Effect Of Hfq On Klebsiella Pneumoniae Resistance And Virtual Screening Of Hfq Inhibitors

Klebsiella pneumoniae(K.pneumoniae), a gram- negative bacterium, is distributed wildly in nature, causing a variety of human and animal diseases such as pneumonia, urinary tract infection, bacterial meningitis, septicemia, respiratory disease and gastrointestinal disease. It is an important zoonosis pathogen. Especially recently, K.pneumoniae was the great threat to human and animal safety and health, raising significant public health concerns owing to its multi-drug resistance and globe reach. The situation of the resistance was really serious. K.pneumoniae was one of the main pathogen which contained carbapenemases, and it was more resistance to the third generation cephalosporins. It is almost has no effective drug for treat K.pneumoniae infection. Explore the resistance mechanism for developing a new approach for clinical treatment of K.pneumoniae infections is highly significant.Hfq, a key post-transcriptional regulator in bacteria, can mediate the interaction between s RNA and m RNA, influencing the stability of m RN A, facilitating or impeding m RN A translation. This schema of controlling the gene expression is very important for bacteria. It is closely related to variety cellular processes, physiological and biochemical reaction, especially the growth, metabolism, virulence, and so on. In K. Pneumoniae, Hfq has greater influence of the gene expression. So, it is highly significance for understanding the formation process of resistance by investigating the mechanism of how Hfq regulate the resistance.K.pneumoniae hfq mutant was constructed by using gene knockout method which based on the Red-recombination system. The drug susceptibility was determined by broth microdilution method accordance with C LSI guidelines. In the tested 16 antibiotics, ?hfq was more resistance to cefazolin sodium, ceftazidime, cefotaxime, cefuroxime sodium, meropenem, levofloxacin, erythromycin, azithromycin, aztreonam, and more sensitivity to streptomycin sulfate, tobramycin sulfate and amikacin sulfate. There is no change for polymyxin B, colistin and rifampicin. Further time-killing curve study was performed to verify the enhancement of resistance to cephalosporins. And the results showed that the hfq mutant could survive under higher concentrations of cephalosporins than the wild-type strain could. In light of Hfq is global regulator, the relative m RN A levels of acr B, oqx B, ram A, mar A, sox S, omp35 and omp36 were determined. The biofilm were quantified using the crystal violet method. Quantitative RT-PCR showed that expression levels for acr B, oqx B, ram A, mar A, sox S and omp36 were upregulated in the hfq mutant. And the biofilm were markedly enhanced. In addtion, the present study was test the relative expression levels of penicillin-binding proteins, which are the targets of cephalosporins. The results shows that except PBP4, expression levels for 8 out of the 9 penicillin-binding proteins were downregulated in the hfq mutant, especially for PBP6 b and PBP7 that have significant difference(p<0.05). It indicate that PBP6 b and PBP7 may be important part of the pathway by which Hfq regulate the resistance.The regulation of Hfq is mediated by interaction between s RNA and m RNA. Finding new s RNA or s RNA new function will help address the regulation of Hfq on K.pneumoniae resistance and stand a change of drug discovery. In the present study, 59 candidate s RN A sequences were predicted by bioinformatics methods. Using PCR and real-time quantitative PCR, 32 s RNAs were verified. For determining which s RN A was related to resistance, K.pneumoniae was treated by cefazolin sodium, levofloxacin, tobramycin, meropenem and morpholine oxazole, and then the total RN A was isolated respectively. Semi-quantitative method was implemented to test the s RNA expression changes from each total RNA for ascertain the related s RNA.Cephalosporin-aminoglycoside combination was a common scheme which used to treat K.pneumoniae infection in clinical. Although hfq mutant was more resistance to cephalosporin, the susceptibility to aminoglycoside was decreased. In addition, the level of drug resistance between K.pneumoniae biofilm and suspended was the same. Meanwhile, the virulence and invasiveness of hfq mutant were significantly weaker. Therefore, Hfq inhibitor combine with aminoglycoside may provide a effective strategy for the prevention and treatment of K.pneumoniae infection. In the present study, we used the structure-based drug design method which involved with homology modeling, activity pocket prediction, virtual screening to identify the potential inhibitors against Hfq. The approach applied here has been successful in obtaining ten compounds base on the binding energy and ADMET analysis. ZINC25147445 possess the lowest molecular binding energy(-11.4kcal/mol). Furthermore, the docking simulation analysis provides useful information to demonstrate the molecular mechanism of the inhibition. The result showed that hydrophobic force, hydrogen bond and ?-? stacking occurs in hits-Hfq complexes. GLN-8, GLN-41, PHE-42, TYR55, LYS56 and HIS57 were the major binding sites. This insilico study helps us to discover potential inhibitors against K.pneumoniae Hfq using a structure-based drug design protocol, and prepare for further cell-based analysis.In summary, Hfq play a very important role in K.pneumoniae resistance. The effects of Hfq on resistance were achieved by regulating t he expression of efflux pump, porin and the antibiotic targets. The forming ability of biofilm was also involved with. Bioinformatics and experiments methods were implemented to identify s RNAs which has to do with the resistance. In addition, we discover potential inhibitors against K.pneumoniae Hfq using a structure-based drug design protocol in silico, and prepare for further cell-based analysis. Our study will provide basis for better understanding the mechanism of K.pneumoniae resistance and also provide novel strategy for developing anti- infection drugs.