| BackgroundAerospace environmental factors include weightlessness,radiation,low pressure,hypoxia and so on.Since special environmental factors such as low pressure and hypoxia,solar ultraviolet radiation and other special environmental factors can be alleviated by spacecraft barrier,weightlessness is one of the main factors affecting the astronauts’ health in the current aerospace work,and it is concerned by most of the aerospace medicine researchers.During space activities,astronauts may take some opportunistic pathogens into the space environment.Under the influence of the special environmental factors especially for weightlessness,the infectivity,immune characteristics,pathogenicity and drug resistance of these opportunistic pathogens may be changed accordingly.At the same time,some studies have found that the aerospace environment will also lead to the reduction of astronauts’ immune function,so some opportunistic pathogens may pose a great threaten to astronauts’ health.Escherichia coli(E.coli)is the most common opportunistic pathogen in human body.The colonization in human body makes it enter the spacecraft with astronauts,and it may be affected by weightlessness.Some studies have reported that under weightlessness or simulated weightlessness,E.coli will undergo a series of changes,such as increased growth level,drug resistance and virulence,which will increase its threaten to human health.However,the mechanism of these changes is still unclear.Host factor for RNA phase Q β Replication(Hfq)is an RNA molecular chaperone protein that exists in E.coli,Pseudomonas aeruginosa and other bacteria.Studies found that Hfq can affect the growth level,environmental stress tolerance and drug resistance of bacteria.At the same time,Hfq can also affect bacterial environmental stress tolerance and drug resistance by regulating RNA polymerase sigma factor(RpoS),coproporphyrinogenⅢ dehydrogenase(HemN),and multidrug efflux pump RND permase(AcrB).However,the effect and mechanism of Hfq on environmental stress tolerance and drug resistance of E.coli under simulated weightlessness are still unclear.Objective1.To determine the changes of biological characteristics of E.coli under simulated weightlessness;2.To detect and analyze the expression level of E.coli transcriptome under simulated weightlessness;3.To preliminarily explore theregulation mechanism of Hfq on tolerance changes of E.coli under simulated weightlessness.Methods1.Used horizontal thermostatic oscillator and Gravite simulated microgravity control system to establish normal gravity and simulated weightlessness environment for bacteria.After 14 days of continuous passage and culture of E.coli,the OD600 value was measured by spectrophotometer to observe the growth level;The morphological changes were observed by scanning electron microscope;The biochemical metabolic changes were detected by Biolog GEN Ⅲ microplate;The biofilm was detected by crystal violet staining;And after high temperature,hypertonic,oxidative stress and acid environment culture,observed the changes in environmental stress tolerance by calculating the survival rate by plate coating method;and determine the drug resistance by the paper diffusion method.2.To extract the total RNA of E.coli after continuous passage and culture for 14 days under two gravity conditions,and performed transcriptome sequencing,analyzed the expression changes of E.coli transcriptome level after simulated weightlessness,and screened the differentially expressed genes.3.Based on the results of differentially expressed genes,the change of hfq was verified by RT-qPCR,and constructed the hfq deletion strain(Δhfq-E.coli)by CRISPR-Cas9 method knocked out hfq in the E.coli.After continuous passage and culture under simulated weightlessness conditions for 14 days,the differences in growth curve,environmental stress tolerance and antibiotic sensitivity between the Δhfq-E.coli and the wild strain under two gravity conditions were observed.The mechanism of hemN,rpoS and acrB regulated by hfq under simulated weightlessness was preliminarily explored by RT-qPCR.Results1.Compared with the normal gravity group,the growth level of E.coli under simulated weightlessness were increased(P<0.01);The morphology became smaller(P<0.001);In the experiment of using carbon source,for the carbohydrates experiment,for dextrin,D-galactouronic acid,pectin,sucrose,D-mannose,D-fructose,L-fucose,D-gluconic acid,N-acetyl-D-glucosamine,N-acetyl-β-D-mannosamine and β-the utilization of formyl-D-glucoside were all changed(P<0.05),and the results were mainly increased;In the acid experiment,for D-malic acid,L-malic acid α-Ketoglutaric acid,citric acid,mucic acid α-Hydroxy-butyric acid β-The utilization of hydroxy-D,L-butyric acid,formic acid,acetoacetic acid,p-hydroxy-phenylacetic acid,quinic acid,propionic acid and bromosuccinic acid were all changed(P<0.05),and the results were mainly decreased;In the alcohol experiment,the utilization of D-arabinol and glycerol increased;In the amino acid experiment,the utilization of D-serine,glycine-L-proline,L-alanine,L-serine and L-aspartic acid increased(P<0.05);In the experiment of glycolysis intermediate products,the utilization of D-glucose-6-phosphate were increased(P<0.01);In the ester experiment,the utilization of methyl D-lactate were decreased(P<0.001);The chemical sensitivity to pH5,pH6,clostridic acid,rifamycin,guanidine hydrochloride,amtreonam,sodium butyrate,lithium chloride and tetrazolium blue were decreased(P<0.05);The formation of biofilm did not change significantly(P>0.05);The tolerance to environmental stress of high temperature,hypertonic and hydrogen peroxide were increased(P<0.001);Antibiotic sensitivity to aminoglycoside antibiotics amikacin and kanamycin,quinolone antibiotics ciprofloxacin and levofloxacin,and ceftriaxone were decreased(P<0.05).2.Compared with the normal gravity,the E.coli of simulated weightlessness group has 229 significantly differentially genes expressed(P≤0.05,|log2Fold Change|≥1),of which 158 genes are up-regulated and 71 genes are down-regulated.Gene ontology(GO)gene enrichment analysis results showed that differential genes were enriched into 14 pathways in three major categories,that including biological processes,molecular functions and cell components.The analysis results of the Kyoto Encyclopedia of Genes and Genomes(KEGG)showed that 229 differential genes were mainly enriched in metabolism-related pathways,including alanine,aspartic acid and glutamic acid metabolism pathway and glycine,serine and threonine metabolism pathways,which is consistented with the above amino acid metabolism trends.At the same time,the differentially expressed genes related to environmental stress tolerance and drug resistance were screened,among which the expression of hfq,hemN,rpoS,acrB were increased compared with normal gravity group(P<0.05).3.Under simulated weightlessness conditions,compared with wild plants,the growth level of Δhfq-E.coli were decreased,the tolerance to oxidative stress were decreased,the tolerance to high temperature had not significant difference,the tolerance to acid and the hypertonic stress were increased,and the antibiotic sensitivity were decreased;The expression of hemN related to oxidative stress tolerance and acrB related to drug resistance were decreased(P<0.001),however,the expression of rpoS related to stress tolerance in high temperature and hypertonic environment had not significant difference.Conclusions1.Simulated weightlessness increased the growth level of E.coli,decreases of the morphology,and changes of the utilization for carbon sources(sugars,acids,alcohols,amino acids,glycolytic intermediates and esters).Meanwhile,it also decreased the sensitivity of E.coli to chemical substances and increased the tolerance of environmental stress,and the antibiotic sensitivity was decreased.2.The expression level of E.coli transcriptome was changed under simulated weightlessness conditions,mainly by up-regulated metabolic pathways,including alanine,aspartic acid and glutamic acid metabolism and glycine,serine and threonine metabolism pathways,which were consistent with the above amino acid metabolism trend.Differentially expressed genes were screened,genes related to tolerance among which the expression of hfq,hemN,rpoS and acrB were increased.3.Under simulated weightlessness,hfq regulated the tolerance of E.coli,and this effect might be related to hemN and acrB. |
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