Characterization of a lipopolysaccharide encoded by a recombinant Shigella sonnei plasmid in Escherichia coli K-12

Zbl. Bakt. 277, 419-428 (1992 ) © Gustav Fischer Verlag, Srurrgart/New York

Characterization of a Lipopolysaccharide Encoded by a Recombinant Shigella sonnei Plasmid in Escherichia coli K-12 GUNTRAM SEL TMANN I , YURIY A . KNIREL 2, ALEXANDER S . SHA SHK O V 2 , and HELMUT TS CHApE 1 1

2

Robert -Koch-Institu r des Bundesgesundheitsamts, Bereich Wernigerode, 0-3 700 Wernigerode, Germany N. D. Zelinskij Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia

With 5 Figures· Received February 25, 1992 . Revision received Ma y 11, 1992 . Accepted Jun e 29, 1992

Summary Th e genetic inform ation to synthesize the S-specific region of Shigella sonnei phase I lipopol ysaccharide (LPS) is localized on a 180 kb plasmid which is lost quite readily. A recombinant plasmid derivat ive remaining stable in the bacteri a was shown to determine the S-specific region of the LPS which is completely identical with that of a S. sonnei pha se I strai n following tran sfer in Escherichia coli K-12. However, the length control in polysaccharide biosynthesis is lost at least part ially.

Zusammenfassung Shigella sonnei Phase l-Stamme besitzen ein 180 kb-Plasmid , das fur die Biosynthese der S-spezifischen Region des Lipopolysaccharides (LPS) kodiert. Das Plasmid und damit die Phase I-Eigenschaften gehen im allgemeinen schnell verloren. Ein rekombinantes Plasmidderivat bleibt stabil in den Bakterien. In Escherichia coli K-12 bildet es die serologischen S. sonn ei Phase I-Eigenschaften aus. In dieser Arbeit wird gezeigt, daf das Plasmidderivat die Biosynthese einer S-spezifischen LPS-Region determiniert, die mit der eines S. sonnei Phase l-Srammes vollstandig identisch ist. Allerdings ist die Kontrolle der Lange (= Zahl der "r epeating units" ) bei der Biosynthe se der S-spezifischen Region zumindest teilweise verloren gegangen.

Shigella so nne i strai ns generally occur in two modification s named ph ase I and phase II. Phase I represents an S form and is viru lent, phase II repre sents an R form (rype R1 ; 14 ) and is avirulent. Phase I stra ins rather frequently undergo a spontane ou s con ver-

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G. Seltrnann, Y. A. Knirel, A. S.Shashkov, and H. Tschape

sian to phase II, sometimes one or two passages on nutrient agar surfaces are sufficient to fully produce this effect. This observation is explained by the fact that the genetic determinants encoding for the complete phase I 0 antigen is localized on a 180 kb plasmid (Vir plasmid), which can be lost quite readily (10). The reason for such an instability is unknown. Since similar Vir plasmid segregations have been observed in other bacterial hosts, too (10), either the plasmid replication is insufficient or the plasmid load (e. g. by 0 antigen formation) might be disadvantageous during subcultivation. Observations that phase II segregants often contain deleted Vir plasmid DNA (which is now stably maintained) and that the piece of deleted DNA comprises the genes for invasion (ipa) and for 0 antigen synthesis (ssa; Tscbdpe, unpublished data) would imply that phase I cells of S. sonnei could be overgrown by the rough phase II segregants. However, O-antigen-determining plasmid derivatives have been obtained after cointegrate formation with pTHI0 (18) which are now stably maintained. This acquired stability could be either of plasmid origin or due to an altered 0 antigen structure. In order to decide between both possibilities, we comparatively characterized the 0 antigenic lipopolysaccharides (LPS) synthesized i. by an Escherichia coli K-12 substrain carrying the stable plasmid variant, ii. by the same strain without plasmid, and iii. by the parental S. sonnei strain by several serological and (physico-) chemical methods. Both S-type LPS (i. and iii.) have been shown to be identical in their S-specific but not in their R-specific part. However, the length control in the course of the Sspecific polysaccharide biosynthesis is lost at least partially.

Materials and Methods Strains. E. coli K-12 substrains, S. sonnei wild type strain 9773/63, and the plasmids used throughout these experiments are listed in Table 1. Plasmid transfer procedures. The transfer of S. sonnei Vir plasmid was carried out on solid media at 40°C in the presence of 100 I-lg ampicillin and 50 I-lg kanamycin as described by Watanabe and Nakamura (18), using pTH10 as mobilizing agent. This procedure implies a cointegrate formation via Tn1 which in turn can lead to various recombinant molecules. Recombinant plasmids have been detected in E. coli K-12 strains by agarose gel electrophoresis of plasmid DNA extracted from the respective E. coli K-12 strains encoding for Shigella 0 antigens. These recombinant plasmids were further characterized by standard plasmid methods as described by Tschape et al. (16) and by plasmid DNA fingerprints according to Grinsted and Bennett (5) using the restriction enzymes HindIII, EcoRI, and EcoRV. Furthermore, the recombinant plasmids have been characterized with regard to the presence of the ipa-gene cluster (invasion plasmid antigen) by hybridization studies using the respective gene probe. The plasmid pIE1204 was applied for generation of the ipaB probe (Prager and Tschdpe, unpublished). Hybridization was carried out as described by Grinstead and Bennett (5), under stringent conditions. Origin of sera. All sera (5. sonnei 9773/63 and E. coli IE 1351) were produced according to standard methods (13). E. coli K-12 serum was a kind gift from Lore Brade, Borstel. Isolation of lipopolysaccharides. The bacteria were cultivated in nutrient broth for 6 h at 37°C under intensive stirring and aeration, collected by centrifugation, washed once with saline, and freeze-dried. They were extracted with phenol/water as described by Westphal and ]ann (19). The pooled water phases were dialyzed intensively, then concentrated in vacuo to about 50% and centrifuged 2 hat 105000 g. The sediment was dissolved in water, checked for the absence of materials absorbing at 260 nm, recentrifuged, if necessary and freeze-dried.

LPS Encoded by a Recombinant Plasmid

421

Isolation and purification of a -specific polysaccharides. A 1% solutio n of the LPS in 2% acetic acid was heated for 2 hour s at 100 °C (water bath ). The precipitate was removed by centrifugation. The supernatant was concentrated in vacuo (below 40 °C) and separated by chromatography on a column (46 X 1.7 ern) of Sephadex G-50 in pyridine : acetic acid : water = 4 : 10 : 986 at a flow rate of 1 mllmin. Fractions were collected and analyzed by the orcinol-sulphuric acid reaction with the help of a Technicon Autoan alyzer II sugar detection system at 490 nm. Fract ions conta ining the high-molecular weight material (peak I) were combined, concentrated in vacuo, and freeze-dried. The material s of peak I were dissolved in water and oxidized by 0.1 M sodium metaper iodate (36 h, 20 °C, in the dark ), the excess of oxidizer was destroyed by ethylene glycol, reduced with sodium boro hydride (1 h, 25 °C), dialyzed against distilled wat er and evaporated in vacuo. The residue was heated in 2% acetic acid (2 h, 100 °C), dialyzed against distilled water , and freeze-dried. Immunodiffusion. It was perform ed in agar gels according to Behm (63). 1% solutions of each LP5 (prepared as described by 0rskov and 0rskov, 12) were dropp ed into the outer wells according to the scheme presented in Fig. 1. The centra l wells contained sera against S. sonnei phase I, E. coli K-12, or E. coli IE 1351. The plates were stored at room temperature in a moist chamber for 24 hours and afterwards in saline for 48 hour s to remove unspecific pro teins. After drying, the gels were stained in a 0.5 % solution of Amido black lOB in methanol/acetic acid (8 : 1) and destained in the same solvent . Discontin uous polyacrylamide gel electrophoresis (PAGE) . The method was applied in two modes i.e., - in the presence of sodium dod ecyl sulphate (50 S-PAGE) according to Kusecek et al. (13), however, using 11% acrylamide and 0.2% methylene bisacrylamide without gradient and - in the presence of sodium deoxycholate (DOC-PAGE): the composition of both the gels and the buffer solutions was the same as in the case of SOS-PAGE, however, SOS was substituted by DOC in a fivefold concentration (1). In both cases, bands were visualized by silver staining (18). 13C-NMR spectroscopy. It was performed on a Bruker AM-300 instrument for the solutions in 0 20 at 30 °C using acetone (b = 31.55 ppm) as the internal standard. Result s Characterization of strain s and plasmids S. sonnei 9773 /63 contained a 180 kb DNA mo lecule only, whic h represente d the S. sonnei-spec ific Vir plasmid. Thi s plasmid was transferred, by means of pTH I0, to the E. coli K-12 derivative CV601. Plasmid-containing strains were isolated that expressed the S. sonnei LPS. Among th ese exco njugant E. coli K-12 strai ns, some plasmi d derivatives could be detected that were 125 kb in size only and remained stable under E. coli K-12 conditions. Such a derivat ive plasmid was designated pIE988 (T able 1). As characterized by hybr idization pattern and genetic tests (data not shown), pIE9 88 consists of complete pTHI0 DNA, additionally to a piece of 55 kb Vir plasmid DNA co mprising the det erminant s for LPS synthesis and invasion (ipa). Characterization of LPS by im m unodiffusion On diffusion agai nst a S. sonnet ph ase l-serum, the LPS of both S. sonnei 9773/63 and E. coli IE1351 yielded one single pre cipitation line each, showing complete fusion (Fig. 1). The LPS of E. coli C V60 1 d id not react with this serum. On the oth er han d, this LPS formed a strong line o n diffu sion against the K-12 seru m. Th e same line was formed, with much less intensity by the LPS from E. coli IE1351 , but not by the S. sonnei 977 3/63 LPS. Against seru m lE1351, the LPSs beha ved as against serum 9773/ 63. However, LPS IE 135 1 formed a second (weaker) band , pres um abl y due to the presence of some unsubstituted K-12 LPS and K-12 antibodies.

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G. Seltmann, Y. A. Knire!, A. S.Shashkov, and H. Tschape

Table 1. Strains and plasmids used throughout this study Designation

Relevant properties

References

Plasmids pTHI0 pIE988

57 Kbp IncPl 125 Kbp IncPl

18 this paper

Tc Am Km Tc Am Km ipa ssa

Strains E.coliK-12 CV601 J53 IE1351

plasmid-free; thr leu thi lac rif" plasmid-free pIE 988 in CV601

16 4 this paper

S.sonnei 9773/63

Vir; drug-sensitive

15

Abbreviations: Tc - tetracycline resistance; Am - ampicillin resistance; Km - kanamycin resistance; rifR - rifampicin resistance; Kbp - Kilobase pairs; ipa - invasion plasmid antigen; ssa - S. sonnei antigen (0 antigen); leu -leucine; thr - threonine; thi - thiamine; laclactose; vir - virulence.

PAGE of the LPS

In SDS-PAGE, both S-rype LPS yielded the typical ladder-like patterns. The arrangement of the bands in the case of both S. sonnei 9773/63 and E. coli IE1351 was identical, indicating the same molecular weight of the repeating units (Fig. 2). However, in the case of strain 9773/63, a bimodal structure was much more pronounced than in the case of strain IE 1351. In DOC-PAGE (Fig. 3), it was clearly visible that the

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Fig. 1. Immunodiffusion of LPS from S. sonnei 9773/63 (1), E. coli IE1351 (2), and E. coli CV601 (3) towards sera against S. sonnei 9773/63 (I; absorbed with phase II bacteria), E. coli K-12 (II), and E. coli IE 1351 (III).

LPS Encoded by a Recombinant Plasmid

423

lipid A-core regions (see 12) of S. sonnei 9773/63 on the one hand and E. coli IE1351 on the other were quite different. On the contrary, the lipid A-core region of the E. coli 1E1351 LPS was very similar to that of the parental strain CV601.

1

2

Fig. 2. SDS-PAGE of LPS from S. sonnei 9773/63 (lane 1) and F. coli IE1351 (lane 2).

123 Fig. 3. DOC-PAGE of LPS from E. coli lE1351 (lane 1), E. coli CV610 (lane 2) and S. sonnei 9773/63 (lane 3).

Gel chromatography of the O-specific polysaccharides on Sephadex G-50 Only the polysaccharides of strains S. sonnei 9773/63 and E. coli IE13S1 were separated. The elution profiles of both polysaccharides were nearly identical (Fig. 4). In both cases, peak I contained the O-specific polysaccharide, however, as could be shown by 13C-NMR analysis, they were contaminated by an a-1.4-glucan (carbon signals at 62.0, 72.7, 72.9, 74.6, 78.9, and 101.0 ppm). Peak II was formed by the Rspecific polysaccharides, and the small peak III, by KDO phosphate. The magnitudes of peaks I and II were comparable in both cases; peak III was more significant in the case of the plasmid-containing E. coli K-12 derivative. Purification of the O-specific polysaccharide The polysaccharides present in peaks I were further purified by periodate oxidation and subsequent mild acid hydrolysis as described in "Materials and Methods". The 0-

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G. Seltmann, Y. A. Knirel, A. S.Shashkov, and H. Tschape

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Fig. 4. Ge! chromatography on Sephadex G-50 of the O-specific polysaccharides from strains S. sonnei 9773/63 and E. coli IE 1351 (= 1250). For experimental details see "Materials and Methods".

specific polysaccharide proved to be stable under these conditions. After this purification, the sample was free of the glucan (13C-NMR control). 13C_NMR spectroscopic investigations

In Fig. 5, the 13C-NMR spectra in the range between 13 and 111 ppm and 172 and 180 ppm are shown and it is seen that they are identical. Moreover, they are identical with the spectra presented by Kenne er al. (8), by means of which they elucidated the structure of the S-specific region of the S. sonnei LPS. As shown in Table 2, the chemical shifts were in agreement with the proposed chemical structure (see Discussion).

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100

90

80

70

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50

40

30

20

Fig, 5 . 13C-NM R spectra of the O-specific polysacchar ides from strai ns S. sonnei 9773/63 (lower lane) and E. coli IE l35l (up per lane).

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Table 2. Chemical shifts (o, ppm) for the polysaccharides of both S.sonnei 9773/63 and E. coli IE1351 Unit

C-l

C-2

C-3

C-4

C-5

C-6

-3)-~-D-FucNAc4N-(1­

104.0 102.1

52.2 53.0

76.9 69.2

56.1 79.0

68.4 78.7

16.6 175.0

A)-a-L-AltNacA-( 1-

Chemical shifts for N-acetyl groups: 23.4,23.6 (CH3), 175.5 (CO). Abbreviations: FucNAc4N = 2-acetamidoA-amino-2.4.6-trideoxygalactose, AltNAcA 2-acetamido-2-deoxyaltruronic acid.

Discussion Similarly to other enterobacterial LPS, the LPS of S. sonnei phase I consist of an Rspecific moiety (core plus lipid A) to which an S-specific polysaccharide is bound (9). This polysaccharide consists of repeating units each containing two unusual sugars, namely 2-acetamido-2-deoxy-L-altruronic acid (AltNAcA) and 2-acetamido-4-amino2.4.6-trideoxy-D-galactose (FucNAc4N). The S-specific polysaccharide chain is made up of repeating units, its structure has been elucidated (8) to be 1-+4)-aAltpNAcA-(l-+3)-~FucpNAc4N-(l-]n'

The information to synthesize the S-specific region of S. sonnet LPS has been found to be located on a 12.6 kb DNA sequence of a 180 kb virulence plasmid, which can be encoded readily under E. coli K-12 conditions (6; 11; 20). This plasmid (Vir plasmid) can be transferred to S. f/exneri 2a, Salmonella typhi, S. typhimurium, E. coli K-12, Proteus mirabilis, and Serratia marcescens (11). All transconjugants were observed immunologically to express the phase I 0 antigen besides the antigens of the parental strains. The proof of the intact phase I 0 antigen was given only by means of serological methods. However, examples are known indicating that serological cross-reactivity can be produced by LPS, the S-specific regions of which are chemically different (7). Thus a chemical proof of the structural identity has remained open up to now. It was stated (10) that even under E. coli K-12 conditions, the plasmid remained unstable. Segregants of 50 to 95% of the population have been observed after overnight culture in spite of its belonging to the common IncFI group. A recombinant plasmid derivative of smaller size (125 kb) was obtained determining synthesis of phase I LPS but remaining stable in the bacteria. This plasmid was characterized to consist of the complete pTHI0 genome, additionally to a 55 kb large stretch of Vir plasmid DNA. This piece of DNA encoded the 0 antigen biosynthesis and the invasion properties as demonstrated by hybridization studies (ipa probe) and throughout this study. It was the aim of the present study to examine whether or not this plasmid derivative indeed contained the information to induce synthesis of the complete S-specific region of the 0 polysaccharide. Therefore, the LPS of the plasmid donor strain S. sonnei 9773/ 63 phase 1, of the plasmid free E. coli strain K-12 (CV601), and of strain CV601 containing plasmid pIE 988 (IE 1351) were compared. The results obtained using three completely different methods were in agreement with the assumption that the LPS of the recombinant strarn consisted of the E. coli K-12 core to which the complete S-

427

LPS Encoded by a Recomb inant Plasmid

sp ecific region of S. sonnei phase I was bound. The only visible difference was found to exi st in the length control of the pol ysacch aride. While the LPS of S. sonn ei strain 9773/63 in SDS-PAGE sho wed a p ron ounced bimodal distribution (Fig. 2, lane 1), in th e case of the LPS of E. co li [E135 1 (la ne 2), the int ens ity of the bands, especially in the high er molecular regio n, but also in th e region between ab out 5 and 15 repeating un its, was higher than in the co m pa ra ble regions of strain 9773/63 . Batch elo r et al. (2) found th at a t lea sr in the case of E . co li 0 75 lipopolysac charide, a bim od al distribution was regulated by a 35.5 kD a p rotein. It seems to be logical to ass ume th at such a pr otein is present in strai n S. sonnei 9773/63, ho wever , not at all o r in reduced amo unts in E. co li 1£ 135 1. Our results fav o ur th e assumption th at th e formati on of the co mplete antigen sho uld not be the reason fo r th e instability of S. sonn ei ph ase I.

a

A ckn owledgements. We are indebted to Dr. G un ter Schmidt, Borstel, for valuable discussions and to Brigitte Tannert for excellent technical assistance.

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206- 224 7. Haragu cbi, G. E., U. Zdhringer, B. [ann , K. [a nn, R. A. Hull, and S. I. Hull: Geneti c characterizatio n of the 0 4 po lysacchride gene cluster fro m Escherichia coli. Micro b. Pathogen . 10 (1991) 35 1-3 61 8. Kenne, L., B. Lindberg, K. Peterson , E. Katzenellenbogen, and E. Rom anowsk a: Structu ral studies on the O-spec ific side chains of Shigella sonnei phase I lipo polysaccharide. Ca rbohydr. Res. 78 (1980) 119- 126 9. Kontrobr, T. and B. Kocsis: Struc ture of the hexose region of Shigella sonnei phase II lipopolysaccharide with J- deoxy- Dvmanno-octuloso nic acid as possible immundeterminat and its relation to Escherichia coli R1 core. Eur . J. Biochcm. 88 (1978) 267-273 10. Kopeck o, D. j., O. Washingt on , and S. B. Formal : Genetic and physical evidence for plasmid control of Shigella sonnei form I surface antigen. Infect. Immun. 29 (19 80) 207-2 14 11. Ko pecho , D. [ ; L. S. Baron , and ]. Buysse: Genetic Determinants of Virulence in Shigella and dysent eric strai ns of Escherichia coli: Their involvement in the pa thogenesis of dysentery. CurroTop . Microbial. lmmu nol. 118 (1985) 71- 95 12. Kusecek , B., H. \Vloch, A . Mercer, V. v aisanen, G. Pluschke, T . Korhonen, and M. Acbtman: Lipop olysaccharide, capsule, an d fimbriae as viru lence factors among 01 , 0 7, 0 16, 0 18, or 0 7.5 and K1, KS, or KlOO Escherichia coli. Infect. Immun. 43 (1984)

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13. 0rskov, F. and I. 0rskov: Complex formation between Escherichia coli lipopolysaccharide 0 antigen and capsular K antigen as detected by immunoelecrrophoresis. APMIS 99 (1991) 615-619 14. Schmidt, G.: Basalstrukturen von Lipopolysacchariden verschiedener Enterobakteriaceen. Generische und serologische Untersuchungen an R-Muranren. Zbl. Bakt. Hyg., I. Abr. Orig. A 220 (1972) 427-476 15. Seltmann, G.: Untersuchungen zur Anrigenstruktur der Shigellen. 4. Mitr. Isolierung und Reinigung eines thermolabilen Anrigens von Sh. sonnei. Zbl. Bakt. Hyg., I. Abt. Orig. A 219 (1971) 324-335 16. Tschape, H., E. Tietze, and C. Koch: Characrerizarion of conjugative plasmids belonging ro the new incompatibility group IncU. J. gen. Microbiol. 127 (1981) 155-160 17. Tsai, C. M. and C. F. Frash: A sensirive silver srain for derecting lipopolysaccharides in polyacrylamide gels. Anal. Biochem. 119 (1982) 115-119 18. Watanabe, H. and A. Nakamura: Large plasmids associated wirh virulence in Shigella species have a common function necessary for epithelial cell penetration. Infect. Immun.

48 (1985) 260-262 19. Westphal, O. and K. [ann: Bacteriallipopolysaccharides. Extraction with phenol-water and further applications of the procedure. Meth. Carbohydr. Chem. 5 (1965) 83-91 . 20. Yoshida, Y., N. Okamura, J. Kato, and H. Watanabe: Molecular cloning and characterization of form I genes from Shigella sonnet, ]. gen. Microbiol. 137 (1991) 867-874

Dr. G. Seltmann, Robert-Koch-Institut des Bundesgesundheitsamtes, Bereich Wernigerode, Burgstr. 37, D-O 3700 Wernigerode