Fish and Shellfish Immunology 99 (2020) 424–434

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Fish and Shellfish Immunology

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Full length article ruckeri strain SC09 disrupts proinflammatory activation via Toll/IL- 1 receptor-containing protein STIR-3 T

∗ Tao Liua,1, Liangyu Lib,1, Wenyan Weib,1, Kaiyu Wanga,c, , Qian Yangc, Erlong Wangc a Department of Basic Veterinary, Veterinary Medicine College, Sichuan Agricultural University, Chengdu, Sichuan, China b Institute of Fisheries of Chengdu Agriculture and Forestry Academy, Chengdu, China c Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan, China

ARTICLE INFO ABSTRACT

Keywords: Virulent pathogenic microorganisms often enhance their infectivity through immune evasion mechanisms. Our research on the integrative and conjugative element (ICE(r2)) of the virulent fish pathogen Yersinia ruckeri SC09 STIR-3 led to the identification of genes related to immune evasion (designated stir-1, stir-2, stir-3 and stir-4), among MyD88 which stir-1 and stir-2 were determined as the key contributors to bacterial toxicity and immune evasion. Here, Immune evasion we further examined the ability of stir-3 to mediate immune evasion based on detailed bioinformatic analysis of Secretion ICE(r2) from Y. ruckeri SC09. Interactions among the translated STIR-1, STIR-2, STIR-3 and STIR-4 proteins in the secretory process were additionally explored. STIR-3 was positively correlated with bacterial toxicity and inhibited host toll-like receptor (TLR) signaling by interacting with MyD88, thereby facilitating bacterial sur- vival in host cells. Importantly, our data showed co-secretion of STIR-1, STIR-2 and STIR-3 as a complex, with secretion failure occurring in the absence of any one of these proteins. While stir-1, stir-2, stir-3 and stir-4 genes werespecifictoY. ruckeri SC09, the ICE(r2) region where these genes were located is a mobile component widely distributed in . Therefore, the potential transmission risk of these immune evasion genes requires further research attention.

1. Introduction to a horizontal gene transfer (HGT) element [20], a powerful evolu- tionary adaptation of prokaryotic microorganisms [21] containing Yersinia ruckeri is an important pathogen in the aquaculture industry plasmids and phage in addition to ICEs [22] that significantly affects [1]. Previous studies on this pathogen have focused on clinical case their diversity and adaptability. Microorganisms containing these ele- reports (infection of multiple serotypes [2–8] and biotypes [9–11]) and ments can effectively transfer genes between strains over long phylo- individual virulence genes, such as cdsAB [12], flhDC [13], incAC [14], genetic distances, mediate mutations beneficial for niche adaptation of ZnuABC [15], and BarA-UvrY [16]. However, limited investigations to strains, and promote the formation of new species [22]. Notably, ICEs date have evaluated pathogenicity from a systemic perspective or differ significantly from plasmids and bacteriophages. On the one hand, clarified pathogen-host interactions. Earlier, we identified a large in- they have both plasmid and phage properties and are considered tegrative and conjugative element (ICEr2) containing the type IV se- plasmid-like prophages [23]. These elements can be inherited vertically cretion system in the Y. ruckeri SC09 genome [17,18] encompassing as part of a bacterial chromosome or horizontally via endogenously multiple immune escape-related genes (stir-1, stir-2, stir-3, and stir-4). encoded conjugative elements. Structurally, ICEs include conservative We suggest that this ICE component and its “cargo” genes participate in modules that mediate integration, excision, binding, and regulation Y. ruckeri SC09-mediated evasion of the natural immune system of the [24]. During the conjugation process, ICEs are cyclized and transferred host, facilitating intracellular survival. to new species with retention of a copy in the original donor bacteria Due to significant developments in high-throughput sequencing [22]. On the other hand, plasmids and phages often carry only genes technology, genetic characteristics of bacteria are more comprehen- that are genetically related to themselves while ICEs carry “cargo” sively understood [19]. Integrative and conjugative elements (ICE) in Y. genes associated with niche adaptation of host strains [23], including ruckeri SC09 were recently identified by our group [17,18]. ICEs belong formation of biofilms, pathogenicity, antibiotic resistance and heavy

∗ Corresponding author. Department of Basic Veterinary, Veterinary Medicine College, Sichuan Agricultural University, Chengdu, China. E-mail address: [email protected] (K. Wang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.fsi.2020.02.035 Received 24 October 2019; Received in revised form 11 February 2020; Accepted 16 February 2020 Available online 19 February 2020 1050-4648/ © 2020 Published by Elsevier Ltd. T. Liu, et al. Fish and Shellfish Immunology 99 (2020) 424–434 metal resistance [25]. In addition, these cargo genes are not conserved in Jianyang, Sichuan Province of China, was routinely cultured on between strains and display significant differences in specificity and Luria-Bertani (LB) medium at 28 °C. A virulent Y. ruckeri SC10 strain genetic information, resulting in sizes ranging from 20 to 500 kb [24]. was isolated from the aquatic environment. The cargo genes, stir-1, stir-2, stir-3, and stir-4, associated with im- mune evasion were examined in this study. A common feature of these 2.3. Construction of Y. ruckeri Δstir-3 mutant and complementary strains genes is the presence of a Toll/interleukin-1 receptor domain within the encoded proteins. Our group previously investigated the pathogenicity Gene knockout was performed as described by Luo et al. [26]. The of stir-1 (originally designated tcpA and renamed stir-1 to distinguish it stir-3 gene sequence (gene accession number: NJ56_RS12440) of the Y. from similar genes found in other bacteria) and stir-2 in the Y. ruckeri ruckeri SC09 strain (gene accession number: NZ_CP025800) is available SC09 infection process [17,18] and established the immune evasion in GenBank. The left and right homology arm primer sequences of stir-3 functions of the corresponding translated proteins in vivo and in vitro were GGAATCTAGACCTTGAGTCGGTGAAAAATGAGGTGCCTTATGG/ [17,18]. The immune evasion mechanisms of stir-1 and stir-2 genes TATAACCTTCATCGAGCGTCCAGGCCATGAATCAACTCCTTTTG (up- were shown to be mainly achieved through direct interactions of the stream, A) and CAAAAGGAGTTGATTCATGGCCTGGACGCTCGATGAA encoded proteins with MyD88 in infected cells [17,18], which induced GGTTATA/ACAGCTAGCGACGATATGTCACACCAAGAGTCAAACACAC an inhibitory effect on the Toll-like receptor signaling pathway, even- CGA (downstream, B), respectively. Left and right homology arms (AB) tually reducing the ability of innate immunity to recognize bacterial of stir-3 were constructed and cloned into pLP12 (Guangzhou KnoGen infections. We propose that this pathway of immune evasion represents Biotech Co., Ltd.) to generate the pLP12-stir-3 construct, which was a universal mechanism. Here, we focused on the immune evasion transformed into the competent E. coli strain β2163 (Guangzhou property of stir-3 and its associations with stir-1, stir-2 and stir-4 in KnoGen Biotech Co., Ltd.) via electroporation. A positive strain re- addition to the collective effects of all four genes on immune evasion. sistant to chloramphenicol, designated pLP12-stir-3-β2163, was subse- quently isolated. Co-culture of β2163 cells containing pLP12-stir-3-po- 2. Materials and methods sitive clones with Y. ruckeri SC09 resulted in conjugation and allowed screening for the first homologous recombinants of the mutant SC09 2.1. Bioinformatics analysis of ICE(r2) in genomes strain on LB plates (20 μg/mL CM + 0.3% D-glucose). SC09 strains with the insertion were screened on LB plates (0.4% L-arabinose) to obtain a ICE(r2) homologous regions in other bacterial genomes were de- Δstir-3 strain with a second homologous recombination. As described tected using MegaBlast (https://blast.ncbi.nlm.nih.gov/Blast.cgi). previously [26], SC09 and SC10 strains were re-transformed with the Individual hits were retrieved and manually searched for ICE(r2) hall- stir-3-pBAD33cm-rp4 vector [27] and expression of stir-3 induced with mark genes in close proximity, such as integrase and tRNA, located near arabinose. the other end of ICEs. Putative ICE regions were isolated in silico from the host genome and pairwise compared with ICE(r2) using WebACT 2.4. Fish infection model (http://www.webact.org/WebACT/home). Regions with > 79% nu- cleotide sequence similarity were exported and displayed on the local At the logarithmic growth phase, wild-type Y. ruckeri SC09 and gene map using DNAPlotter. TIR domain analysis of STIR-1, 2, 3, and 4 recombinant SC09Δstir-3 were inoculated intraperitoneally in SC09 was performed using BlastX (https://blast.ncbi.nlm.nih.gov/ (5 × 107 CFU) into 15 random rainbow trout (60–100 g), and mortality Blast.cgi). in fish assessed. Rainbow trout survival curve analysis and mapping were performed using GraphPad Prism software version 8.0. The 2.2. Bacterial strains growth curves of Y. ruckeri SC09 and SC09Δstir-3 were compared to eliminate the potential effect of differences in growth ability of the The strains and plasmids used in this study are listed in Table 1. knockout strain in the infection model. To investigate infection and Wild-type Y. ruckeri SC09 isolated from diseased fish in a reservoir farm histological differences in immune organs post-infection, liver, spleen,

Table 1 Strains and plasmids used in this study.

Strain or plasmid Description Reference

Strains Y. ruckeri SC09 GenBank (CP025800.1) with ICE (r2) Liu et al. [19] Y. ruckeri SC10 Does not carry ICE (r2) Liu et al. [19] − – + – E. coli DH5α F endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG purB20 ϕ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17 (rK mK ), λ Clontech β2163 (F−) RP4-2-Tc::Mu ΔdapA::(erm-pir) KnoGen Biotech SC09 Δstir-1 stir-1 deletion Liu et al. [17] SC09 Δstir-2 stir-2 deletion Liu et al. [18] SC09 Δstir-3 stir-3 deletion This work SC09 Δstir-4 stir-4 deletion This work Plasmids R pLP12 oriTRP4 oriVR6K vmi480 PBAD (Cm ) KnoGen Biotech R pBAD33 orip15A araC PBAD (Ap ) KnoGen Biotech pET28a ori f1, KanR, T7 pRomoters Invitrogen pGEX-6P-1 Expression vector/GST Novagen pCMV-HA-MyD88 Expression vector/CMV promoter Miaoling pCMV-Flag-2B Expression vector/CMV promoter Stratagene pBIIXLuc NF-κB Luciferase Reporter Plasmid GeneCreate Biological Engineering pCMV-Tag 2B Expression vector/CMV promoter Miaoling pGADT7 Expression vector/T7 promoter Clontech pGBKT7 Expression vector/T7 promoter Clontech pTLR-4 Expression vector/T7 promoter Miaoling pTLR-2 Expression vector/T7 promoter Miaoling

425 T. Liu, et al. Fish and Shellfish Immunology 99 (2020) 424–434 and kidney of dead rainbow trout were homogenized under aseptic 2.7. Construction of prokaryotic expression vectors conditions. Homogenates were diluted 10 times and bacterial numbers determined via conventional plate counting. Briefly, bacterial cells Full-length Y. ruckeri SC09 stir-3 was cloned into pET28a were counted on a plate containing nutrient agar supplemented with (Invitrogen) to generate pET28-stir-3 using the primers 5′-ATGGCCAA triphenyl tetrazolium chloride (TTC can stain bacteria to red, which can GAAGGTGTT-3′ and 5′-TTTTAACCACGATGAAA-3′. The pET28-stir-3 help people to better distinguish and count bacteria) and the bacterial construct was used for production of STIR-3 recombinant protein. Full- load in each organ determined. Liver, kidney, and spleen of rainbow length Y. ruckeri SC09 stir-3 was additionally cloned into pGEX-6P-1 trout were additionally harvested mid-infection (5 days after onset) for (Invitrogen) to generate pGEX-stir-3 carrying the tac promoter, N- routine paraffin embedding and HE staining. terminal GST tag and an ampicillin resistance gene, using the primers 5′-CGGGATCCATGGCCAAGAAGGTGTT-3′ and 5′- CGCTCGAGTTTTAA 2.5. Bacterial adherence, invasion and intracellular survival assays CCACGATGAAA-3′. The pGEX-stir-3 was employed for production of recombinant STIR-3-GST protein. Head kidney macrophages of rainbow trout were separated ac- cording to the method of Jiang et al. [28] and used to determine the 2.8. Construction of eukaryotic expression vectors effects of Y. ruckeri stir-3 on bacterial adherence, invasion, and in- tracellular survival capacities. Cells were seeded at a density of The pCMV-HA-MyD88 plasmid was purchased from Wuhan ~2 × 105 cells per well in 24-well plates and grown in Medium 199 Miaoling Bioscience & Technology Co., Ltd. The stir-3 gene fragment (M199) (Hyclone) with 10% fetal bovine serum (FBS) (Hyclone) at was cloned into the eukaryotic expression plasmid pCMV-Flag-2B 20 °C for 24 h. For the adherence assay, the cell monolayer was washed (Stratagene) to generate pSTIR-3-Flag using the following primers: twice with M199 and infected with Y. ruckeri SC09 or Y. ruckeri 5′-ACCATGGATTACAAGGATGAATGGCCAAGAAGGTGTT-3′ and 5′- SC09Δstir-3 at a multiplicity of infection (MOI) of 1.5. Bacteria were GGGCCCCCCCTCGAGGTCGATTTTAACCACGATGAAA-3'. centrifuged onto cells at 400 × g for 5 min, followed by incubation at 20 °C for 1 h. Nonadherent bacteria were removed by rinsing wells twice with D-PBS (Hyclone). Cells were released from the plate by 2.9. Transfection and NF-κB-dependent luciferase reporter assay adding 100 μL of 0.2% Triton X-100 in sterile water, followed by 900 μL M199. The cell suspension was serially diluted 10-fold with D-PBS and Rainbow trout head kidney macrophages were transiently trans- spread onto LB plates to determine the number of viable bacteria. For fected using Lipofectamine™ 3000 Transfection Reagent (Life) for 12 h the invasion assay, cell culture, bacterial infection and bacterial counts using a total of 0.3 μg DNA consisting of 50 ng TLR-4 and TLR-2 were performed as described for the bacterial adherence assay, except plasmids (Wuhan Miaoling Bioscience & Technology Co., Ltd), 200 ng that extracellular bacteria were killed by treatment of monolayers with pBIIXLuc (Wuhan GeneCreate Biological Engineering Co., Ltd.) reporter M199 containing gentamicin (100 μg/mL) for 1 h after incubation and plasmid and 50 ng FLAG-STIR-3 expression vector, according to the two washes with D-PBS. For the bacterial intracellular survival assay, manufacturer's instructions. The total amount of DNA was kept constant cell culture and bacterial counting were performed as described for the by adding a quantity of empty vector. Where indicated, cells were adherence assay. Specifically, rainbow trout head kidney macrophages treated with E. coli LPS (Invitrogen) and Pam2CSK4 (Invitrogen) for 8 h, were infected with Y. ruckeri SC09 or Y. ruckeri SC09Δstir-3 at MOI of lysed, and luciferase activity measured using the Dual-Glo® Luciferase 1.5 and incubated in M199 containing 3% FBS and 50 g/mL genta- Assay System (Promega). The stir-3 constructs were obtained from SC09 micin. Next, cells were washed and lysed at 1, 4, 8, 12, 18, 24, 48, and genomic DNA by treatment with recombinase Exnase II (ClonExpress II; 72 h post-infection (hpi) to determine the extent of bacterial recovery. Vazyme) and cloned into pCMV-Tag 2B vector (Miaoling). All assays were performed in sextuplicate wells, and the results re- present the average of at least six independent experiments. The in- 2.10. Co-immunoprecipitation (Co-IP) assay tracellular survival state of cells 48 h after wild-type bacterial infection was examined via transmission electron microscopy. To establish Rainbow trout head kidney macrophages were infected with wild- whether gene knockout resulted in loss of STIR-3 protein in cells, rabbit type SC09 for 12 h. Cells were washed twice in ice-cold PBS, harvested, anti-STIR-3 antibody was employed to examine protein production of and treated with cell lysis buffer. Cell lysis and processing for co-im- knockout, wild-type and replenished strains. To ascertain the efficacy of munoprecipitation were performed using the Pierce™ Co- replenished strains, the stir-3 expression plasmid was further com- Immunoprecipitation Kit (Thermo Scientific, Shanghai, China). Eluted plemented in SC10 lacking stir-3 in the chromosome and rabbit anti- IP samples were detected via western blot using rabbit anti-STIR-3 STIR-3 employed to assess protein production. (prepared for this study) or anti-MyD88 (Abcam) antibody. 2.6. Enzyme-linked immunosorbent and qPCR assays 2.11. Pulldown from cell extracts To quantify IL-6, IL-1β, and TNF-α levels in culture supernatants, ELISA was performed (Jiangsu Meimian industrial Co., Ltd). Tissue Rainbow trout head kidney macrophages were transiently trans- homogenates and supernatant fractions from infected cells were pre- fected with pCMV-HA-MyD88 using Lipofectamine™ 3000 Transfection pared for extraction of total RNA with RNAiso Plus (TaKara, Dalian, Reagent (Life). At 18 h after infection, cells were washed in ice-cold China) according to the manufacturer's protocol. For qPCR, cDNA was PBS, harvested and resuspended in RIPA buff er (Sigma). Extracts were synthesized from extracted RNA using the Prime Script RT reagent Kit centrifuged at 16,000 × g at 4 °C for 20 min. The supernatant was (TaKaRa). The expression levels of immune-related genes (IL-6, IL-1β, incubated with STIR-3-GST recombinant protein for 3 h at 4 °C, fol- TNF-α) were analyzed using real-time PCR. β-Actin (F: CCGCTGCCTC lowed by application to a gravity flow column containing Glutathione- CTCTTCCTCTC/β, R: TCTCGTGGATACCGCAAGACTCC) of rainbow Sepharose (GE) washed in water and pre-equilibrated in equilibrium trout was selected as the reference gene due to low variability. The buffer (20 mM Tris–HCl) 1 h beforehand. The column was washed twice following primers were used: IL-1β-F: TGAGAACAAGTGCTGGGTCC successively in equilibration buffer and eluted in PBS containing 1% and IL-1β-R: GGCTACAGGTCTGGCTTCAG (148 bp product), IL-6-F: Triton. Eluted proteins were separated via SDS–PAGE, transferred to GAGTTTCAGAAGCCCGTGGA and IL-6-R: AGCTGGTACACTTGCAGACC PVDF membrane, and incubated with anti-HA (Abcam), anti-MyD88 (149 bp product); TNF-α-F: CACACTGGGCTCTTCTTCGT and TNF-α-R: (Abcam) or anti-STIR-3 antibodies for 50 min, followed by detection CAAACTGACCTTACCCCGCT (155 bp product). with horseradish peroxidase (HRP)-conjugated secondary antibodies.

426 T. Liu, et al. Fish and Shellfish Immunology 99 (2020) 424–434

Fig. 1. Genomic localization of ICE(r2) and identical TIR domains of STIR-1, STIR-2, STIR-3 and STIR-4 proteins.

2.12. Antisera and polyclonal antibodies microporous membrane. Filtered supernatant was mixed with solid ammonium sulfate to 75% saturation and allowed to stand at 4 °C To obtain rabbit polyclonal sera against rENO, six male New overnight. After centrifugation at 12,000 rpm for 10 min at 4 °C, the Zealand white rabbits were immunized with a subcutaneous injection of pellet obtained was dissolved in 0.02 mol/L Tris-HCl (pH 7.4). The 2 mg purified STIR-3 mixed with Freund complete adjuvant (1:1) resuspended liquid was loaded into a dialysis bag with a cut-off of (Sigma-Aldrich, St, Louis, MO, USA). After 3 weeks, each rabbit re- 8–14 kDa to remove ammonium sulfate. An extracellular bacterial se- ceived a booster injection with the same antigen concentration emul- cretory protein (BSP) sample was secreted during bacterial culture. sified with Freund incomplete adjuvant (1:1) (Sigma-Aldrich). A second Western blot was performed on BSP using rabbit anti-STIR-1, anti-STIR- booster injection was administered after 3 days. Serum samples were 2, anti-STIR-3 and anti-STIR-4 antibodies. Bacteria were cultured under collected 3 days after the second booster injection. All surgical proce- similar conditions, cells collected via centrifugation at 12,000 rpm for dures were performed under isoflurane anesthesia. Rabbit polyclonal 10 min at 4 °C and the intracellular product, bacterial intracellular anti-STIR-3 serum (1/1000 dilution) along with secondary anti-rabbit- protein (BIP), obtained by lysing cells with lysozyme. WB was per- HRP antibodies (1/5000 dilution) were utilized for western blot. Two formed on BIP using rabbit anti-STIR-1, anti-STIR-2, anti-STIR-3 and purification steps were required for antibodies for CO-IP and pull-down anti-STIR-4 antibodies. The culture supernatant of the wild-type strain experiments. Antibody purification was performed using Protein G af- was used as a positive control. finity chromatography.

3. Results 2.13. Yeast two-hybrid assay 3.1. ICE(r2), a novel Y. ruckeri integrative and conjugative element with a The plasmids used for Y2H were constructed with the aid of re- type IV secretion system, encodes a virulence factor containing four TIR combinase Exnase II (ClonExpress II, Vazyme). The myD88 and Y. domain-containing proteins ruckeri SC09 stir-3 genes were amplified via PCR from the pCMV-HA- MyD88 vector and SC09 genomic DNA, respectively, with Exnase II The mobile element, ICE(r2), in SC09 (NJ56_RS12595- primers. Amplified products were separately cloned into the pGBKT7 NJ56_RS12425), integrated between an intact or partial tRNA-Asn copy entry vector downstream of the Gal4 DNA-binding domain (BD) was further identified in Y. ruckeri strains RS41, OMBL4 and CSF007- (Clontech) with the screening marker gene trp and pGADT7 down- 82, with extensive similarities in sequence, gene content and gene ar- stream of the Gal4 activation domain (AD) with the screening marker rangements (Figs. 1 and 2). ICE(r2) was flanked on one side by and an gene leu. Y2HGold Yeast competent cells were transformed with BD and integrase gene and encompassed a type IV secretion system operon AD fusion protein vectors. Diploid yeast carrying both plasmids was (T4SS, NJ56_RS12550-NJ56_RS12510) that may mediate conjugation obtained by mating and selected on synthetic dextrose medium (SD) from donor to recipient cells analogous to conjugative plasmid trans- lacking leucine (leu) and tryptophan (trp). Protein interactions were location (Fig. 1). However, details of this transfer require further ex- assessed on medium lacking histidine (his). The β-galactosidase ex- perimental validation. A class of bacterial proteins displaying homology pression filter assay was performed using the LacZ reporter gene. The to the Toll/IL-1 receptor (TIR) domain encoded by SC09-ICE(r2) that primers used for two-hybrid constructs were: GWMyD88F- CATATGG act as “accessory genes” has additionally been identified. These pro- CCATGGAGGCCGAGCTGCAGGAGGTCCCGGCGC, GWMyD88R- GCGG teins, designated STIR-1, STIR-2, STIR-3 and STIR-4, are encoded by CCGCTGCAGGTCGACGTCAGGGCAGGGACAAGGCCT; GWstir-3F- CAT NJ56_RS12465 (stir-1), NJ56_RS12445 (stir-2), NJ56_RS12440 (stir-3) ATGGCCATGGAGGCCAGATGGCCAAGAAGGTGTT, GWstir-3R- CTGCA and NJ56_RS12430 (stir-4), respectively (genes colored in red, Fig. 1), GCTCGAGCTCGATGGTTTTAACCACGATGAAA. which are specific to SC09 and absent from Y. ruckeri strains RS41, OMBL4 and CSF007-82. 2.14. Studies on secretion of STIR-1, STIR-2, STIR-3, and STIR-4 To ascertain the distribution of this mobile element across a broader bacterial population, we applied SC09-ICE(r2) to a BLAST search of the Bacteria (Δstir-1, Δstir-2, Δstir-3 and Δstir-4) were inoculated in LB, NCBI Genome database and manually searched for the nearby presence cultured at 28 °C for 18 h (OD600 = 1), and centrifuged at 12,000 rpm of ICE(r2) hallmark genes, such as integrase, tRNA-Asn, and T4SS. for 10 min at 4 °C, followed by collection of supernatant at OD600 of 3. Interestingly, comparative analysis of SC09-ICE(r2) and related mobile The supernatant was subsequently filtered through a 0.22 μm islands in other prokaryotes revealed widespread occurrence of the ICE

427 T. Liu, et al. Fish and Shellfish Immunology 99 (2020) 424–434

Fig. 2. Detailed comparison of ICE(r2) with putative ICE regions in representative bacterial genomes. A-C. Comparison with SC09-ICE(r2) identified in draft genomes of Y. ruckeri RS41, OMBL4 and CSF007-82; D. Comparison with an ICE identified in the draft genome of D. zeae CSL Rw192; E. Idem to P. carotovorum BC T5; F. Idem to Y. enterocolitica YE09/03; G. Idem to Y. intermedia IP10066; H. Idem to C. neteri ND14b. Regions with > 69% nucleotide identity are connected by red windows using a color intensity gradient based on identity scores of Blastn comparisons in WebACT. Hallmark genes of SC09-ICE(r2) are presented in panel A and annotations of other genomes are available in each panel (int, integrase; tra, transfer elements; tnp, transposons). The locations of ICE(r2)-like elements are provided on the right-hand side. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

(r2)-like island across the Enterobacteriales (Fig. 2). These proteins share microevolutionary diversification via gene insertion. a core of conserved modular structures (integrase, upstream regulatory region, T4SS, mobB and mobC) that are integrated at the end of tRNA- 3.2. Stir-3 is required for Yersinia ruckeri SC09 virulence during infection in Asn and possess accessory regions associated with changes in functional vivo and in vitro requirements or adaptation to specific niches (Fig. 2). Genes associated with TIR domain-containing proteins were additionally identified in The immune evasion function of stir-1 and stir-2 has been previously Dickeya zeae CSL RW192 and YE09/03 but ex- confirmed by our group. Here, we focused on whether stir-3 ad- hibited high diversity in terms of sequence and arrangement, compared ditionally contributes to pathogenicity and immune evasion of SC09. To to stir-1234 from SC09 [29,30]. Within Yersiniaceae, in addition to Y. analyze the effects of stir-3 on virulence during infection, we con- ruckeri, Y. enterocolitica strains YE09/03 isolated from mammals and structed a stir-3 deletion mutant of SC09 and Δstir-3+ pSTIR-3 mutant, strains IP10066 isolated from water contained an which was complemented with a plasmid containing stir-3 controlled by ICE(r2)-related element (Fig. 2). Specific transposons (tnp), integrins its promoter. An acute in vivo infection model of rainbow trout was (int) and sporadic conjugative transfer elements (tra) were identified in employed to evaluate the involvement of stir-3 in Y. ruckeri-induced fish Y. ruckeri OMBL4, D. zeae CSL Rw192, Pectobacterium carotovorum BC disease. The SC09 wild-type strain caused 50% lethality 6 days after T5 and Cedecea neteri ND14b (Fig. 2). These observations supported the inoculation (Fig. 3A). Notably, the SC09Δstir-3 mutant induced a slight ongoing and active distribution of ICE(r2)-like elements and further but significant (P < 0.01; **) attenuation of virulence in fish (Fig. 3A).

428 T. Liu, et al. Fish and Shellfish Immunology 99 (2020) 424–434

Fig. 3. Stir-3 is required for Yersinia ruckeri SC09 virulence in vivo. A. Survival curves of rainbow trout (There are 15 fish in each group). Fish were infected with Y. ruckeri SC09Δstir-3 and wild-type Y. ruckeri SC09. The Mantel-Cox test was used for comparison of curves (**P < 0.01). Fish infected with E. coli DH5α with low toxicity were used a negative control. B. Growth curves of wild-type Y. ruckeri SC09 and SC09Δstir-3. C-E. Rainbow trout were infected with Y. ruckeri SC09 or SC09Δstir-3 strains (n = 15/group). Bacterial loads in liver (C), spleen (D) and kidney (E) were assessed through culturing tissue homogenate. The non-parametric two-tailed t-test was conducted for (C), **P < 0.01, (D) **P < 0.01 and (E) **P < 0.01. F–H. Pathological lesions of rainbow trout infected with wild-type Y. ruckeri SC09. Necrotic areas in liver (F, white dotted line); glass-like substrate in kidney tubules (G, white dotted line); edema in spleen medulla (H, asterisk).I–K. Histology observation of rainbow trout infected with SC09Δstir-3 mutant.

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Fig. 4. The stir-3 deletion mutant from Yersinia ruckeri SC09 affects intracellular survival but not adherence and invasion capacities of pri- mary rainbow trout head kidney macrophages. Adherence (A), invasion (B), and invasion ratios (C) for wild-type SC09, SC09Δstir-3 and Δstir- 3 + pSTIR-3 mutants in primary rainbow trout head kidney macrophages showed no significant differences in infection at an MOI of 1.5. The in- vasion ratio was evaluated as the number of in- ternalized bacteria/number of adherent bacteria (ns, no significant difference). (D) Intracellular bacterial survival of the SC09Δstir-3 mutant was significantly decreased, compared to that of wild- type SC09 and Δstir-3 + pSTIR-3 mutants, at 18, 24, 48, and 72 hpi following infection at an MOI of 1.5 (**P < 0.01). (E) Transmission electron micrographs of primary rainbow trout head kidney macrophages fixed 48 h after infection in vitro with wild-type Y. ruckeri SC09 or SC09Δstir-3 mutant. (F) Western blots from representative in- ocula used for infection experiments showing STIR-3 (18 kDa) expression in different Y. ruckeri strains visualized using polyclonal rabbit anti- STIR-3, along with a control blot against the standard cytoplasmic protein EF-Tu (43 kDa).

Fish infected with E. coli DH5α with low toxicity were used as a ne- protein accumulation in diseased cells or interstitial tissue that is gative control (Fig. 3A). We established that these differences were not homophilic in HE-stained sections) in kidney tubules (Fig. 3G). Spleen due to an in vitro growth defect of the mutant (Fig. 3B). SC09Δstir-3- medulla of fish became loose and exhibited edema (Fig. 3H). Our results infected fish showed decreased cell recruitment and enhanced bacterial collectively support the involvement of stir-3 for Y. ruckeri SC09 in- clearance in liver, spleen and kidney, compared to those infected with fection in vivo. Interestingly, in experiments to determine the effects of the wild-type strain (Fig. 3C–E, P < 0.01; **). Tissue damage was stir-4, Δstir-4 did not appear to attenuate the pathogenicity of SC09 additionally detected in fish infected with wild-type SC09 but not the (data not shown). These results require further validation. SC09Δstir-3 mutant (Fig. 3I–K). Histopathological examination of in- The evasion molecule, STIR-3, is hypothesized to interfere with the fected fish revealed marked changes in the liver, kidney, and spleen innate immune response of the host, leadig to enhanced intracellular (Fig. 3F–H). Severe vacuolar degeneration and necrosis of hepatic cells survival capacity of bacteria. To examine this hypothesis, we in- was evident in liver (Fig. 3F), along with a glass-like substrate (also vestigated its effects on bacterial adherence, invasion and intracellular known as hyalinization, indicating uniform, unstructured, translucent survival in vitro. To this end, rainbow trout head kidney macrophages

430 T. Liu, et al. Fish and Shellfish Immunology 99 (2020) 424–434

Fig. 5. STIR-3 reduces cytokine secretion and expression in macrophage cells. (A–C) ELISA analysis of IL-6, IL-1β and TNF-α secretion by rainbow trout head kidney macrophages not infected or infected with Y. ruckeri wild-type SC09, Y. ruckeri SC09Δstir-3 or Δstir-3 + pSTIR-3 mutant. Cytokine levels were determined at 2, 4, 8, 12, 24, and 48 h of infection. (D–F) The qPCR analysis of IL-6, IL-1β and TNF-α expression in macrophages not infected or infected with Y. ruckeri wild-type SC09, Y. ruckeri SC09 Δstir-3 or Δstir-3 + pSTIR-3 mutant. Cytokine levels were determined at 2, 4, 8, 12, 24, and 48 h of infection. Error bars indicate s.d. of six individual cultures. **P < 0.01, one-way ANOVA. were infected with wild-type SC09, SC09 Δstir-3 mutant or Δstir- higher IL-6 (Fig. 5A), IL-1β (Fig. 5B) and TNF-α responses (Fig. 5C) in 3 + pSTIR-3 mutant pathogens. We observed no significant differences macrophages at 18, 24 and 48 hpi, respectively, relative to wild-type between wild-type SC09 and SC09Δstir-3 mutant in terms of bacterial SC09 or Δstir-3 + pSTIR-3-complemented mutant strain. To further adherence (Fig. 4A), invasion (Fig. 4B) or invasion ratio (number of establish whether the effect of stir-3 was due to suppression of cytokine invasive bacteria/number of adherent bacteria) (Fig. 4C), indicating no transcription, we examined IL-6, IL-1β and TNF-α expression in involvement of stir-3 in Y. ruckeri adherence and invasion into macro- rainbow trout head kidney macrophages. Infection with the Δstir-3 phages of rainbow trout. However, data from the intracellular survival mutant induced markedly higher IL-6 (Fig. 5D), IL-1β (Fig. 5E) and assay demonstrated reduced survival of Δstir-3 mutant cells in rainbow TNF-α responses (Fig. 5F) in macrophages at 18, 24, and 48 hpi, re- trout head kidney macrophages, compared to wild-type SC09 and Δstir- spectively, than wild-type SC09 or the Δstir-3 + pSTIR-3-com- 3 + pSTIR-3 mutant cells after 18 hpi (Fig. 4D), indicating a role of stir- plemented mutant strain, supporting the hypothesis that STIR-3 reg- 3 in intracellular survival. It is worth noting here that Δstir-3 mutant ulates the secretion and transcription of proinflammatory cytokines. and Δstir-3 + pSTIR-3 mutant apparently decreases by two logs during the first hour, but then remains stable for the next 18 h. We have no 3.4. Yersinia ruckeri STIR-3 affects TLR signaling clear explanation for this phenomenon. As visualized using TEM, the intact wild-type SC09 strain was detected within macrophages at 48 h Based on data from combined bioinformatics analysis showing that post-infection and several bacteria detected in autophagocytic vacuoles STIR-3 belongs to the TLR family, we investigated its ability to speci- (Fig. 4E). On western blots, STIR-3 protein was not detected in the Δstir- fically interfere with TLR signaling with the aid of an in vitro NF-κB- 3 mutant, as expected, but detectable in the Δstir-3 + pSTIR-3 mutant dependent luciferase reporter system. Stir-3 was ectopically expressed (Fig. 4F). STIR-3 protein was not detected in SC10 (non-stir-3 Y. ruckeri in rainbow trout head kidney macrophages transfected with the NF-κB strain) but observed upon ectopic expression in SC10 (Fig. 4F). Our luciferase reporter vector and plasmids encoding TLR4 or TLR2 and collective results supported the requirement of STIR-3 for Y. ruckeri stimulated with LPS or PAM. Notably, STIR-3 inhibited the TLR4- SC09 infection in vitro. mediated NF-κB response to LPS (Fig. 6A) and impaired the NF-κB re- sponse to the potent TLR2 agonist, PAM, in cells transfected with TLR2 3.3. STIR-3 affects the secretion and transcription of proinflammatory (Fig. 6B). These results suggested that STIR-3 interfered with a common cytokines molecule of TLR pathways, such as MyD88. To examine the potential interactions between STIR-3 and MyD88, rainbow trout head kidney The intracellular survival assay was performed on macrophages to macrophages were infected with wild-type SC09. Co-im- establish whether survival was due to immune evasion or inactivation munoprecipitation of the two molecules was observed, indicating that of innate immunity. To examine this hypothesis, innate responses to STIR-3 and MyD88 were part of the same complex (Fig. 6C). This as- wild-type SC09, SC09Δstir-3 and Δstir-3 + pSTIR-3 mutants were fur- sociation was confirmed by retention of HA-MyD88 on purified GST- ther evaluated in rainbow trout head kidney macrophages. IL-6, IL-1β tagged STIR-3 immobilized on Ni-NTA resin (Fig. 6D). We additionally and TNF-α levels in culture supernatants were examined via ELISA. observed interactions between STIR-3 and MyD88 in a directed yeast Notably, infection with the Δstir-3 mutant stimulated significantly two-hybrid assay (Fig. 6E).

431 T. Liu, et al. Fish and Shellfish Immunology 99 (2020) 424–434

Fig. 6. STIR-3 impairs TLR signaling via binding to MyD88. (A) Luciferase reporter and TLR4-expressing plasmids were transiently transfected into rainbow trout head kidney macrophages with or without the STIR-3 plasmid. After 24 h, cells were stimulated with LPS for 6 h and luciferase activity evaluated. The white, black and gray bands represent the negative control (empty vector), LPS-stimulated cells, and cells transfected with the STIR-3 plasmid and stimulated by LPS, respectively. Data correspond to median ± standard error of relative luciferase activity from five independent experiments. (B) This experiment was similar to (A), except that the TLR2 plasmid was used and cells were stimulated with PAM. (C) Co-immunoprecipitation (co-IP) experiments were performed on cells infected with wild-type SC09 or PBS (control). The anti-STIR-3 antibody was subsequently used to detect protein interactions and anti-MyD88 antibody to detect proteins bound to beads, and the respective inputs detected with anti-MyD88 and anti-STIR-3 antibodies. (D) Pulldown experiments were performed using in vitro expressed HA-MyD88 protein and prokaryotically expressed GST-STIR-3 protein immobilized onto Ni-NTA resin with empty vector as a control. Anti-HA and anti-MyD88 antibodies were employed to detect protein interactions (lower blot) on western blots and the anti-GST antibody to detect GST-STIR-3 protein binding to the resin. Flowthrough (FT), two washes (W) and elution (E) are shown in each lane. (E) Recombinant plasmids containing Gal4 BD and AD were co-transformed into yeast cells and screened on plates lacking leucine (Leu) and tryptophan (Trp) (left panel). Gal4 BD- and Gal4 AD-recombinant plasmids were co-transformed into yeast cells and screened on plates lacking histidine (His) in the presence of 20 mM 3AT (middle panel). Transformants grown on this medium present interactions between STIR-3 and MyD88. Expression of the reporter β-galactosidase gene in yeast produced blue yeast indicative of protein interactions (right panel). Empty vectors for AD and BD plasmids served as negative controls and MyD88 homodimerization as a positive control. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.5. Yersinia ruckeri STIR-3 is co-secreted with STIR-1 and STIR-2 intracellular STIR-2, STIR-3, or STIR-4. Similarly, deletion of stir-2, stir- 3 and stir-4 did not affect expression of the remaining intracellular STIR To further ascertain whether deletion of a single stir gene can affect proteins in bacteria. Notably, background expression of STIR-4 was the expression and secretion of proteins encoded by other stir genes, extremely low, compared to the three other proteins. Furthermore, secreted and cytoplasmic proteins encoded by Δstir-1, Δstir-2, Δstir-3 wild-type STIR-1, STIR-2, STIR-3, and STIR-4 were detected in extra- and Δstir-4 mutants were detected via WB. Deletion of any one stir gene cellular secretions and levels of secreted STIR-4 were extremely low, resulted in lack of production of the corresponding protein (Fig. 7), as compared to the other proteins. Interestingly lack of stir-1 resulted in expected. However, the absence of stir-1 did not affect expression of absence of STIR-2 and STIR-3 in extracellular secretions. Deletion of

432 T. Liu, et al. Fish and Shellfish Immunology 99 (2020) 424–434

Fig. 7. STIR-3 is co-secreted with STIR-1 and STIR-2. Y. ruckeri SC09 Δstir-1, Δstir-2, Δstir-3, and Δstir-4, pRAB11-EsaE-HA or pRAB11-EsaD

(H528A)-His-EsaE-HA was cultured to OD600 of 1 and collected at OD600 of 3. Bacteria were lysed and the bacterial intracellular protein (BIP) ana- lyzed via western blot with anti-STIR-1, 2, 3 or 4. Culture supernatants of bacteria were collected and the bacterial secretory protein (BSP) analyzed via western blot with anti-STIR-1, 2, 3, and 4. The wild-type strain was used as a positive control.

stir-2 resulted in the absence of secreted STIR-1 and STIR-3 and lack of aureus and P. aeruginosa, the Y. ruckeri SC09 strain contained multiple stir-3 was associated with non-secretion of STIR-1 and STIR-2. In con- different copies of TIR domain-containing proteins (designated STIR-1, trast, Δstir-1, Δstir-2, and Δstir-3 did not affect STIR-4 secretion and STIR-2, STIR-3 and STIR-4). Despite the common presence of TIR do- Δstir-4 did not affect secretion of STIR-1, STIR-2, and STIR-3. mains, amino acid similarities among the proteins were relatively low. In the current study, we further examined the contribution of STIR-3 to 4. Discussion bacterial pathogenicity and mediation of immune evasion. Our results were consistent with prior observations that along with fi The TIR domain is essential for Toll-like receptor (TLR) and adaptor STIR-1 and STIR-2 [17,18], STIR-3 contributes signi cantly to patho- fi interactions and onset of a signalling cascade resulting in nuclear genicity during infection in vivo. In keeping with our expected ndings, κ translocation of the transcription factor, NF-κB, followed by production STIR-3 interacted with MyD88 and inhibited NF- B signaling, facil- of inflammatory cytokines and type I interferons [31]. We hypothesized itating Y. ruckeri SC09 infection of host cells and evasion of natural that Y. ruckeri SC09 targets TLR signalling to facilitate escape from the immune killing. In contrast, STIR-4 was not involved in virulence and host innate immune response and enhance virulence. Thus, TIR do- could not interact with MyD88 (since the sample was inadequate, data main-containing proteins possibly contribute to Y. ruckeri SC09 infec- for STIR-4 are not shown). These results led to confusion in two di- tion of fish. rections, as it remains unclear why: (1) STIR-4 carrying a similar do- Bacterial strategies using TIR homologs to evade recognition by TLR main to STIR-1, STIR-2, and STIR-3 has no immune evasive function signaling pathways in innate immunity are ubiquitous in microorgan- and (2) deletion of stir-3 achieves loss of immune evasion, even in the isms [32]. The earliest organism shown to encode the TIR domain presence of stir-1 and stir-2. In view of these results, we propose that containing-protein (Tcp) is the TcpC protein of Escherichia coli CFT073 STIR-1, STIR-2, and STIR-3 are co-secreted by a complex (such as a [33]. TcpC promotes the survival of pathogenic E. coli in kidney and terpolymer). Additionally, STIR-4 may contain a residual TIR domain inhibits TLR signaling by interacting with myeloid differentiation factor that does not interact with MyD88. To examine this hypothesis, we 88 (MyD88) adaptor protein [33]. Subsequently, highly pathogenic performed WB analysis of secreted products and precipitated lysates of Brucella melitensis was reported to secrete a Toll/interleukin-1 receptor four knockout strains. We were unable to observe secretion of STIR-1, Δ (TIR) domain-containing protein, designated TcpB [34,35]. TcpB in- STIR-2, and STIR-3 in the stir-1 strain but detected low levels of STIR- Δ teracts directly with MyD88 and suppresses TLR signaling [34,35]. 4. Similarly, STIR-1, STIR-2, and STIR-3 were not secreted in stir-2 or Δ Recent reports suggest that pathogenic Staphylococcus aureus produces a stir-3 strains. SC09 continuously produced and secreted STIR-4 at a similar protein that functions in natural immune evasion, designated low level relative to STIR-1, STIR-2, and STIR-3. These results suggest staphylococcal TIR domain protein (TirS) [36,37]. TirS downregulates that STIR-1, STIR-2, and STIR-3 are secreted as a complex and depletion ff the NF-κB pathway through inhibiting TLR2, TLR4, TLR5, and TLR9 of any one inhibits secretion of the other two, thereby a ecting immune [36,37]. Moreover, TirS interacts with not only MyD88 but also the evasion function. Expression of STIR-4 encoded by stir-4 did not appear ff ff Toll-interleukin 1 receptor (TIR) domain-containing adaptor protein to be a ected by knockout of stir-1, stir-2, and stir-3 and had no e ect on (TIRAP) [36,37]. Interestingly, recent research on the Pseudomonas expression or secretion of these three genes. However, a number of aeruginosa TIR effector (PumA) revealed interactions with not only issues, such as whether STIR-1, STIR-2, and STIR-3 form a terpolymer, TIRAP and MyD88 but also ubiquitin-associated protein 1 (UBAP1) how these three proteins are secreted outside bacteria, and whether this [38], suggestive of a novel immune evasion strategy. We previously secretion is related to the adjacent type IV secretion system (T4SS) identified Toll/interleukin-1 receptor (TIR) domain-containing proteins remain to be established. STIR-1 (previously designated TcpA) and STIR-2 in the fish pathogen Y. Integrative and conjugative elements (ICEs) are a newly recognized ruckeri SC09 [17,18]. Similar to TcpC of E. coli, TcpB of B. melitensis, class of mobile DNA elements in prokaryotes [24]. Numerous ICEs fl TirS of S. aureus and PumA of P. aeruginosa, STIR-1 and STIR-2 ap- harbor a tyrosine recombinase gene and are anked by direct repeats ′ peared significantly associated with pathogenicity in vivo [17,18]. corresponding to the 3 end of conserved genes (e.g., a tRNA gene), fi Moreover, STIR-1 and STIR-2 have been shown to inhibit the NF-κB strongly suggesting integration via site-speci c recombination [24]. pathway via interactions with MyD88, leading to a weakened in- Furthermore, ICEs typically contain a core of conserved modular flammatory signal during infection and bacterial escape from natural structures that mediate their integration, excision, conjugation and “ ” immune killing [17,18]. However, in contrast to E. coli, B. melitensis, S. regulation interspersed with accessory regions that are variably

433 T. Liu, et al. Fish and Shellfish Immunology 99 (2020) 424–434 present across members of a species [21,23]. Although stir-1, stir-2, stir- [13] A.K.S. Jozwick, J. Graf, T.J. Welch, The flagellar master operon flhDC is a pleio- 3, and stir-4 genes are specific for Y. ruckeri SC09, the ICE(r2) where tropic regulator involved in motility and virulence of the fish pathogen Yersinia ruckeri, J. Appl. Microbiol. 122 (3) (2017) 578–588. these genes are located is a mobile component widely distributed [14] W.F. Fricke, T.J. Welch, P.F. McDermott, M.K. Mammel, J.E. LeClerc, D.G. White, among bacteria. Plant pathogens possessing this mobile island have T.A. Cebula, J. Ravel, Comparative genomics of the IncA/C multidrug resistance – been shown to infect leaves of host plants. For instance, Pectobacterium plasmid family, J. Bacteriol. 191 (15) (2009) 4750 4757. [15] I. Dahiya, R.M.W. Stevenson, The ZnuABC operon is important for Yersinia ruckeri carotovorum BC T5 infects Brassica rapa ssp. pekinensis and Dickeya zeae infections of rainbow trout, Oncorhynchus mykiss (Walbaum), J. Fish. Dis. 33 (4) CSL RW192 causes pathogenicity in rice (microbe sample information (2010) 331–340. fi [16] I. Dahiya, R.M.W. Stevenson, The UvrY response regulator of the BarA-UvrY two- obtained from NCBI). ICE sequences have been identi ed in these component system contributes to Yersinia ruckeri infection of rainbow trout bacteria although the toxicity and immune evasion properties of the (Oncorhynchus mykiss), Arch. Microbiol. 192 (7) (2010) 541–547. component genes are yet to be determined. Similar tir genes are in- [17] T. Liu, E. Wang, W. Wei, K. Wang, Q. Yang, X. Ai, TcpA, a novel Yersinia ruckeri TIR-containing virulent protein mediates immune evasion by targeting MyD88 corporated in the ICE regions of these pathogens. Further studies are adaptors, Fish Shellfish Immunol. 94 (2019) 58–65. therefore warranted to establish the potential transmission risk of stir-1, [18] T. Liu, W.Y. Wei, K.Y. Wang, E.L. Wang, Q. Yang, A Yersinia ruckeri TIR Domain- containing protein (STIR-2) mediates immune evasion by targeting the MyD88 stir-2, stir-3, and stir-4. adaptor, Int. J. Mol. Sci. 20 (18) (2019). [19] T. Liu, K.Y. Wang, J. Wang, D.F. Chen, X.L. Huang, P. Ouyang, Y. Geng, Y. He, CRediT authorship contribution statement Y. Zhou, J. Min, Genome Sequence of the Fish Pathogen Yersinia ruckeri SC09 provides insights into niche adaptation and pathogenic mechanism, Int. J. Mol. Sci. 17 (4) (2016) 557. Tao Liu: Conceptualization, Methodology, Software, Visualization, [20] F. Delavat, R. Miyazaki, N. Carraro, N. Pradervand, J.R. van der Meer, The hidden Writing - original draft, Validation, Investigation, Funding acquisition, life of integrative and conjugative elements, FEMS Microbiol. Rev. 41 (4) (2017) 512–537. 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