Genetic Loci of Major Antigenic Protein Genes of Edwardsiella tarda

Seven antigenic proteins of Edwardsiella tarda were identifiedby using a rabbit polyclonal antiserum. Four of these proteinsalso reacted with a Japanese flounder antiserum. The amino acidsequences had identity to lipoproteins, periplasmic proteins,and exported and secreted proteins with roles in transport ofmetabolites across the cell membrane, stress response, and motility.These genes and their products are useful for developing DNAor recombinant subunit vaccines to control edwardsiellosis.

INTRODUCTION

Top

Introduction

Edwardsiella tarda is a gram-negative bacterium that inducesdisease mostly in fish (33) but also in humans (18). The diseasein fish, termed edwardsiellosis, is characterized by skin lesionsthat progress into suppurative abscesses, hyperemia, hemorrhages,swelling, and necrosis, all of which are disseminated systemically(29, 33). Among a number of factors related to E. tarda virulence(5, 14, 26, 34), only hemolysin has been associated with invasionand cytotoxicity (17, 38). At present, the pathogenesis of thedisease, as well as the antigenic proteins that could induceprotective immunity, is unknown.

In this study, we constructed a cosmid DNA library of E. tarda54, a virulent strain isolated from an outbreak of disease inJapanese flounder (Paralichthys olivaceus). The library wasscreened for clones expressing antigenic proteins using a rabbitanti-E. tarda (NG8104) polyclonal antiserum as the first antibodyand a goat anti-rabbit immunoglobulin G (IgG) bound to ¹²⁵Ias the second antibody. Six different cosmid clones were foundand analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western blot analyses (24). The proteins weretransferred to polyvinyldifluoride membranes and then incubatedwith the same first antibody. A goat anti-rabbit IgG conjugatedwith alkaline phosphatase was used as the second antibody (Fig.1). The immune reaction was revealed with the 5-bromo-4-chloro-3-indolylphosphate-Nitro Blue Tetrazolium substrate.

View larger version (111K):
[in this window]
[in a new window]
FIG. 1. Western blot analysis of total cell proteins of six Escherichia coli cosmid clones expressing Edwardsiella tarda antigenic proteins. Lane M, marker; lane Et, E. tarda strain 54; lane Ec, E. coli XL1-Blue MR carrying Supercos I vector; lane A, cosmid 54-1 carrying the Et 49 gene; lane B, cosmid 54-2 carrying the Et 38 gene; lane C, cosmid 54-3 carrying the Et 18, Et 28, and Et 76 genes; lane D, cosmid 54-4 carrying the Et 18 and Et 76 genes; lane E, cosmid 54-5 carrying the Et 46 gene; lane F, cosmid 54-6 carrying the Et 32 gene. Arrows indicate the antigenic protein band.

Cosmid DNA from each clone was digested with several restrictionenzymes, and the DNA fragments were cloned in plasmid vectorsfor immunoscreening and sequencing. IPTG (isopropylthio-ß-D-galactoside)at 1 mM was added to induce expression of the protein. Nucleotidesequences were determined by the cycle sequencing method usingThermo Sequenase. Then, specific primers were designed to amplifythe putative open reading frames (ORFs) and each gene was clonedinto pBluescript SK(+) vector. Recombinant proteins were analyzedby sodium dodecyl sulfate-polyacrylamide gel electrophoresisand Western blotting with the same antibodies (Fig. 2). Theproteins were also analyzed by using a Japanese flounder serumraised against the bacteria (first antibody), a rabbit polyclonalantiserum against Japanese flounder IgM as the second antibody,and the goat anti-rabbit IgG conjugated with alkaline phosphataseas the third antibody (Fig. 3). All sera used in this studywere previously incubated with the Escherichia coli strainscarrying empty vectors to remove unspecific antibodies. DNApreparations were carried out by using standard procedures (37).Sequence data were compared with those in GenBank by using theBLAST program. Putative signal peptides were determined by usingthe Signal P program (4). The proteins were named Et 18 throughEt 76 based on their molecular masses (Table 1).

View larger version (75K):
[in this window]
[in a new window]
FIG. 2. Western blot analysis of Edwardsiella tarda recombinant antigenic proteins reacting with a rabbit serum anti-E. tarda. Lane M, marker; lane Et, E. tarda strain 54; lane Ec, Escherichia coli JM109 carrying pBluescript vector; lane A, E. coli carrying the Et 49 gene; lane B, E. coli carrying the Et 38 gene; lane C, E. coli carrying the Et 28 gene; lane D, E. coli carrying the Et 18 gene; lane E, E. coli carrying the Et 46 gene; lane F, E. coli carrying the Et 32 gene; and lane G, E. coli carrying the Et 76 gene. Arrows indicate the antigenic protein band.

View larger version (69K):
[in this window]
[in a new window]
FIG. 3. Western blot analysis of Edwardsiella tarda recombinant antigenic proteins reacting with a Japanese flounder anti-E. tarda serum. Lane M, marker; lane Et, E. tarda strain 54; lane Ec, Escherichia coli BL21 codon plus carrying pET32 expression vector; lane A, E. coli carrying the Et 49 gene; lane B, E. coli carrying the Et 38 gene; lane C, E. coli carrying the Et 28 gene; lane D, E. coli carrying the Et 18 gene; lane E, E. coli carrying the Et 32 gene; lane F, E. coli carrying the Et 46 gene; lane G, E. coli carrying the Et 76 gene. Arrows indicate the antigenic protein band.

View this table:
[in this window]
[in a new window]
TABLE 1. General features of the Edwardsiella tarda antigenic proteins and oligonucleotide primers used for the amplification of each ORF

Et 18 and Et 28 (putative lipoproteins), together with Et 76,were expressed in clones 54-3 and 54-4 (Fig. 1 and 4). Theiramino acid sequence identities are compared in Table 2. Et 18had identity to Salmonella enterica periplasmic lipoprotein(YraP) (31), whereas Et 28 had identity to Shigella flexneriand E. coli putative lipoprotein (YaeC) (3, 20). Both proteinspossess signal peptides at the N terminus and the consensuscleavage site {LA(G,A) ${downarrow}$ C} of bacterial lipoproteins for peptidaseII, where the arrow indicates the position of cleavage betweenthe signal peptide and the mature protein, the amino acid cysteineis absolutely required in position +1, alanine, glycine, orserine is acceptable in position –1, and alanine and leucineare preferred in positions –2 and –3, respectively(12). Bacterial lipoproteins are one of the components of pathogen-associatedmolecular patterns produced by microorganisms (19). They possessimmunostimulatory activities, i.e., they trigger the innateimmune response via Toll-like receptors 2 and 6 (TLR2 and TLR6)in mammals (1, 15, 39). In all cases, a tripalmitoyl-S-glyceryl-modifiedcysteine (Pam₃Cys) is required for these stimulatory properties(8, 44). Japanese flounder also possess a functional homologof the human TLR2 (13). Therefore, it is reasonable to assumethat the immunostimulatory properties of these proteins in Japaneseflounder and human are similar. The genetic location of theEt 28 gene in the chromosome (Fig. 4) was the same as that reportedfor S. flexneri and was designated an O-island (O-island no.4) in E. coli O157 (32). These antigenic protein genes and othersgenes at the same locus, for instance, the arylsulfate sulfotransferasegene, should be overexpressed during the infection process infish, just as some Vibrio vulnificus genes (yaeC) are overexpressedduring the infection process in humans (22). The arylsulfatesulfotransferase gene has been described as a potential E. tardavirulence gene (26). Thus, this locus could represent a pathogenicisland in E. tarda.

View larger version (30K):
[in this window]
[in a new window]
FIG. 4. Schematic representation of the genetic loci of major antigenic proteins of Edwardsiella tarda. Open reading frames (ORFs) and the transcription directions are indicated by arrows. Sizes of ORFs in base pairs are in parentheses. Black arrows indicate antigenic protein genes. Restriction sites are as follows: A, PstI; B, HincII; C, EcoRV; D, BamHI; E, SacII; F, SacI; G, EcoRI; and H, HindIII. Gene names and product are as follows: dgt, deoxyguanosine triphosphate triphosphohydrolase; drpA, prolyl-tRNA synthetase; fbpA, ferric binding protein A; fbpB, transmembrane permease (iron III); fbpC, ABC transporter, ATP binding protein; fliA, RNA polymerase, regulator of flagellar operon, sigma factor 28; Et 46 gene (fliC), flagellin FliC; fliD, flagellar capping protein, hook-associated protein 2; fliJ, flagellar protein FliJ; fliL, flagellar biosynthesis protein FliL; fliM, flagellar motor switch protein FliM; fliN, flagellar motor switch protein FliN; fliO, flagellar protein fliO; fliP, flagellar biosynthesis protein FliP; fliS, flagellin-specific chaperone FliS; fliT, repressor of class 3a and 3b operon FliT; fliZ, regulator of FliA; fnr, fumarate and nitrate reduction regulatory protein; Et 49 gene (gsrA), global stress requirement protein A; lamB, maltoporine LamB; Et 38 gene (malE), maltose binding periplasmic protein MalE; malK, maltose ATP binding protein MalK; malM, maltose binding protein MalM; metI, permease protein MetI; metN, D-methionine transport, ATP binding protein MetN; mtgA, monofunctional biosynthetic peptidoglycan transglycosylase; pntA, pyridine nucleotide transhydrogenase, alpha subunit; pntB, pyridine nucleotide transhydrogenase, beta subunit; rcsF, regulator of colanic acid synthesis RcsF; yaeB, hypothetical protein YaeB; Et 28 gene (yaeC), putative lipoprotein YaeC; yaeD, putative phosphatase YaeD; ydaA, universal stress protein YdaA; ydcP, putative protease, collagenase YdcP; Et 32 gene (ydgH), putative exported protein; yjjK, ATPase component of ABC transporter YjjK; yraL, putative tetrapyrrole methylase YraL; Et 76 gene (yraM), putative glycosylase YraM; yraN, hypothetical protein, endonuclease YraN; yraO, probable phosphosugar isomerase YraO; Et 18 gene (yraP), possible lipoprotein YraP. Hp, hypothetical protein; IF-2, translation initiation factor 2.

View this table:
[in this window]
[in a new window]
TABLE 2. Comparison of predicted amino acid sequence identities of Edwardsiella tarda antigenic proteins and their homologues in other bacteria

Et 32 and Et 76 (putative exported proteins) amino acid sequenceshad identities to Yersinia pestis putative exported proteinYPO2305 and the glycosylase YraM (Table 2) (6, 30). The geneticlocation of the Et 32 gene suggests that it is a component ofan operon involved in arginine or pyrimidine nucleotide uptake(Fig. 4). This observation agrees with the expression of a gene(pyrH) involved in the synthesis of pyrimidine nucleotides duringinfections by V. vulnificus in humans (22). This pathway islinked to the synthesis of arginine (21).

As mentioned above, Et 76, Et 18, and/or Et 28 was expressedby two clones carrying overlapping DNA fragments. The geneticlocation (Fig. 4) is the same as that reported in Y. pestis(30). Although their functions are unknown, bacterial cell envelope-associatedand secreted proteins are immunogenic, with roles in pathogenesis.These proteins comprise 6 to 37% of the gram-positive proteomesand 12 to 36% of the gram-negative proteomes (36). Secretedand exported proteins are considered important virulence factorsand vaccine candidates. Therefore, we believe these genes andproteins may be useful to vaccine development.

Et 38 and Et 49 (putative periplasmic proteins).

Top

Et 38 and et...

The Et 38 amino acid sequence had 84% identity to that of theperiplasmic maltose binding protein (MalE) of E. coli (32),which is involved in energy-dependent translocation of maltosethrough the cytoplasmic membrane (16). The Signal P programpredicted a 26-residue signal peptide, which would produce amature protein of 40.9 kDa. This size did not correlate wellwith the antigenic band detected by Western blotting (Fig. 1and 2). The fish serum reacted with two antigenic bands thatmight correspond to the immature and mature proteins (Fig. 3).The differences in protein size may be due to an inaccurateprediction of the signal peptide cleavage site.

The Et 49 gene nucleotide sequence had 76% identity to thatof the gene for the global stress requirement protein (GsrA)of Yersinia enterocolitica (45). The protein conserved the majorfeatures of heat shock proteins (HSPs), such as a putative signalpeptide, a Ser/His/Asp active site, and a sequence similar tothe ${sigma}$ ^E type heat shock promoter consensus (35). HSPs preservecellular functions and are necessary for growth and survivalduring exposure to environmental stress, including the intramacrophagicstate of pathogens such as Y. enterocolitica (45) and Brucellaabortus (2, 23). HSPs are immunogens that induce both cellularand humoral immunity (2). E. tarda is considered an intracellularpathogen. It survives and replicates in phagocytes (34). Italso grows on media with 3% NaCl and at temperatures as highas 40°C (33). Thus, it was not unexpected that the E. tardaheat shock proteins have immunostimulatory activity in a mammalianhost. On the other hand, because the flounder serum did notreact with this protein, it may not have the same role in fish.

Et 46 (flagellin, a secreted protein).

Top

Et 46 (flagellin, a...

Flagellins (H antigens) belong to the pathogen-associated molecularpattern that, in mammals, activates the innate immune systemvia TLR5 (11, 25). Bacterial adhesion to and invasion of animals'cells are necessary for virulence and depend on the presenceof flagellum in Aeromonas hydrophila (28) and S. enterica (42).E. tarda flagellin was described as an extracellular virulence-associatedprotein (40). As expected, the E. tarda flagellin of this studyhad 95% amino acid identity with the E. tarda flagellin reportedby Tan et al. (40). On the other hand, it had moderately lowidentity (64%) to other bacterial flagellins, particularly inthe antigenic domain (Table 2). This domain is a nonconservedregion and is very different in bacteria belonging to differentspecies (9). The protein probably does not have strong immunostimulatoryfunctions in fish, since it was recognized by only the rabbitantiserum (Fig. 1 and 2).

Finally, based on the sequence identities and other bioinformaticstools, the E. tarda major antigenic proteins are normal componentsof the bacterial cell wall and are involved in aromatic aminoacid, sugar, and probably nucleotide uptake, stress response,and motility. Some E. tarda antigenic proteins in the mammalimmune system may not be in the fish counterpart. These genesand their products are useful for developing DNA or recombinantsubunit vaccines to control edwardsiellosis.

Nucleotide sequence accession numbers.

Top

Nucleotide sequence accession...

The sequences for the genes encoding Et 18, Et 28, Et 32, Et38, Et 46, Et 49, and Et 76 were deposited in GenBank underthe accession numbers AB195503, AB195504, AB195505, AB195506,AB195507, AB195508, and AB195509, respectively.

ACKNOWLEDGMENTS

This study was supported in part by Grants-in-Aid for ScientificResearch from the Ministry of Education, Science, Sports andCulture of Japan.

FOOTNOTES

* Corresponding author. Mailing address: Laboratory of Genome Science, Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato, Tokyo 108-8477, Japan. Phone: 81-3-5463-0556. Fax: 81-3-5463-0690. E-mail: aoki{at}s.kaiyodai.ac.jp.

REFERENCES

Top