Avian Pathology Identification of Riemerella

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Identification of Riemerella anatipestifer genes differentially expressed in infected duck livers by the selective capture of transcribed sequences technique a

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Zutao Zhou , Juan Zheng , Wenxia Tian a

a

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a b

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, Jianli Li , Wanpo Zhang , Jinliang a

Zhang , Xianrong Meng , Sishun Hu , Dingren Bi & Zili Li

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State Key Laboratory of Agricultural Microbiology , College of Veterinary Medicine, Huazhong Agricultural University , Wuhan, 430070, Hubei, China b

College of Animal Science and Technology , Shanxi Agricultural University , Taigu, Shanxi, 030801, China Published online: 03 Aug 2009.

To cite this article: Zutao Zhou , Juan Zheng , Wenxia Tian , Jianli Li , Wanpo Zhang , Jinliang Zhang , Xianrong Meng , Sishun Hu , Dingren Bi & Zili Li (2009) Identification of Riemerella anatipestifer genes differentially expressed in infected duck livers by the selective capture of transcribed sequences technique, Avian Pathology, 38:4, 321-329, DOI: 10.1080/03079450903071311 To link to this article: http://dx.doi.org/10.1080/03079450903071311

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Avian Pathology (August 2009) 38(4), 321329

Identification of Riemerella anatipestifer genes differentially expressed in infected duck livers by the selective capture of transcribed sequences technique Zutao Zhou1, Juan Zheng1, Wenxia Tian1,2, Jianli Li1, Wanpo Zhang1, Jinliang Zhang1, Xianrong Meng1, Sishun Hu1, Dingren Bi1 and Zili Li1* 1

Downloaded by [Ingenta Content Distribution (Publishing Technology)] at 18:01 08 October 2014

State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei, China, and 2College of Animal Science and Technology, Shanxi Agricultural University, Taigu, Shanxi 030801, China

Riemerella anatipestifer is the causative agent of duck septicaemia. Determination of R. anatipestifer virulence mechanisms will help us to effectively control this contagious agent. The differentially expressed gene profile of R. anatipestifer in infected duck livers was therefore identified and compared with in vitro cultures by selective capture of transcribed sequences analysis. A total of 48 genes were identified, of which 43 were genes that encode enzymes for amino acid biosynthesis and metabolism, intermediary metabolism, and energy metabolism, or proteins for regulatory adaptive responses, general microbial stress response, transport proteins and secreted proteinases. Five were unknown, novel genes. Eight genes representing the categories were randomly chosen and verified by real-time reverse transcriptase-polymerase chain reaction analysis. All were upregulated by R. anatipestifer in infected duck livers, with changes ranging from 1.44-fold to 4.62-fold compared with in vitro cultures. The results from the present study revealed a gene expression profile of R. anatipestifer in infected duck livers. The unknown but novel genes may be potential novel virulence factors for R. anatipestifer. In conclusion, the data from this study will provide a molecular basis for further study of R. anatipestifer pathogenesis.

Introduction Riemerella anatipestifer, a Gram-negative, non-motile, non-spore-forming, rod-shaped bacterium, is the causative agent of duck septicaemia (Segers et al., 1993). The latter is a contagious disease for domestic ducks, turkeys, and various other birds, and the disease can occur in an acute or chronic form of septicaemia, characterized by fibrinous pericarditis, perihepatitis, and airsacculitis. R. anatipestifer infection accounts for major economic losses for duck farmers (Sandhu & Rimler, 1997; Ryll et al., 2001; Sarver et al., 2002), especially in China and Southeastern Asia countries (Huang et al., 1999; Pathanasophon et al., 2002; Cheng et al., 2003). Such an infection causes high mortality and morbidity rates, typically ranging from 10% to 30% (Guo et al., 1982; Loh et al., 1992; Subramanism et al., 1997). Microbiologically, R. anatipestifer has been classified to the Flavobacteriaceae of rRNA superfamily V according to 16S rRNA gene analysis (Segers et al., 1993; Subramaniam et al., 1997). R. anatipestifer can be differentiated into 21 serotypes based on the slide and tube agglutination tests with antisera (Loh et al., 1992; Pathanasophon et al., 1994, 1995). Currently, DNA fingerprints and repetetive sequence based-polymerase chain reaction (rep-PCR) profiles are widely used for subtyping R. anatipestifer in epidemiological studies

(Rimler & Nordholm, 1998; Huang et al., 1999). Vaccines based on inactivated bacteria offer certain levels of protection against homologous strains or serotypes, but bacterins prepared from heterogeneous serotypes do not provide cross-protection (Sandhu, 1979; Pathanasophon et al., 1996). To date, little work has been done on the molecular basis of the pathogenesis of R. anatipestifer. So far, only two potential virulence factors, VapD and CAMP, have been identified. The data showed that VapD has some homology to virulence-associated proteins from other bacteria (Weng et al., 1999) and that CAMP cohaemolyin has high homology to those of O-sialoglycoprotein endopeptidases (Crasta et al., 2002). For a successful infection, bacterial pathogens must be able to acquire sufficient nutrients for their growth and replication but to avoid the host immune system. Therefore, bacterial pathogens continually adjust their gene expression profiles according to the changed environments and host defence system. Thus, identification of genes, which are differentially expressed during infection, may shed a light on the molecular pathogenesis of R. anatipestifer. The selective capture of transcribed sequences (SCOTS) technique has been successfully used to

*To whom correspondence should be addressed. Tel: 86 27 87280208. Fax: 86 27 87280208. E-mail: [email protected] Received 24 February 2009 ISSN 0307-9457 (print)/ISSN 1465-3338 (online)/09/040321-09 # 2009 Houghton Trust Ltd DOI: 10.1080/03079450903071311

322 Z. Zhou et al.


identify bacterial genes differentially expressed during infection (Graham & Clark-Curtiss, 1999; Dozois et al., 2003; Faucher et al., 2006). This technique is based on generation of cDNA from mRNA that is harvested from bacteria grown in two different environments (e.g. infection tissues and in vitro growth) and then use of the cDNA for DNADNA hybridization to identify differential gene expression. In the present study, we adopted SCOTS to detect R. anatipestifer genes differentially expressed between duck liver and in vitro culture. After that, real-time reverse transcriptase (RT)-PCR was performed to verify the data obtained from SCOTS analysis. Materials and Methods Genomic DNA preparation. R. anatipestifer RA-YM, a serovar 1 strain, was isolated from YunMeng county of Hubei province in China (Li et al., 2004). R. anatipestifer RA-YM genomic DNA was prepared using a DNeasy Tissue Kit following instructions provided by the manufacturer (QIAGEN, Germany). Escherichia coli strain DH5a was used for construction and maintenance of R. anatipestifer RA-YM cDNA libraries in pMD18-T vector (TaKaRa Biotech, Dalian, China). Cloning of the R. anatipestifer rDNA operon. The published sequence for R. anatipestifer 16S rDNA (AY871819) was used to design primers (16Sa/16Ss) (Table 1) for the amplification of 16S rDNA of R. anatipestifer RA-YM. Because the 23S rDNA sequence of R. anatipestifer is not available, the 23S rDNA sequences of S. pyogenes (NC_002737), Pasteurella multocida (NC_002663), E. coli (NC_000913) and Capnocytophaga canimorsus strain 24231 (AY661855) were aligned to identify conservative regions for designing three sets of primers (23S2a/23S-2s, 23S-1a/23S-1s, 23S-3a/23S-3s) (Table 1) for the amplification of 23S rDNA of R. anatipestifer RA-YM. The primers were designed by

Table 1. Primer 16Sa 16Ss 23S-2a 23S-2s 23S-1a 23S-1s 23S-3a 23S-3s SCOTSN601 SCOTSN602 SCOTS01 SCOTS02 gcvT hmgA aspC cat ptfp dpp IV m28 RA46 16S

following an online program (www.ncbi.nlm.nih.gov/tools/primer-blast/ index.cgi?LINK_LOCBlastHomeAd). The primers were customsynthesized by Invitrogen Biotechnology Ltd (Shanghai, China). These primers (Table 1) were used to amplify DNA fragments using PCR with TaqTM polymerase (TaKaRa Biotech) and R. anatipestifer RA-YM genomic DNA as a template. The amplified DNA fragments were then ligated into pMD18-T vector and confirmed by sequence analysis. Finally, the confirmed plasmids (i.e. pMD-16rRNA, pMD23S1rRNA, and pMD-23S3rRNA) were amplified and stored for further use in the following experiments. Infection of R. anatipestifer RA-YM, RNA purification, and cDNA synthesis. R. anatipestifer RA-YM was grown at 378C to the midexponential phase (OD600, 0.6) in 100 ml tryptic soy broth (TSB) (Difco, Michigan, USA). In order to keep cellular RNA intact, cultured bacteria were rapidly mixed with a cold RNA degradation stop solution (10% buffer-saturated phenol in ethanol), which can keep the bacterial transcriptome intact (Bernstein et al., 2002). To infect duck livers, an animal study protocol was reviewed and approved by the Institutional Animal Experimental Committee of the Veterinary Faculty of Huazhong Agricultural University, in accordance with the China regulation on experimental animals. Specifically, healthy white Pekin ducks (Anas platyrhynchos), not previously exposed to R. anatipestifer, were purchased from a commercial farm (Wuhan, China). The ducks (n7, 9 days old) were inoculated via their feet with 0.5 ml suspension containing 1.0 x 107 colony-forming units of R. anatipestifer RA-YM, and 24 h later the liver samples from these dusks were collected and immediately put into liquid nitrogen. These liver samples, confirmed by bacterium culture, contained approximately 108 colonyforming units per gram of liver tissue. Total RNA from R. anatipestifer RA-YM-infected livers or from TSB controls was isolated using TRIzol reagent (Invitrogen, Carlsbad, California, USA). The total RNA samples were then treated with RNase-free DNase I (Promega, Madison, Wisconsin, USA) and RNA concentrations and integrity were measured by spectrophotometer for the A260/A280 ratio, followed by agarose gel electrophoresis. First-strand

Primers used in the present study Sequence 5?-CTCGAGACGGCTACCTTGTTACGACTTAGC-3? 5?-GGATCCAGAGTTTGATCCTGGCTCAGGATG-3? 5?-GTCGGAACTTACCCGACAAGGAATTTCG-3? 5?-ACAGCCAGGATGTTGGCTTAGAAGCAG-3? 5?-GTGCTTGAGTAACCAAAAATATTAG-3? 5?-GATTAGCATGGCTGAGGAAACCTTAGTC-3? 5?-CTAAGGTTTCCTCAGCCATGCTAATCAG-3? 5?-CCTGCTTAGATGCTTTCAGCACTTATC-3? 5?-GACACTCTCGAGACATCACCGGTACCNNNNNN-3? 5?-TGCTCTAGACGTCCTGATGGTTC NNNNNN-3? 5?-GACACTCTCGAGACATCACCGGTACC-3? 5?-TGCTCTAGACGTCCTGATGGTTC-3? Forward: 5?-AGTGGTGTCATCAATATCGTTTCC-3? Reverse: 5?-GCTTATCCCTTGTGGGTTGG-3? Forward: 5?-GAGTACCGTGTCTTCTGTTTCAA-3? Reverse: 5?-TTCCGTAGGCGACTATCTTATT-3? Forward: 5?-CGTCGTCTATAAGAGCGGCTAA-3? Reverse: 5?-GGGGAACCCGATTTTGATGT-3? Forward: 5?-CTACCTTTCTCACCACCAACC-3? Reverse: 5?-ACTACGAATACGAAGACTCACAGA-3? Forward: 5?-CTACCTTTCTCACCACCAACC-3? Reverse: 5?-ACTACGAATACGAAGACTCACAGA-3? Forward: 5?-GAATACTGAAACATGAGCAAAGG-3? Reverse: 5?-CAATGATGCAGGAGACCAAA-3? Forward: 5?-TTTCCCAAGAACGCCACTCA-3? Reverse: 5?-CCCTAAAATGCAACAAGCTCAC-3? Forward: 5?-AGCATCATTAGTGCGTATCTCAA-3? Reverse: 5?-CCCTTCCCTCTTTATCCATTT-3?? Forward: 5?-CGTTTACGGCGTGGACTACC-3? Reverse: 5?-CAGAACACCGATTGCGAAGG-3?

Gene expression of Riemerella anatipestifer 323


cDNAs were synthesized by Superscript III reverse transcriptase (Invitrogen) with a random primer set. After that, the second-strand cDNA was synthesized using Klenow enzyme (NEB, Beverly, Masachusetts, USA) in a mixture of the first-strand cDNA from 5 mg total RNA reverse transcriptase reaction plus 1 mg SCOTSN601 primers for cDNA obtained from the bacteria grown in the duck liver, or 1 mg SCOTSN602 primers for cDNA obtained from the bacteria grown in TSB. After that, the double-stranded cDNA was purified using the E.Z.N.A Cycle-pure Kit (Omega, USA) and amplified by PCR as previously described (Hou et al., 2002) with appropriate primers, For preparing SCOTS analysis, three standard 50 ml PCRs were carried out for 25 cycles (SCOTS01, 958C for 30 sec, 578C for 90 sec, and 728C for 50 sec; SCOTS02, 958C for 30 sec, 498C for 90 sec, and 728C for 50 sec) in parallel for each cDNA.

SCOTS analysis. Genomic DNA from R. anatipestifer RA-YM was first biotinylated as described previously (Hou et al., 2002). The biotinylated R. anatipestifer RA-YM genomic DNA (12 mg) was then mixed with 200 mg pMD-16rRNA, pMD-23S1rRNA, and pMD23S2rRNA plasmid DNA (R. anatipestifer RA-YM rrnA DNA cloned into pMD18-T vector) and sonicated for 10 sec at an output setting of 2 using a microprobe (Fisher Scientific 100). After sonication, the samples were precipitated and resuspended in 320 ml of 10 mM N-(2hydroxyethyl) piperazine-N-(3-propanesulfonic acid)-1 mM ethylenediamine tetraacetic acid and then aliquoted into 40 tubes with 8 ml in each. These aliquots were then subjected to three rounds of SCOTS analysis. For each round of SCOTS, an 8 ml sample of the mixture (containing 0.3 mg R. anatipestifer RA-YM genomic DNA and 5 mg rrnA DNA) was denatured by incubation at 988C for 3 min. Two microlitres of 1 M NaCl were added to the mixture and incubated at 648C (calculated as Tm minus 208C for 35% GC DNA content) for 30 min. This would allow the plasmid rrnA DNA to bind to the rrnA sites of the R. anatipestifer RA-YM genomic DNA, thereby rendering these sites unavailable for hybridization to ribosomal DNA present in the cDNA mixtures. Similarly, 5 mg PCR amplified cDNA from either infected liver or control culture in 8 ml of 10 mM N-(2-hydroxyethyl) piperazine-N-(3-propanesulfonic acid)-1 mM ethylenediamine tetraacetic acid were also denatured at 988C for 3 min, followed by the addition of 2 ml of 1 M NaCl. The denatured cDNA mixture was added to the tubes containing the biotinylated chromosomal DNArrnA prehybridized mixture (final volume, 20 ml), and hybridization continued at 648C for 20 h. Bacterial cDNAgenomic DNA hybrids were bound to streptavidin-coated M280 magnetic beads (Invitrogen), collected, and washed as instructed by the manufacturer. Captured cDNAs were then eluted, precipitated, and amplified using primer SCOTS01 or SCOTS02 in triplicate, parallel 50 ml PCRs for the second amplification and for all subsequent PCR amplifications as described above. In the first round of SCOTS, seven separate samples of cDNAs from R. anatipestifer RA-YM of duck liver or TSB were captured by hybridization to the biotinylated rDNA-blocked genomic DNA in parallel reactions, respectively. This was done to enhance the likelihood of recovering cDNA molecules corresponding to a more complement of transcripts present at the time of RNA preparation for each growth condition. After the first round of SCOTS, the seven amplified cDNA preparations were then combined, denatured, and again hybridized to fresh aliquots of rDNA-blocked, biotinylated genomic DNA. Three rounds of SCOTS were done for each growth condition (infection versus control).

Enrichment of cDNA molecules from infected liver-grown R. anatipestifer and preparation of liver-specific cDNA libraries. To identify gene transcripts that were upregulated during growth of R. anatipestifer RA-YM in the infected duck liver, an additional step was included. In particular, cDNA mixtures obtained from R. anatipestifer RA-YMinfected liver after three rounds of SCOTS were added to biotinylated genomic DNA that had been prehybridized with both rDNA and cDNA preparations from TSB-grown R. anatipestifer RA-YM after three rounds of SCOTS and then hybridizd for 20 h at 648C. After that, the hybridized molecules were recovered by binding to streptavidincoated beads. After elution of the cDNA molecules from the bound genomic DNA, these cDNA molecules were amplified by PCR with SCOTS01 primers. After three rounds of this enrichment procedure, the

cDNA molecules were then cloned into pMD18-T vectors (TaKaRa Biotech) to generate R. anatipestifer RA-YM-infected liver-specific cDNA libraries. Dot-blot hybridization. cDNA obtained from infected livers and TSBgrown bacteria after three rounds of SCOTS were used as templates for probe preparation for the hybridization experiments. Digoxigeninlabelled probes were generated using DIG High Prime DNA Labeling and Detection Starter Kit I (Roche). Individual SCOTS clones obtained from liver-specific cDNA libraries were verified by PCR with SCOTS01 primers, and the PCR products of these SCOTS clones were spotted on positively charged membranes (Hybond, Amersham) in duplicate. These membranes were then prehybridized and hybridized at 658C with digoxigenin-labelled probes (infection or control). The positive signals were detected using a DIG Luminescent Detection Kit and photographed by using a gel imaging system. Sequencing of the SCOTS clones. The positive clones obtained from the dot-blot analysis were identified and then sequenced by Invitrogen Biotechnology Ltd. BLASTn was performed to identify sequences of genes, and the Swiss-Prot database was performed to identify sequences of translated products. Real-time RT-PCR. To confirm the data obtained from SCOTS analysis, eight genes (gcvT, hmgA, aspC, cat, ptfp, dpp IV, m28, and RA46) were randomly selected for real-time RT-PCR analysis with their primers (see Table 1) and 16S rRNA as a loading control. Briefly, total RNA from three individual R. anatipestifer-infected duck livers or TSBgrown bacteria was subjected to real-time RT-PCR analysis with the SYBR Premix Ex TaqTM (TaKaRa Biotech) in ABI PRISM model 7900 (Applied Biosystems). The following cycles were performed: 958C for 10 sec, 40 cycles at 958C for 5 sec, 548C to 608C for 20 sec (temperatures depended on the different gene primers), 728C for 15 sec. Relative quantification of gene amplification was calculated using the cycle threshold (Ct) values. All RT-PCRs amplified a single product as determined by melting curve analysis. Statistical analysis. Data from the real-time RT-PCR (Ct values) were analysed by Student’s t test using the SPSS15.0. P B0.05 was considered significant.

Results Nucleotide sequence of the R. anatipestifer RA-YM rDNA operon. The sequence of 23S rRNA of R. anatipestifer RA-YM was obtained by three sets of primers (23S-2a/23S-2s, 23S-1a/23S-1s, 23S-3a/23S-3s) (Table 1) designed after comparing with several bacterial sequences of 23S rRNA. The sequence data of R. anatipestifer RA-YM rDNA are accessible with GenBank accession numbers FJ031240 and FJ031241. SCOTS analysis. After R. anatipestifer RA-YM was first inoculated in seven ducks or in TSB, respectively, RNA from duck liver and TSB culture was isolated and cDNA was synthesized for SCOTS analysis. Three rounds of hybridization of rDNA-blocked R. anatipestifer RA-YM genomic DNA fragments with PCR-amplified cDNAs obtained from bacteria growth at either in vivo or in TSB cultures were used to remove rRNA sequences and to normalize the abundance of cDNAs representing the entire RNA populations obtained from R. anatipestifer RA-YM grown at the two different conditions. Three rounds of differential hybridization of the normalized cDNAs from bacteria grown in vivo and in TSB to the biotinylated genomic DNA fragments were performed to identify the differentially expressed genes in the duck livers. These liver-specific cDNAs were then ligated into


324 Z. Zhou et al.

the pMD18-T vector (TaKaRa Biotech) and amplified by PCR. A total of 187 positive SCOTS clones were obtained and sequenced, of which 171 corresponded to 48 genes and an additional 16 clones had no similarity to or low similarity to Genbank entries (Table 2). Because R. anatipestifer belongs to the family Flavobacteriaceae, the nucleotide sequence similarity alignment indicated that most of the 48 genes had high identity to those of bacterial species that belong to the Cytophaga FlavobacteriumBacteroides group. Therefore, there were up to 48 genes that were differentially expressed in the duck livers after R. anatipestifer infection compared with the control cultures. Among the 48 known genes, 43 were involved in amino acid biosynthetic and metabolism, intermediary metabolism, energy metabolism, regulatory adaptive response, general microbial stress response, protein transport or they were secreted proteinase. The remaining five genes had identity with genes of unknown functions in R. anatipestifer (Table 2). Dot-blot analysis. To confirm the differentially expressed genes of R. anatipestifer RA-YM in infected duck livers, the PCR products of these 48 known genes were spotted onto nylon membranes and subjected to dot-blot analysis. The data shown in Figure 1 demonstrate that these 48 genes were all expressed strongly in the duck livers after R. anatipestifer infection compared with the control cultures. Real-time RT-PCR analysis. To further confirm these differentially expressed genes by R. anatipestifer RA-YM in infected duck livers, eight genes (gcvT, hmgA, aspC, cat, ptfp, dpp IV, m28, and RA46) were randomly selected and amplified using real-time RT-PCR. The expression levels of all eight genes were upregulated in infected duck livers compared with the control cultures, with changes ranging from 1.44-fold to 4.62-fold, as seen from Figure 2. Discussion In the current study, R. anatipestifer was first inoculated in ducks and, 24 h later, total cellular RNA was isolated and cDNA was synthesized from the RNA and subjected to SCOTS analysis for identification of differential gene expression. The data showed that up to 48 genes were differentially expressed in duck livers compared with the control cultures. Among these expressed genes, 43 were known and functional genes and five were of unknown function. None of the genes had homology to genes from R. anatipestifer. Eight genes were verified by real-time RT-PCR analysis. This is the first data to show a gene expression profile of R. anatipestifer during infection, which may provide a molecular basis for future study of R. anatipestifer pathogenesis. R. anatipestifer is an avian pathogen of ducks and turkeys, and it is a major economic burden in the duck industry worldwide (Huang et al., 1999; Ryll et al., 2001; Pathanasophon et al., 2002; Sarver et al., 2002; Cheng et al., 2003). Limited information about the molecular basis of the pathogenesis is available for this organism, and so far only two potential bacterial virulence or virulence-associated gene have been identified (Weng et al., 1999; Crasta et al., 2002). Determination of bacterial genes differentially expressed during infection

in the host may provide valuable information for increasing our understanding of pathogenesis of R. anatipestifer. This is because bacteria express special genes in response to the environment encountered in their host while the products of these genes would be unnecessary for the organism in in vitro culture (Chiang et al., 1999). To this end, researchers in the field have applied a variety of approaches to identify genes expressed by bacterial pathogens in vivo, such as signature-tagged mutagenesis (Hensel et al., 1995), in vivo expression technology (Mahan et al., 1993), differential fluorescence induction (Valdivia & Falkow, 1997), in vivo-induced antigen technology (Handfield et al., 2000), and SCOTS and DNA microarray analyses (Wilson et al., 1999). In the current study, we have compared these techniques and performed SCOTS analysis to identify R. anatipestifer RA-YM genes differentially expressed in duck liver tissue. The advantage of this technique is to utilize the bacterial cDNA prepared directly from infected tissues to hybridize to biotinylated bacterial genomic DNA, and followed by a PCR-based subtractive hybridization with cDNA from the control cultures. By using this technique, we have identified up to 48 differentially expressed genes of R. anatipestifer in infected duck liver compared with the controls. Among these genes, 43 were known proteins and can be classified into five functional groups: metabolism, regulatory, stress, transporter and proteinase*the remaining five had unknown functions, which may be potential novel virulence factors for R. anatipestifer. Future study will determine their identities and functions. Bacteria will regulate and adapt their biosynthetic and metabolic pathways in vivo to acquire necessary nutrients, including necessary amino acids and carbohydrates. This is because many of these nutrients are present at lower concentrations in the host. In the current study, most differentially expressed R. anatipestifer RA-YM genes obtained by SCOTS analysis were involved in amino acid and carbohydrate metabolism. For example, GdhA converts ammonia to glutamate. In E. coli, this reaction is one of the primary pathways for the assimilation of nitrogen and the glutamate, which is required directly or indirectly for synthesis of almost all of amino acids. Although glutamate can be synthesized by two different pathways, the GdhA pathway is the preferred one under energy-limited conditions (Helling, 1998). In an environment containing ammonia with amino acid and energy limitations, the GdhA pathway is of critical importance. The gene aspC is predicted to encode aspartate transaminase, which catalyses the conversion of glutamate to aspartate. In E. coli this reaction is the primary mechanism of aspartate formation. In addition, glutamate can also function as a major carbon source in E. coli and the conversion of glutamate to aspartate by AspC is a part of the glutamate energy utilization pathway. Indeed, gdhA and aspC of P. multocida were also reported to be highly upregulated in chicken livers during infection of fowl cholera. It is likely that gdhA and aspC play an important role in conversion of host-derived ammonia to amino acids (Boyce et al., 2002). The hmgA gene encodes the homogentisate dioxygenase that is responsible for the conversion of homogentisate into fumarate and acetoacetate, which enter the Krebs cycle and provide the cell with energy and substrates. The acquisition of the

Category Metabolism

Stress response

Homologue

Genea

Proteina

Function or property

acpP cynT iscS bioF fahA trmU gcvT

Bacteroides vulgatus Cytophaga hutchinsonii Flavobacterium johnsoniae Gramella sp. F. johnsoniae Flavobacterium psychrophilum F. psychrophilum

acpPb (186, 80), CP000139 cynT (123, 70), CP000383 iscS (200, 66), CP000685 bioF (120, 68), CU207366 fahA (117, 73), CP000685 trmU (215, 75), AM398681 gcvT (183, 69), AM398681

(62, 88), A6KZ78 (NIb, 75), Q11YC2 (59, 60), A5FKQ2 (71, 57), A0M7A4 (51, 76), A5FED7 (140, 66), A5FHA9 (NI, 75), A6GXW3

hmgA lipB

F. johnsoniae F. psychrophilum

hmgA (160, 68), CP000685 lipB (118, 78), AM398681

(75, 62), A5FM05 (54, 72), A6GYW1

aspC gdhA aroB pckA pgk cat2

Gramella sp. F. johnsoniae F. psychrophilum B. vulgatus F. psychrophilum F. psychrophilum

aspC (230, 68), CU207366 gdhA (231, 76), CP000685 aroB (237, 67), CP000685 pckA (198, 71), CP000139 pgk (58, 82), AM398681 cat2 (191, 68), AM398681

(70, 54), Q64YT2 (72, 84), A5FM33 (89, 62), A6H0A1 (65, 69), A6KZ70 (NI, 78), A6GVL5 (42, 58), A6GWR2

adhS rok

F. johnsoniae B. vulgatus

adhS (100, 71), CP000685 rok (101, 75), CP000139

(NI, 60), A5FLW9 (41, 85), A6KWA5

mal kduD

F. johnsoniae F. psychrophilum

mal (52, 76), CP000685 kduD (54, 77), AM398681

(42, 54), A5FJD9 (49, 60), A6GY85

Metabolism of lipids Specific carbohydrate metabolic pathway Cysteine desulfurase 8-Amino-7-oxononanoate synthase Fumarylacetoacetase tRNA-Methyl transferase Metabolism of amino acids and related molecules Homogentisate 1,2-dioxygenase Metabolism of coenzymes and prosthetic groups Aspartate transaminase Glutamate dehydrogenase (NADP ) 3-Dehydroquinate synthase Phosphoenolpyruvate carboxykinase Main glycolytic pathways Metabolism of coenzymes and prosthetic groups Iron-containing alcohol dehydrogenase Putative ROK family transcriptional repressor Malate dehydrogenase Metabolism of lipids

mutS recA dnaJ ppk

Bacteroides fragilis F. johnsoniae F. psychrophilum Campylobacter jejuni (DNA), Kocuria rhizophila (protein) F. johnsoniae Helicobacter pylori B. vulgatus F. johnsoniae

mutS (166, 84), CR626927 recA (207, 75), CP000685 dnaJ (53, 77), AM398681 ppk (120, 71), AL111168

(49, 57), Q64Z14 (NI, 75), A5FLA2 (42, 50), A6GVN5 (NI, 76), B2GIH6

DNA mismatch repair protein MutS RecA protein HSP70 family cofactor Putative polyphosphate kinase

FI166208 FI166226 FI166227 FI166228

lon (91, 79), AM398681 katA (236, 73), AE001439 mfd (104, 70), CP000139 rpmB (283, 68), CP000685

(53, 64), A5FG89 (64, 69), B2USV5 (NI, 67), AOM658 (NI, 57), A5F9Z1

FI166229 FI166230 FI166231 FI166232

FI166236 FI166237

lon katA mfd rpmB rplX rplB rplN Transporter

Organism

Accession number from current investigation

secA ABC transporter

F. psychrophilum (DNA), Porphyromonas gingivalis (protein) P. gingivalis F. psychrophilum

rplX (237, 67), AM398681

(NI, 55), B2RLY1

rplB (103, 73), AE015924 rpsN (238, 76), AM398681

(55, 65), Q7MTL6 (NI, 85), A6GZ86

ATP-dependent protease La Catalase Transcription-repair coupling factor Ribosomal protein L33; ribosomal protein L28 50S ribosomal protein L24; 50S ribosomal protein L14 Ribosomal protein L2 30S ribosomal protein S14

B. vulgatus Gramella sp.

secA (72, 79), CP000139 RA33 (117, 75), CU207366

(40, 74), Q64XF8 (41, 78), A0M3M6

Protein translocase subunit secA ABC transporter, ATP-binding protein

FI166206 FI166209 FI166210 FI166211 FI166212 FI166213 FI166214 FI166207 FI166215 FI166216 FI166217 FI166218 FI166220 FI166219 FI166221 FI166222 FI166223 FI166224 FI166225

FI166233 FI166234 FI166235



Table 2. R. anatipestifer cDNA clones identified by SCOTS


Category

Homologue

Organism

Genea

Proteina

Function or property

Accession number from current investigation

ptfp Phosphate transporter

F. psychrophilum F. johnsoniae

ptfp (182, 75), AM398681 RA35 (107, 70), CP000685

(65, 78), A6GYC1 (36, 59), A5FGR2

Transport/binding of inorganic ions phosphate transporter

FI166238 FI166239

pst

Gramella sp.

pst (124, 79), CU207366

(56, 50), A0LXL2

FI166240

rpoB

F. psychrophilum

rpoB (185, 70), AM398681

(46, 75), Q596K6

pir

Nitrobacter hamburgensis

pir (128, 69), CP000319

(48, 77), Q1QQ58

Two-component system response regulatory protein involved in phosphate regulation DNA-directed RNA polymerase beta subunit Pirin family protein

Proteinase

dpp IV dpp IV Peptidase Peptidase Peptidase Metalloprotease

Flavobacterium meningosepticum F. psychrophilum Gramella sp. Gramella sp. B. fragilis Flavobacterium columnare (DNA), Tetrahymena thermophila (protein)

dpp IV (194, 74), D42121 dpp IV (154, 72), AM398681 m28 (135, 74), CU207366 RA41 (110, 70), CU207366 RA42 (79, 74), CR626927 mop (66, 95), AY387596,

(66, 78), Q47900 (55, 70), A6GYM7 (39, 61), A0M6Z3 (71, 59), A0M7C7 (NI, 62), Q5L886 (30, 50), Q22S27

Dipeptidyl peptidase IV Xaa-Pro dipeptidyl-peptidase Secreted peptidase, family M28 Secreted peptidase, family M16 Putative peptidase Membrane-associated zinc metalloprotease gene

FI166205 FI166243 FI166244 FI166245 FI166246 FI166247

Proteins with unknown functions

Iojap-like protein

F. johnsoniae

RA48 (149, 71), CP00685

(57, 68), A5FGN5

Iojap-like protein

FI166252

Unknown

Trichodesmium erythraeum (DNA), F. psychrophilum (protein) F. psychrophilum F. psychrophilum Neisseria meningitidis

RA44 (84, 77), CP000393

(80, 57), A6GYI2,

Protein of unknown function

FI166248

RA45 (183, 66), AM398681 RA46 (98, 70), AM398681 RA47 (52, 78), AM421808

Unknown (71, 66), A6H2F9 Unknown (68, 58), A6GYC4 Unknown

Protein of unknown function Protein of unknown function Putative integral membrane protein

FI166249 FI166250 FI166251

Regulation

Unknown Unknown Unknown a

FI166241 FI166242

Results of BLASTn with the length of matching region and percenta geidentity in parentheses to sequences labelled with accession number and source organism. bNI, length of matching region not available.

326 Z. Zhou et al.

Table 2 (Continued)



Figure 1. Dot-blot analysis of the selected SCOTS clones. The 48 SCOTS clones selected from the first-round dot-blot analysis were spotted onto nylon membranes in duplicate and subjected to hybridization using two different probes. 1a: Probes prepared from R. anatipestifer-infected duck liver-derived cDNAs after three rounds of normalization. 1b: Probes prepared from TSB-grown bacteriaderived cDNAs after three rounds of normalization.

homogentisate operon may represent a special adaptation to meet carbon source requirements under conditions of environmental stress (Milcamps & de Bruijn, 1999; Arias-Barrau et al., 2004; Adams et al., 2006). Thus, R. anatipestifer will increase expression of genes involved in different metabolic pathways to acquire nutrients during growth in the host, and keep the efficiency of energy production, in order to retain maximal growth rates. Furthermore, there are a number of genes involved in the microbial adaptive response and correlated regulation, which enable the bacteria to flexibly adapt to the drastically different conditions in the host. Two-component systems are signal transduction proteins that respond to the changed environmental conditions. The pst operon is regulated by the two-component regulatory system; PhoBR monitors the external P concentration, which is part of a complex network important for both bacterial virulence and stress response. The gene ppk encodes poly P kinase, the enzyme that is responsible for the synthesis of poly P from ATP. Poly P is important for growth and survival of both bacterial and eukaryotic species, and the ppk mutants of P. aeruginosa are deficient in motility, quorum sensing, biofilm formation, and virulence in mouse models (Rashid et al., 2000). Vibrio cholerae ppk mutants also show defects in growth,

Figure 2. Real-time RT-PCR analysis of the differentially expressed genes of R. anatipestifer grown in infected livers and in TSB cultures. Eight genes were selected and amplified using real-time RT-PCR. Relative quantitation was performed using the Ct values. Statistical differences were analysed by Student’s ttest. *P B0.05, **P B0.01.

motility, and surface attachment, features linked to virulence (Ogawa et al., 2000). In addition, catalase can help bacteria to protect themselves from oxygen toxicity and any insult by host phagocytosis. The catalase of Heliobacter pylori was identified in response to interactions with mammalian gastric mucosa (Graham et al., 2002). The dnaJ transcription is induced by a range of stresses, including oxidative stress, osmotic stress, and amino acid starvation. The heat shock-induced chaperone DnaJ of P. multocida was upregulated in vivo (Boyce et al., 2002). In addition to eliminating the irreversibly damaged proteins, Lon appears to perform important functions in the bacterial cell through its ability to degrade proteins that regulate gene expression. The Lon in connection with bacterial pathogenesis has demonstrated that the Brucella abortus lon homologue was shown to be required for wild-type virulence during the initial stage of infection in mice (Takaya et al., 2002). Lastly, bacterial proteinases are mainly involved in providing peptide nutrients for the microorganism. However, the production of bacterial proteinases could also contribute to the pathogenesis of infections, and therefore they could be considered virulence factors. In the current study, five genes of R. anatipestifer RA-YM that encode different proteinases were identified These had high identity with those of other bacteria. DPP IV is a serine protease that cleaves X-Pro or X-Ala dipeptide from the N-terminal ends of polypeptide chains. DPP IV is an important virulence factor of Porphyomonas gingivalis by contributing to the degradation of connective tissues, and also mediates the adhesion of P. gingivalis to fibronectin (Kumagai et al., 2000, 2005). The proteolysis of substance P by Sg-xPDPP was observed, and the concerted action of an extracellular Arg aminopeptidase and Sg-xPDPP produces a truncated form of bradykinin. The combined effect of these modifications may result in local changes in vascular permeability and smooth muscle contraction at the infected endothelium (Goldstein et al., 2001). The fibrinous exudate is the prominent pathological characterization of R. anatipestifer infection, so the DPP IV may serve as a critical virulence factor of this organism for pathogenicity. Inactivation of the DPP IV gene in R. anatipestifer RA-YM using genetic tools should make it possible to determine the contribution of this protease to bacterial growth and pathogenicity. In the future, studies will focus on the isolation, expression and knockout of this gene or more of the in vivo-expressed genes in order

328 Z. Zhou et al.

to evaluate their relative contributions to R. anatipestifer RA-YM virulence and survival at the site of infection.


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