Effects Of Salmonella Outer Membrane Protein Related Genes And Mutant Strains On Pathogenicity

Salmonella is a member of the Enterobacteriaceae and Salmonella genus.It can cause a variety of human and animal diseases.It occurs all over the world and causes serious economic losses.Outer Membrane Proteins(OMPs),as one of the main components on the outer membrane of bacteria,are great significance to the life activities and affect the survival and pathogenicity of bacteria.RNA is an important regulatory factor that can regulate m RNA.It participates in a variety of bacterial life processes,such as regulation of bacterial virulence,adaptation to environmental changes,include regulation of the expression of outer membrane protein genes,and the regulation of m RNA by s RNA often requires the participation of the chaperone protein Hfq.In many bacteria,Hfq not only cooperate with s RNA,but also regulate the expression of multiple genes independently in bacteria,which has an impact on bacterial growth,virulence,and invasiveness.In this study,Salmonella typhimurium LT2 was used as a template to construct Salmonella typhimurium omp N,fad L,ybf M,ryb B gene deletion strains,omp N,fad L,ybf M color indicator strains,and spot-deleted color strains.Changes are detected in the m RNA and protein levels of fad L,omp N,ybf M in Salmonella typhimurium after deletion,to explore the regulatory effects of sRNA RybB and Hfq on outer membrane proteins.The omp N,fad L,ybf M,ryb B,and hfq gene deletion strains were used to conduct a mouse infectivity test to evaluate the pathogenicity,and to study the effect of outer membrane protein-related genes on the pathogenicity of Salmonella typhimurium,and hope for buliting the foundation on the development of live vaccines and laid new antibacterial drugs.1.Construction and identification of a strain of Salmonella typhimurium with deletion of genes related to outer membrane proteinBased on the red-homologous recombination and FLP/FRT system,fad L,ybf M,omp N,and ryb B were knocked out in Salmonella typhimurium.PCR sequencing results showed that fad L,ybf M,omp N,ryb B and ryb B(GGAGCCCATC)were successfully knocked out.The Salmonella typhimurium deletion strains?fad L,?omp N,?ybf M were made into corresponding competent cells,and the plasmid p CE40 was transferred into the corresponding competent cells through electrotransformation,base the FLP/FRT system,?fad L,?omp N,?ybf M strains remove the remaining FRT sites and the Kn resistant fragments were construct and color indicator strains were built.The sequencing results and amino acid sequence indicate that the mud K fragment has been inserted into LT2?fad L,LT2?omp N,LT2?ybf M,and can translate Lac Z successfully.2.The effect of ryb B and hfq gene deletion in Salmonella typhimurium on fad L,ybf M and omp NIn order to understand the effects of sRNA RybB and RNA chaperone Hfq on the outer membrane proteins gene fad L,ybf M,and omp N,Salmonella typhimurium ryb B,hfq,ryb B&hfq deletion strains were used as donor bacteria,and the fragment?hfq::Tet Ra,?ryb B::cat,?hfq::Tet Ra?ryb B::cat are transferred to the color indicator strain LT2 fad L::Mud K,LT2 omp N::Mud K,LT2 ybf M::Mud K by P22 phage.The?-galactosidase activity experiment and q PCR results showed that:Hfq and Ryb B strengthened and inhibited the expression of fad L respectively at the protein level.Hfq's regulatory effect on fad L was greater than that of Ryb B on fad L;Hfq and Ryb B both up-regulated fad L transcription Level of expression.Hfq and Ryb B can inhibit the expression of omp N at the protein level and transcription level.Hfq and Ryb B can inhibit the expression of ybf M at the protein level,and they can work together to enhance the inhibitory effect on ybf M;and at the transcription level,Ryb B and Hfq respectively promote and inhibit the expression of ybf M.The regulatory effect of Hfq on ybf M is greater than that of Ryb B on ybf M.Regulation.In general,Hfq can inhibit the expression of ybf M and omp N and up-regulate the expression of fad L;Ryb B has an inhibitory effect on omp N and fad L,but has an up-regulation effect on ybf M.When Hfq and Ryb B regulate the common ybf M and fad L,the regulation effect of Hfq is greater than that of Ryb B.In order to understand the influence of the(ARN)n sequence in fad L,omp N,and fad L on the expression of fad L,omp N,and fad L proteins,Salmonella typhimurium LT2RNA was used as a template to determine the transcription initiation positions of fad L,ybf M,and omp N through the 5'RACE kit.And through RNAstructure to predict the secondary structure of fad L,ybf M,omp N 5'-UTR,and screen possible fad L,ybf M,omp N 5'-UTR(ARN)n sites.Based on the screening results,the fad L,ybf M,omp N 5'-UTR(ARN)n point-directed deletion color indicator strains were constructed.ARN-1(AAAAATAAT)and ARN-2(AAAAAA)were selected from fad L.Two(ARN)n-sequence deletions reduced the protein expression of fad L by 5.4 and 2.3 times respectively;?(AAAAAA)strengthened the?hfq pair The down-regulation of fad L protein levels was changed from 2.33 times to 3.4 times,and?(AAAAATAAT)attenuated the down-regulation of fad L protein levels by?hfq,from 2.33 times to 2.16times.Screening of ARN-1(GTTTTT),ARN-2(TCTTTT),ARN-3(ATTATT),ARN-4(TTTGCT)in omp N,(ARN)n deletion can slightly change the protein expression of omp N,which are 1.29,1.3,0.9,1.07.ARN-1(AAGAGG),ARN-2(AGCAAT),ARN-3(AGTAAT),ARN-4(AAAAATAGT),ARN-5(AACAGAAAG)were screened from ybf M.The deletion of(ARN)n caused changes in ybf M protein levels.They are 4.2,3.99,10.9,227,and 46 times respectively.Both?(AAAAATAGT)and?(AACAGAAAG)can attenuate the up-regulation of?hfq on the level of ybf M protein,from 134.6 times to 4.12 and 7.25 times respectively.In order to explore the effect of ryb B sequence on Hfq and outer membrane protein related genes,ryb B(GGAGCCCATC)was used as the donor bacteria,and the ryb B(GGAGCCCATC)::cat fragment was transferred into LT2?Hfq::Tet Ra,and the strain was indicated by color Based on LT2 ybf M::Mud K,the LT2 ybf M::Mud K strain LT2ybf M::Mud K was used as the basis for the deletion of all ryb B,ryb B?(GGAGCCCATC),ryb B and hfq,ryb B(GGAGCCCATC)and hfq.The results show that both?ryb B and ryb B?(GGAGCCCATC)can improve the regulation of Hfq on the protein expression of ybf M,but in the process of changing the regulation of Hfq on the protein expression of ybf M,ryb B?(GGAGCCCATC is not as effective as?ryb B.ryb B.Deletion of GGAGCCCATC can upregulate the transcription level of ybf M,omp N,and fad L,and weaken the regulation of?hfq on the transcription level of ybf M and fad L,and increase the regulation of?hfq on the transcription level of omp N;while deletion of ryb B can reduce the transcription level and upregulate the transcription level of fad L and ybf M.omp N transcription level,and reduce the regulatory effect of?hfq on the transcription level of ybf M,omp N,and fad L.Deletion of ryb B?GGAGCCCATC may affect the binding of Ryb B and Hfq and weaken the competitive regulation of m RNA between hfq and ryb B.3 Effect of deletion of outer membrane protein-related genes on the pathogenicity of Salmonella typhimuriumIn order to explore the influence of fad L,omp N,ybf M,ryb B,and hfq gene deletion on the pathogenicity of Salmonella typhimurium,some biological characteristics and virulence of Salmonella deleted strains were determined,and the genes related to the outer membrane protein of Salmonella typhimurium were pathogenic to Salmonella.The impact of sex.The results of growth curve determination showed that,compared with wild-type LT2,?omp N,?ybf M,and?hfq had no significant effect on the growth rate of Salmonella typhimurium.?ryb B reduced the growth rate of Salmonella typhimurium.The results of mouse LD50 showed that the LD50 of KM mice infected with the wild strain of Salmonella typhimurium LT2 was 3.97×10~7 CFU,and the LD50 of LT2?omp N,LT2?fad L,and LT2?ybf M were 1.58×10~5 CFU,6.29×10~5 CFU,9.97×10~5CFU,which are about 251 times,63 times,and 40 times that of wild type strain LT2;LT2?ryb B::cat LD50,LT2?hfq::Tet Ra,LT2?ryb B::cat?hfq::Tet Ra is greater than5×10~7 CFU.In addition,the measurement of the bacterial load of the liver and spleen showed that the LT2?ryb B,LT2?omp N,LT2?fad L,and LT2?ybf M groups were infected with mice for 6 h and 48 h,and only the LT2?omp N and LT2?ybf M groups were infected for 48 h The bacterial load in the liver increased,1.27 and 1.1 times respectively.In the LT2?hfq::Tet Ra,LT2?ryb B::cat?hfq::Tet Ra group,the bacterial load on the liver and spleen showed a downward trend after 6 h and 48 h in the infection of mice.After 6 h of infection,LT2?hfq::Tet Ra,LT2?ryb B::cat?hfq::Tet Ra group had a bacterial load of 0.65 and 0.79 times respectively in the liver,LT2?hfq::Tet Ra group had a bacterial load of 0.789 times in the spleen;48 h after infection,LT2?hfq::Tet Ra,LT2?ryb B::cat?hfq::Tet Ra had 0.86 and 0.85 times the bacterial load in the liver,and 0.88 and 0.83 times the spleen bacterial load in the Tet Ra group,respectively.In general,the deletion of hfq and ryb B can reduce the virulence of Salmonella typhimurium,while the loss of fad L,omp N,and ybf M can increase the virulence of Salmonella typhimurium.