The Molecular Mechanism Of Netilmicin Sulfate Against Burkholderia Pseudomallei And Study On The Gut Microbiota Of Marmot

Objective:Zoonosis has always been a global public health problem.Burkholderia pseudomallei is a zoonotic pathogen,causative agent of melioidosis that can affect any organs of the body.This bacterium has the characteristics of natural resistance to multiple antibiotics and easy resistance in later stage,which leads to poor clinical effect in its treatment.Melioidosis has high mortality rate,therefore finding relevant inhibitors and treatment schemes is extremely important.In addition,epidemiological studies have shown that Yersinia pestis infected by human beings are transmitted by Tibetan sheep,while Yersinia pestis of Tibetan sheep originate from wild marmots in this area,indicating that wild marmots in this area are also hosts of Yersinia pestis.Furthermore,studies have suggested that zoonotic pathogens-flavivirus have been detected in ticks,cattle,sheep and people in Hulun Buir in Inner Mongolia,China.However,whether wild marmots in the region also carry zoonotic pathogens.Currently,no research has been documented on this aspect.This study,consists of two major parts,one is mainly analysis the structure of Hcp protein of T6SS-4 in B.pseudomallei,screening antibacterial drugs using Hcp protein as a target and research on the molecular mechanism of the antibacterial drug against B.pseudomallei;and the other is investigating the background of gut microbiota of wild marmots in Hulun Buir Grassland,Inner Mongolia,China,including bacteria,fungi,viruses and archaea in order to understand whether there are zoonotic pathogens and the role of gut microbiota.Methods:1.Analysis of the Hcp protein crystal structure of T6SS-4 in B.pseudomallei,screening antibacterial drugs using Hcp protein as a target and research on the molecular mechanism of the antibacterial drug against B.pseudomallei.（1）The prokaryotic expression vector p ET-30a（+）-Hcp containing His-tag sequence was constructed and transformed into BL21（DE3）.The target protein expression was induced by IPTG.The target protein was purified by His Trap HP 5 ml columns（GE Healthcare）and molecular sieve.The expressed protein was identified by SDS-PAGE and western blot.The Hcp protein crystal was screened,and the crystal data was collected by the Shanghai Synchrotron Radiation Center（SSRF）,and the protein structure was analyzed and refined by HKL2000,CCP4i,COOT and Phenxi and other software.（2）The virtual screening was performed using the Yin Fu Computing Platform,and the inhibitors that really bind to Hcp protein stably and possessing inhibitory effect on B.pseudomallei were screened.（3）Construction of infected A549 cells and C.elegans model（including early events and late events）to test the toxicity of the inhibitor to the host and the protective effect of the inhibitor on the infected host model.（4）The molecular docking method performed by Autodock Vina simulates the interaction between Hcp and inhibitors,and then uses Discovery Studio Client 2019 software to draw different forms of interaction.In addition,the combination of Hcp protein and inhibitor was simulated by molecular dynamics using GROMACS 2019 software.2.Investigation of the background of gut microbiota of wild marmot in Hulun Buir Grassland,Inner Mongolia,China.We selected five locations in the Hulun Buir Plateau in Inner Mongolia,China.Three fecal samples were randomly taken from each location,with a total of 15 samples.The three fecal samples from each location were mixed into one group,giving a total of five groups.Then,we created a comprehensive database of bacterial,fungal,viral,and archaeal genomes and aligned metagenomic sequences against the database.Results:1.Achievement of the Hcp protein crystal structure of T6SS-4 in B.pseudomallei,screening antibacterial drugs using Hcp protein as a target and research on the molecular mechanism of the antibacterial drug against B.pseudomallei.（1）The crystal structure of Hcp protein of T6SS-4 has been obtained.The crystal structure of Hcp shows oneα-helix and eightβ-fold.The dimer structure is formed through the interaction of amino acids on the contact surface,and the contact surface of the dimer is mainly composed of a monomerα-helix,β₆-fold and another monomerβ₂-fold and other amino acid residues.Six monomers are aggregated into a hexamer ring,which can be stacked into tubes.（2）Virtual screening finally screened 16 small molecule inhibitors for binding validation and activity experiment validation,and Netilmicin sulfate（NET）binding to Hcp protein was obtained through experimental screening,and its affinity KD（M）=1.94E-05.（3）Evaluated the inhibitory effect of NET on B.pseudomallei:On infected A549cells,with the increase of the concentration of NET,its bacteriostatic effect increased and the cells survival rate increased.In the early event,compared with NET or CAZ alone,the combination of NET and CAZ enhanced the cell protection rate and bacteriostatic effect.On infected C.elegans,with the increase of the concentration of NET,its antibacterial effect increased and the survival rate of nematode increased.Compared with the control group,the survival rate of treatment groups was significantly higher,and the difference was statistically significant.（4）NET and Hcp protein were linked to each other,and the formation of the complex is spontaneous.For the Hcp-NET complex system,residues 54LYS,70MET,74LEU,75THR,76GLY,105ILE,108ARG,109VAL,and 156LYS may promote binding.The amino acid sites of NET binding to Hcp protein are the same as the main amino acid sites of the monomer indirect contact surface of Hcp protein dimer.2.The background of gut microbiota of wild marmot in Hulun Buir Grassland,Inner Mongolia,China.We delineated the detailed and distinct gut microbiota structures of marmots.A total of 5,891 bacteria,233 viruses,236 fungi,and 217 archaea were found.The dominant bacterial phyla were Firmicutes,Proteobacteria,Bacteroidetes and Actinomycetes.The viral families were Myoviridae,Siphoviridae,Phycodnaviridae,Herpesviridae and Podoviridae.The dominant fungi phyla were Ascomycota,Basidiomycota and Blastocladicmycota.The dominant archaea were Biobacteria,Omoarchaea,Nanoarchaea and Microbacteria.Furthermore,the gut microbiota was affected by host species and environment,and environment was the most important factor.There were 36,989 glycoside hydrolase genes in the microbiota,with 365 genes homologous to genes encodingβ-glucosidase,cellulase,and celluloseβ-1,4-cellobiosidase.Additionally,antibiotic resistance genes such as mac B,bcr A and msb A were abundant.Conclusion:In this study,we have found that Netilmicin sulfate inhibits the aggregation of Hcp proteins into circular and tubular structures by binding to some amino acid sites on the indirect contact surface of the monomer of T6SS-4’s Hcp protein dimer,which affect the functional structure of T6SS-4 and against B.pseudomallei,Thus,this novel mechanism plays the role of anti-B.Pseudomallei.This study provides a new research and development approach for studying inhibitors,promoting treatment,preventive use,and targeted therapeutic drug development.In addition,Netilmicin sulfate possess the activity of inhibiting B.pseudomallei in the body,and its activity is better than the current clinical drug Gentamicin and Sulfamethoxazole/Trimethoprim.Netilmicin sulfate combined with Ceftazidime could not only enhance the protective effect on host cells,but also reduce the drug concentration.Netilmicin sulfate and Ceftazidime have a synergistic effect to reduce the intake of drug load,which is conducive to reducing the drug resistance rate of B.pseudomallei to antibiotics.We suggest that Netilmicin sulfate and Ceftazidime could be used in the treatment of melioidosis caused by B.pseudomallei.Besides,we found that no zoonotic pathogens were found in the gut microbiota of the wild marmots under study,but their gut microbiota had population diversity and functional diversity,which provides a basis for further research on the regulatory effects of the gut microbiota on the host.In addition,metagenomics revealed that the gut microbiota of marmots can degrade cellulose and hemicellulose.However,no zoonotic pathogens were found in the gut microbiota of this wild marmot.