| Salmonella is an important zoonotic pathogen,which seriously threatens human health and the development of animal husbandry.Salmonella enterica serovar typhimurium is one of the serotypes with the highest isolation rate in Salmonella.Since S.Typhimurium was reported,the colony morphology was mostly smooth,and only a small number of colonies were reported as rough.During the monitoring of intestinal pathogens,our group identified a novel rough type variant of S.Typhimurium.It has unique phenotypic and genotypic characteristics,shows an upward trend in prevalence,and evolves into a new epidemic clone.This new epidemic variation trend of S.Typhimurium and its potential threat to public safety cannot be ignored.At present,it is still unclear that the biological characteristics and variation mechanism of rough type S.Typhimurium.On the basis of previous work,this project intends to carried out in-depth and systematic research on the biological characteristics and phenotypic variation mechanism of rough type S.Typhimurium,thus providing a scientific basis for hazard assessment and treatment prevention and control of S.Typhimurium in our country and possibly worldwide..Objective1.To clarify the morphological characteristics and growth characteristics of rough type S.Typhimurium,rough strains and smooth strains from the same evolutionary branch were selected for morphological characteristics analysis,growth curve characteristics under different acid-base and osmotic pressure environments,and biofilm formation ability analysis.2.Through proteomics and metabolomics analysis,the expression differences of proteins and metabolites between rough type strains and smooth type strains were clarified,and the differences of functions and pathways between rough type strains and smooth type strains were clarified by GO analysis and KEGG analysis,thus lay the foundation for the further study of the phenotypic variation mechanism of S.Typhimurium.3.By means of transmission electron microscopy,Western-Blot(WB),construction of gene deletion strains and bioinformatics analysis,the differences of main components between the two types strains were verified,and the key mechanism of rough type variation was explored.Methods1.Using rough diagnostic soft agar and Congo red cellulose medium method to determine the morphological characteristics of different types of S.Typhimurium.The growth curves under different PH conditions and osmotic pressure were measured by real-time microbial growth analysis system.The biofilm formation ability was evaluated by crystal violet staining and laser scanning confocal microscopy.2.The protein products of rough and smooth type strains were identified by DIA proteomics.The function of differential proteins was described by domain analysis and GO function analysis.The main differential protein pathways were found by KEGG pathway analysis.3.The metabolites of rough and smooth type strains were identified by UHPLC-MS.The significant differential metabolites were screened by OPLS-DA.The main differential metabolites pathways were found by KEGG pathway analysis.4.The joint omics analysis was used to find the KEGG pathway with more differentially expressed proteins and metabolites.5.WB experiment was used to verify the difference in flagellar protein expression among different types strains.The flagella content and length of different morphological strains were observed by transmission electron microscopy;construction of csgD knockout strain verified that csgD is a key gene involved in rough type variation of S.Typhimurium;bioinformatics analysis showed that the gene mutation affecting the expression level of csgD gene revealed the key variation mechanism in rough strains.Results1.On the soft agar medium for the diagnosis of Salmonella asperity,the rough type strain showed the characteristics of "fried eggs",and the whole colony could be picked up by inoculation ring,while the smooth strain showed the morphological characteristics of full center,smooth edge and regular pattern.On the Congo red Coomassie brilliant blue medium,the rough type strain showed red,dry and rough morphological characteristics,while the smooth type strain showed brown and smooth morphological characteristics.On the automatic colony real-time culture counting station,it was found that the growth morphology of rough strains was significantly greater than that of smooth strains from the logarithmic phase.Scanning electron microscopy showed that the rough strain were scattered and wrapped by a large number of filamentous substances,while smooth strain were scattered with only a small amount of filamentous substances.Compared with smooth type strain,rough type strain has stronger survival ability under acid(PH=5.5),alkali(PH=8.5)and low osmotic pressure(NaCl=0.1%),and it also has stronger biofilm formation ability,which indicated that rough type strain has stronger survival and adaptability under environmental stress.This may be an important factor in the formation of clonal advantage of rough strains2.The results of DIA differential proteomics analysis showed that the expression levels more than 3 times in rough strains compared with smooth strains mainly included:proteins CsgD,CsgG,CsgE,and CsgF related to the production of bacterial fimbriae and cellulose,proteins FliD,FlgL,FlgK,FlgB,and FlgC related to flagellar formation,and proteins SipD,SipC,and SipA related to virulence proteins and type III secretion system;the expression levels more than 10 times in rough strains compared with smooth strains mainly included:CsgD、CsgG、CsgE、FliD;CsgD is the most differentially expressed protein,up to 28.82 times.The results of functional enrichment analysis showed that flagellar motor-related functions were the most prominent functions.The results of KEGG pathway analysis showed that there were more differentially expressed proteins in flagellar assembly pathway,biofilm formation pathway,bacterial chemotaxi pathway,two-component system pathway and ABC transport system pathway;the flagellar assembly is the most prominent pathway for differential protein enrichment3.The results of UHPLC-MS analysis showed that Organic acids and derivatives accounted for the highest proportion of metabolites identified by rough and smooth strains.The expression levels more than 3 times in rough strains compared with smooth strains mainly included:carboxylic acids and derivatives(creatinine and Tyr-Val-Arg),benzene and substituted derivatives,quinolines and derivatives,flavonoids,organooxygen compounds,pyridines and derivatives,peptidomimetics,creatinine and tyr-val-arg;creatinine is the most differentially expressed metabolite,up to 96.17 times.The results of KEGG pathway analysis showed that there were more differentially expressed metabolites in the ABC transport pathway and amino acid biosynthesis pathway;the ABC transport pathway is the most prominent pathway for differential metabolite enrichment.4.The results of joint omics analysis showed that 59 KEGG pathways were involved in both proteomics and metabolomics.The most common pathways involved in proteins and metabolites were two-component system pathway,ABC transport system pathway,flagellar assembly pathway,bacterial chemotaxi pathway pathway and biofilm formation pathway,among which bacterial chemotaxi pathway was the most significant pathway for enrichment of differentially expressed proteins and metabolites.5.WB experiment and transmission electron microscopy results showed that the expression level of flagellin in rough type strains were higher than smooth type strains,which verified that the differential expression level of flagellin in proteomics results was high.After csgD gene was knocked out,the morphology of rough strain became smooth;bioinformatics analysis showed that both rough type strains and smooth type strains carried csgD gene,but the point mutation(199bp position)occurred in the csgABC-csgDEFG operon region(755bp)of 20 rough type strains,from C to A,which belonged to the csgD promoter region,but the smooth type strain did not occur this point mutation,suggesting that the point mutation in the csgD promoter region of rough type strains made csgD strongly activated and overexpressed,which may be the key mechanism of phenotypic variation.This study further revealed the variation mechanism of rough type strains.ConclusionsRough S.typhimurium has stronger survival ability and biofilm formation ability than smooth S.typhimurium in acid environment,alkali environment and low osmotic pressure environment,suggesting that rough S.typhimurium has stronger survival and adaptability to environmental stress.And this may be an important factor in the formation of clonal advantage of rough strains.The biological characteristics of rough strains may be significantly up-regulated in flagellar pathway,biofilm formation pathway,and bacterial chemotaxis pathway,and the flagellar expression is high.The point mutation in the promoter position of csgD in the rough type strain makes csgD strongly activated and overexpressed,which may be the key mechanism leading to phenotypic variation. |
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