| Streptococcus suis is an important but neglected zoonotic pathogen,which can lead to acute septicemia,meningitis,arthritis,endocarditis,pneumonia and so on,causing great economic losses to the pig industry.Moreover,S.suis can also cause meningitis and toxic shock syndrome in human,posing a serious public health concern.With the extencive use of antibiotics in human and veterinary,antimicrobial resistance and even multi-drug resistance is becoming increasingly common in S.suis,especially to that of macrolides and tetracyclines.In recent years,Integrative and conjugative elements（ICEs）,a kind of mobile genetic elements（MGEs）located on bacterial chromosome and capable of self-transfer,have attracted extensive attention dure to the role in horizontal transfer of antimicrolbial resistance genes.However,little is known about the multi-site integration,transfer diversity and the regulation mechanisms in ICE transfer.In this paper,we focused on the distribution,intergration,and regulation mehanisms in horizontal transfer of ICESsuYZDH1 in S.suis.The main research abstract are follows.1 Multi-site integration and distribution diversity of ICESsuYZDH1 in S.suisIn this chapter,we confirmed the transferability of ICESsuYZDH1,a novel ICE with triplet serine integrases,and evaluated the fitness cost of integration of ICESsuYZDH1 into recipient.First,through mating experiments and DNA probe hybridization assays,we found that ICESsuYZDH1 could be transferred to recipient strain P1/7 with a frequency from1.19×10-6to 1.90×10-6,and were integrated into two bands with the sizes of～530 kb and～600 kb in transconjugant.The integration,excision and circularization of ICESsuYZDH1 at primary locus（SSU0877）,secondary locus（SSU1797）and pseudo-locus（SSU0468 and SSU1262）were further detected by PCR.The results showed that ICESsuYZDH1 could be excised form SSU0877 and SSU1797 loci.Sequence analysis of the att sites at SSU0877and SSU1797 revealed the presence of incomplete direct repeats 2-/4-nt（5’-TC-3’/5’-TCCC-3’）and（5’-GC-3’/5’-TCCC-3’）.The fitness cost of the transconjugant was evaluated after the integration of ICESsuYZDH1 into P1/7RF by in vitro competition and stability assays.The results showed that the transconjugant suffered a weak fitness cost with stunted growth and less competition with recipient strain（W=0.89）.Successive passages indicated the ICESsuYZDH1 could be persist（94/100）and endued stable resistant phenotype.Among 1334 WGS of S.suis from Gen Bank,ICESsuYZDH1-like ICE was presented at four sites（SSU0877,SSU0468,SSU1262,and SSU1797）.Among them,168strains had one ICE at one of the four integration sites;72 strains carried two ICEs into SSU0877+SSU1797 genes（44/72）,SSU0877+SSU0468（24/72）,SSU0877+SSU0468（2/72）,and SSU0468+SSU1797（2/72）;and nine strains contained three ICEs into SSU0468+SSU0877+SSU1797（7/9）and SSU0877+SSU1262+SSU1797（2/9）.We further detected 101 ICESsuYZDH1-like ICEs in 330 swine isolates stored in our lab.Similarly,the ICESsuYZDH1-like ICEs could be detected in single integration site（61/101）,in two integration sites（36/101）,and in three integration sites（4/101）.The integration rates of ICESsuYZDH1-like ICE into primary site SSU0877,secondary site SSU1797,and pseudo-loci SSU0468 and SSU1262 were 65%,6%,18%,and 11%,respectively.These results showed that ICESsuYZDH1-like ICEs can be integrated into multi-sites and are widely distributed in S.suis.2 Transfer diversity and fitness evolution of ICESsuYZDH1 in S.suisIn this chapter,the transfer diversity and fitness evolution trend of ICESsuYZDH1 is further explored.Firstly,The diversify transfer of ICESsuYZDH1 was evaluated by mating experiments and DNA hybridization.The results showed ICESsuYZDH1 could be transferred at different frequencies by integrating into SSU0877(7.42×10-7～1.77×10-6),into SSU1797(6.22×10-7～9.00×10-7)and into both sites(1.19×10-6～1.90×10-6),which resulting three types of transconjugants,ICESsuYZDH1＿SSU0877,ICESsuYZDH1＿SSU1797 and YP20190405（co-exist in both sites）.The differences in macrolides and tetracyclines resistance phenotype were further tested between the three transconjutants and no MIC changes were observed.The fitness was slightly lower by integrating ICE into SSU0877（w=0.93）than into SSU1797 site（w=1.01）.The changes in loss of ICE and switch of integration sites were further evaluated by successive passages for 30 days.The results showed that the loss of ICE at SSU0877 site was 2/48,and the number of clones switched from SSU0877 single site to SSU0877+SSU1797 sties was 7/48;the loss of ICE at SSU1797site was 3/48,and the number of clones switched from SSU1797 single site to SSU0877+SSU1797 sites was 17/48;in addition,the number of YP20190405 clones switched to ICESsuYZDH1＿SSU0877 and ICESsuYZDH1＿SSU1797 was 2/48 and 2/48,and 6/100 clones lost two ICEs completely.The results showed that the ICE trends to integrating into both sites of SSU0877 and SSU1797 during successive passages with the interaction between ICESsuYZDH1 and host bacteria.3 Function of Hdi R-I and its role in horizontal transfer of ICESsuYZDH1Transmission of ICE is regulated by complex regulatory networks,particular the SOS response.In order to further analyze the molecular mechanism in regulating horizontal transfer of ICESsuYZDH1,we identified an Lex A-like transcriptional factor,Hdi R-I,was encoded by ICESsuYZDH1.The Hdi R-I protein exhibited 48%homology with the reported Hdi R,a Lex A-like protein in Streptococcus mutans.Further analysis of the domain of Hdi R-I showed that its C-terminal peptidase domain was relatively conservative.The active sites A115,G116,S149 and K185 in S.suis,were similar with that of A84,G85,S119 and K156 in Lex A of Escherichia coli,respectively,suggesting similar self-cleavage function to Lex A which was confirmed by followed self-cleavage assay.We further evaluated the selective stresses that induce the transfer of ICESsuYZDH1.The results showed that both ciprofloxacin and mitomycin C could induce the expression of Hdi R-I（P<0.05）,promote the excision/circularization and increase the horizontal transfer of ICE.The transcriptome results further confirmed that the expression of Hdi R-I was significantly increased under mitomycin C treatment.Interestingly,although the Hdi R-I was up-regulated in nutrition limitation（cultured with NB medium）,the transfer frequency was significantly decreased（P<0.01）.We further explored the function of Hdi R-I.Recombinant Hdi R-I protein of wild type（r Hdi R-I）and A115 to D mutant（A115D）,and verify the self-cleavage function at different p H in vitro.The results comfirmed that the self-cleavage function was abolished to A115D,while r Hdi R-I remain acvtive.In vitro EMSA assays verified the DNA binding fuction of r Hdi R-I while abolished to A115D.By using a varity of different lengh of DNA in promoter region of hdi R-I,we comfirmed the 26-bp binding sequence was5’-TTTTGTTAGTTAAAAACAACAACAAAAAA-3’.The results further indicate that Hdi R-I could response to DNA damage,thus promoting the excision/circularization and horizontal transfer of ICE.In conclusion,this dissertation focuses on the transferability,multi-sites integration,distribution,and transfer diversity of ICESsuYZDH1 after transfer and integrate into new bacterial host,and systematically studied the fitness evolution and regulatory mechanism,which provide a foundation for the final elucidation of the molecular mechanism of horizontal transfer of antimicrobial resistance genes mediated by ICE. |
