Veterinary Microbiology 250 (2020) 108857

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Development of signature-tagged mutagenesis in Riemerella anatipestifer to identify genes essential for survival and pathogenesis Minjie Tao, Jialing Wang, Ke Li, Yafei Xue, Xinxin Xu, Xiaoli Du, Xiaohua He, Xiangqiang Tian, Zuocheng Zou, Zhonghao Hu, Nazrul Islam, Qinghai Hu * Shanghai Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, 518 Ziyue Road, Shanghai 200241, China

A R T I C L E I N F O

A B S T R A C T

Keywords: Riemerella anatipestifer Signature-tagged transposon mutagenesis (STM) Complemented strain Bacterial loads Virulence

Riemerella anatipestifer causes epizootic infectious disease in ducks, geese, turkeys and other birds, and serious economic losses especially to the duck industry. However, little is known about the molecular basis of its pathogenesis. In this study, signature-tagged transposon mutagenesis based on Tn4351 was developed in R. anatipestifer to identify genes essential for survival and pathogenesis. Seventeen tagged Tn4351 random mutation libraries of the R. anatipestifer strain WJ4 containing 5100 mutants were screened for survive using a duckling infection model. Twenty mutants that could not be recovered from the infected ducklings, were identified, and 17 mutated genes were identified by inverse PCR or genome-walking PCR. Of these genes, FIP52_03215, FIP52_04350 and FIP52_09345, were inserted into two mutant strains, and FIP52_03215 and FIP52_03175 were found exclusively on the chromosome of serotype 1 R. anatipestifer strains. Twelve out of 17 genes encoding for proteins were predicted to be involved in amino acid, nucleotide, coenzyme, or lipid transport and metabolism, one gene was predicted to be involved in signal transduction, one gene was predicted to be involved in DNA replication, recombination and repair, the other three genes had an unknown function. Animal experiments showed that the virulence of mutants 16-284, 7-295, 24-231, 9-232 and 19-214 were significantly attenuated compared to that of the wild-type WJ4. Moreover, the median lethal dose of mutant 16-284 was greater than 1010 CFU, and its virulence to ducklings was partially restored when it was complemented with the shuttle expression plasmid pRES-FIP52_09345. The results in this study will be helpful to further study the molecular mechanisms of the pathogenesis of R. anatipestifer infection.

1. Introduction Riemerella anatipestifer is a member of the genus Riemerella in the family Flavobacteriaceae. R. anatipestifer infection occurs as an acute or chronic septicemia characterized by fibrinous pericarditis, perihepatitis, airsacculitis, caseous salpingitis, and meningitis. It is probably the most economically important infectious disease of farmed ducks worldwide. Once the disease invades a flock of ducks or geese, it can become endemic. Eradication can be difficult, with repeated episodes of infec­ tion possible. Currently, at least 21 serotypes have been identified with very little or no cross protection among different serotypes (Ruiz and Sandhu, 2013; Sandhu, 1979). There are strong variations in virulence between different serotypes and even within a given serotype of R. anatipestifer (Subramaniam et al., 2000). Several bacterial factors associated with the virulence of R. anatipestifer, have been identified, including outer membrane proteins * Corresponding author. E-mail address: [email protected] (Q. Hu). https://doi.org/10.1016/j.vetmic.2020.108857 Received 11 July 2020; Accepted 13 September 2020 Available online 19 September 2020 0378-1135/© 2020 Elsevier B.V. All rights reserved.

(Hu et al., 2011; Yi et al., 2017), iron acquisition systems (Lu et al., 2013; Miao et al., 2015; Tu et al., 2014), and lipopolysaccharide com­ plexes (Dou et al., 2018), as well as the Type IX Secretion System (Chen et al., 2019; Hu et al., 2019; Yuan et al., 2019), etc. However, the mechanisms underlying pathogenicity remain unclear. Random transposon mutagenesis, which allows screening for atten­ uated mutants in an animal model of infection at the genome wide level, is one of the most powerful technologies for mining of potential virulence-associated gene. Signature-tagged mutagenesis (STM) is a refinement of the classical transposon mutagenesis system involving transposons that carry unique DNA tags, enabling the identification either by hybridization or by PCR of individual mutants in a mixture of mutants carrying different tags (Lehoux et al., 1999; Sheehan et al., 2003). The use of STM minimizes the number of animals used, by pooling mutants and permits the identification of genes essential in vivo and negative selection in a mixed population of bacterial mutants. The

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sequence tags within the mutant “input” population are compared to those mutants recovered from the host, and those that represent muta­ tions in essential in vivo genes that fail to survive or are undetectable. So far, STM has led to the identification of hundreds of new genes required for virulence in a broad range of bacterial pathogens (Autret and Charbit, 2005; Saenz and Dehio, 2005), and genes essential for growth in vivo (Sheehan et al., 2003). In our previous studies, the Bacteroides transposon Tn4351 was successfully used in R. anatipestifer strains to construct random trans­ poson mutagenesis libraries. Moreover, some genes involved in R. anatipestifer biofilm formation or virulence have been identified (Hu et al., 2012; Ni et al., 2016; Wang et al., 2015). To improve our un­ derstanding of the pathogenesis of R. anatipestifer infection and to identify genes essential for its virulence, a PCR-based STM was devel­ oped in this study to screen and identify genes essential for survival and pathogenesis of R. anatipestifer in a duck infection model. Seventeen putative virulence associated genes were screened using 17 tagged Tn4351 mutant libraries containing over 5100 mutants.

(with BamH I and Ned I site at 5′ ) and Tag1 P2 (with Afl II and Sac I site at 5′ ) through denaturation at 97 ◦ C for 10 min and annealing at room temperature for 30 min. Then double strand Tag1 DNA was ligated to BamH I- and Sac I-digested T-cfxA to generate T-cfxA-Tag1. Subse­ quently, cfxA-Tag1 was cut from T-cfxA-Tag1 with PpuM I and Afl II, and ligated into pEP4351 to generate pEP4351-cfxA-Tag1, in which part of ermF and tetX was replaced with cfxA expression box (Figure S1). Plasmid pEP4351-cfxA-Tag1 was digested with Nde I and Pst I, and Tag1 was respectively replaced with Tag2 to Tag24 double-strand DNA fragments, to generate pEP4351-cfxA-Tagn (n = 2 … 24). The cross reactivity between different Tags was detected by PCR using Forward universal primer C plus Pn (n = 1 … 24, depending on which Tag to be detected) (Table S1), on a mixture containing 23 pEP4351-cfxA-Tagns plasmids except the one carrying a tag used as the reverse primer. The genomic DNAs of the R. anatipestifer strain WJ4 were used as a control to confirm the specificities of tags. 2.4. Construction of 17 signature-tagged transposon mutant libraries of R. anatipestifer strain WJ4

2. Materials and methods

Tagged R. anatipestifer transconjugant libraries were made by conjugation using procedures previously described (Ni et al., 2016) with minor modifications. Briefly, the recipient strain R. anatipestfier WJ4 and the donor strain E. coli BW19851(pEP4351-cfxA-Tagn), were mixed in 10 mM MgSO4 and 100 μl of the mixture of bacteria was added on TSA agar and incubated at 30 ◦ C for 8 hours. The transconjugants were selected on TSA containing cefoxitin and kanamycin, with the 16S rRNA+cfxA+ transconjugants as the positive mutants. Each tagged Tn4351 mutant library contained over 300 mutants. In this study, there were 17 mutant libraries containing over 5100 mutants generated.

2.1. Bacterial strains, plasmids, and culture conditions R. anatipestifer serotye 1 strain WJ4 was isolated from the heart blood of a sick duckling in China in 2000. The Escherichia coli strain BW19851 (pEP4351), which carries the plasmid pEP4351, was provided gener­ ously by Professor Mark J. McBride at the University of WisconsinMilwaukee in the United States. The E. coli–R. anatipestifer shuttle plasmid pRES was constructed by Qinghai Hu et al (Hu et al., 2013). The R. anatipestifer strain WJ4 and its derivatives were cultured at 37 ◦ C in tryptic soybean broth (TSB, Difco, Detroit, MI, USA) or tryptic soybean agar (TSA) and E. coli strains were grown routinely in Luria-Bertani (LB) broth (Difco, Detroit, MI, USA) or LB agar at 37 ◦ C. For the selective growth of bacterial strains, antibiotics were added at the following concentrations: ampicillin (100 μg/ml), chloramphenicol (50 μg/ml), erythromycin(1 μg/ml), kanamycin (50 μg/ml), or cefoxitin (1 μg/ml).

2.5. STM screen of tagged mutants in ducklings One-day-old Cherry Valley ducklings were obtained from the Lijia Duck Farm (Wuxi, Jiangsu province, China), and no antibodies against R. anatipestifer in the serum of ducklings was detected by indirect ELISA using strain WJ4 whole cells as coating antigen. The ducklings were housed in cages with a controlled temperature of 24–26 ◦ C, under a 12 h light/dark cycle and with free access to food and water during the study period. Animal experiments in this study were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Institutional Animal Care and Use Com­ mittee guidelines set by the Shanghai Veterinary Research Institute, the Chinese Academy of Agricultural Sciences (CAAS; Shanghai, China). The study protocol was approved by the Committee on the Ethics of Animal Experiments of the Shanghai Veterinary Research Institute, CAAS (Permit No.: SHVRI-SZ-20160312-02). Seventeen colonies, one from each mutant library of individual tags, were inoculated into individual tubes containing 5 mL of TSB broth with cefoxitin and kanamycin and shakn at 37 ◦ C to OD600≈1.0. Then, 3 mL of culture for each transconjugant from each of the 17 signature- tagged libraries was combined to created input pools for infecting ducklings. Each input pool containing 2 × 109 CFU bacteria were inoculated intramuscularly into eight-day-old duckling, with three ducklings per input pool. At 36 h post-inoculation, blood collected from the infected ducklings (containing about 105 CFU bacteria) was spread on the TSA agar supplemented with cefoxitin and kanamycin, and incubated at 37 ◦ C under 5% CO2 overnight and the bacteria on the TSA agar were collected as output pools. Genomic DNAs was isolated from each input pool and output pool using TIANamp Bacteria DNA Kit (TIANGEN Biotech, Beijing, China), and PCR was performed to detect each specific tag DNA (Lehoux et al., 1999). The tagged mutants present in the input pools, but not in the output pools, were screened out. The mutants selected in the first screen were combined into new input pools and inoculated into ducklings for the second screen. After three rounds of STM screens, all the selected mutants were subjected to inoculate

2.2. Full genome sequence of R. anatipestifer strain WJ4 The complete sequence of strain WJ4 was sequenced using the Pac­ Bio RS II long-read sequencing method. A 10-kb genomic DNA library was constructed using the PacBio SMRTbell template prep kit (PacBio, Menlo Park, CA, USA) and sequenced with a PacBio RS II sequencer (PacBio). To fill the gaps between contigs, an additional 300 bp genomic DNA library was constructed using the NEBNext UltraTM DNA library prep kit (New England BioLabs, Ipswich, MA, USA) and sequenced with a HiSeq4000 sequencer (Illumina, Inc., San Diego, CA, USA). The sequencing information was assembled with HGAP V3.0, with a genome coverage of 160 folds. Putative coding sequences (CDSs) were identified with Grammer version 3. Functional annotations of CDSs were per­ formed by searching against the nonredundant protein database using the BLASTP algorithm. The tRNA and rRNA genes were predicted using tRNAscan-SE v1.31 and RNAmmer v 1.2, respectively. 2.3. Insertion of different signature tag DNA into transposon Tn4351 Our previous results showed that 53.8% of the tested R. anatipestifer strains showed resistance to erythromycin (Xing et al., 2015), but none was cefoxitin-resistant. So, in this study, erythromycin-resistant gene ermF in the transposon Tn4351 was replaced with cefoxitin-resistant gene cfxA. The cfxA expression box was amplified from plasmid pCP29 by PCR using primers cfxA P1 and cfxA P2, and ligated into pGEM-T easy vector (Promega, Madison, WI, USA) generating T-cfxA. Twenty-four different tag sequences reported by Sanschagrin et al (Sanschagrin et al., 2008) were synthesized and designated as Tag1 to Tag24. Tag1 double-strand DNA fragments were generated with Tag1 P1 2

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ducklings separately, and the mutant was recovered from the blood and confirmed by detecting the respective tag using PCR.

2.10. The virulence of the mutants to ducklings To determine whether the virulence of screened mutants 16-284, 7295, 24-231, 9-232 and 19-214 were attenuated, eight-day-old duck­ lings were challenged with 1 × 109 CFU and 1 × 1010 CFU of each mutant, with 10 ducklings for each dose. The mortality of the ducklings was recorded daily for a period of 10 days after challenge. To further determine whether the virulence-attenuated phenotype of 16-284 was due to an inactivated FIP52_09345 gene, the median lethal dose (LD50) of the wild-typeWJ4, mutant 16-284 and its complemented strain 16-284 (pRES-FIP52_09345) were measured as described previ­ ously (Hu et al., 2011). For each strain, eight-day-old Cherry Valley ducklings were divided evenly into four groups with 10 ducklings per group and injected intramuscularly with 107, 108, 109, or 1010 CFU of bacteria respectively. The mortality of the ducklings was recorded daily for a period of 10 days after challenge. The LD50 was calculated using the Reed–Muench formula. In addition, the blood was collected from 109 CFU of bacteria infected ducklings with eight ducklings per group at 24 h post inocu­ lation, and bacterial loads in the blood in these ducklings were measured as described previously (Hu et al., 2011).

2.6. Southern blot Southern blot analysis of the Tn4351 insertions was used for the identification of the mutants. The genomic DNAs of the tagged Tn4351 insertion mutants were digested with Xba I, separated by gel electro­ phoresis and transferred to nylon membranes essentially as described previously (Hu et al., 2012). The cfxA gene, which is present on pEP4351-cfxA-Tagn, but not on the genome of the wild-typeWJ4, was used to detect transposons for identification. The DIG DNA labeling and detection kit (Roche Diagnostics USA, Indianapolis, IN, USA) was used to prepare probes and perform hybridization. The number of cfxA bands on nylon membrane represented the number of transposon insertion sites in one mutant. 2.7. Identification of transposon insertion sites The nucleotide sequence flanking the tagged Tn4351 insertion site was determined using inverse PCR or genome-walking PCR (Genomic walking kit, Takara, Dalian, China) as described previously (Hu et al., 2012). Briefly, genomic DNAs were digested with the restriction enzyme Hind III and then re-ligated, which resulted in the formation of circular molecules. Primer pairs specific for Tn4351 (primers 340 plus 341; primers TN-1 plus IS4351-F, respectively) (Alvarez et al., 2006) were used to amplify the sequences adjacent to the insertion site using TaKaRa LA PCR kit (TaKaRa, Dalian, China). Genomic walking was performed with a genomic walking kit (TaKaRa) using a variety of arbitrary primers (AP1, AP2, AP3, and AP4) provided in the kit and three specific primers (SP1, SP2 and SP3), according to the manufac­ turer’s instructions. The sequences of the identified genes were used to search for other known homologous sequences and putative functions using the BLASTX server (http://www.ncbi.nlm.nih.gov/BLASTX/) and the online PSORT v.3.0 program (http://www.psort.org/) was used to predict the subcellular localization of the proteins.

2.11. Statistical analysis The statistical significance of the data was determined using one-way ANOVA and Tukey’s Multiple Comparison test within GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA), or using x2 test by SPSS 22.0 (IBM, NY, USA). A probability (p) value of < 0.05 was considered statistically significant. 3. Results 3.1. Full genome sequence of R. anatipestifer strain WJ4 The complete genome sequence of serotype 1 Riemerella anatipestifer strain WJ4 consisted of a single circular chromosome of 2,240,780 bp containing 2,141 putative open reading frames (ORFs), 3 ncRNAs, and 41 tRNA genes. The complete genome sequence of strain WJ4 has been deposited in GenBank under the accession number CP041029.

2.8. Construction of the complemented strain of mutant 16-284 An E. coli–R. anatipestifer shuttle plasmid pRES was used to construct the complemented strain of mutant 16-284. The FIP52_09345 open reading frame (ORF), which was inactivated by the insertion of taggedTn4351 in mutant 16-284, was amplified and subcloned into the pRES, and the expression of FIP52_09345 in the complementing plasmid was under the control of the putative promoter pompA (Hu et al., 2013). Then, the plasmid pRES-FIP52_09345 was introduced into the mutant 16-284 by conjugation, to generate the complemented strain 16-284 (pRES-FIP52_09345). The growth curves of the wild-typeWJ4, mutant 16-284, and com­ plemented strain 16-284 (pRES-FIP52_09345) in TSB, were determined as described previously (Hu et al., 2002).

3.2. Cross reactivity of tagged transposons The cross-reactions among 24 tagged transposons were identified by PCR. The results showed that there were cross-reactions among Tag3, Tag4, Tag5, and Tag8, and among Tag20, Tag21, and Tag22 (Figure S2A) when the annealing temperature was 60 to 65 ◦ C. Next, the cross reactions among Tag3, Tag4, Tag5, and Tag8 was detected one by one, as were the cross reactions among Tag20, Tag21, and Tag22. The results showed there was cross-reaction between Tag 3 and Tag 8, and Tag21 could reaction with Tag 20 and Tag 22 (Figure S2B). Therefore, Tag 8 and Tag 21 were discarded as a result, and the other 22 tags could be used to construct the transposon mutation libraries of R. anatipestifer strain WJ4.

2.9. Real-time PCR analysis To determine whether FIP52_09345 gene insertion had a polar effect on transcription of adjacent genes, total RNA was isolated from the wildtype WJ4, mutant 16-284 and complemented strain 16-284 (pRESFIP52_09345) cells at the logarithmic growth phase (OD600 was about 1.0) with RNeasy Mini kit (Qiagen, Hilden, Germany), followed by cDNA synthesis using Sensiscript RT kit (Qiagen). The relative mRNA levels of FIP52_09345, upstream FIP52_09340 and downstream FIP52_09350 were measured by Real-time RT-PCR on a PRISM 7900 sequence detection system (Applied Biosystems, UK) with primer pairs using SYBR® green PCR master mix (Applied Biosystems). Relative quantification of gene expression was calculated according to the 2− ΔΔCT method using 16S rRNA gene of strain WJ4 for normalization.

3.3. In vivo negative selection of attenuated mutants R. anatipestifer infection, is also known as duck septicemia, anati­ pestifer septicemia. It commonly causes an acute septicemic disease in younger bids (Brodgen, 1989), and high bacterial loads in blood can be detected in R. anatipestifer infected ducklings (Hu et al., 2011). In addition, intramuscular, rather than oral or intranasal challenge resul­ ted in clinical signs of infection and caused 100% mortality (Hatfield and Morris, 1988). In this study, therefore, the ducklings were inocu­ lated with input pools by intramuscular injection, and bacteria (output pools) were recovered from the blood of infected ducklings. In this study, seventeen signature-tagged transposons including Tags 3

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only found to be on the chromosome of three serotype 1 R. anatipestifer strains (WJ4, accession no. CP041029.1; CH3, accession no. CP006649.1; and RA-CH-1, accession no. CP003787.1), but not on the other R. anatipestifer strains whose full genomes have been released on the GenBank. The nucleotide sequence of gene FIP52-03175 gene is 100% homologous among serotype 1 R. anatipestifer strains CH3, WJ4 and CH-1, as is FIP52-03215 gene. The subcellular locations of the protein products of these 17 identified genes were predicted with PSORTb software. Thirteen of 17 proteins (76.47%) were predicted to be cytoplasmic proteins, one (5.88%) was predicted to be an outer mem­ brane protein, and the other three proteins had unknown locations. These proteins were further categorized on the basis of their putative functions. Twelve of 17 proteins (70.59%) were classified into the “metabolism” category (E, F, H, I and C), cellular processes -related category contained one protein (T, 5.88%), information storage and processing category contained one protein (L, 5.88%), and three pro­ teins could not be categorized.

1-4, Tags 6-7, Tags 9-16, Tag19, Tags 23-24, were used to construct Tn4351 mutant libraries of R. anatipestifer strain WJ4. One signaturetagged R. anatipestifer mutant from each library was included in each input pool and 5100 mutants (300 mutants per tagged Tn4351) were arranged into 300 input pools. Each input pool was used to infect three ducklings and 36 h post inoculation, bacteria (output pools) were recovered from the blood of input pool-infected ducklings and each tag was detected by PCR. During the first round STM screen, 101 mutants which were detected in input pools but not in the output pools, were identified. These mutants were reassembled into new input pools, and each new pool was used to infect a further three ducklings. Following this second and third round screen, 32 mutants were found to be consistently absent from the output pools. Then, each mutant was used to infect three ducklings separately and 20 mutants that could not be recovered from the blood of inoculated ducklings were identified. The results of southern blot showed that only one transposon Tn4351 insertion site on the genome of all 20 mutants had been detected. During the second and third round of STM screening and subsequent inoculations of mutant strains to ducklings, 81 mutants can be detected or recovered again. Thirteen of these removed mutants were randomly selected to determine the bacterial loads in blood of ducklings 36 h after infection. As shown in Fig. 1, the bacterial loads in blood of all the tested mutants infected ducklings were decreased significantly (p < 0.01, Fig. 1) compared to those in blood of wild-type WJ4 infected ducklings. In addition, among 20 mutants screened by STM in this study, 9 mutants showed a significantly reduction in growth in TSB compared to the wildtypeWJ4 (p < 0.01, Figure S3).

3.5. Pathogenicity of the mutants to ducklings Ducklings were challenged with 1 × 109 CFU and 1 × 1010 CFU of mutants 16-284, 7-295, 24-231, 9-232, 19-214, and the wild-type WJ4 respectively. As shown in Table 2, the results showed that all five mu­ tants caused zero mortality when administered at a dosage of 1 × 109CFU, while one of 10 (10%) mutant 7-295 infected ducklings and two of 10 (20%) mutant 16-284 infected ducklings died during 10 days post-inoculation period when the challenge dosage was 1 × 1010 CFU. In contrast, eight out of 10 (80%) and 10 out of 10 (100%) wild-type WJ4 infected ducklings died when they were challenged with 1 × 109 CFU and 1 × 1010 CFU respectively. Moreover, no 1 × 1010 CFU of 24-231, 9232 or 19-214 infected ducklings died, so the results suggested that the pathogenesis of these five mutants to ducklings was significantly attenuated (p < 0.01). To further determine whether the attenuated virulence of mutant 16284 was due to the Tn4351 insertion inactivated FIP52_09345 gene, the complemented strain 16-284 (pRES-FIP52_09345) was constructed. The growth curves showed that there was no significant difference in growth in TSB between the wild-type WJ4, mutant 16-284, and its com­ plemented strain M16-284 (pRES-FIP52_09345) (p > 0.05, Fig. 2A). The calculated LD50 values of the wild-type WJ4, mutant M16-284 and its complemented strain 16-284 (pRES-FIP52_09345) were 6.38 × 107 CFU, > 1.0 × 1010 CFU, and 7.57 × 108 CFU, respectively. The 156fold difference in the LD50 between the wild-type WJ4 and mutant 16284 indicated that disruption of the FIP52_09345 gene resulted in attenuation of R. anatipestifer virulence. The LD50 of the complemented strain 16-284 (pRES-FIP52_09345) was increased by 13-times compared to that of the mutant 16-284, suggesting that the virulence to ducklings was only partially restored when the mutant was complemented with the shuttle expression plasmid pRES-FIP52_09345. In addition, as shown in Fig. 2B, the bacterial loads in the blood of the mutant 16-284 infected ducklings at 24 h post-inoculation were zero, which was significantly decreased compared to that of the wild-type WJ4 (p < 0.01), which was 1.61 ± 0.44 × 106 CFU/ml, while that of the complemented strain 16284 (pRES-FIP52_09345) was restored partially. The results of realtime PCR showed that the relative mRNA level of FIP52_09345 in the complemented strain 16-284 (pRES-FIP52_09345) was about 17 folds higher than that in the wild type WJ4 (Fig. 2C), and the relative mRNA levels of upstream gene FIP52_09340 and downstream gene FIP52_09350 in the mutant 16-284 were 2-3 folds higher than those in the wild type WJ4. The results suggested that the high levels of FIP52_09345 expression in the complemented strain 16-284 (pRESFIP52_09345) may be due to its partial restored virulence and bacterial loads in the infected ducklings.

3.4. Identification of genes disrupted by Tn4351 insertion The nucleotide sequence of the DNA flanking the site of transposon Tn4351 insertion obtained for each of the 20 mutants was used to search the GenBank databases for homologous genes. The results of this anal­ ysis are shown in Table 1, along with details regarding the 17 mutated genes we identified. Of these, FIP52_03215, FIP52_04350 and FIP52_09345, were inserted into two mutant strains. In addition, 15 of 17 identified genes were carried by different serotypes of R. anatipestifer strains, for which the full genome sequences have been released on the GenBank. The other two genes, FIP52_03215 and FIP52_03175, mutated by Tn4351 insertion in three mutants 3-144, 24-225 and 24-292, were

Fig. 1. Blood bacterial loads in ducklings infected with the wild-type WJ4 and removed mutants during the second, third STM screen and the following indi­ vidual infection. Thirteen of these removed mutants were randomly selected to determine the bacterial loads in blood of ducklings 36 hours after infection. The bacterial loads in blood of all the tested mutants infected ducklings were decreased significantly (p < 0.01) compared to those in blood of wild-type WJ4 infected ducklings. The error bars represent means ± standard deviations from three ducklings. 4

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Table 1 Riemerella anatipestifer strain WJ4 virulence-related genes identified by STM. Functional family

Mutants

Disrupted gene in strain WJ4a

Putative function/ product

Subcellular locationb

COGc

Metabolism

6-254

FIP52_00315

Cytoplasmic

COG0127 F

7-242 7-295 9-232

FIP52_04485 FIP52_01425 FIP52_06345

non-canonical purine NTP pyrophosphatase, rdgB/HAM1 family glycosyl transferase group 1 Adenylosuccinate synthetase aminodeoxychorismate synthase, subunit I

Cytoplasmic Cytoplasmic Cytoplasmic

11-66

FIP52_01395

Cytoplasmic

11-187 15-199 16-290 19-58 19-214 24-231, 24296 24-234 16-284, 4231 15-200

FIP52_04205 FIP52_04080 FIP52_06240 FIP52_07380 FIP52_09955 FIP52_04350

phosphoribosylaminoimidazole-succinocarboxamide synthase Xaa-Pro aminopeptidase putative hemin receptor adenosine deaminase biotin synthase BioB aminotransferase class-iii (BioA) NADH-quinone oxidoreductase subunit C

COG0438 E COG0104 F COG0147 EH COG0152 F

Cytoplasmic Outer Membrane Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic

COG0006 COG2067 COG1816 COG0502 COG0161 COG0852

FIP52_01390 FIP52_09345

phosphoribosylformylglycinamidine synthase putative transcriptional regulator, Crp/Fnr family

Cytoplasmic Cytoplasmic

COG0046 F COG0664 T

FIP52_03010

bacterial nucleoid protein Hbs

Cytoplasmic

COG0776 L

24-9 3-144, 24225 24-292

FIP52_02870 FIP52_03215

hypothetical protein ATPase family associated with various cellular activities (AAA) YecR-like lipoprotein

Unknown Unknown

-

Unknown

-

Cellular processes Information storage and processing unknown

FIP52_03175

E I F H H C

a

The complete genome sequence of strain WJ4 has been deposited in GenBank under the accession number CP041029. Subcellular locations were predicted by the PSORTb v.3.0.2 software (http://www.psort.org/). c COG functional categories: (1) Information storage and processing: (A. RNA processing and modification; B. Chromatin structure and dynamics; J: Translation, ribosomal structure and biogenesis; K: Transcription; L: Replication, recombination and repair); (2) Cellular processes: (D: Cell cycle control, cell division and chromosome partitioning; M: Cell wall/membrane/envelope biogenesis; N: Cell motility; O: Posttranslational modification, protein turnover, chaperons; T: Signal transduction mechanisms; U: Intracellular trafficking, secretion, and vesicular transport); (3) Metabolism: (C. Energy transport and conversion; E: Amino acid transport and metabolism; F: Nucleotide transport and metabolism; G: Carbohydrate transport and metabolism; H. Coenzyme transport and metabolism; I: Lipid transport and metabolism; P: Inorganic ion transport and metabolism; Q: Secondary metabolites biosynthesis, transport and catabolism). (4) Poorly characterized: (R: General function prediction only; S: Function unknown). b

4. Discussion

Table 2 Pathogenicity of the mutants to ducklings. Strains

Infection dosage

The number of ducklings used

The number of infected ducklings died

Mortality rate

7-295

1 × 109 CFU 1 × 1010 CFU 1 × 109 CFU 1 × 1010 CFU 1 × 109 CFU 1 × 1010 CFU 1 × 109 CFU 1 × 1010 CFU 1 × 109 CFU 1 × 1010 CFU 1 × 109 CFU 1 × 1010 CFU

10

0

0a, **

10

1

10% **

10

0

0 **

10

0

0 **

10

0

0 **

10

2

20% **

10

0

0 **

10

0

0 **

10

0

0 **

10

0

0 **

10

8

80%

10

10

100%

9-232

16284 19214 24231 WJ4

In this study, STM was developed for R. anatipestifer to identify virulence factors using three ducks per pool and three rounds of screening, which may avoid or reduce the individual differences of ducklings on bacterial proliferation, and stochastic loss of tagged mu­ tants in the infected ducklings (Grant et al., 2005). During the first round screening, 101 mutants were screened, but 81 mutants were excluded in the following screening. This may due to the bacterial loads in the blood of some mutants infected ducklings being very low (Fig. 1). Even for some mutants, the bacterial loads in the blood of one to three infected ducklings were zero. Moreover, the growth of some mutants was significantly decreased compared to that of the wild-type WJ4, and these mutants, which proliferated slowly in the infected ducklings, may have low bacterial loads in blood. The mutants with very low bacterial loads in blood were also easy to be eliminated by the immune system in ducks. Therefore, PCR may have given negative results from recovered pools when the bacterial loads in blood were zero or very low. In most studies, both predicted and unsuspected virulence factors have been identified by STM and include proteins involved in cellsurface structures, transport, metabolism, secretion, stress response and transcriptional regulation, as well as open reading frames (ORFs) with unknown function or with no homology to other genes in GenBank (Mecsas, 2002). In this study, 12 of 17 identified genes were predicted to be involved in metabolism. The capsule plays an important role in the virulence of many path­ ogenic bacteria. Inactivating the wza gene, which was involved in capsule biosynthesis in R. anatipestifer, affected its pathogenicity to ducklings and biofilm Formation (Yi et al., 2017). In this study, the mutant 7-242, with the transposon inserted in FIP52_04485, showed no significantly difference in growth compared to the wild-type WJ4 in TSB. Glycosyl transferase encoded by FIP52_04485, may be involved in

a The mortality rate was significantly decreased compared to that of WJ4(**, p < 0.01). The statistical significance of the data was determined using x2 test by SPSS 22.0.

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Veterinary Microbiology 250 (2020) 108857

Fig. 2. Characterization of the mutant strain 16-284. (A) Growth curves of the wild-type WJ4, 16-284 and its complemented strain 16-284 (pRES-FIP52_09345). Growth of each bacterial strain in TSB at 37 ◦ C with shaking was monitored by measuring the OD600 values. This experiment was repeated twice and the data from one representative experiment is shown. (B) Blood bacterial loads in ducklings infected with the wild-typeWJ4, 16-284 and its complemented strain 16-284 (pRESFIP52_09345). The bacterial loads in blood from ducklings infected with 16-284 was zero 24 h post-inoculation, and that of the complemented strain 16-284 (pRESFIP52_09345) was partly restored. The error bars represent means ± standard deviations from six ducklings. Asterisks indicate statistically significant differences between two groups (**p < 0.01). (C) Real-time PCR analysis. The relative mRNA levels of FIP52_09345, upstream FIP52_09340 and downstream FIP52_09350 in the wild-type WJ4, 16-284 and its complemented strain 16-284 (pRES-FIP52_09345) were measured. The changes of mRNAs were expressed as fold expression and calculated using the comparative CT (2− ΔΔCT) method. Error bars represent SD from three replicates.

cell wall biosynthesis (COG0438) and capsular polysaccharide biosyn­ thesis (KEGG: cat:CA2559_13003). It suggested that FIP52_04485 insertion in the mutant 7-242 lead to its decreased pathogenicity to ducklings possibly by affecting the bacterial capsular polysaccharide or cell wall biosynthesis. Mutants 19-58 and 19-214 contained an insertion in biotin synthesis related genes bioB (locus tag: FIP52_07380) and bioA (locus tag: FIP52_09955) respectively. Biotin is an essential cofactor for the biotindependent enzymes that are involved in important metabolic pathways such as membrane lipid synthesis, replenishment of the tricarboxylic acid cycle and amino acid metabolism (Salaemae et al., 2016). The universal biotin biosynthetic pathway, converts a pimeloylthioester to biotin through the activity of four enzymes encoded by bioF, bioA, bioD and bioB respectively (Salaemae et al., 2011). Previously, Tn4351 insertion in R. anatipestifer Yb2 bioB gene (AS87_05545) resulted in a significant decrease in pathogenicity of the mutant to ducklings (Wang et al., 2015). In addition, R. anatipestifer bioF gene (AS87_RS09170) was involved in biotin synthesis, bacterial morphology and virulence (Ren et al., 2018). These results suggested that biotin biosynthesis associated proteins played a critical role in R. anatipestifer survive and virulence in ducklings. In the mutant 15-199, Tn4351 was inserted into gene FIP52_04080, which was encoded for the putative hemin receptor. Heme is the most abundant iron source in the infection host for bacterial pathogens. Our previous results showed that R. anatipestifer strain CH3 could utilize hemin as the sole iron source for its growth in vitro (Lu et al., 2013). The FIP52_04080 had 100% homology to R. anatipestifer strain CH-1 B739_1208, which was proven to be involved in iron uptake and viru­ lence in strain CH-1 (Wang et al., 2017). In addition, Tn4351 inserted genes in two mutants 7-295 and 16-290 were involved in purine metabolism. FIP52_01425, inserted in mutant 7295, encoded adenyl succinate synthetase (AdSS), an enzyme at regu­ latory point of purine metabolism. In mutant 16-290, FIP52_06240 encoded for adenosine deaminase, was inserted by Tn4351. Adenosine deaminase is a critical enzyme in purine metabolism that regulates intra and extracellular adenosine concentrations by converting it to inosine. In this study, the growth of mutants 7-295 and 16-290 was significantly decreased compared to that of the wild-type WJ4, so whether the attenuated virulence of these two mutants were related to the growth defect or other mechanism should be further uncovered. Except for metabolism, FIP52_09345, inserted in two mutants 16284 and 4-231, were predicted to be involved in cellular processes and signaling. FIP52_09345 encoded for putative transcriptional regu­ lators of the crp/fnr family. Members of Crp/Fnr superfamily, both in Gram-negative and Gram-positive bacteria, are involved in regulation of a vast range of physiologic functions such as metabolism, anaerobic and

aerobic respiration, resistance to oxidative stress, and virulence (Smith et al., 2017). The regulatory mechanisms of FIP52_09345 identified in this study remain to be clarified in the future study. In addition, the inserted gene FIP52_03010 in mutant 15-200, was involved in DNA replication, recombination and repair, but Tn4351 insertion in FIP52_03010 in mutant 15-200 had no significant effect on its growth in TSB medium. That how the insertion in genes FIP52_03010 and FIP52_05160 identified in this study affected the survival and pathogenesis of R. anatipestifer remains unclear. So far, there are two reports on screening virulence related genes of R. anatipestifer by random transposon mutation (Ni et al., 2016; Wang et al., 2015), and 77 genes were identified, but only one gene (AS87_05545) among these was also identified in this study (FIP52_07380). The reasons for this phenomenon may be as follows: Firstly, in this study, STM was used to screen out the mutants that could not be recovered or detected in the blood of infected ducks, and the identified genes may play a critical role in bacterial survival in vivo and pathogenesis, while conventional random transposon mutation used in two previous reports, could screen any mutants with reduced pathoge­ nicity to ducklings. Secondly, this may be related to the capacity of the mutant libraries used. Obviously, the library capacity used in two pre­ vious reports was not large enough, so few genes were screened out again by STM in this study. In fact, both random transposon mutation and STM have limitations of insufficient capacity of libraries, more an­ imals used, and high time and cost consumption. These defects can be overcome by transposon insertion sequencing, such as Tn-seq and Tra­ DIS, a combination of random transposon mutation and high-throughput sequencing (van Opijnen et al., 2009). Moreover, transposon insertion sequencing, could unlock data missed by STM screens (Eckert et al., 2011). In conclusion, in this study, Tn4351-based STM was developed, and 17 genes involved in R. anatipestifer survival in vivo and pathogenesis were identified. The results will be helpful to understand the molecular mechanisms of R. anatipestifer pathogenesis. Moreover, Tn4351-based STM mutagenesis strategy developed in this study is expected to be applicable to other Bacteroides. Acknowledgement This work was supported by the National Natural Science Foundation of China (31772770, 31472224 and 31272590). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.vetmic.2020.108857. 6

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Veterinary Microbiology 250 (2020) 108857

References

Ren, X., Wang, X., Shi, H., Zhang, X., Chen, Z., Malhi, K.K., Ding, C., Yu, S., 2018. Riemerella anatipestifer AS87_RS09170 gene is responsible for biotin synthesis, bacterial morphology and virulence. Sci. Rep. 8, 14645. Ruiz, J.A., Sandhu, T.S., 2013. Rimerella anatipestifer infection. In: Swayne, D.E., Glisson, J.R., McDougald, L.R., Nolan, L.K., Suarez, D.L., Nair, V.L. (Eds.), Diseases of Poultry, 13th ed. Wiley-Blackwell Press, pp. 823–828. Saenz, H.L., Dehio, C., 2005. Signature-tagged mutagenesis: technical advances in a negative selection method for virulence gene identification. Curr. Opin. Microbiol. 8, 612–619. Salaemae, W., Azhar, A., Booker, G.W., Polyak, S.W., 2011. Biotin biosynthesis in Mycobacterium tuberculosis: physiology, biochemistry and molecular intervention. Protein Cell 2, 691–695. Salaemae, W., Booker, G.W., Polyak, S.W., 2016. The Role of Biotin in Bacterial Physiology and Virulence: a Novel Antibiotic Target for Mycobacterium tuberculosis. Microbiol. Spectr. 4. Sandhu, T., 1979. Immunization of White Pekin ducklings against Pasteurella anatipestifer infection. Avian Dis. 23, 662–669. Sanschagrin, F., Kukavica-Ibrulj, I., Levesque, R.C., 2008. Essential genes in the infection model of Pseudomonas aeruginosa PCR-based signature-tagged mutagenesis. Methods Mol. Biol. 416, 61–82. Sheehan, B.J., Bosse, J.T., Beddek, A.J., Rycroft, A.N., Kroll, J.S., Langford, P.R., 2003. Identification of Actinobacillus pleuropneumoniae genes important for survival during infection in its natural host. Infect. Immun. 71, 3960–3970. Smith, L.J., Bochkareva, A., Rolfe, M.D., Hunt, D.M., Kahramanoglou, C., Braun, Y., Rodgers, A., Blockley, A., Coade, S., Lougheed, K.E.A., Hafneh, N.A., Glenn, S.M., Crack, J.C., Le Brun, N.E., Saldanha, J.W., Makarov, V., Nobeli, I., Arnvig, K., Mukamolova, G.V., Buxton, R.S., Green, J., 2017. Cmr is a redox-responsive regulator of DosR that contributes to M. tuberculosis virulence. Nucleic Acids Res. 45, 6600–6612. Subramaniam, S., Huang, B., Loh, H., Kwang, J., Tan, H.M., Chua, K.L., Frey, J., 2000. Characterization of a predominant immunogenic outer membrane protein of Riemerella anatipestifer. Clin. Diagn. Lab. Immunol. 7, 168–174. Tu, J., Lu, F., Miao, S., Ni, X., Jiang, P., Yu, H., Xing, L., Yu, S., Ding, C., Hu, Q., 2014. The siderophore-interacting protein is involved in iron acquisition and virulence of Riemerella anatipestifer strain CH3. Vet. Microbiol. 168, 395–402. van Opijnen, T., Bodi, K.L., Camilli, A., 2009. Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nat. Methods 6, 767–772. Wang, M., Zhang, P., Zhu, D., Wang, M., Jia, R., Chen, S., Sun, K., Yang, Q., Wu, Y., Chen, X., Biville, F., Cheng, A., Liu, M., 2017. Identification of the ferric iron utilization gene B739_1208 and its role in the virulence of R. anatipestifer CH-1. Vet. Microbiol. 201, 162–169. Wang, X., Ding, C., Wang, S., Han, X., Yu, S., 2015. Whole-genome sequence analysis and genome-wide virulence gene identification of Riemerella anatipestifer strain Yb2. Appl. Environ. Microbiol. 81, 5093–5102. Xing, L., Yu, H., Qi, J., Jiang, P., Sun, B., Cui, J., Ou, C., Chang, W., Hu, Q., 2015. ErmF and ereD are responsible for erythromycin resistance in Riemerella anatipestifer. PLoS One 10, e0131078. Yi, H., Yuan, B., Liu, J., Zhu, D., Wu, Y., Wang, M., Jia, R., Sun, K., Yang, Q., Chen, S., Liu, M., Chen, X., Cheng, A., 2017. Identification of a wza-like gene involved in capsule biosynthesis, pathogenicity and biofilm formation in Riemerella anatipestifer. Microb. Pathog. 107, 442–450. Yuan, H., Huang, L., Wang, M., Jia, R., Chen, S., Liu, M., Zhao, X., Yang, Q., Wu, Y., Zhang, S., Liu, Y., Zhang, L., Yu, Y., You, Y., Chen, X., Zhu, D., Cheng, A., 2019. Role of the gldK gene in the virulence of Riemerella anatipestifer. Poult. Sci. 98, 2414–2421.

Alvarez, B., Secades, P., Prieto, M., McBride, M.J., Guijarro, J.A., 2006. A mutation in Flavobacterium psychrophilum tlpB inhibits gliding motility and induces biofilm formation. Appl. Environ. Microbiol. 72, 4044–4053. Autret, N., Charbit, A., 2005. Lessons from signature-tagged mutagenesis on the infectious mechanisms of pathogenic bacteria. FEMS Microbiol. Rev. 29, 703–717. Brodgen, K.A., 1989. Pasteurella anatipestifer infection. In: Adlam, C., Rutter, J.M. (Eds.), Pasteurella and Pasteurellosis. Academic Press Inc., London, p. 15. Chen, Z., Wang, X., Ren, X., Han, W., Malhi, K.K., Ding, C., Yu, S., 2019. Riemerella anatipestifer GldM is required for bacterial gliding motility, protein secretion, and virulence. Vet. Res. 50, 43. Dou, Y., Yu, G., Wang, X., Wang, S., Li, T., Tian, M., Qi, J., Ding, C., Yu, S., 2018. The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis. Vet. Res. 49, 93. Eckert, S.E., Dziva, F., Chaudhuri, R.R., Langridge, G.C., Turner, D.J., Pickard, D.J., Maskell, D.J., Thomson, N.R., Stevens, M.P., 2011. Retrospective application of transposon-directed insertion site sequencing to a library of signature-tagged miniTn5Km2 mutants of Escherichia coli O157:H7 screened in cattle. J. Bacteriol. 193, 1771–1776. Grant, A.J., Coward, C., Jones, M.A., Woodall, C.A., Barrow, P.A., Maskell, D.J., 2005. Signature-tagged transposon mutagenesis studies demonstrate the dynamic nature of cecal colonization of 2-week-old chickens by Campylobacter jejuni. Appl. Environ. Microbiol. 71, 8031–8041. Hatfield, R.M., Morris, B.A., 1988. Influence of the route of infection of Pasteurella anatipestifer on the clinical and immune responses of white Pekin ducks. Res. Vet. Sci. 44, 208–214. Hu, D., Guo, Y., Guo, J., Wang, Y., Pan, Z., Xiao, Y., Wang, X., Hu, S., Liu, M., Li, Z., Bi, D., Zhou, Z., 2019. Deletion of the Riemerella anatipestifer type IX secretion system gene sprA results in differential expression of outer membrane proteins and virulence. Avian Pathol. 48, 191–203. Hu, Q., Chen, H., Liu, X., Zhan, M., Zhang, Z., Deen, S., Zhang, Y., 2002. Determination of growth curve of Riemerella anatipestifer. Anim. Husband. Vet. Med. 34, 8–9. Hu, Q., Han, X., Zhou, X., Ding, C., Zhu, Y., Yu, S., 2011. OmpA is a virulence factor of Riemerella anatipestifer. Vet. Microbiol. 150, 278–283. Hu, Q., Miao, S., Ni, X., Lu, F., Yu, H., Xing, L., Jiang, P., 2013. Construction of a shuttle vector for use in Riemerella anatipestifer. J. Microbiol. Methods 95, 262–267. Hu, Q., Zhu, Y., Tu, J., Yin, Y., Wang, X., Han, X., Ding, C., Zhang, B., Yu, S., 2012. Identification of the genes involved in Riemerella anatipestifer biofilm formation by random transposon mutagenesis. PLoS One 7, e39805. Lehoux, D.E., Sanschagrin, F., Levesque, R.C., 1999. Defined oligonucleotide tag pools and PCR screening in signature-tagged mutagenesis of essential genes from bacteria. Biotechniques 26 (473-478), 480. Lu, F., Miao, S., Tu, J., Ni, X., Xing, L., Yu, H., Pan, L., Hu, Q., 2013. The role of TonBdependent receptor TbdR1 in Riemerella anatipestifer in iron acquisition and virulence. Vet. Microbiol. 167, 713–718. Mecsas, J., 2002. Use of signature-tagged mutagenesis in pathogenesis studies. Curr. Opin. Microbiol. 5, 33–37. Miao, S., Xing, L., Qi, J., Yu, H., Jiang, P., Sun, B., Cui, J., Ou, C., Hu, Q., 2015. Roles of the TonB1 and TonB2 proteins in haemin iron acquisition and virulence in Riemerella anatipestifer. Microbiology 161, 1592–1599. Ni, X., Jiang, P., Xing, L., Ou, C., Yu, H., Qi, J., Sun, B., Cui, J., Wang, G., Hu, Q., 2016. Genome-wide mining of potential virulence-associated genes in Riemerella anatipestifer using random transposon mutagenesis. Vet. Microbiol. 189, 52–58.

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