| Streptococcus suis serotype 2(SS2)is an important zoonotic disease with high isolation rate and strong pathogenicity,which causes a huge economic losses to the pig industry.It can also infect humans and cause diseases such as meningitis,arthritis and even toxic shock syndrome,which is a serious threat to human public health security.Zn is the second most abundant transition metal in biological systems.Zn uptake has been reported to regulate the virulence of a variety of bacteria.On the one hand,it can participate in bacterial physiological catalytic reaction as a cofactor,it can also affect central carbon metabolism and peptidoglycan biosynthesis,and Zn homeostasis is associated with many bacterial pathogenicity.However,the function and mechanism of Zn in SS2 remain unclear.This study analyzed two potential Zn uptake proteins SSUSC84＿0110(AdcA)and SSUSC84＿0296(Lmb)in SS2 through bioinformatics,and explored their functions in SS2 pathogenicity.Specific research contents are as follows:1.Bioinformatics analysis of AdcA and LmbIn order to excavate the Zn uptake related proteins in SS2 genome,this study compared the amino acid sequences of Zn uptake proteins AdcA and Lmb in different Gram-positive bacteria with Streptococcus suis genome.The results showed that the homology between Streptococcus suis SSUSC84＿0110 protein and AdcA protein in Streptococcus pneumoniae,Streptococcus pyogenes,Streptococcus agalactiae and Staphylococcus aureus was 71%,60%,57% and 38%,respectively.The amino acid homology of SSUSC84＿0296 protein with Streptococcus pneumoniae,Streptococcus pyogenes,Streptococcus agalactiae and Escherichia coli Lmb protein was 63%,58%,56% and 24%,respectively.The phylogenetic tree was used to analyze the clustering relationship between the homologous proteins of Streptococcus suis SSUSC84＿0110 and SSUSC84＿0296 and other Gram-positive bacteria.It was found that the AdcA and Lmb proteins in Streptococcus pneumoniae had the highest similarity.Therefore,we named Streptococcus suis type 2 SSUSC84＿0110 and SSUSC84＿0296 AdcA and Lmb,respectively.2.Study on the basic biological functions of the AdcA and Lmb of SS2In order to study the function of adcA and lmb in SS2,we constructed the deletion strainsΔadcA,Δlmb and ΔadcAΔlmb by homologous recombination.We constructed the complement strains CΔadcA and CΔlmb by shuttle plasmid p SET2,and studied the basic biological characteristics of these strains.The Gram staining showed that the chain length of ΔadcAΔlmb was longer than that of SC19.Through scanning electron microscopy,compared with SC19,the chain length of ΔadcAΔlmb was longer,the cell shape was irregular,and some of the cell division was abnormal.Compared with SC19,there was no significant difference in the growth of ΔadcAΔlmb in normal TSB medium.Compared with SC19,the ability of ΔadcAΔlmb to adhere to h BMEC cells and invade Hep-2 cells was significantly decreased by cell adhesion and invasion tests.3.Function of AdcA and Lmb in Zn uptake of SS2In order to study the function of AdcA and Lmb in Zn uptake of SS2,we formulated chemical synthetic medium(CDM)based on previous studies,and found that the expression levels of adcA and lmb genes of wild strain SC19 were significantly increased in Zn-restricted medium by qRT-PCR.By comparing the growth curves of the mutant and wild strains in the CDM medium with different concentrations of Zn ion,we find that the growth of ΔadcAΔlmb is obviously weakened in the Zn-limited medium,while the growth ability of single gene mutant(ΔadcA and Δlmb)has no obvious difference compared with the wild strain.Only when0.01 m M Zn ions are added,the growth of ΔadcAΔlmb can recover to the level of wild strain,indicating that AdcA and Lmb have synergistic effect on Zn uptake.In order to investigate the role of AdcA and Lmb in resistance to host nutritional immunity,the growth of mutant and wild strains supplemented with different concentrations of calprotein(CP)was compared,and the tolerance of ΔadcAΔlmb to CP was significantly reduced compared with SC19.After sitespecific mutation of CP,there was no significant difference in the growth of all strains,indicating that the Zn ion binding sites in CP had antibacterial activity.4.Functional study of AdcA and Lmb in virulence of SS2The function of AdcA and Lmb in the virulence of SS2 was evaluated in a mouse animal model.We first peformed the survival test and tissue bacteria load test in mice,the survival rate of ΔadcAΔlmb group after infection was 100% compared with SC19 group.After 48 hours of infection,the bacterial load in heart,liver,spleen,lung,kidney and brain of mice infected withΔadcAΔlmb was significantly decreased.The blood bacterial load and inflammatory factors assays showed that the blood bacterial load of mice infected with ΔadcAΔlmb decreased significantly at 6 h,9 h and 12 h after infection compared with SC19.The levels of IL-6,IL-1βand TNF-α in blood of mice infected with ΔadcAΔlmb were significantly decreased at 9 h and12 h after infection.Pathological section test showed that the organ damage of heart,liver,spleen and lung of mice infected with ΔadcAΔlmb decreased significantly after 7 days of infection.5.Function of AdcA and Lmb in drug resistance of SS2The function of AdcA and Lmb in SS2 drug resistance was investigated by measuring biofilm formation and drug sensitivity tests.The biofilm formation capacity of ΔadcA,Δlmb and ΔadcAΔlmb decreased significantly compared with SC19.The expression levels of virulence and adhesion genes related to biofilm formation(gdh,gor,ccpa,fbps,gapd H,gdp P,srt A and per R)in ΔadcAΔlmb were significantly decreased by qRT-PCR.Among these genes,gdh and gor were most significantly down-regulated.Drug screening assays showed thatΔadcAΔlmb showed increased sensitivity to bactericin,vancomycin,fosfomycin and ampicillin that targeted cell wall compared with SC19.6.Effect of SS2 AdcR on Zn homeostasisIn order to investigate the role of AdcR in Zn homeostasis of SS2,we constructed adcR deletion strains(ΔadcR)and complement strains(CΔadcR).The results showed that the deletion of adcR gene attenuated the virulence of SS2.In order to further explore how AdcR responds to external Zn ion signals and regulates related genes,qRT-PCR,gel migration test(EMSA)and biolayer interference technology(BLI)were used in this study.The binding site of AdcR was predicted by JASPAR and BPROM.The results showed the transcription levels of adcA and lmb genes in ΔadcR are significantly upregulated compared with SC19.We found that the transcription factor AdcR can directly bind to the promoter regions of adcA and lmb in the presence of Zn ions in vitro by EMSA and BLI assays,indicating that AdcR is a Zn-dependent negative regulator.The transcription levels of adcR,adcA and lmb under different Zn ion concentration signals were measured by qRTPCR.The results showed that adcA and lmb may act as a system to respond to external Zn ion concentration changes,indicating that AdcR plays an important role in Zn homeostasis and pathogenesis of SS2. |
PDF Full Text Request