Printed in Great Britain
Microbiology (1997), 143, 3481-3489
Expression of Campylobacter hyoilei lipo-oligosaccharide (LOS) antigens in Escherichia coli Victoria Korolik,’ Ben N. Fry,2 Malcolm R. Alderton,’ Bernard A. M. van der Zeijst’ and Peter J. Coloel Author for correspondence: Victoria Korolik. Tel : + 61 396602796. Fax : e-mail: [email protected]
Department of Applied Biology and Biotechnology, RMIT, GPO Box 2476V, Melbourne 3001, Australia Department o f Bacteriology, Institute of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Universiteit Utrecht, Yalelaan 1 3584 CL Utrecht, The Netherlands
+ 61 396623421.
Campylobacter spp. are well recognized as primary pathogens in animals and in people. To isolate and define the genetic regions encoding major surface antigens of Campylobacter hyoilei, genomic DNA of the type strain of the species, RMIT-32A, was cloned into a cosmid vector, plA2917, in Escherichia coli and the resulting genomic library was screened using antiserum raised to the parent C. hyoilei strain. Six cosmid clones were found to express a series of immunoreactive bands in the 15-25 kDa range. These bands were proteinase Kresistant and were found in the LPS fraction of the cells, suggesting that the recombinant cosmids expressed C. hyoirei lipo-oligosaccharide (LOS) antigen(s). The minimum DNA insert size required for expression of C. hyoilei LOS [email protected]
) in E. coli was 11.8 kb. This region was subcloned into the plasmid vector pBR322. The partial sequencing of the 11.8 kb region showed that it contains two ORFs, designated rfbf and rfbP, showing homology with the rfbF gene from Serratia marcescens and the rfbP gene from Salmonella typhimurium. Both genes are involved in LPS synthesis. The region also contained a sequence homologous to the rfaC gene of E. coli and Sal. typhimurium which is involved in core oligosaccharide synthesis. Keywords : Campylobacter, lipo-oligosaccharide (LOS), LPS, cloning and expression
Campylobacter spp. are known to colonize and cause disease in both humans and animals (Skirrow, 1982; Blaser & Reller, 1981). Campylobacter hyoilei, a thermophilic species identified by Alderton et al. (1995), is closely related to both C. jejuni and C. coli. It has been shown to be one of the causative agents of Porcine Proliferative Enteritis (PPE) (Alderton et al., 1991). As with other Campylobacter spp., the mechanisms of pathogenicity of this organism are poorly understood. However, as for other bacterial pathogens of the gastrointestinal tract of humans and animals, such as enteropathogenic Escherichia coli, Salmonella spp. and Shigella spp., the pathogenic mechanisms are thought to be related to surface antigens such as LPSs (Luderitz et al., 1982). The lipo-oligosaccharides (LOSs) of other GramAbbreviation: LOS, lipo-oligosaccharide. The GenBank accession numbers for the sequences reported in this paper are X91081 for the sequence containing the rfbF and the rfbP genes, and X91082 for the partial sequence of rfaC.
negative bacteria such as Neisseria spp., Haemophilus spp. and Bordetella spp. are also thought to play a major role in their pathogenicity (Preston et al., 1996). LPS antigens of E. coli and Salmonella spp. have been well studied and consist of three components : a glucosamine-based phospholipid known as lipid A, a nonrepeating core oligosaccharide and distal repeating oligosaccharide chains known as the O-antigen (Luderitz et al., 1982; Reeves, 1993). Campylobacter spp. LPS appears to have a structure similar to that of other members of the Enterobacteriaceae, containing a high variety of O-polysaccharides. Both smooth and rough LPS types of Campylobacter spp. have been identified. As in other enteric bacteria, those with smooth LPS show serum resistance while strains with rough LPS are serum-sensitive (Perez-Perez & Blaser, 1985). It has been suggested that the low molecular mass LPSs of many ‘rough’ C. jejuni strains may be similar to LOSs characteristic of Neisseria spp. and Haemophilus spp. in that they lack O-antigen but contain a variety of core structures. This is in contrast to Salmonella spp. LPS which contains a conserved LPS core. Finally, the
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LPS of Campylobacter spp. has been shown to share antigenic determinants with core regions of other Gramnegative bacteria (Perez-Perez et al., 1986).
RMIT-32A genomic library. E . coli DH1 (Frey et al., 1983) was used in construction of subclones expressing C. hyoilei antigen(s) in the plasmid vector pBR322 (Bolivar et al., 1977). The E. coli strain DH5a (Sambrook et al., 1989) was used as a host for the subcloned EcoRV fragments in pUC18 (YanischPerron et al., 1985) for DNA sequencing.
The structure of LPS core molecules isolated from several C. jejuni strains, several C. lari strains and a C . coli strain has been determined by chemical analysis Culturing conditions. C. hyoilei RMIT-32A was grown in a (Aspinall et al., 1995a, b, c, 1994a, b, 1993a, b, c; gaseous mixture of 50% (v/v) CO, and 50% (v/v) H, on Salloway et al., 1996). Common sugars for the core Columbia Agar (Oxoid) supplemented with 7 Yo (v/v) defibare 3-deoxy-~-manno-octulosonicacid,~-g~ycero-~rinated horse blood for 48-72 h at 37 "C. E. coli was grown in manno-heptose, ethanolamine, D-glucose, D-galactose, Nutrient Broth No. 2 (Oxoid) supplemented with 0.5 '/o (w/v) yeast extract (Oxoid) overnight at 37 "C, or on agar plates N-acetyl-D-glucosamine and N-acetyl-D-galactosamine. using the same broth composition supplemented with 1.5 '/o One notable feature is the presence of N-acetylneura(w/v) Bacteriological Agar (Oxoid). Tetracycline hydrochlominic acid (sialic acid; NeuNAc) in the core molecules ride (CSL) and ampicillin (CSL) were used at 25 and 200 pg isolated from the C . jejuni strains. NeuNAc is a rarely ml-', respectively, in both solid and liquid media. encountered sugar in LPS molecules, but is commonly Antisera. Anti-C. hyoilei RMIT-32A polyclonal mouse ascites found in mammalian glycoproteins and glycolipids. It is fluid to whole-cell antigen was prepared as described prenormally not antigenic and may therefore play a role in viously (Alderton et al., 1991).Prior to use this antiserum was escaping the immune system, for instance by masking adsorbed with E. coli HBlOl cells carrying cosmid vector LPS epitopes or creating steric hindrance to antibody pLA2917 [E. coli HBlOl(pLA2917)l as follows. One volume of binding. This would explain the described non-speciantiserum was added to 9 vols of a solution of total E. coli cell ficity of the agglutination slide test for Campylobacter antigen. The solution was prepared by growing E. coli (Penner & Hennessy, 1980). NeuNAc residues attached HBlOl(pLA2917) until confluent on three agar plates, harvia 2,3 linkages to P-D-galactose resemble gangliosides vesting into 20 ml 10 mM Tris/HCl (pH 7.5) and lysing by (Aspinall et al., 1992,1993b). This molecular mimicry is sonication. Unlysed cells were removed by centrifugation. The thought to play a role in the neuropathological diseases mixture was incubated for 24 h at 4 "C with gentle shaking. The adsorbed antiserum was diluted fivefold before use. Guillain-Barre syndrome and Miller-Fisher syndrome (Salloway et al., 1996; Schwerer et al., 1995). In Neisseria DNA manipulations. Total genomic DNA of C. hyoilei RMITand Haemophilus sialylation of LOS has been shown to 32A was prepared by harvesting bacterial cells from agar play a role in pathogenicity by enhancing serum plates into 10 m M Tris/HCl (pH 7.5). Then the DNA was resistance (Demarco de Hormaeche et al., 1991 ; Moxon extracted as described previously (Korolik et al., 1995). Plasmid DNA extraction, purification, restriction endo& Maskell, 1992). A complementary approach to purification and chemical analysis for the study of LPS and LOS structure, synthesis and function is the cloning and expression of genomic sequences encoding the enzymes involved in LPS and LOS synthesis. Identification and characterization of genes involved in the synthesis of carbohydrate surface antigens of micro-organisms such as Salmonella spp., Vibrio cholerae and Klebsiella pneumoniae has contributed to a better understanding of the mechanisms of bacterial pathogenesis (Collins & Hackett, 1991; Manning et al., 1988; Arakawa et al., 1991). In this paper we report on the molecular cloning and expression in E. coli of a DNA region encoding the synthesis of LOS antigen(s) from a newly identified species, Campylobacter hyoilei strain RMIT-32A (Alderton et al., 1995). C . hyoilei RMIT-32A had been shown to possess a low molecular mass glycolipid component (Kuan et al., 1992) which is likely to be LOS rather then traditional LPS characteristic of E. coli and Salmonella spp. METHODS Bacterial strains and plasmids. C. hyoilei RMIT-32A was isolated from a pig with PPE (Alderton et al., 1991, 1995). Cosmid vector pLA2917 (TcR KmR) has been described by Allen & Hanson (1985). E. coli HBlOl (Maniatis et a/., 1982) was used as the host strain in the construction of the C. hyoilei
nuclease cleavage, agarose gel electrophoresis and enzyme usage were as described by Korolik et al. (1988) and Collins & Ross (1984). Restriction endonucleases EcoRV, BglII, ClaI and Sau3AI (Promega) were used as recommended by the manufacturer, except that 20 U enzyme was used to cleave 1 pg Campylobacter spp. DNA.
Construction of the C. hyoilei RMIT-32A genomic library. High molecular mass genomic DNA of RMIT-32A was partially cleaved using Sau3AI and fragments were sizefractionated by sucrose gradient centrifugation (Maniatis et al., 1982). Fragments of 25-35 kb were selected and cloned into an alkaline phosphatase-treated unique BglII site in the kanamycin resistance gene of a 21 kb cosmid vector, pLA2917 (Allen & Hanson, 1985). The ligation mixture (2 vector:l chromosomal fragments) was then packaged (0-5pg ligation mix per 50 pl, one packaging mix) into a Packagene A-particle packaging mix (Promega) and the mixture was transduced into the host E . coli strain HBlOl as recommended by the manufacturer. Subcloning of the C. hyoilei LOS coding region. Recombinant cosmid pBT9101 DNA was partially cleaved with EcoRV to give fragments of 4-20 kb which were then subcloned into EcoRV-cleaved, alkaline phosphatase-treated plasmid vector pBR322 (Bolivar et al., 1977) and transformed into the E . coli DH1 host. Immunological screening. E. coli HBlOl transductants comprising the C. hyoilei RMIT-32A genomic library were grown for 16 h, then the colonies were lifted onto nitrocellulose filters (Bio-Rad) and screened for expression of RMIT-32A antigens with adsorbed mouse anti-C. hyoilei RMIT-32A ascites fluid as described previously (Korolik et al., 1988).
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Cloning of Campylobacter LOS genes LPS and LOS extraction. E. coli HBlOl cells carrying the recombinant plasmids expressing the C. hyoilei antigens and C. hyoilei RLMIT-32Acells were tested for the presence of LPS/LOS by treatment with 20 mg proteinase K for 16 h at 37 "C per 20 mg sample. Bacterial LPS and/or LOS was also extracted using a modified hot phenol procedure (Kuan et al., 1992).
SDSPAGE. The antigens were resolved by discontinuous SDSPAGE using a 4.5 '/o stacking gel and 12 and 15.5'/o resolving gels where appropriate. (Laemmli, 1970).Low molecular mass protein and polypeptide standards (Bio-Rad)were used as size markers.
Western blotting. The antigens resolved by SDS-PAGE were
transferred to nitrocellulose membrane (Bio-Rad) according to Towbin et al. (1979). Primary antibody (adsorbed mouse anti-C. hyoilei RMIT-32A ascites fluid, adsorbed as described previously; Korolik et al., 1988) was bound to the antigen for 16 h at room temperature. The antigen-antibody reaction was detected colorimetrically using horseradish-peroxidase-conjugated anti-mouse IgG second antibody. DNA sequencing and analysis. Fragments of plasmid pBT91OS were subcloned after digestion with EcoRV by ligation of selected fragments into pUC18 digested with HincII. The sequence of three cloned DNA fragments was determined by the dideoxy chain-termination method (Sanger et al., 1977) with an Automated Laser Fluorescent DNA Sequencer (Pharmacia), the Autoread Sequencing Kit using T7 DNA polymerase (Pharmacia) and fluorescein-labelled nucleotide primers (Pharmacia).To confirm the continuity between the 1000 and 1600 bp fragments, this EcoRV region was sequenced on plasmid pBT91OS to bridge the sequence. PC/GENE 6.70 (Korn & Queen, 1984) was used to analyse nucleotide and amino acid sequences, which were compared to databases available at GenomeNet using the program BLAST (Altschulet al., 1990). The MACAW program (Lawrence et al., 1993) was used for multiple sequence alignment.
RESULTS Immunological screening of the C. hyoilei RMIT-32A genomic library and recovery of cosmid clones expressing RMIT-32A antigen(s) Twenty-five randomly selected TcR KmS transductants were screened for the presence of recombinant cosmids and were found to carry a DNA insert with a mean size of 27 kb. A total of 800 such transductants were chosen randomly and constituted the C. hyoilei RMIT-32A genomic library. All E. coli transductants carrying recombinant cosmids with C. hyoilei RMIT-32A DNA inserts were then screened for expression of C. hyoilei antigens in E. coli using colony blotting with anti-C. hyoilei RMIT-32A mouse ascites fluid pre-adsorbed with E. coli HBlOl(pLA2917). Strongly reactive transductants were selected and further examined for expression of C. hyoilei antigens. Whole-cell lysates of E. coli transductants were separated using SDS-PAGE, Western blotted onto nitrocellulose membrane filters and probed with the same antiserum. Western blot analysis showed six E. coli transductants expressing a low molecular mass anti-
Fig. 1. Western blot analysis of whole-cell lysates using adsorbed C. hyoilei RMIT-32A polyclonal ascites fluid with horseradish-peroxidase-conjugated secondary antibody. Lanes: 1, E. coli HB101 transductant carrying recombinant cosmid expressing RMIT-32A LOS antigen(s); 2, E. coli HBlOl carrying cosmid vector plA2917; 3, pre-stained molecular mass standards.
gen(s) of 15-25 kDa which was strongly reactive with anti-C. hyoilei RMIT-32A serum. Fig. 1 shows one such transductant displaying a ladder-like formation in the 15-25 kDa region. N o reaction was observed with LPS of the E. coli HBlOl(pLA2917)control (Fig. 1).Although additional bands appearing at 14 and 30 kDa were detected in all samples, these are most likely due to the binding of the E. coli-specific antibody following incomplete adsorption of the antiserum as whole-cell lysate of the E. coli HBlOl(pLA2917) control also shows the same reaction. Western blot analysis using both 12 Yo polyacrylamide gels (as shown in Fig. 1)and 15.5YO gels were repeated following extended treatment of samples at 100 "C. This did not change the ladder-like appearance and the distribution of the apparent size of the reacting antigen(s) (data not shown). Restriction endonuclease analysis of the recombinant plasmids expressing RMIT-32A antigen(s) Transductants expressing RMIT-32A antigen(s) in E. coli were analysed using restriction endonucleases BglII, EcoRV and ClaI and fragment maps were generated (Fig. 2a). The analysis showed that four of these carried recombinant cosmids with unique but overlapping inserts. They were named pBT9101, pBT9102, pBT9103 and pBT9104 (Fig. 2a). The insert of pBT9103 was found in the vector in the opposite orientation to that of the other three recombinant cosmids, yet still expressed recombinant antigens.
To determine the minimum region of C. hyoilei genomic DNA required for the expression of the antigen, the C. hyoilei genomic DNA insert of pBT9101 was partially digested with EcoRV and the resulting fragments were subcloned into the plasmid vector pBR322 in E. coli DH1. Two-hundred transformants carrying subcloned C. hyoilei DNA were then screened immunologically for expression of the reactive antigen(s). Ten E. coli transformants that reacted with the antiserum were 3483
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u3: E E E
CCC E E E
EE I I
E E E I I I
E E E I
w pBT9103 (29 kb)
pBT9104 (24 kb)
pBT9105 (1 1.8 kb)
Fig. 2. Restriction endonuclease cleavage map o f recombinant cosmids carrying C. hyoilei RMIT-32A genomic sequences. (a) Restriction maps o f four recombinant cosmids expressing RMIT32A antigen($ originally isolated from the genomic library. (b) EcoRV subclone (1 1.8 kb) expressing RMIT-32A LOS antigen(s). Only insert DNA is shown. Abbreviations: B, Bglll; C, Clal, E, EcoRV.
16.9 14.4 10.7 8.1 -
pBT9102 (28 kb)
pBT9101 (26 kb)
Figrn4. (a) Western blot analysis of whole-cell lysates treated with proteinase K using adsorbed C. hyoilei RMIT-32A polyclonal ascites fluid with horseradish-peroxidase-conjugated secondary antibody. Lanes: 1, RMIT-32A; 2, E. coli HB101 transductant pBT9101 carrying recombinant cosmid expressing RMIT-32A antigen(s); 3, E. coli DH1 carrying subclone pBT9105 expressing RMIT-32A antigen(s); 4. E. coli HBlOl control carrying cosmid vector pLA2917. Molecular size markers are indicated o n the left. (b) Western blot analysis o f purified LOS using adsorbed C. hyoilei RMIT-32A polyclonal ascites fluid with horseradish-peroxidase-conjugated secondary anti body. Lanes: 1, pre-stained molecular mass standards; 2, RMIT-32A; 3, prestained molecular mass standards; 4, E. coli D H l carrying subclone pBT9105 expressing RMIT-32A antigen(s); 5, E. coli HBlOl control carrying plasmid vector pBR322.
700 Rsal Asp700 Rsal RSdl
Fig. 3. Western blot analysis o f whole-cell lysates using adsorbed C. hyoilei RMIT-32A polyclonal ascites fluid with horseradish-peroxidase-conjugated secondary anti body. Lanes: 1, E. coli DH1 control carrying vector pBR322; 2, E. coli DH1 carrying pBT9105 with an LOS-expressing 11.8 k b EcoRV C. hyoilei RMIT-32A fragment subcloned in pBR322; 3, E. coli HBlOl carrying pBT9101, an original LOS expressing cosmid; 4, E. coli DH1 carrying a pBR322 subclone with an 8.6 k b EcoRV fragment internal t o pBT9105 b u t n o t expressing C. hyoilei RMIT-32A LOS antigen; 5, pre-stained molecular mass standards; 6, C. hyoilei RMIT-32A.
further analysed by Western blotting. All showed the presence of antigens of 15-25 kDa, equivalent to that expressed by the original cosmid clone pBT9101. Analysis of the subclones showed that the minimal DNA region required for the expression of C. hyoilei RMIT32A LOS antigen(s) is 11.8 kb (Fig. 2b). Fig. 3 shows a Western blot of one such clone, pBT9105. Thirty-six clones with smaller inserts, both internal and outside the region defined by pBT9105, were tested and in these no
Fig. 5. Structure o f plasmid pBT9105 showing the genes found in the sequenced parts. The single line indicates the 11.8 k b insert; the open bar indicates the vector pBR322. The arrows indicate the putative genes rfbF and rfbP. The available part o f the rfaC gene is also indicated.
expression could be detected. Fig. 3 shows a Western blot of one such subclone internal to pBT9105, carrying a major EcoRV fragment of 8.6 kb, included as a control.
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Cloning of Carnpylobacter LOS genes 1
GTATGATTTA GTGATTAATG CTAGAGATGC AAAAGTTTTT rm-+ TTTACAATGA GAGTAGGCTT TTTAACTCAC GCAGGAGCAA n~ V G F L T H A G A S
GTATATATCA TTTTAGATTG CCTATAATTA AAGCTTTOAT TGCAAGAGGA GATGAGGTTT I Y H F R L P I I K A L I A R G D E V F
TTGTCATAGT GCCACAAGAT GAATATACCG AAAAATTAAA GGCTTTAAAT TTAAATATCG V I V P Q D E Y T E K L K A L N L N I V
TTGTTTATGA GCTTTCAAGA GCGAGTTTAA ATCCTTTAAT TC-TTTTTAAA AATTTTTTAC V Y E L S R A S L N P L I V F K N F L H
ATCTTAAAAA TGTPTTAAAA AACTTAAATT TGGATCTTTT GCAAAGTGGA GCTCATAAGA L K N V L K N L N L D L L Q S G A H K S
GCAATACCTT TGGTTTAATA GCTGCAAAAT ATGCCAAAAT TCCTTATAAA ATCGGTCTTG N T F G L I A A K Y A K I P Y K I G L V
TTGAGGGACT TGGATCTTTT TATATAGACA AGGGTTTTAA AGCAAATTTA GTGCGTTTTG E G L G S F Y I D K G F K A N L V R F V
TTATCAATAC TCTTTATAAA CTTAGTTTTA AAATCGCTGA TTCTTTCATC TTTGTAAATC I N T L Y K L S F K I A D S F I F V N Q
AAGCTAATGC GGATTTTATG CGAAATTTAG GACTTAAGGA AAATAAAATT TGCGTGATTA A N A D F M R N L G L K E N K I C V I K
AATCCGTGGG TATCAATCTT AARAAATTCT TTCCTATGAG AGTGGAGCAA GAAGCTAAAA S V G I N L K K F F P M R V E Q E A K K
661 AAGCTTTTTG GCAARATTTA AARA'ITGATG AARAGCCTAT TGTTTTGATG ATAGCTAGGG A
CTTTGTGGCA TAAAGGTGTA AAAGAATT" ATGAAAGTGC AGAGTATTTA AAAGATAGGG L W H K G V K E F Y E S A E Y L K D R A
CAAATTTTGT TTTAGTGGGC GGAAGAGATG ATAATCCTTC TTGTGCGAGT TTGGAATTTT N F V L V G G R D D N P S C A S L E F L
TAAATTCAGG CAAGGT"T N S G K V F
ATTGCGATAT CTTTGTPTTG CCAAGCTATA AAGAAGGATT TCCAGTAAGT GTTTTAGAGG P S Y K E G F P V S V L E A C D I F V L
CGAAAGCTTG TGGCAAAGTT ATTGTAGTAA GTGATTGTGA GGGTTGTGTT GAGGCAATTT I V V S D C E G C V E A I S K A C G K V
CAAATGCTTA TGATGGGCTT TGGGCTAAGA CCAAAGATAG TAAAGATTTA ATAGAAAAU W A K T K D S K D L I E K I N A Y D G L
TACAAGTTTT ATTAGAAGAT GAAAGTTTAA GAATCAACCT AGGTAAAAAT GCAGCTAAGG E S L R I N L G K N A A K D Q V L L E D
ATGCTTTACA ATACGATGAA AATGTTATCG CGCAGCGTTA TTTAGAACTT T A T G A T E N V I A Q R Y L E L Y D R V A L Q Y D E
rfbp-, TGATTAAAU TGTATGAGAA ATGGATAAAA AGAATTTTTG ATTTTGTTTT GGCTTTGTTT n Y E K W I K R I F D F V L A L F I K N V -
TA'N'TGGGTG CTAGAAGTGA TATAGTAGAA CTTTTGCAAA Y L G A R S D I V E L L Q N
CTTTTGGTGC TTTTTTCGCC TCTTATTTTA ATCACTGCTT TGCTTTTAAA AATAACTCAA L I L I T A L L L K I T Q L L V L F S P
GGTAGTGTGA TTTTTACTCA AAATCGTCCA GGTTTAAATG AAAAAATTTT TAAAATTTAT N R P G L N E K I F K I Y G S V I F T Q
AARTTTAAAA CCATGAGTGA TGAAAGAGAT GAAAAGGGCG AGCTCTTAAG TGATGAGTTG E R D E K G E L L S D E L K F K T M S D
CGTTTGAAGG CTTTTGGGAA GATTGTTAGA AGTTTAAGCT TAGATGAGCT TWACAGCTT I V R S L S L D E L L Q L R L K A F G K
'I"AATGTAT TAAAGGGTGA TATGAGTTTT GTGGGTCCAA GACCTCTTTT AGTAGAATAC M S F V G P R P L L V E Y F N V L K G D
TTACCCCTTT ACAATGAAGA GCAAAAACTC CGCCATAAAG TGCGTCCTGG TATCACAGGA L P L Y N E E Q K L R H K V R P G I T G
TGGGCACAGG TAAATGGTAG AARTGCGATT TCTTGGCAAA AAAAATTTGA ACTTGATGTG W A Q V N G R N A I S W Q K K F E L D V
TATTATGTAA AAAATATTTC T m C T A C T A GATTTAAAAA TCATGTTCTT AACAGCCTTA D L K I M F L T A L Y Y V K N I S F L L
GGCAAGAACT GAAAAAATTT ACATTTATGG TGCGAGCGGA CATGGGCTTG TTTGTGCTGA G K N -
TGTCGCTACG AArPTGGGCT ATAAAGAATG T A T T T T m A GATGATTTTA AGGGTAAAAA
ATTTGAAAGC TCTTTGCCAA AATATGATAT C
AAAGAAGCGG AGTAAGCAAA GAAGGTCATG TTACAACGGA GAAATTTAAT R S G V S K E G H V T T E K F N
Fig. 6. The nucleotide and amino acid sequences of rfbF and rfbP. Possible ribosome-binding sites are underlined.
Determination of the nature of the cloned antigens
T o further elucidate the nature of the expressed antigen(s), whole-cell lysates of c. hyoilei strain RMIT-32A
and E. coli carrying the recombinant cosmid pBT9101 or plasmid pBT9105 expressing C. hyoilei antigen(s) were treated with proteinase K, resolved by SDS-PAGE and analysed by Western blotting (Fig. 4a). Proteinase Kresistant antigens of C. hyoilei RMIT-32A reacted with the anti-RMIT-32A antiserum at a single low molecular mass band with an apparent size of 7.5-8.5 kDa. Proteinase K-resistant antigens encoded by recombinant cosmid (or plasmid) reacted with a band of 13.5 kDa, indicating the glycolipid nature of the antigen. N o ladder-like pattern evident in Western blots of untreated total cell antigen could be detected. The molecular mass of the Campylobacter antigen(s) in proteinase K-treated E. coli transductants differed slightly from that of similarly treated cell lysates of C. hyoilei strain RMIT32A (Fig. 4a). Proteinase K-digested cell lysate of E. coli HBlOl carrying the cosmid vector pLA2917 with no insert did not react with the antiserum. T o determine if the antigen(s) encoded by the cloned DNA fragment were C. hyoilei LPS or LOS, the liposaccharides of the parent strain, RMIT-32A, and E. coli DH1 carrying recombinant plasmid pBT9105 (harbouring the 11.8 kb C. hyoilei RMIT-32A subfragment in pBR322 required for expression) were extracted using hot phenol, resolved by SDS-PAGE and the antigens analysed by Western blotting (Fig. 4b). Antisera reacted with the liposaccharides extracted from C. hyoilei RMIT-32A at a broad band of apparent molecular mass 7-5-10 kDa and E. coli DH1 carrying recombinant plasmid pBT910.5 reacted with a broad band of 13.515.5 kDa. N o ladder-like pattern was detected in the original C. hyoilei strain RMIT-32A or the recombinants. This showed that the cloned C. hyoilei antigen(s) were likely to be LOS rather then 'smooth' LPSlike structures typical of some C. jejuni strains (Aspinall et al., 1992). N o reaction was detected with the control E. coli strain DH1 (pBR322). Sequence homology of cloned C. hyoirei LOS genes with known LPS and LOS genes
The three outer EcoRV fragments, 600, 1000 and 1600 bp in length, from pBT9105 were cloned into pUC18 and their sequences were determined. The 1000 and 1600 bp fragments are situated adjacent to each other on clone pBT9105 (Fig. 5 ) . They contain two ORFs, designated rfbF and rfbP (Figs 5 and 6). Gene rfiF encodes 376 aa, and has a G C content of 31.5 o/' . It is homologous to a number of galactosyltransferases (Table 1).The rfbP gene encodes 200 aa and has a G C content of 32.3 %. The derived protein sequence shows homology with transferases involved in the coupling of the first sugar, in most cases a galactose residue, of 0antigen to a lipid carrier (Table 1).
The sequence of the 600 bp fragment revealed part of a potential ORF. The protein product is homologous to those encoded by the 3'-terminal regions of the rfaC genes of E. coli (Chen & Coleman, 1993) and Sal. 3485
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V. K O R O L I K a n d O T H E R S
Table 7. Comparison of C. hyoilei gene products with those of homologous genes
was used to compare the deduced gene products. Open gap cost, 6 ; unit gap cost, 2.
C . hyoilei
Similarity (To 1
39.0 39.0 38.5 37.0 35.0 26.9 25.8 25.0 23.1 23.1
5 6.0 535 57.5 51.5 53.5 43.4 40.7 40.7 41.1 40.0
Klebsiella pneumoniae Haemophilus influenzae Salmonella typhimurium Rhizobium sp. Erwinia amylovora Shigella dysenteriae Serratia marcescens Klebsiella pneumoniae Salmonella typhimurium Escherichia coli
gaisqdnie fdy-vdvlpr fayvevlpk
tpsy r n a f t i n l
_ - - -- - - - ---------
Arakawa et al. (1995) Fleischmann et al. (1995) Jiang et al. (1991) Gray et al. (1990) Bugert & Geider (1995) Klena et al. (1992) Szabo et al. (1995) Szabo et al. (1995) Sirisena et al. (1992) Chen & Coleman (1993)
cosmids was found to carry C. hyoilei RMIT-32A DNA inserted into the cosmid KmR gene in the opposite orientation to the others, negating the possibility of the initiation of transcription from the KmR gene promoter. Transcription initiation of Campylobacter spp. promoter(s) in E. coli has previously been suggested for the expression of the cloned gene encoding serine hydroxymethyl transferase (Chan et al., 1988). The identification of a strong Campylobacter promoter functional in E. coli is likely to be a helpful tool in the study of heterologous expression of cloned Campylobacter genes. The electrophoretic mobility and antigenicity of C. hyoilei RMIT-32A LOS have previously been reported to be similar in size to C. coli with low molecular mass LOS antigens (Kuan et al., 1992; Mandatori & Penner, 1989). Whether C. hyoilei RMIT-32A LOS is similar to that of the LPS of a rough phenotype of Salmonella spp. or similar to LOS of Neisseria spp. and Haemophilus spp. remains to be determined. However, as with some C. jejuni strains for which the latter has previously been suggested (Aspinall et al., 1992), the C . hyoilei liposaccharide is more likely to be a genuine LOS. In this study, following proteinase K treatment and hot phenol extraction, C. hyoilei RMIT-32A LOS antigen(s) and the recombinant antigen(s) expressed by pBT910.5 reacted with C. hyoilei antiserum with a single broad band of low molecular mass, as expected. N o ladder-like pattern characteristic of ‘smooth ’ C. jejuni was produced. The slight laddering effect which was consistently produced following Western blot analysis of untreated whole-cell lysates may be due to the aggregation of low molecular mass material. The recombinant C. hyoilei RMIT-32A LOS antigen(s) expressed by both pBT9101 and pBT9105 reacted with C. hyoilei antiserum with a band of higher molecular mass than that of the parent strain (Fig. 4). There are a number of possible events which can account for this difference in size. (a) The expression of the LOS gene cluster in a foreign host leads to imprecise assembly of the recombinant antigen. (b). Not all genes required for
ChRfaC lglts StRfaC plasl EcRfaC plasl
Fig. 7. Alignment of the C-terminal part of the tfaC gene products from Sal. typhimurium (StRfaC) and E. coli (EcRfaC) with the predicted translation product (ChRfaC) of the region of the rfaC gene of C. hyoilei that is present in recombinant plasmid pBT9105. Identical amino acids in the three sequences are marked (black boxes).
typhimurium (Table 1, Fig. 7) (Sirisena et al., 1992). These rfaC genes are involved in the synthesis of the LPS core oligosaccharide. They encode LPS heptosyltransferase-1. DISCUSSION
A C. hyoilei DNA sequence encoding the expression of LOS antigen(s) in an E. coli host has been cloned. The immunogenicity of the gene products expressed by the cloned C. hyoilei sequences, together with genetic similarity to rfb and rfa genes of other Gram-negative bacteria confirms that C. hyoilei LOS genes have indeed been cloned and expressed in E. coli. The expression of this Campylobacter spp. antigen in E. coli is a significant finding in view of the difficulties in expressing Campylobacter spp. genes in E. coli (Labigne-Roussel et al., 1987). Expression of the cloned LOS antigen(s) in E. coli from the native Campylobacter promoter appears possible. One of the four LOS-expressing recombinant -
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Cloning of Campylobacter LOS genes correct assembly of C. hyoilei RMIT-32A LOS are present in E. coli, instead some of the host E. coli cell LPS core components are being used. (c). Although the whole C. hyoilei RMIT-32A LOS gene cluster is present, some of the host cell LPS core components are being included regardless. The distribution of LOS extracted by hot phenol over a wider size range is probably due to the slight size alteration of some LOS molecules during the procedure. The minimum region required for expression of the C. hyoilei LOS antigen(s) identified in this study is 11.8 kb. Limited sequencing of the cloned 11.8 kb region showed sequence homology with rfbF, rfbP and rfaC genes from several bacteria. The proteins RfbF and RfbP are galactosyl transferases and the latter is involved in the coupling of the first sugar of an 0-antigen subunit to a lipid carrier. RfaC proteins are involved in biosynthesis of the LPS core of E. coli and Sal. typhimurium (Table 1, Fig. 7) (Chen & Coleman, 1993; Sirisena et al., 1992). The presence of galactose and heptose residues in all analysed Campylobacter spp. core molecules is in agreement with the genes encoding galactose and heptose transferases found within the cloned 11.8 kb region. Clustering of genes involved in LPS 0-antigen synthesis ( r f b ) or LOS outer core synthesis in the same chromosomal loci as genes involved in the inner core synthesis (rfa),is unusual. In E. coli the chromosomal map positions of rfb and rfa genes are 45 and 81 min, respectively (Bachmann, 1987). In Sal. typhimurium these genes can be found at map positions of 42 and 79 min (Sanderson & Hurley, 1987). In other Gramnegative pathogens which express LOS, such as Neisseria spp. and Haemophilus spp., LOS biosynthetic genes are not clustered at all (Preston et al., 1996). However, cloning of a gene cluster involved in LOS biosynthesis, also showing genes involved in inner core synthesis, has been reported for Bordetella pertussis (Allen & Maskell, 1995). The cloned gene cluster expressing C. hyoilei LOS antigen(s) may have a similar arrangement and possibly all the genes involved in LPS or LOS synthesis in Carnpylobacter are situated together in one cluster. Since Campylobacter spp. have a relatively small genome (Taylor, 1992) when compared with E. coli and other enteric bacteria, it could be of significant advantage for the organism to have all the LOS genes in one cluster. However, further characterization of the genes encoding C. hyoilei LOS is needed to facilitate the analysis of the molecular events involved in LOS synthesis in Campylobacter spp. and to clarify why the LOS genes are clustered and whether this clustering is also characteristic of other Campylobacter spp. REFERENCES Alderton, M. R., Borland, R. & Coloe, P. 1. (1991). Experimental
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