Shigella flexneri type-specific antigen V: cloning, sequencing and characterization of the glucosyl transferase gene of temperate bacteriophage SfV

Gene 195 (1997) 207–216

Shigella flexneri type-specific antigen V: cloning, sequencing and characterization of the glucosyl transferase gene of temperate bacteriophage SfV Pham thi Huan a,b, Belinda L. Whittle a, David A. Bastin a, Alf A. Lindberg b, Naresh K. Verma a,* a Division of Biochemistry and Molecular Biology, Faculty of Science, School of Life Sciences, The Australian National University, Canberra, ACT 0200, Australia b Department of Immunology, Microbiology, Pathology and Infectious Diseases, Division of Clinical Bacteriology, Karolinska Institute, Stockholm, Sweden Received 4 November 1996; accepted 29 January 1997; Received by M. Salas

Abstract With lysogeny by bacteriophage SfV, Shigella flexneri serotype Y is converted to serotype 5a. The glucosyl transferase gene ( gtr) from bacteriophage SfV of S. flexneri, involved in serotype-specific conversion, was cloned and characterized. The DNA sequence of a 3.7 kb EcoRI–BamHI fragment of bacteriophage SfV which includes the gtr gene was determined. This gene, encoding a polypeptide of 417 aa with 47.67 kDa molecular mass, caused partial serotype conversion of S. flexneri from serotype Y to type V antigen as demonstrated by Western blotting and the sensitivity of the hybrid strain to phage Sf6. The deduced protein of the partially sequenced open reading frame upstream of the gtr showed similarity to various glycosyl transferases of other bacteria. Orf3, separated from the gtr by a non-coding region and transcribed convergently, codes for a 167 aa (18.8 kDa) protein found to have homology with tail fibre genes of phage lambda and P2. © 1997 Elsevier Science B.V. Keywords: Bacteriophage SfV; Glucosyl transferase gene; Phage conversion; Shigella flexneri

1. Introduction Shigella flexneri is a major cause of bacillary dysentery in developing countries (Bennish and Wojtyniak, 1991; Mikhail et al., 1990; Zaman et al., 1991) and is responsible for high morbidity and mortality, particularly in children under 5 years of age ( Ferreccio et al., 1991; Kagalwalla et al., 1992). Shigella flexneri is divided into various serotypes based on the combination of antigenic determinants present in the O polysaccharide chains of the cell envelope lipopolysaccharide (LPS ). This O polysaccharide is a polymer of tetrasaccharide repeating units: [-2)-a--RhaI-(1-2)-a--RhaII-(1-3)-a--RhaIII(1-3)-b--GlcNAcp-(1-] shared by all serotypes except * Corresponding author. Tel. +61 6 2492666; Fax: +61 6 2490313; e-mail: [email protected] Abbreviations: aa, amino acid; AB, Applied Biosystems; bp, base pair; GCG, Genetic Computer Group; gtr, glucosyl transferase; kb, kilobase; LPS, lipopolysaccharide; ORF, open reading frame; Sf6, bacteriophage 6; SfV, bacteriophage V; SfX, bacteriophage X. 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 1 44 - 3

serotype 6 ( Fig. 1). The basic tetrasaccharide polymer is the O-antigen of S. flexneri serotype Y and is designated as having group 3,4 antigen specificity ( Kenne et al., 1978). Antigenic differences arise by the addition of a glucosyl and/or O-acetyl residue to a specific position on the basic tetrasaccharide repeating unit. Thus, strains expressing type V antigen have a glucosyl residue attached to the RhaII of the tetrasaccharide repeating unit (Fig. 1) ( Kenne et al., 1977). Genetic studies have revealed that the rfb (Macpherson et al., 1991) gene cluster and the rfc (Morona et al., 1994) gene coding for tetrasaccharide O-unit biosynthesis and polymerization, respectively, map adjacent to the his locus. Genes coding for Oacetylation and glucosylation, carried by temperate phages, are integrated near the pro-lac region on the S. flexneri chromosome (Petrovskaya and Licheva, 1982). Bacteriophage V (SfV ) is responsible for type V antigen of S. flexneri. Shigella flexneri serotypes 5a and 5b are a consequence of the lysogenization of S. flexneri serotype Y and X by SfV, respectively.

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Fig. 1. Shigella flexneri O-antigen structures. Rha, rhamnose; GluNAc, N-acetylglucosamine.

Although a number of candidate S. flexneri vaccine strains have been constructed by various groups in the last 10 years, to date there is no vaccine available to control S. flexneri infections. SFL124 (DaroD), belonging to serotype Y, is an attenuated candidate vaccine strain (Lindberg et al., 1988, 1990) which has recently been shown to be safe and immunogenic in human volunteers (Li et al., 1993). It has been reported that natural S. flexneri infections in humans result in the production of an antibody response specific to the serotype of the infecting strain. Therefore it is likely that a vaccine must incorporate different serotypespecific antigens in order to induce an immune response against other serotypes of S. flexneri. This could be achieved by cloning the O-acetyl or glucosyl transferase ( gtr) gene from temperate phages and then introducing them to SFL124 to create a hybrid vaccine strain which

would have modified O-antigens on its surface. The Oacetyl and the gtr genes of phage Sf6 and SfX, respectively, have been cloned and characterized previously ( Verma et al., 1991, 1993). In this paper we describe the cloning and sequencing of the bacteriophage SfV gene encoding glucosyl transferase, and the expression of the type V antigenic determinant in a serotype Y strain.

2. Materials and methods 2.1. Bacterial strains, phages and plasmids Bacterial strains, phages and plasmids used in this study are shown in Table 1.

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P.t. Huan et al. / Gene 195 (1997) 207–216 Table 1 Bacterial strains, phages and plasmids Strain/plasmid/phage Strains JM109 SFL124 EW 595/52 SFL1161 SFL1164 SFL1070 Y53 Y379 F2291 Plasmids pUC19 pNV143 pNV307 pNV318 pNV320 pNV335 Phages SfV Sf6 SfX

Relevant characteristics

Source/Reference

supE44, nalA96, recA1, relA1, endA1, thi, hsdR17, D( pro-lac) [F ∞, traD36, proAB, lacIq, zDM15] Serotype Y DaroD Serotype 5a lysogen SFL124/pNV307 SFL124/pNV318 S. flexneri 2a DaroD S. flexneri 1a S. flexneri 3a S. flexneri 5a

Yanisch-Perron et al. (1985) Lindberg et al. (1988) P. Gemski Jr. This study This study Verma and Lindberg (1991) SIIDC SIIDC SIIDC

gtr gene gtr gene (7.2 kb EcoRI ) gtr gene (3.7 kb EcoRI/BamHI ) gtr gene (3.7 kb opposite orientation) 2.0 kb EcoRI/PstI

Yanisch-Perron et al. (1985) Verma et al. (1993) This study This study This study This study This study Verma et al. (1991) Verma et al. (1993)

SIIDC, Swedish Institute for Infection Disease Control.

2.2. Bacterial growth conditions Bacteria were grown in Luria broth (LB) and Luria agar (LA) supplemented with ampicillin 50 mg/ml, 5-bromo-4-chloro-3-indolyl b--galactopyranoside ( Xgal ) and isopropyl b--thiogalactopyranoside (IPTG) 20 mg/ml as necessary. 2.3. Chemicals, restriction endonucleases and antibodies Restriction endonucleases, T4 DNA ligase, DNase I and RNase I were obtained from Pharmacia. Proteinase K and alkaline phosphatase were from BoehringerMannheim and Erase-a-Base kit was purchased from Promega. Monoclonal antibodies MASF V (monoclonal antibody recognizes a--Glu-1-3-a-L-Rham II, the antigenic determinant of type V antigen of S. flexneri) and MASF Y-5 (monoclonal antibody against group antigen 3,4 of S. flexneri) (Carlin and Lindberg, 1983, 1987) were a gift from Nils Carlin. Anti-mouse Ig alkaline phosphatase conjugate was purchased from Sigma. 2.4. Phage methods SfV temperate phage was induced from its host strain EW 595/52 by UV irradiation. In brief, 5 ml of a EW 595/52 overnight culture in LB was spun down for 8 min at 3000×g in a Sorvall RT6000B refrigerated centrifuge,

and the pellet was resuspended in 2.5 ml of 10 mM MgSO . The bacterial suspension was exposed under a 4 germicidal lamp at 260 nm wavelength for 30 s. The suspension was diluted in LB, protected from light by wrapping the tube with aluminium foil and then incubated at 37°C with shaking for 5 h. The culture supernatant was collected by filtration through a 0.45 mm microfilter. The supernatant was mixed with the SFL124 culture, in the presence of 0.01 mM CaCl , and incu2 bated at 37°C for 20 min. The mixture was then mixed with 3 ml of soft agar and poured on a LA plate. Plates were incubated at 37°C overnight. Single plaques were picked, purified and used to infect SFL124 to check for serotype conversion. The DNA of phage stock which converted SFL124 from serotype Y to serotype 5a was extracted according to the method for bacteriophage l (Maniatis et al., 1982). 2.5. Phage sensitivity test A plate flooded with an overnight bacterial culture was left to dry at 37°C for 20 min. One drop of each phage was applied to the lawn of bacteria and allowed to absorb completely, then the plates were incubated face down at 37°C overnight. The strain was said to be sensitive to the phage if a clear zone of no growth was seen in the bacterial lawn where the phage drop had been placed.

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2.6. Plasmid DNA preparation, transformation and electroporation

to the recommendation of the producer. Sequencing was performed by an AB 373A automatic sequencer.

To clone SfV DNA fragments, EcoRI-digested SfV DNA was ligated with the EcoRI-digested, dephosphorylated pUC19 vector DNA and transformed into an E. coli strain (JM109). Plasmid DNA was prepared by alkaline lysis according to the method of Birnboim and Doly (1979). Plasmid DNA for sequencing was treated with RNAse before running a reaction according to the recommendation of the supplier. Transformation by the CaCl procedure was performed as described according 2 to the recommendations of the Erase-a-Base kit’s supplier (Promega). Electroporation was performed in 0.2 cm cuvettes with a Gene-pulser (Bio-Rad ), following the manufacturer’s instructions.

2.9. DNA sequence analysis Sequence data were stored and analysed by the University of Wisconsin Genetic Computer Group (GCG) sequence analysis program. Amino acid similarities were determined with the program BESTFIT and multiple alignments displayed in PILEUP. BLAST and FASTA, available at the National Center for Biotechnology Information, were used to detect protein and nucleotide similarities. Hydropathy plots were achieved with the program PEPPLOT ( Kyte and Doolittle, 1982).

2.7. Antibodies and immunoblotting

3. Results and discussion

LPS whole cell lysates were prepared as described by Hitchcock and Brown (1983). LPS was analysed by SDS–PAGE on 13.5% acrylamide separating gels. Whole-cell immunoblotting was performed as described earlier (Huan et al., 1995).

3.1. Cloning and expression of the gtr gene of SfV

2.8. DNA sequencing The Erase-a-Base method was used to create a set of nested deletions from each end of the insert according to the recommendations of the supplier (Promega). The deletion plasmids were sequenced by using reverse or forward dye primer kits purchased from Applied Biosystems (AB). The gaps in the sequence were filled by using appropriate primers and the dye terminator kit. Oligonucleotides were synthesized by a 380B AB DNA synthesizer and purified by 1-butanol according

EcoRI-digested SfV DNA was ligated with pUC19 vector DNA and transformed into E. coli JM109. Four plasmid clones containing SfV DNA fragments of different size were identified. The plasmids were then transformed into a S. flexneri serotype Y strain, SFL124, expressing group 3,4 antigen. Monoclonal antibodies of type V antigen specificity (MASF V ) and group 3,4 antigen specificity (MASF Y-5) were used in agglutination tests to detect serotype conversion of the recombinant strains. One of the four recombinant clones, harbouring a 7.2 kb EcoRI fragment of SfV DNA (pNV307), expressed type V antigen as well as group antigen 3,4 and was designated SFL1161. To locate further the gene responsible for type V antigen, several restriction enzymes were used to map

Fig. 2. Expression of phageV gtr by plasmid clones. (A) Restriction map of plasmid clones. Restriction enzymes used: EI, EcoRI; Ba, BamHI; EV, EcoRV. (B) Serotype conversion to type V as determined by slide agglutination, and sensitivity to phage V and phage 6. S, Sensitive.

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the 7.2 kb fragment (Fig. 2). A plasmid (pNV318), derived from pNV307 and carrying a 3.7 kb EcoRI–BamHI insert, expressed the type V antigen when it was transformed into SFL124. The resulting strain was named SFL1164. Immunoblotting was used to check the expression of type V antigen and to confirm the conversion of serotype Y to 5a of the hybrid strains. The expression of type V antigen detected in Western blots showed that MASF V bound to F2291 (5a wild-type strain) and two recombinant strains but did not bind to SFL124. In contrast, MASF Y-5 bound to SFL124, SFL1161 and SFL1164 but did not bind to F2291 ( Fig. 3). The binding of both monoclonal antibodies to extracted LPS from the two recombinants revealed that the strains are hybrids of type V and factor 3,4 specificity and that plasmids pNV307 and pNV318 confer the glucose modification characteristic of type V antigenic conversion. As the group 3,4 antigen is considered to be the receptor for phage Sf6 (Lindberg et al., 1978), sensitivity to Sf6 was used to check the elimination of 3,4 antigen determinants on the cell surface of the hybrid strains. Both recombinant strains SFL1161 and SFL1164 were sensitive to SfV and phage Sf6, while F2291 was resistant to both SfV and Sf6 ( Fig. 2). These data, as well as the immunoblotting results, strongly indicated that the gtr gene carried by both plasmids (pNV307 and pNV318) could add the glucosyl residue to only a portion of

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repeating units of strain SFL124 (partial serotype conversion). Plasmid pNV318 was subsequently transformed into strains SFL1070 (serotype 2a) ( Verma and Lindberg, 1991), Y53 (serotype 1a) and Y379 (serotype 3a) to see whether the gtr gene of another S. flexneri serotype would interfere with the function of the gtr gene of SfV; surprisingly, each of the recombinant strains expressed both serotype V antigen and its native serotype antigen, as tested by slide agglutination. 3.2. Sequence analysis The nucleotide sequence of the 3.7 kb EcoRI–BamHI fragment in pNV318 was determined ( Fig. 4). Three complete and two incomplete open reading frames (ORFs) were found ( Table 2). The orf1 distal portion (orf1∞), orf2 and orf3 are located in the upper strand, transcribed from left to right. The orf4 with 1251 nucleotides and the orf5 distal portion (orf5∞) are transcribed from right to left. All three complete ORFs begin with the ATG codon, while the termination codons are TAA for orf1∞, orf 3 and orf4, TAG for orf2 and TGA for orf5∞. The 137 aa and 112 aa deduced from orf1∞ and orf2, respectively, showed no homology with any known protein. orf2 is separated from orf3 by a non-coding region of 223 nucleotides. The start codon of orf3 is at

Fig. 3. (A) Immunoblotting of S. flexneri LPS. Lanes 1–4 show LPSs from S. flexneri SFL1164, SFL1161, SFL124 and F2291. Monoclonal antibody specific to group antigen 3,4 (MASF Y-5) was used as first antibody. (B) Immunoblotting of S. flexneri LPS. Lanes 1–4 show LPSs from SFL1164, SFL1161, SFL124 and F2291. Monoclonal antibody specific to type V antigen (MASF V ) was used as first antibody.

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Fig. 4. Nucleotide sequence of the 3.7 kb BamHI–EcoRI phage SfV fragment. The gtr sequence starts from nucleotide position 2878 from the 3∞ end and stops at position 1628. The putative regulatory regions are marked as follows: S.D. (underlined ), Shine-Dalgarno (ribosome-binding) site; ATG, start codon; −10 and −35 (underlined and italics), potential promoter-binding region. A potential terminator is denoted by italics and underlining. The sequence has been deposited in GenBank under accession No. U82619.

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Fig. 4. (continued )

Table 2 Summary of ORF/gene EcoRI/BamHI clone

properties

Gene

% G+C content

Number of aa

orf1∞ orf2 orf3 orf4 orf5∞

58.1 55.1 40.6 33.4 40.12

137 112 167 417 283

of

phage

Gene

gtr

SfV

3.7 kb

Position in sequence 1–412 439–774 997–1497 2878–1628 3726–2877

position 997. The 167 aa deduced from orf3 showed 30%, 33% and 34% identity to the protein products of bacteriophage P2 gene G (Haggard-Ljungquist et al., 1992), S. boydii orfB175 ( Tominaga et al., 1991) and to phage l tfa (Montag and Henning, 1987) (formerly called orf194), respectively, all of which are thought to be involved in the assembly of tail fibres. Interestingly, Orf3 has higher identity at the carboxy terminal of the protein to all of the mentioned genes. It is 50%, 52% and 56% identical at the Y region (Haggard-Ljungquist et al., 1992) of P2 gene G, l tfa and S. boydii orfB175,

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Fig. 5. Alignment of SfV Orf3 with product of bacteriophage P2 gene G (P2 G), S. boydii orfB175 and l tfa. PILEUP program was used for alignment with gap weight 6.000 and gap length weight 0.100. Bold letters indicate the identical aa residues among them, and asterisks denote conserved substitutions.

respectively (Fig. 5). Based on the similarity of the SfV orf3 product with those of l tfa and P2 G, especially in the carboxy termini, it appears that orf3 of SfV may be involved in tail fibre assembly. The genes encoding tail fibres in bacteriophage l and P2 have been studied and found to be in a cluster (Lengyel et al., 1974; Lindahl, 1974; Temple et al., 1991). Although Orf1 and Orf2 do not show homology with any known protein, their location indicates that they might also code for tail fibre proteins. However, further studies are required to confirm the function of these ORFs. A potential promoter was found upstream of orf4 inside orf5∞ with a −10 region (5∞ TATATT ) and −35 region ( TGTTCT ); the distance between the two regions is 22 bp. As the lac promoter of the vector is in the opposite direction to orf4 in pNV318 it suggests that the gtr gene is driven by its own promoter in the insert or by another vector promoter which is reading from the other end. A long palindromic sequence was also found at the 3∞ end of orf4, suggestive of a transcriptional stem loop structure ( Fig. 4). Orf4 was 35.3% identical to the product of the gtr gene of SfX (Fig. 6). The PEPPLOT of Orf4 was hydrophobic and its pattern looked very similar to the PEPPLOT pattern of gtr of SfX (data not shown). orf5∞ is 849 nucleotides in length and its stop codon has a 4 nucleotide overlap with the start codon of orf4. Interestingly, the 283 aa orf5∞ is 49% identical to a hypothetical protein of Synechocystis sp. ( Kaneko et al., 1995), and has a low level of similarity to known or

likely glycosyl transferases of the other bacteria (data not shown). It is interesting to note the variation (33–55%) in the G+C percentage of all five ORFs ( Table 2). While orf2 and orf3 have 55.1% and 40.6% G+C, respectively, orf4 has the lowest G+C of 33.4%. This seems to suggest that genes such as gtr might have been imported into the phage SfV genome from some other microorganism of low G+C content DNA. In conclusion, we have identified and characterized the gtr gene from bacteriophage SfV. The gtr gene alone could not completely convert S. flexneri SFL124 from serotype Y to serotype 5a. These observations suggest that more than one gene may be required in order to have full conversion to the type V antigen. In fact, characterization of DNA present downstream of orf5∞ has revealed that at least three genes are needed for complete antigenic conversion. The results are presented in the following paper ( Huan et al., 1997).

Acknowledgement We would like to express our sincere thanks to Kathy Smith and Wafa El Adhami for their help in sequence analysis, and to P. Gemski Jr. for providing the strain EW595/52. This work was supported in part by grants from the Lederle-Praxis Biologicals, and the Swedish Agency for Research Co-operation with Developing Countries (SAREC ).

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Fig. 6. Alignment of phage SfV (upper line) and SfX ( lower line) deduced gtr amino acid sequences. The sequence alignment was obtained with the BESTFIT program, with gap creation penalty 5.00 and gap extension penalty 0.30.

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