1 Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762-6100, USA
2 Complex Carbohydrate Research Center, 220 Riverbend Road, Athens, GA 30602-4712, USA
Correspondence
Mark L. Lawrence
lawrence{at}cvm.msstate.edu
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ABSTRACT |
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The GenBank accession number for the Ed. ictaluri OPS biosynthesis gene cluster is AY057452.
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INTRODUCTION |
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OPS is composed of a series of repeating oligosaccharide subunits that form chains of varying length. It is the outermost component of LPS, which forms the outer leaflet in the Gram-negative outer membrane. OPS is attached to a core oligosaccharide (COS), which in turn is attached to the nonpolar component of LPS, lipid A. OPS composition and structure is highly species- and serotype-specific, and OPS is the immunodominant antigen of most Gram-negative bacteria, including Ed. ictaluri (Baldwin et al., 1997; Moore & Thune, 1999
). Ed. ictaluri expresses long OPS chains that are antigenically and structurally homogeneous between isolates (Bertolini et al., 1990
; Newton & Triche, 1993a
), indicating that the species is composed of a single O serotype.
OPS is an important virulence factor in many Gram-negative species, and it often mediates resistance to complement-mediated killing (Allen et al., 1998; Amaro et al., 1997
; Merino et al., 1994
, 2000
) and opsonization/killing by phagocytes (Burns & Hull, 1999
; Engels et al., 1985
; Price et al., 1990
). Ed. ictaluri is resistant to the catfish alternative complement pathway (Ourth & Bachinski, 1987
). There is direct evidence that it is capable of surviving in catfish neutrophils (Ainsworth & Dexiang, 1990
), and there is indirect evidence that it can survive in macrophages (Baldwin & Newton, 1993
; Miyazaki & Plumb, 1985
).
We previously reported the construction of an isogenic Ed. ictaluri OPS-negative mutant (93-146 R6) using transposon mutagenesis and demonstrated that the mutant strain is avirulent in channel catfish (Lawrence et al., 2001). In the present work, we identify the mutated gene in 93-146 R6, and we report the sequence of a 12 kb region from the Ed. ictaluri chromosome containing OPS biosynthesis genes. We also report the results of compositional analysis of LPS derived from wild-type Ed. ictaluri and the OPS mutant, and the results of a study comparing the resistance of wild-type and OPS mutant Ed. ictaluri to normal catfish serum and to catfish neutrophils.
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METHODS |
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LPS purification.
Ed. ictaluri LPS was isolated from strains 93-146 and 93-146 R6 by the hot aqueous phenol extraction method (Newton & Triche, 1993a). Two litres of stationary-phase bacterial broth culture were used for each extraction. Lyophilized LPS samples were resuspended in distilled water (1 mg ml-1) for either analysis by SDS-PAGE or glycosyl composition analysis (Complex Carbohydrates Research Center, Athens, GA, USA).
Glycosyl composition analysis.
Purified LPS was first treated with mild acid hydrolysis to remove the lipid A fraction. Acetic acid was added to a final concentration of 1 % (v/v), and the samples were heated at 100 °C for 2 h. Lipid A was removed by centrifugation, and the remaining LPS in solution was lyophilized and dissolved in distilled water. Gel-permeation chromatography was used to fractionate the LPS samples by molecular mass using a BioGel P4 (Bio-Rad, fine grade 115 cmx1 cm) with a flow rate of 8 ml h-1. The column eluant was monitored with a Hewlett Packard 1037A refractive index detector, and the fractions were collected manually.
Fractions from LPS samples were hydrolysed with 1·0 M methanolic HCl for 16 h at 80 °C. The released methyl glycosides were dried and N-acetylated using methanol and acetic anhydride (1 : 1, v/v) for 15 min at 45 °C. The acetylated samples were trimethylsilylated using Tril-Sil and resolved on a 30 m DB-1 column (0·25 mm0·25 µm inner diameter; J &W Scientific) in a Hewlett Packard 5985 GC-MS system using myo-inositol as an internal standard. An initial temperature of 160 °C was used, which was then increased to 200 °C at 2° min-1, followed by increasing to 260 °C at 10° min-1. Derivatized methyl glycosides were run alongside the samples as standards for identification (York et al., 1986).
DNA preparations.
Ed. ictaluri 93-146 R6 genomic DNA was isolated from a 100 ml overnight culture by phenol/chloroform extraction followed by precipitation with 2-propanol (Ausubel et al., 1994). Plasmid DNA was prepared by alkaline lysis and purified using Qiagen-tip 100 columns (Qiagen). Small-scale preparations of plasmid DNA were isolated using Qiagen spin-prep columns.
Cloning and sequencing the OPS biosynthesis gene cluster.
In preparation for cloning the mutation in 93-146 R6, chromosomal DNA from this strain was digested with SalI. Our previous Southern hybridization results had indicated that the Tn903 kanamycin-resistance gene was located on a 11 kb SalI chromosomal fragment (Lawrence et al., 2001
). SalI-digested chromosomal DNA was ligated into SalI-digested pGEM-3Z (Promega) that had been dephosphorylated with shrimp alkaline phosphatase (Amersham Biosciences), and the ligation was electroporated into E. coli XL-1 Blue MRF' (Ausubel et al., 1994
) using a Gene Pulser II (Bio-Rad). After recovery, E. coli was cultured on LB plates with kanamycin to select for a clone containing the kanamycin-resistance gene. The resulting clone was designated pMBEi1.
The 12 kb pMBEi1 insert was sequenced on both strands using custom-synthesized oligonucleotide primers (Sigma Genosys) and the ABI Prism Big Dye Terminator cycle sequencing Ready Reaction (Applied Biosystems) protocol for double-stranded plasmid DNA. To obtain the sequence of the gene that was mutated in 93-146 R6, PCR primers based on the pMBEi1 sequence data (MBEi1PCRM1, CGCCACAGTCATAGGATA, bp 1068310700; MBEi1aP1, TGGTAGGTGATGGTCCTC, bp 85448561) were used to amplify an intact copy of the gene from wild-type chromosomal DNA (93-146). A 2·1 kb amplicon was produced under the following conditions: 95 °C, 2 min; 35 x (95 °C, 30 s; 50 °C, 30 s; 72 °C, 30 s); 72 °C, 10 min. The amplicon was cloned into pT7Blue using the Perfectly Blunt Cloning Kit (Novagen), and the resulting plasmid was designated pMBEi2.
Sequencing results from pMBEi1 and pMBEi2 were compiled using SeqMan v5.0 (DNAStar) and used to generate the final sequence of the Ed. ictaluri OPS biosynthesis gene cluster. The final 12 215 bp DNA sequence is in GenBank (accession no. AY057452).
Sequence analysis.
Homology searches were conducted using the BLAST algorithm (Altschul et al., 1997). Multiple sequence alignments were performed to quantify identities between derived Ed. ictaluri amino acid sequences and selected sequences in the protein database using CLUSTAL W (Thompson et al., 1994
) with MegAlign v5.0 (DNAStar). Membrane-spanning domains were predicted using transmembrane helices Markov model or TMHMM (Sonnhammer et al., 1998
), and hydropathy profiles were performed using the KyteDoolittle method (Kyte & Doolittle, 1982
) with Protean v5. (DNAStar).
Complementation of the wbiT mutation in 93-146 R6.
To determine whether the insertion mutation in wbiT was causing a polar effect on downstream genes, the wbiT gene was subcloned into a pBluescript derivative (pMBRP4) under the control of the lacZ promoter and transferred into Ed. ictaluri strain 93-146 R6. First, because electroporation into Ed. ictaluri has very low efficiency, a plasmid was constructed that would allow transfer into Ed. ictaluri by conjugation. The RP4 origin of transfer was isolated from pGP704 (Miller & Mekalanos, 1988) on a 1·7 kb BamHI fragment, blunt-ended using the Klenow fragment of DNA polymerase I (Promega), and ligated into the NaeI site of pBluescript SK(-) to create pMBRP4. The multiple cloning site of pBluescript remains intact in this plasmid, allowing bluewhite screening to detect clones with inserts.
The 2·0 kb insert from pBMEi2 was removed by digestion with BamHI and HindIII, gel purified, and subcloned into BamHI- and HindIII-digested pMBRP4. This ligation oriented the wbiT gene so that it could be expressed from the lacZ promoter in pMBRP4. The resulting plasmid, designated pMBRP4Ei2, was transferred into Ed. ictaluri 93-146 R6 from E. coli SM10 pir by conjugation using a filter-mating technique (Miller & Mekalanos, 1988
), followed by selection using ampicillin (to select Ed. ictaluri transconjugates) and colistin (to counterselect E. coli donors). Ampicillin-resistant Ed. ictaluri colonies were screened by colony blots (described below) and by plasmid preparations.
Colony blots, SDS-PAGE and immunoblots.
Ampicillin-resistant Ed. ictaluri were transferred onto nitrocellulose membranes by colony lifts, and colony blots were done as previously described (Lawrence et al., 2001) using the mAb Ed9 (Ainsworth et al., 1986
), which is specific for Ed. ictaluri OPS. LPS was purified from one of the Ed9-positive colonies, and 12 %/4 % SDS-PAGE gels were used as described by Laemmli (1970)
to resolve and compare LPS isolated from wild-type 93-146, mutant 93-146 R6, and complemented mutant 93-146 R6/pMBRP4Ei2. Gels for silver staining were fixed, oxidized in periodic acid, and stained using the Bio-Rad Silver Stain Kit following the manufacturer's protocol. For immunoblotting, LPS was transferred from gels to nitrocellulose and incubated with Ed9 using the method described for colony blots.
Serum resistance assay.
Serum was collected from 21 adult channel catfish and pooled. To remove anti-Ed. ictaluri antibodies, the serum was first absorbed with wild-type Ed. ictaluri. Stationary-phase 93-146 culture was pelleted, resuspended in an equal volume of catfish serum, and incubated for 90 min on ice. Bacteria were removed by centrifugation and filtration (0·45 µm pore size). Half of the serum was heated at 56 °C for 30 min to inactivate complement, and normal serum (NS) and heat-inactivated serum (HIS) were aliquoted and stored at -80 °C.
Ed. ictaluri strains 93-146 and 93-146 R6 and E. coli strain 11229 were grown to stationary phase in broth culture and diluted to OD5400·4. Bacteria and serum were mixed in equal volumes (0·1 ml each) and incubated at room temperature for 1 h. Controls consisted of 0·1 ml bacteria mixed with 0·1 ml 0·9x phosphate-buffered saline (CF-PBS). Bacteria were quantified in duplicate by serial dilution and plate counts. Five independent replicates from separate cultures were run for each strain and serum treatment.
Percentage killing was calculated by averaging duplicate plate counts for each replicate and dividing the number of bacteria surviving the serum treatments by the percentage of bacteria in the CF-PBS control for each replicate. Percentage killing for each of the five replicates was then averaged, and the mean serum killing rates were compared by analysis of variance (ANOVA) for a randomized complete block design with run as the blocking factor. If significant differences among strains were found at the 5 % level of significance, means were separated using the least significant difference test and 95 % confidence intervals were calculated to characterize the biological importance of those differences. The homogeneity of variances and normality assumptions necessary for valid application of ANOVA were examined by Levene's test and by stem-and-leaf and normal probability plots, respectively. Statistical computations were performed using the SAS System for Windows, Version 8 (SAS Institute).
Serum inhibition of luminescence.
The serum assay was repeated with the same bacterial strains expressing bacterial luciferase. Because bacterial luciferase requires FMNH2 for the luminescence reaction, bacteria must be metabolically active to luminesce (Unge et al., 1999). Therefore, this assay provides a more sensitive measure of inhibition of bacterial metabolic activity rather than cell death, and it allows real-time monitoring.
To express bacterial luciferase in Ed. ictaluri, the luxCDABE operon from Photorhabdus luminescens was removed from pCGLS-1 (Frackman et al., 1990) on a 10 kb fragment by digestion with EcoRI and ligated into the EcoRI site of pMBRP4. The resulting plasmid, pMBRP4lux, was transferred into Ed. ictaluri strains 93-146 and 93-146 R6 by conjugation (Maurer et al., 2001
); the resulting Ed. ictaluri transconjugates luminesce constitutively without addition of substrate. The same plasmid was also transferred into E. coli 11229 by electroporation.
The serum inhibition assay was conducted as described for the serum killing assay, except the assays were conducted with 0·09 ml each of the luminescent bacteria and serum in white 96-well plates (Nunc Nalge International). Luminescence was measured immediately after addition of serum to bacteria and at 10 min intervals for 60 min using end-point analysis with a 1 s integration time on an Lmax luminometer (Molecular Devices). Four individual replicates were run for each treatment. For each replicate, relative percentage luminescence at each time point was calculated by dividing the measured luminescence from bacteria with serum by the luminescence from the same strain in saline. The relative percentage luminescence from the four replicates for each treatment at each time point was then averaged.
Polymyxin B resistance.
Resistance of Ed. ictaluri strains 93-146 and 93-146 R6 to polymyxin B were determined as a model for resistance to cationic antimicrobial peptides. Minimum inhibitory concentrations (MICs) for polymyxin B were determined using a standard two-fold macrobroth dilution method in MuellerHinton broth. Bacteria were suspended at 5x105 c.f.u. ml-1 in broth containing appropriate antibiotic doses, and after 24 h incubation, turbidity of the cultures was assessed visually by comparison with uninoculated media. Each MIC was determined in triplicate, and the MIC determinations for the two Ed. ictaluri strains were repeated.
Resistance to catfish neutrophils.
Neutrophils were isolated from channel catfish anterior kidneys using a discontinuous Percoll gradient technique (Waterstrat et al., 1988) and adjusted to 5x106 cells ml-1. The percentage of neutrophils was determined by staining with Sudan black B; all neutrophil suspensions used in this assay contained at least 70 % neutrophils. Bacteria were grown overnight to stationary phase, washed in 0·9x Hanks' balanced salt solution (CF-HBSS), and adjusted to OD540 0·40 (approx. 2x108 c.f.u. ml-1). Catfish serum was obtained and stored as described for the serum resistance assay.
Equal volumes (100 µl each) of bacterial and neutrophil suspensions were mixed, and 40 µl of NS, HIS or CF-HBSS was added. Following 45 min incubation at 25 °C, 20 µl samples were removed and diluted in 80 µl sterile distilled water to release engulfed and adsorbed bacteria. Samples were serially diluted and spread on BHI plates to determine c.f.u. ml-1. Normal bacterial growth in 200 µl CF-HBSS with 40 µl NS or HIS (no neutrophils) for each treatment was also determined, and percentage survival was determined by dividing c.f.u. ml-1 of surviving phagocytosed bacteria into the c.f.u. ml-1 of normal bacterial growth in the absence of phagocytes. Seventeen replicates from separate fish were run on the Ed. ictaluri strains, and 10 replicates were run on the E. coli strains. Percentage survival for each strain and serum treatment was averaged, and statistical analysis was performed as described for the serum killing assay.
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RESULTS |
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In summary, compositional analysis confirmed the absence of OPS in 93-146 R6 and identified the composition of Ed. ictaluri OPS as GalNAc and Gal in a 2 : 1 ratio. Compositional analysis also indicated that Ed. ictaluri COS contains KDO, Hep, GalA, Gal, Glc, GalNAc, GlcNAc, NANA, and an unknown sugar. In addition, the mutation in 93-146 R6 appeared to affect COS in addition to OPS, as evidenced by the absence of NANA in the mutant and fewer COS variants (three in 93-146 R6 compared to at least five in wild-type 93-146).
Sequence results
The 12 215 bp sequence obtained from pMBEi1 and pMBEi2 contained eight complete ORFs (wzx, wzy, wbiB, wbiM, wbiH, wbiT, galF, gne) and two partial ORFs (dcuC and ugd) (Fig. 2). Seven of the eight proteins encoded by complete ORFs were similar to polysaccharide biosynthesis enzymes from other bacterial species (Table 3
). The remaining protein, Wzy, had no significant homology with previously reported sequences, but it had features (discussed below) that suggested it was also a polysaccharide biosynthesis enzyme. The predicted amino acid sequence from the incomplete ORF at the 3' end of the pMBEi1 insert was also similar to a polysaccharide biosynthesis protein (Ugd). The other incomplete ORF at the 5' end of the pMBEi1 insert was located upstream of the OPS biosynthesis genes and had high homology with dcuC from other bacterial species. In E. coli, DcuC is a C4 dicarboxylate carrier (Zientz et al., 1999
) and is not involved in OPS biosynthesis.
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Similar to polysaccharide biosynthesis gene clusters in other bacteria, the mol% G+C of the Ed. ictaluri OPS biosynthesis cluster is low compared to the reported overall mol% G+C content of the Ed. ictaluri chromosome (53 %) (Fig. 2) (Hawke et al., 1981
). Low mol% G+C content is a common feature of bacterial polysaccharide biosynthesis clusters and is thought to reflect a heterologous origin due to lateral transfer of genetic material between species or strains (Reeves, 1993
).
The Tn903 insertion mutation in 93-146 R6 was located in a gene whose derived amino acid sequence was similar to glucose-4-epimerases from other bacterial species. This gene was designated wbiT and is discussed below.
Sequence features
Between wzx and wzy is a 560 bp intergenic space, and between wzy and wbiB is a 75 bp intergenic space. Intergenic spaces between wbiB, wbiM, wbiH, wbiT, galF, gne and ugd are less than 25 bp or the genes overlap. A rho-independent transcriptional terminator was identified 27 bp downstream (GGCAGGCC-3 bp-GGCCTGCCTTTTTT) of dcuC, but no terminator sequences were identified in the OPS biosynthesis gene cluster. ShineDalgarno sequences for initiation of translation were identified within 15 bp upstream of the start codons for wzx, wzy, wbiB, wbiM, wbiH, wbiT, galF, gne and ugd.
A 39 bp sequence similar to JUMPstart consensus sequences was identified 403 bp upstream of the IS1 element (1310 bp upstream of wzx). JUMPstart sequences are located in noncoding sequence upstream of many bacterial polysaccharide biosynthesis gene clusters (Hobbs & Reeves, 1994), and they are required for regulation of OPS gene expression (Wang et al., 1998
). It has also been suggested that they serve as a site for recombinational events for exchange of polysaccharide genes (Hobbs & Reeves, 1994
). The regulatory function of JUMPstart sequences is best characterized in Salmonella enterica and is mediated by RfaH, which appears to function as a transcriptional antiterminator (Wang et al., 1998
). In fact, RfaH has a greater regulatory effect on promoter-distal genes in the S. enterica serotype Typhimurium OPS operon. Therefore, the Ed. ictaluri JUMPstart sequence could have a regulatory effect on the OPS operon despite its location more than 1 kb upstream of the first gene in the OPS cluster. This would require that the promoter for the OPS operon be located upstream of the JUMPstart sequence; it is possible that the OPS genes are expressed from the IS1 element promoter.
UDP-Glc synthesis
UTP Glc-1-phosphate uridylyltransferase catalyses the formation of UDP-Glc from Glc-1-phosphate, and UDP-Glc can subsequently be used to form activated sugar precursors for polysaccharide biosynthesis. Several enterobacterial species have two similar proteins, GalU and GalF, that are both designated as UTP Glc-1-phosphate uridylyltransferases. However, in E. coli, GalU was shown to be a functional UTP Glc-1-phosphate uridylyltransferase, while GalF appears to be a non-catalytic subunit that modulates activity of the GalU enzyme (Marolda & Valvano, 1996). The eighth ORF in the Ed. ictaluri OPS biosynthesis cluster was designated galF primarily because its predicted amino acid sequence shows highest identity to GalF proteins sequenced from other Enterobacteriaceae (59·663·3 %; Table 3
). However, Ed. ictaluri GalF also has significant identity with GalU proteins in Enterobacteriaceae (58·358·5 %). Functional analysis will be required to determine whether Ed. ictaluri GalF is a non-catalytic modulator or a functional uridylyltransferase.
Formation of activated sugar precursors
We identified three genes within the Ed. ictaluri OPS biosynthesis cluster encoding proteins that appear to be involved in the production of activated sugar precursors for Ed. ictaluri LPS. The first of these genes encodes a protein that is very similar to UDP-GlcNAc-4-epimerase (Gne) from Yersinia enterocolitica O : 8, which converts UDP-GlcNAc into UDP-GalNAc (Bengoechea et al., 2002). In Y. enterocolitica O : 8, Gne is encoded by a gene located in the Y. enterocolitica OPS biosynthesis cluster, and its expression is required for synthesis of LPS (Pierson & Carlson, 1996
). Y. enterocolitica Gne has significant similarity to UDP-Glc-4-epimerases (GalE) in other bacteria and has weak UDP-Glc-4-epimerase activity; however, it is primarily a UDP-GlcNAc-4-epimerase because of two amino acids located in its active site, Leu136 and Cys297, that allow UDP-GlcNAc to bind its active site (Bengoechea et al., 2002
). Bacterial UDP-Glc-4-epimerase proteins have bulky Tyr residues at these locations, which apparently exclude GlcNAc from binding in the active site, while the smaller side groups of Leu136 and Cys297 in Gne allow GlcNAc to bind in the active site (Bengoechea et al., 2002
). The predicted Ed. ictaluri protein that we have designated Gne has Leu and Cys residues at positions 136 and 299 that correspond to Leu136 and Cys297 in Y. enterocolitica Gne, suggesting that the active site in the Ed. ictaluri Gne protein could accommodate binding of UDP-GlcNAc.
The second gene is the partial ORF at the 3' end of the pMBEi1 insert, which has a predicted amino acid sequence that is similar to UDP-Glc dehydrogenase (Ugd) proteins in other bacterial species. Ugd converts UDP-Glc into UDP-glucuronic acid. In the Enterobacteriaceae, ugd is often located within the OPS biosynthesis gene cluster adjacent to a gene encoding an OPS chain length determinant (wzz) (Amor & Whitfield, 1997; Bastin et al., 1993
).
The third gene was designated wbiT, and its function cannot be predicted as easily based on comparisons with other bacterial enzymes. Homology searches indicated that WbiT is similar to bacterial UDP-Gal-4-epimerases, but it has lower identity with other bacterial epimerases than Ed. ictaluri Gne. WbiT had the highest identity with a putative UDP-Gal-4-epimerase from E. coli O157 : H7 (Perna et al., 2001) and a GalE homologue from Vibrio cholerae O139 that was designated WbfT (Yamasaki et al., 1999
). The enzymic functions of these two proteins are not known.
Glycosyltransferases
Three glycosyltransferases, which mediate the covalent bonding of activated sugar precursors to form oligosaccharide subunits, were identified in the Ed. ictaluri OPS gene cluster. None of these putative glycosyltransferases contained predicted transmembrane helices.
The first putative glycosyltransferase was designated WbiB, and BLAST searches indicated similarity to galactosyltransferases from other bacteria. One of the enzymes to which it is most similar is a galactosyltransferase (WaaX) responsible for addition of Gal in a -1,4 configuration in the R4 subtype of E. coli COS (Heinrichs et al., 1998a
). WaaX is a member of a family of
-glycosyltransferases that contains conserved motifs within the N-terminal 100 amino acids (Heinrichs et al., 1998b
). The motifs, FXFFD located
3040 residues from the N terminus and EDD located
90 residues from the N terminus, were identified in WbiB. WbiB is also similar to Lic2A, which is required for production of the phase-variable
Gal-1,4-
Gal epitope in Haemophilus influenzae type B lipooligosaccharide (High et al., 1993
), and to Haemophilus ducreyi lipooligosaccharide biosynthesis enzyme LbgA, which is a
-1,4-galactosyltransferase (Tullius et al., 2002
).
The second glycosyltransferase, designated WbiM, was similar to either galactosyl- or N-acetylgalactosaminyltransferases (Table 3). The functions of E. coli O111 WbdM and Streptococcus thermophilus EpsG are not known, but E. coli O111 OPS contains Gal and GlcNAc, and Strep. thermophilus extracellular polysaccharide contains Gal and GalNAc. Y. enterocolitica WbcN was reported to be either a galactosyl- or N-acetylgalactosaminyltransferase (Skurnik et al., 1995
). Pseudomonas aeruginosa WbpT was proposed to be an
-N-acetylgalactosaminyltransferase (Belanger et al., 1999
) based on sequence similarity and on the presence of the strictly conserved motif EX7E, which is present in the carboxy termini of
-D-glycosyltransferases (Geremia et al., 1996
). EX7E is also present in Ed. ictaluri WbiM.
The third glycosyltransferase, WbiH, also had the conserved EX7E motif in its C terminus. One of the enzymes WbiH was similar to, WbcQ from Y. enterocolitica O : 3, is proposed to function as either a galactosyl- or N-acetylgalactosaminyltransferase (Skurnik et al., 1995). Interestingly, WbiH was 19·9 % identical to WbiM.
Translocation and polymerization
Genes were identified in the Ed. ictaluri OPS biosynthesis cluster that encode proteins for transfer of the completed oligosaccharide subunits across the cytoplasmic membrane into the periplasmic space and subsequent polymerization of the subunits into chains. The first ORF in the Ed. ictaluri OPS gene cluster has low similarity with Wzx proteins in other bacterial species, and it has multiple predicted transmembrane regions, similar to other Wzx proteins (Table 3; Fig. 3
). Wzx proteins transport completed undecanyl phosphate-linked O subunits across the cytoplasmic membrane by flipping them from the cytoplasm into the periplasm (Liu et al., 1996
). Except for a few cases where an ATP transporter is used (Kido et al., 1995
; Zhang et al., 1993
), most OPS biosynthesis gene clusters in other species contain a Wzx homologue. However, Wzx proteins from different species and strains generally have low or no detectable sequence similarity because the sugar subunits they bind are unique to each O type (Reeves, 1993
).
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Construction of pMBRP4
An effective Ed. ictaluri shuttle vector was constructed in this study that is transferred from E. coli donors into Ed. ictaluri with high efficiency. Because it is a member of the family Enterobacteriaceae, Ed. ictaluri recognizes the ColE1 origin of replication in pBluescript. However, our previous experience in attempting to transfer plasmid DNA into Ed. ictaluri indicated that electroporation has very low efficiency, possibly due to the presence of an endogenous restriction endonuclease system (Maurer et al., 2001). Previously, we reported efficient transfer of suicide plasmids derived from pGP704 into Ed. ictaluri by conjugation for the purpose of constructing isogenic mutants (Lawrence et al., 1997
, 2001
; Maurer et al., 2001
). To our knowledge, this present paper is the first report of high-efficiency transfer of a ColE1 plasmid into Ed. ictaluri and subsequent expression of a gene from the plasmid.
Complementation of the wbiT mutation in 93-146 R6
Following conjugal transfer of pMBRP4Ei2 into 93-146 R6, plasmid preparations were performed on four randomly selected ampicillin-resistant colonies. One was selected that was kanamycin-resistant, had the confirmed presence of pMBRP4Ei2, and had a strong positive reaction to Ed9 on colony blots. Purified LPS from this colony had the same profile as LPS isolated from wild-type parent strain 93-146 both on silver-stained SDS-PAGE gels and on immunoblots (Fig. 4). Similar to previously published studies of Ed. ictaluri LPS (Lawrence et al., 2001
; Newton & Triche, 1993a
), silver staining only detected lower molecular mass LPS bands, while the more sensitive immunoblotting method detected the presence of low, medium and high molecular mass bands. This phenomenon has been attributed to a predominance of lower molecular mass LPS molecules in Ed. ictaluri LPS (Newton & Triche, 1993a
). Our results indicate that the OPS-negative phenotype in 93-146 R6 is due to the single effect of the kanamycin-resistance gene insert in wbiT rather than due to a polar effect on downstream genes in the OPS biosynthesis operon.
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Resistance to catfish neutrophils
There was no significant difference in the abilities of wild-type and OPS-negative Ed. ictaluri to resist killing by catfish neutrophils with either NS or HIS (Table 5). By contrast, E. coli controls had significantly lower survival rates than the Ed. ictaluri strains in the presence of neutrophils plus NS or HIS. Both wild-type and OPS-negative Ed. ictaluri had significantly decreased survival rates in the presence of neutrophils with NS compared to neutrophils with HIS.
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DISCUSSION |
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Comparison of the sugar composition of LPS from wild-type Ed. ictaluri and OPS mutant 93-146 R6 indicates that Ed. ictaluri OPS contains GalNAc and Gal in a 2 : 1 ratio. This finding is supported by the presence of a gene encoding an enzyme required for the synthesis of UDP-GalNAc in the OPS biosynthesis gene cluster as well as genes encoding glycosyltransferases that are similar to previously reported galactosyl- or N-acetylgalactosaminyltransferases. These findings are also supported by a previous report that Ed. ictaluri could be agglutinated by galactose-specific lectin derived from Ricinus communis (Ainsworth, 1993). Agglutination with the galactose-specific lectin could be prevented by preincubation of the lectin with galactose, and the author suggested that galactose on the cell surface could play a significant role in adherence in vivo. In addition, a separate study demonstrated that preincubation of catfish olfactory mucosa with soluble D-galactose significantly reduced adhesion of Ed. ictaluri to cell surfaces (Wolfe et al., 1998
). In some bacterial species, OPS is known to mediate adherence and invasion of host cells (Kohler et al., 2002
; Merino et al., 2000
; Paton & Paton, 1999
); perhaps Ed. ictaluri OPS plays a role in adherence to host mucosal surfaces as well.
The compositional analysis also detected the presence of COS variants in Ed. ictaluri. COS variants have been reported for Campylobacter jejuni, where the variants represented eight different ganglioside mimics (Gilbert et al., 2002). Interestingly, COS in C. jejuni contains NANA, which our compositional analysis indicated is a component of Ed. ictaluri COS. The C. jejuni COS variants result from a combination of genetic mechanisms, including different gene complements, phase variation, and various mutations. At this point it is unknown whether the Ed. ictaluri COS variants are a result of phase variation or concurrent expression of multiple forms of COS. Ed. ictaluri also expresses two flagellar proteins that could be the result of phase variation or concurrent expression (Newton & Triche, 1993b
).
Prediction of bacterial polysaccharide biosynthesis protein functions based on sequence data is often difficult due to low homology of the amino acid sequences to other sequences in GenBank. Enzymes responsible for synthesis of sugar precursors tend to be the most conserved, so our predictions of Ugd and Gne functions are the most reliable. Even though Wzx and Wzy had low or no homology with orthologous proteins, these predictions are also reliable based on their locations in the gene cluster and their similar structures to other Wzx/Wzy proteins.
Predicting functions of the glycosyltransferases is the most difficult. Based on identified conserved motifs, WbiB is likely a -glycosyltransferase, and based on sequence similarities, its sugar specificity is most likely UDP-Gal or UDP-GalNAc. We propose that WbiM is an
-N-glycosyltransferase based on the presence of the EX7E motif, with sugar specificity for either UDP-GalNAc or UDP-Gal. Due to its lower similarities with other bacterial glycosyltransferases compared to WbiB and WbiM, we cannot predict the sugar specificity for WbiH, but we predict it is a
-glycosyltransferase. However, despite the identification of conserved motifs within WbiB, WbiM and WbiH that suggest the types of glycosidic linkages they form, functional analyses will be required to elucidate the functions of these proteins.
We have determined that the OPS-negative phenotype of 93-146 R6 is due to the single effect of the insertion mutation in wbiT, and sequence alignments indicate that WbiT is similar to galactose-4-epimerases. Our compositional analysis indicated that Ed. ictaluri OPS contains GalNAc and Gal. An epimerase for the synthesis of UDP-GalNAc can be accounted for by Gne, and Ed. ictaluri has the ability to utilize galactose as a carbon source (Waltman et al., 1986), which indicates that Ed. ictaluri should have a functional gal operon encoding a UDP-Glc-4-epimerase (GalE). Perhaps WbiT is a second UDP-Glc-4-epimerase that allows efficient production of UDP-Gal for LPS biosynthesis.
Alternatively, WbiT could be involved in the biosynthesis of a COS component that is essential for attachment of OPS. Compositional analysis indicated that 93-146 R6 has altered COS, as evidenced by the absence of NANA and fewer COS variants in the mutant. Although OPS and COS biosynthesis genes are usually located in separate clusters (Schnaitman & Klena, 1993), there is precedent for clustering of OPS and COS biosynthesis genes together in at least one other species (Carlson et al., 1995
). Ugd, which could be involved in biosynthesis of UDP-GalA (an Ed. ictaluri COS sugar), is also located in the Ed. ictaluri OPS gene cluster. Further analysis will be required to determine the function of WbiT.
Our results indicated that Ed. ictaluri OPS contributes to serum resistance. However, it is not the only component that mediates resistance to serum, because 93-146 R6 still had >50 % survival in normal catfish serum. By comparison, other Gram-negative pathogens demonstrate complete or nearly complete serum sensitivity when OPS is not expressed (Allen et al., 1998; Amaro et al., 1997
; Merino et al., 1994
, 2000
). However, OPS is not always the most important component in serum resistance: OPS-negative (and S-layer-negative) Aeromonas sp. O11 mutants with intact COS retain serum resistance (Merino et al., 1996
), and OPS is not the major factor in serum resistance for Yersinia enterocolitica O3 (Pilz et al., 1991
).
In fact, the relatively small decrease in serum resistance of 93-146 R6 compared to wild-type Ed. ictaluri could be accounted for by its failure to express sialic acid. A previous study demonstrated that wild-type Ed. ictaluri that had been treated with neuraminidase had a relatively small, but significant, decrease in serum resistance (70 % survival in normal catfish serum compared to 92 % survival for untreated wild-type) (Ourth & Bachinski, 1987). Therefore, Ed. ictaluri OPS may play only a minor role in serum resistance, while NANA and the other COS sugars (Glc, Hep, KDO, GalA, Gal, GalNAc, GlcNAc, and unknown) may significantly contribute to serum resistance. However, the role of Ed. ictaluri COS in serum resistance requires confirmation, and we cannot exclude the possibility of other Ed. ictaluri surface structures (e.g. capsular polysaccharide, outer-membrane proteins) contributing significantly to serum resistance.
Based on chelation studies using MgEGTA and EDTA, the channel catfish alternative complement pathway is more important than the classical pathway and lysozyme in killing Ed. ictaluri and other Gram-negatives (Ourth & Wilson, 1981, 1982
; Ourth & Bachinski, 1987
). Therefore, the decreased serum resistance in 93-146 R6 is probably due to increased susceptibility to the alternative complement cascade. In agreement with this premise, 93-146 R6 was not killed by heat-inactivated serum. However, it is also possible that the OPS-negative mutant's increased susceptibility to serum was actually due to cationic antimicrobial peptides. Therefore, we tested 93-146 R6 for its sensitivity to polymyxin B, which is commonly used as a model for sensitivity to cationic antimicrobial peptides (Allen et al., 1998
). Our results showed that 93-146 R6 was not more sensitive to polymyxin B than 93-146, which indicates that the modest increase in serum susceptibility of 93-146 R6 was not due to the effect of cationic antimicrobial peptides.
The serum luminescence inhibition assay supported the results of the serum killing assay, demonstrating that 93-146 R6 is more susceptible to serum than wild-type Ed. ictaluri. It also suggested that other heat stable serum factor(s), although not apparently killing either Ed. ictaluri strain, caused a very rapid inhibition of luminescence, probably due to increased energy demands on the bacteria. Such inhibition, which could be mediated by other known bacterial inhibitors in serum such as lysozyme, transferrin, or cationic antimicrobial peptides, may be important in slowing bacterial growth and allowing clearance by the host immune system.
In contrast, the phagocytosis assay indicated that Ed. ictaluri OPS does not play a role in resistance to catfish neutrophils. At this time, the mechanisms used by Ed. ictaluri to resist killing by phagocytic cells remain unresolved.
In conclusion, the compositional analysis of Ed. ictaluri OPS and predicted functions of enzymes encoded within the Ed. ictaluri OPS biosynthesis gene cluster indicate that Ed. ictaluri OPS contains GalNAc and Gal. The genetic basis for the OPS-negative phenotype in 93-146 R6 was determined to be the single effect of insertion of the kanamycin-resistance gene in wbiT, which encodes an epimerase of unknown specificity. Finally, Ed. ictaluri OPS was shown to contribute to the ability of Ed. ictaluri to resist killing by normal catfish serum, but not to its ability to resist killing by catfish neutrophils.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr Russell Carlson for valuable discussions and assistance with interpretation of glycosyl compositions. Monoclonal antibody Ed9 was used with the permission of Dr Jerald Ainsworth. We thank Bobbie Boyd for maintaining the cell lines and supplying monoclonal antibody for this study. We thank Marian Hughlett for technical assistance in DNA sequencing, and we thank Dr Carolyn Boyle for assistance in statistical analysis. We are also grateful to Drs Shane Burgess, Jerald Ainsworth and Chinling Wang for their critical review of this manuscript. This paper is MAFES publication J10182.
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Received 20 November 2002;
revised 24 February 2003;
accepted 27 February 2003.
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