibit the expression of the toxical gene or formation of biofilm. In order to guide the clinical medication , we also would make the similarity analysis of gene expression between the strain isolated from patients and the PAO1 strain. Aimed to screen the drugs which could blockade the gene expression relating to the QS system and formation of biofilm in gene level ,our research applies the gene chips technique ,which is fit to the mode of multi-gene (at least 39 genes ) modulatory in QS system., and we believe it would realise our purpose that we could rapidly detecting PA , and then screening drugs which could treat PA effectively.1. To obtain the QS related gene fragmentsAfter search the latest literatures abroad , we induct all of the genes fragments, which have been confirmed to be related with the QS system and take these sequences depend on the results of bacteria genome. Then making the bacteria genome DNA as the template Resigning the specific primers by the biology software, amplification,cloning, sequencing and identification these production fragments.2. Manufacture of QS related gene cDNA chipsCollaborating with SHANGHAI HUAGUAN BIOCHIPS CO., Ltd, we developed the research of "Detection drug resistance mutation of pathogenic bacteria such as Bacillus tuberculosis by DNA chips". To draw assistance from this research and the equipments, we prepare to make cDNA chips of OS signal system related genes.3. Under the administration, the disparity of gene expression of QS related and formation of biofilmAfter the administration of tenebrimycin, comparing the growth state of PA01 between different drugs and to observe gene expression variance of QS related gene under a certain dose of tenebrimycin, further more to investigate which kinds of gene are inhibited or stimulated by the drugs.Comparing the expression difference of QS related gene of the same bacte ria during the different growth periods, platform phase and exponential phase, we found that: 9 genes exhibited to up-regulatory (PA2300,PA2144,PA1216,PA1212, PA2566,PA1319,PA2564,PA3189) and 6 genes presented to down- regulatory (PA2250,PA0399,PA0447,PA1658, PA1669,PA1999).CDNA chip is a new method to research the bacterial virulence and mechanism of drugs resistance. Until now , we prepare the QS related gene cDNA chips first time domestic and it would provide a possibility to build a theory and practical base of control cluster effect signal system , generation of bacteria virulence factors, elevation the drugs sensitivity and finding new drugs. It would supply an effective way to rapidly detect and control PA infection, also could construct a technical platform to investigate PA gene expression spectrum and gene function analysis.
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