Shigella dysenteriae type 1 carrying LPS biosynthesis genes of Salmonellatyphimurium a�ects both Invasive Plasmid AntigenH (IpaH) secretion and invasion

Debabrata Biswas1, Michael Bagdasarian2 and Ranajit Kumar1,*1National Institute of Cholera and Enteric Diseases, P-33, C.I.T. Road, Scheme XM, Beliaghata,Calcutta - 700 010, India2Michigan State University, Department of Microbiology, S-110 Plant Biology Bldg, East Lansing, MI 48824, USA*Author for correspondence: Fax: 91-33-350-5066, E-mail: icmrnicd@ren.nic.in

Received in revised form 22 February 1999; accepted 2 July 1999

Keywords: Invasion, IpaABCD, lipopolysaccharides, Plasmid pPR1347, Sereny, triparental cross

Summary

A Shigella dysenteriae 1 strain isolated from an epidemic in West Bengal, India. The strain contained six plasmidsincluding a large virulence plasmid. A plasmid, pPR1347 carrying both the rfb gene cluster and the rfc gene ofSalmonella typhimurium has been transferred to this invasive Shigella dysenteriae 1 strain by triparental cross with avery low frequency. Only ®ve stable (100%) clones were isolated after examining several thousand colonies. All ®vetransconjugants were Sereny negative and were unable to invade the HeLa cells. Transconjugants exhibited strongcross reactivity with S. dysenteriae 1 antisera but they showed weak reaction with Salmonella typhimurium antisera.Plasmid pro®les of the transconjugants were unaltered as compared with the wild type strain except for the presenceof pPR1347. The transconjugants regained their invasive property after elimination (curing) of pPR1347. However,Shigella-infected convalescent phase serum was able to detect IpaABCD proteins from whole cell lysate and culturesupernatant of transconjugants and cured (pPR1347) transconjugants. A 60 kDa IpaH protein was not secretedinto the culture supernatant by the transconjugants. Synthesis of lipopolysaccharides (LPS) of the hybrid strainswas increased within the region of 43 to 67 kDa in comparison with the wild type S. dysenteriae 1 strains.

Introduction

The invasive genes of Shigella spp. are located on a37-kb segment of the large invasive plasmid (140±180 MDa), which contains more than 30 genes (Sans-onetti et al. 1981, 1982). Immunoblot analysis with theserum isolated from monkeys or humans infected withShigella spp. showed a consistent serum immuneresponse which recognizes four invasive plasmid anti-gens (IpaABCD) along with VirG protein (120 kDa)(Hale et al. 1985; Kato et al. 1989; Watanabe et al.1990). The gene products IpaABCD are 78, 62, 43and 38 kDa, respectively (Buysse et al. 1995; Haleet al. 1985). However, the Invasive Plasmid AntigenH,IpaH (60 kDa) gene is present as multicopies in theinvasion plasmid as well as in the chromosome of theShigella spp (Hartman et al. 1990). Multicopies ofIpaH are also present in the chromosome and plasmidof EIEC Escherichia coli strains (Venkatesan et al.1989). However, the exact function of IpaH protein isunknown (Buysse et al. 1987; Hartman et al. 1990;Venkatesan et al. 1989).The lipopolysaccharide (LPS) is a major cell surface

antigen which is responsible for a wide range ofpathophysiological host reactions. The mechanism of

LPS biosynthesis is not the same in all bacteria.Synthesis of LPS in Shigella dysenteriae 1 is encodedby a plasmid (Watanabe & Timmis 1984). O-antigen-negative mutants of S. dysenteriae 1 are avirulent andfail to produce the Sereny reaction (Binns et al. 1985).Genes associated with O-antigen biosynthesis of Shigellahave been transferred to heterologous hosts by severalinvestigators. A large invasive plasmid (120 MDa) ofS. sonnei has been transferred into E. coli K-12 andS. typhi Ty21a where the O-polysaccharide of S. sonneiwas expressed in both the strains (Formal et al. 1981;1984; Baron et al. 1987). The expression of Shigella LPSis very weak in both the hybrid strains.Formal et al. (1981) reported that the weak expression

of LPS was observed when Shigella plasmid wastransferred into E. coli or S. typhi Ty21a. In the presentstudy we have transferred a plasmid vector pPR1347carrying rfb and rfc gene clusters of Salmonella typhi-murium into an invasive S. dysenteriae 1. The expressionof the Salmonella LPS within S. dysenteriae type 1(hybrid strain) was investigated in relation to invasion ofthe colonic epithelial cells. Another aim of our work isto investigate the possibility of producing bivalentvaccine strains to protect against S. typhimurium andS. dysenteriae.

World Journal of Microbiology & Biotechnology 15: 693±698, 1999. 693Ó 1999 Kluwer Academic Publishers. Printed in the Netherlands.

Materials and Methods

Media, culture condition and chemicals

Tryptic soy broth (TSB) or tryptic soy agar (TSA)(Difco, USA) were used as culture media (Biswas et al.1991). Bacterial cells were grown over night at 37 °Cwith constant shaking. Antibiotics were added 30 lgampicillin (Am)/ml and 100 lg Kanamycin (Km)/ml.

Bacteria and plasmids

Multidrug resistant highly virulent Shigella dysenteriaetype 1 strain isolated from an epidemic in West Bengal(India) (Palchaudhuri et al. 1985) was used as recipientstrain. Tetracycline resistant (Tcr) E. coli R64-drd 11was used as helper strain in triparental cross. Kanamy-cin resistant (Kmr) E. coli strain carrying a cosmid(pPR1347) which contained both the rfb gene clusterand rfc gene of a Salmonella group B serovar was usedas a donor strain of this study (Neal et al. 1993).

Triparental cross

The cells grown for 4 h in TSB were used in triparentalcross. The triparental cross was performed following astandard method (Bogosian & Kane 1991). The trans-conjugants were selected on Am (30 lg/ml), Km(100 lg/ml) plates.

Plasmid isolation

Plasmids were isolated from 5 ml cultures grown over-night in TSB at 37 °C with constant shaking. Theprocedure of Kado & Liu (1981) was followed forisolation.

Electrophoresis of DNA and protein

Agarose gel electrophoresis was performed using hori-zontal slab gels in TAE bu�er (Maniatis et al. 1982).Polyacrylamide gels were run as vertical slabs accordingto the method of Laemmli (Laemmli 1970). Proteinsamples were boiled for 5 min in sample bu�er beforeseparation on 10% acrylamide gels. Proteins werestained with Coomassie Brilliant blue R (Sigma).

Isolation of secretary proteins and whole cell lysis

Secretory proteins were isolated from the mid-log phaseof the wild type strain (invasive), transconjugant andplasmid (pPR1347) cured strains by ammonium sul-phate precipitation (80% saturation). After extensivedialysis (72 h in 0.025 M Na-phosphate bu�er, pH 7.4at 4 °C), the proteins were concentrated by ultracentrif-ugation (Sorvall Ultracentrifuge, Model, OTD Combi)at 100,000 ´ g for 1 h at 4 °C. The bacterial pellet from1 ml culture (TSB) was suspended in 1 ml of cold waterand one third of a volume of 24% TCA was added.

After centrifugation, the precipitated pellet was dis-solved in 100 ll of SDS sample bu�er and boiled for5 min. Fifteen micrograms of each extract was subjectedto an SDS-polyacrylamide gel (10%) electrophoresis(SDS-PAGE) (Watarai et al. 1995).

Immunoblotting

Secretory proteins and whole cell lysate of virulent wildtype, transconjugant and cured strains were subjected toSDS-PAGE. The separated proteins were transferredonto nitrocellulose ®lters according to the methoddescribed by Towbin et al. (1979). Human convalescentphase sera (sera were obtained from the acute bacteri-ologically con®rmed shigellosis patients admitted to theInfectious Diseases Hospital, Calcutta) was also usedand the conjugate was alkaline phosphatase-conjugatedanti-human IgG (Sigma) diluted 1:2000. The colour wasdeveloped with p-nitroblue tetrazolium chloride (NBT;033 mg/ml, 5-bromo-4-chloro-3-indolyl phosphate(BCIP; 0.165 mg/ml) as substrate in alkaline phospha-tase bu�er (0.1 M Tris-0.1 M NaCl-0.005 M MgCl2:pH9.5).

Lipopolysaccharide isolation

The lipopolysaccharide (LPS) extraction and analysiswere followed as described by Slauch et al. (1995). LPSof transconjugants and S. dysenteriae 1 were isolatedfrom whole cells grown overnight at 37 °C in tryptic soybroth (100 ml) with constant shaking. The cells werewashed with distilled water and resuspended in 20 ml of150 mM NaCl containing 20 mM MOPS (3-[N-mo-rpholino]propanesulphonic acid), pH 6.9. An equalvolume of bu�er saturated (20 mM MOPS; pH 6.9)phenol was added into the mixture and incubated at65 °C for 30 min with occasional shaking. The mix-ture was kept in ice for 10 min and centrifuged for20 min at 1500 ´ g. The top aqueous phases werecollected and four volumes of chilled ethanol wereadded to each sample and they were kept overnight at)20 °C. The precipitated LPS was collected by centrif-ugation. The isolated LPS were preserved (Slauch et al.1995) and were analysed on 12% SDS-PAGE. The gelwas silver stained (Tsai & Frasch 1982) to visualize theLPS.

Sereny test

Wild type, transconjugant and cured transconjugantstrains (5 ´ 108 bacteria) were installed into the con-junctival sac of guinea pigs (Sereny 1955). The animalswere observed for 5 days for signs of keratoconjuncti-vitis.

Curing of plasmid

Overnight cultures of the transconjugant were diluted to103 cells/ml in Penassay broth containing 5% SDS and

694 D. Biswas et al.

shaken at 37 °C for 72 h. (Tomoeda et al. 1968).Kanamycin sensitive strains were screened on TSAcontaining kanamycin (100 lg/ml).

Plaque assay

Plaque assay was done according to the methoddescribed by Oaks et al. (1985) with minor modi®ca-tions. HeLa cells were grown to con¯uence in 35 mmtissue culture petri dishes (Gibco) at 37 °C in ahumi®ed atmosphere at 5% CO2. In preparation forthe plaque assay the monolayer was washed twicewith antibiotic-free MEM supplemented with glucose(4.5 g/l). After aspirating the second wash approxi-mately 5 ´ 107 c.f.u. of virulent S. dysenteriae 1 (grownat 37 °C for 4 h) suspended in MEM (antibiotic-free)were added to the monolayer, which was subsequentlyincubated at 37 °C for 90 min. During this adsorptionand attachment phase, the plates were rocked every30 min to ensure uniform distribution of plaque-forming bacteria. After incubation the extracellularbacteria were aspirated, the monolayer was washedfour times with MEM and ®nally 2 ml of MEMsupplemented with 40 lg of gentamicin/ml was addedand kept at 37 °C in a humi®ed atmosphere of 5%CO2 incubator. After 48 h of incubation the infectedmonolayer was ®xed and stained simultaneously with25% methanol and 1% crystal violet (w/v) for countingplaques.

Results

Transfer of pPR1347 into virulent S. dysenteriae 1

Kanamycin resistant pPR1347 was transferred to avirulent Shigella dysenteriae 1 (recipient) by triparentalcross with a frequency of 1.8 ´ 10)6 and the transcon-jugants were selected on TSA plates containing Am(30 lg/ml) and Km (100 lg/ml). The Kmr transconjug-ants were highly unstable due to loss of their plasmidvector pPR1347 within a few generations. We haveisolated ®ve stable Kmr clones after screening 4000transconjugants. These ®ve transconjugants are highlystable even grown after more than 100 generations. Theplasmid pro®les of donor recipient and transconjugantsare shown in Figure 1. The transconjugant clearlyshowed a 43-kb plasmid (pPR1347) on agarose gel(Figure 1). The plasmid pro®les of wild type S. dysen-teriae 1 and the transconjugants were similar except forthe presence of pPR1347. Transconjugants showedstrong cross reactivity with S. dysenteriae 1 antiserabut weak with Salmonella typhimurium antisera.

Curing of pPR1347 from transconjugants

Two hundred SDS treated colonies were screened onTSA plates containing Km (100 lg/ml) and found 8%strains became Km sensitive which have lost pPR1347.

They have regained their virulence property (invasive)like wild type S. dysenteriae 1 strains.

Sereny reaction and plaque assay

Transconjugants and donor E. coli K12 carrying cosmidvector pPR1347 did not show keratoconjunctivitiswithin 96 h. However, wild type S. dysenteriae 1 andplasmid-cured (pPR1347) strain show strong keratocon-junctivitis within 24 h. No plaque formation was de-tected on HeLa cell monolayers when they were treatedwith the transconjugant however, wild type S. dysenter-iae 1 and cured transconjugant showed clear zone ofplaque formation throughout the plates.

Protein pro®les of whole cell lysate and secreted proteinson SDS-PAGE

Whole cell lysate and secretory proteins were isolatedfrom wild type, transconjugant and plasmid cured(pPR1347) transconjugant strains of S. dysenteriae 1and their protein pro®les were compared on SDS-PAGE(Figure 2). Only a 60-kDa (IpaH) protein was notsecreted by the transconjugant, however, synthesis ofthis protein was detected in the whole cell lysate(Figure 2). Otherwise, there were no di�erences in theprotein pro®les among the strains (Figure 2).

Immunoblot analysis using human convalescent sera

The invasive plasmid antigen genes encode the fourmajor antigenic polypeptides IpaA (78 kDa), IpaB

Figure 1. Agarose gel electrophoresis of plasmid DNA isolated from

donor E. coli k12 carrying plasmid vector pPR1347, recipient

S. dysenteriae 1, transconjugant and plasmid cured (pPR1347) trans-

conjugant have shown in this ®gure. Lane 1, donor; Lane 2, recipient

S. dysenteriae 1; Lane 3, transconjugant carrying a 43-kb pPR1347;

Lane 4, transconjugant after curing of pPR1347. Inv. ± invasive

plasmid. The arrow indicates the plasmid DNA of pPR1347.

IpaH secretion and invasion of S. dysenteriae 695

(62 kDa), IpaC (43 kDa) and IpaD (38 kDa), detectedby post-shigella infection convalescent-phase humansera in the whole cell lysate and secretory proteins ofwild type, transconjugant and plasmid cured (pPR1347)transconjugant strains (Figure 3). Therefore, there wereno di�erences in the synthesis and secretion of the ipaprotein pro®les between the invasive S. dysenteriae 1with non-invasive transconjugant (except IpaH).

Lipopolysaccharide analysis

The synthesis of LPS by the transconjugants (hybridstrains) were increased at the region of 67 kDa to43 kDa (Figure 4, lane 1 & 2) in comparison with theinvasive S. dysenteriae 1 strain (lane 3 & 4). A 67-kDa

band was very weak in wild type S. dysenteriae 1 strainon 12% SDS-PAGE. However, increased LPS synthesisat the region of 67 to 43 kDa of the hybrid strains wasdue to expression of plasmid vector pPR1347 carryingrfb and rfc gene clusters of S. typhimurium. Transcon-jugants also showed weak cross reactivity with Salmo-nella antisera.

Discussion

Gaudio et al. (1997) reported that the IpaH gene wasassociated with dysentery. We have also reported thesigni®cance of IpaC in relation to invasion of colonicepithelial cells (Shaikh et al. 1995). The hydrophilicand antigenic nature of IpaH are exposed on thesurface of the Shigella spp. which does not have atypical signal peptide in its aminoacid sequence (Hall &Silhavy 1981; Watson 1984). An alternative secretorymechanism or some other unknown factor(s) may berequired for the transport of the IpaH protein on thebacterial cell surface (Hartman et al. 1990). Secretionblocked mutants of Ipa proteins became non-invasivein Shigella (Watarai et al. 1995). Our newly construct-ed transconjugants carrying plasmid vector pPR1347failed to secrete IpaH protein in the culture superna-tant. However, other Ipa proteins ABCD were detectedin the culture supernatant of the transconjugants.

Figure 2. SDS-PAGE (10%) analysis of the secretory proteins of wild

type S. dysenteriae 1, transconjugant and plasmid cured (pPR1347)

strains of S. dysenteriae 1 have shown in this gel. Lane 1, wild type

strain; Lane 2, transconjugant; Lane 3, plasmid cured (pPR1347)

transconjugant and Lane 4, molecular mass markers. The arrow

indicates the 60 kDa IpaH protein.

Figure 3. Western blot analysis of the whole cell lysate as well as the

secreted proteins of wild type. S. dysenteriae 1 and the transconjugant

have been shown in this ®gure. Labels A±D indicate IpaA±IpaD. Lane

1, molecular mass markers; Lane 2, whole cell lysate of the wild type;

Lane 3, whole cell lysate of the transconjugant; Lane 4, secretory

protein pro®les of the wild type; Lane 5, secretory protein pro®les of

the transconjugant.

Figure 4. Comparison of LPS from invasive strains of S. dysenteriae 1

and transconjugants (hybrid strains) carrying a plasmid vector

pPR1347 of S. typhimurium by SDS-PAGE with a 12% acrylamide

gel concentration. After electrophoresis, the gels were silver stained.

Lanes: 1 & 2, LPS from the transconjugants (hybrid strains); 3 & 4,

invasive S. dysenteriae strains; 5, Molecular mass markers; 97 kDa

phosphorylase, b; 68 kDa, bovine serum albumin; 43 kDa, ovalbumin;

25 kDa, a-chymotrypsin.

696 D. Biswas et al.

O-antigen biosynthesis genes (rfb-rfp) of Shigellatransferred to E. coli K-12, Salmonella typhimuriumand S. typhi Ty21a resulted in expression of bothhomologous and heterologous LPS among the hybridstrains for the development of bivalent vaccine strains(Sturm & Timmis 1986; Mills et al. 1988). The expres-sion of Shigella LPS in the hybrid strains were weak.However, antibiotic selection pressure was required tomaintain the plasmid.We have transferred the plasmid vector pPR1347

from Salmonella typhimurium into an invasive Shigelladysenteriae 1 to examine the expression of LPS inShigella. The transconjugants (hybrid strains) showed astrong reaction with Shigella antisera and a weakreaction with Salmonella. These hybrid strains becameSereny negative and also incapable of invading HeLacells. The secretion of IpaH protein (60-kDa) may beinhibited due to production of new LPS synthesized bypPR1347 (rfb and rfc gene product of S. typhimurium)which make the transconjugants non-invasive. However,the exact mechanism of secretion block of IpaH proteinof the transconjugants is not clear to us. MulticopyIpaH gene might play a signi®cant role in cellularinvasion of Shigella spp. Probably, the host cell inter-action with secreted from of IpaH protein is necessary asone of the factor for invasion into colonic epithelialcells. Antibiotic selection pressure is not required forproduction of both LPS to maintain the plasmid and thehybrid strain is also 100% stable. Therefore, this strainmay be considered as a bivalent vaccine strain.

Acknowledgements

Thanks to Mr S.K. Das for secretarial assistance.

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