effect of mutations in shigella flexneri chromosomal and plasmid-encoded lipopolysaccharide genes on...

Effect of mutations in Shigella flexneri chromosomaland plasmid-encoded lipopolysaccharide genes oninvasion and serum resistance

Mei Hong and Shelley M. Payne *Department of Microbiology, University of Texas atAustin, Austin, Texas 78712, USA.

Summary

This study shows that both length and distributionof lipopolysaccharide (LPS) are important for Shigellaflexneri invasion and virulence. Mutants were gene-rated in the chromosomal LPS synthesis genes rfa,rfb , and rol , and in a plasmid-encoded O-antigenchain-length regulator, cld pHS-2 . LPS analysis showedthat mutations in rfb genes and in a candidate rfaLgene either eliminated the entire O-antigen side chainsor produced chains of greatly reduced length. Mutationin a previously unidentified gene, rfaX, affected theLPS core region and resulted in reduced amounts ofO-antigen. Mutants defective in cld pHS-2 or rol had dif-ferent distributions of O-antigen chain lengths. Theresults of tissue-culture cell invasion and plaqueassays, the Sere´ny test, and serum-sensitivity assaysuggested roles for the different LPS synthesis genesin bacterial survival and virulence; rfaL, rfaX and rfbloci are required for serum resistance and intercellularspread, but not for invasion; cld pHS-2 is required forresistance to serum killing and for full inflammationin the Sere ny test, but not for invasion or intercellularspread, while rol is required for normal invasivenessand plaque formation, but not for serum resistance.Thus, O-antigen synthesis and chain-length regula-tion genes encoded on both the chromosome and thesmall plasmid pHS-2 play important roles in S. flex-neri invasion and virulence.

Introduction

Shigella species are the aetiologic agents of bacillary dys-entery in humans (Hale, 1991; Sansonetti, 1992; Menardet al., 1996). The essential steps of pathogenesis includeinvasion of the colonic epithelial cells, intracellular multi-plication, spread to adjacent cells, and elicitation of aninflammatory response. The large 180–230 kb plasmid

encodes most of the essential virulence genes, whichinclude invasion genes ipaBCD, intra- and intercellular-spread genes icsA (virG) and icsB, the type III secretoryapparatus genes mxi–spa, and the virulence regulatorsvirF and virB. Several chromosomal loci also affect viru-lence, among them the virulence-gene repressor virR,the aerobactin synthesis/utilization locus iuc–iut, and lipo-polysaccharide (LPS) synthesis alleles rfa and rfb (Hale,1991; Sansonetti, 1992).

LPS is a major component of the Gram-negative bac-terial surface. As in other smooth organisms, the LPS ofShigella spp. contains a lipid A region, a core oligosac-charide, and an O-antigen polysaccharide side chainwhose composition varies among different serotypes ofShigella (Kenne et al., 1978; Simmons and Romanowska,1987; Brahmbhatt et al., 1992) (LPS of Shigella flexneri 2ais shown in Fig. 1A). Earlier studies showed that unchar-acterized rough mutants of S. flexneri lacking LPS O-anti-gen side chains were avirulent in the Sereny test (Okamuraand Nakaya, 1977; Okamura et al., 1983), an assay thatmeasures bacterial ability to invade epithelial cells, tospread intercellularly, and to elicit an inflammatory response(Sereny, 1957). These rough strains did retain the abilityto invade tissue-culture cells but could not spread to adja-cent cells. In other experiments, S. flexneri hybrid strainsthat express different LPS O-antigens, such as Escheri-chia coli O8 antigen, were less virulent in the Sereny test(Gemski et al., 1972; Sandlin et al., 1996). These resultssuggested a critical role for LPS O-antigen side chains inShigella virulence, in addition to the general role of LPSlipid A as an endotoxin to provoke an inflammatory response(Lindberg et al., 1991; Brahmbhatt et al., 1992).

LPS also has been shown to play a role in serum resis-tance in several enteric species. LPS molecules in Sal-monella spp. and E. coli are required for the bacterialresistance to complement-mediated serum killing (Rowley,1968; Joiner, 1988; Taylor, 1995). The relationship betweenLPS and the susceptibility of Shigella spp. to serum killinghas not been reported.

In S. flexneri, most LPS biosynthesis genes are locatedon the chromosome. These genes include the locus rfb–rfc–rol at 44 min for O-antigen synthesis (Macpherson etal., 1994; Morona et al., 1994; 1995; Rajakumar et al.,1994) (Fig. 1B) and a potential LPS core synthesis rfalocus which maps near 81 min (Okada et al., 1991a,b). A

Molecular Microbiology (1997) 24(4), 779–791

Q 1997 Blackwell Science Ltd

Received 13 January. 1997; revised 11 February, 1997; accepted 10March, 1997. *For correspondence. E-mail [email protected];Tel. (512) 471 9258; Fax (512) 471 7088.

m

role for plasmid genes in O-antigen synthesis also hasbeen observed in various Shigella spp. Shigella sonneiO-antigen-synthesis genes are located on the large viru-lence plasmid (Kopecko et al., 1980). In S. dysenteriaeserotype 1, a 9 kb plasmid contains the gene rfp which,in addition to chromosomal genes, is required for LPS O-antigen synthesis (Watanabe et al., 1984). In both cases,loss of the plasmid-encoded O-antigen genes resulted inloss of virulence (Kopecko et al., 1980; Watanabe andTimmis, 1984). S. flexneri serotype 2a isolates often con-tain a 3 kb plasmid, pHS-2 (Stieglitz et al., 1989), whichhas an open reading frame (ORF) for a predicted 368-amino-acid protein (Stieglitz et al., 1989; Stevenson et al.,1995). The predicted protein has 63% amino acid identitywith E. coli FepE (C. Shea-Cleavinger, B. Ozenbergerand M. McIntosh, Genbank Accession No. X74129, 1993).Although fepE is located within the enterobactin biosynthe-sis and transport cluster on the E. coli chromosome (Ozen-berger et al., 1987), it does not play an essential role in thisiron-transport system in E. coli (M. McIntosh, personalcommunication). Recently it was shown that the predictedprotein sequences of both the pHS-2-encoded gene and

fepE have 21–26% identity to those of a family of chromo-somal genes determining LPS O-antigen chain length(Batchelor et al., 1992; Bastin et al., 1993; Morona et al.,1995; Stevenson et al., 1995). This family has been calledcld (O antigen chain-length determinant), rol (regulationof O-antigen chain length), or wzz in different E. coli, Sal-monella, and Shigella species (Reeves et al., 1996; Whit-field et al., 1997). The plasmid pHS-2-encoded gene wasnamed cldpHS-2 (Stevenson et al., 1995). It was shown thatthe absence of this gene caused loss of O-antigen chainsof 90–100 repeating units in S. flexneri serotype 2a(Stevenson et al., 1995). It was not reported if this plas-mid-encoded gene plays a role in S. flexneri virulence.

In this study we determined the roles of various LPS-synthesis genes in S. flexneri pathogenesis. Mutants inrfb genes, candidate rfa genes, the chromosomal rolgene, and cldpHS-2 were constructed and characterized,and the expression and regulation of cldpHS-2 werestudied. These mutant strains were compared to the wild-type strain in terms of LPS structure, serum sensitivity,tissue-culture cell invasion and plaque assays. The resultsindicated that not only the presence of O-antigens, but

Q 1997 Blackwell Science Ltd, Molecular Microbiology, 24, 779–791

Fig. 1. Lipopolysaccharide (LPS) structure ofS. flexneri serotype 2a and some of thegenes involved in the LPS synthesis in S.flexneri (Kenne et al., 1978; Simmons andRomanowska, 1987).A. LPS structure and brief synthesis pathway.KDO, 2-keto-3-deoxy-octonate; Hep,L-glycero-D-manno-heptose phosphate; PEtN,O-phosphoryl-ethanolamine; Glc, D-glucose;Gal, D-galactose; GlcNAc, N-acetyl-D-glucos-amine; Rha, L-rhamnose. The oligosaccharidewithin the parentheses is designated as oneO-antigen repeating unit since it is added ontothe growing chain as one complete unit byRfc, the O-antigen polymerase. The completeO-antigen chain is transferred from ACL(antigen-carrier lipid) and ligated to thecomplete lipid A core by RfaL, the O-antigenligase. ‘rfaX? ’ points to possible targets of themutation in S. flexneri strain SA555-148. ‘rol?cldpHS-2? ’ indicate possible functioning sitesas proposed by others (Schnaitman andKlena, 1993).B. rfb–rfc–rol region map (Morona et al.,1995). Boxes are genes or predicted ORFs.Each arrowhead indicates the position of theTnphoA insertion in one of the mutants whichwere deficient in plaque formation. Lines witharrows are possible transcripts. The transcriptfrom rfbB to rfc and that for rol weresuggested by Morona et al. (Morona et al.,1995). The transcript with a question markwas suggested in this work.

780 M. Hong and S. M. Payne

also the wild-type length distribution and the number ofO-antigen molecules present on cell surface are importantfor full virulence of S. flexneri. rfa loci, rfb loci, cldpHS-2 androl play important, yet different, roles in virulence owing totheir different roles in synthesis of O-antigen or in regula-tion of O-antigen chain length.

Results

Isolation of strains with chromosomal LPS-synthesisgene mutations

Random TnphoA mutagenesis was used to generate a poolof mutants of S. flexneri with potential virulence defects.TnphoA was chosen because PhoA-positive cells arethose in which the transposon has interrupted a geneencoding a protein with a leader sequence and has cre-ated a translational fusion to the phosphatase sequences.Many virulence gene products are membrane- or surfaceproteins and would therefore be targeted by screeningfor colonies expressing the transposon-encoded PhoA.PhoAþ colonies were selected and screened for the abilityto invade and form plaques in HeLa cell monolayers.Forty-five mutants that were invasive but were defectivein plaque formation, indicating mutations affecting intra-cellular multiplication or spread to adjacent cells, wereselected for further study. The DNA sequences adjacentto the TnphoA inserts were analysed in the mutants andamong these were icsA (virG), a gene previously shownto be required for plaque formation (Bernardini et al.,1989; Lett et al., 1989), and a number of genes associatedwith LPS synthesis (Table 1). The S. flexneri LPS O-anti-gen-synthesis genes identified in this screen includedrfbA, -B, -C, -F, and -G. These genes are involved in dif-ferent steps in synthesis of the O-antigen repeating unitand are encoded on the S. flexneri chromosome(Macpherson et al., 1994; Morona et al., 1994) (Fig. 1, Aand B). Other candidate LPS mutants had insertions in

regions not identified previously. Three of these mutantseach had an insertion at a different site within a genehomologous to the Salmonella typhimurium O-antigenligase gene, rfaL, which ligates the complete O-antigenside chain to the LPS core (Fig. 1A) (Schnaitman andKlena, 1993). One insert was in an ORF with homologyto the 58 portion of E. coli rfaY, whose exact function hasnot been identified, but it was speculated that the geneencodes a protein involved in LPS core synthesis (Klenaet al., 1992b; Schnaitman and Klena, 1993). This S. flex-neri gene was named rfaX, since LPS studies (shownbelow) indicated that the LPS core synthesis of this mutantwas affected; it is not known whether the function of thisgene is equivalent to that of rfaY. In the sequenced regionthat was upstream of rfaX, there was a partial ORF pre-dicting 164 amino acids (ending with a stop codon at 54nucleotides upstream of the rfaX start codon) that had sig-nificant homology to the C-termini of both RfaI and RfaJ inS. typhimurium and E. coli (data not shown).

Isolation of strains with plasmid-encoded cldpHS-2 andchromosomal rol mutations

The plasmid-encoded ORF cldpHS-2 was originally chosenfor study because the homology between cldpHS-2 andfepE was related to our interest in iron uptake. The laterfinding of its homology to cld/rol genes, a family of O-anti-gen chain-length determinants (Stevenson et al., 1995),led us to include this gene in our study of Shigella LPS.Several strains were constructed in order to study theexpression and localization of the gene product, and therole of cldpHS-2 in virulence. One of these strains,SA100(pME2), contained a TnphoA insertion fusing thereporter protein PhoA to the 74th amino acid of CldpHS-2

(Fig. 2). The presence of phosphatase activity inSA100(pME2) (207.9 U) indicated that cldpHS-2 was tran-scribed and translated, and RNA dot-blot hybridizationconfirmed that cldpHS-2 is expressed (data not shown).


Table 1. DNA sequence analysis of insertion sites of S. flexneri TnphoA mutants defective in plaque formation.a

Group InvasionPlaqueformationb

Temperatureregulationc Site of insertion Predicted protein sequence identity

I þ ¹ þ icsA(virG) 100%II þ ¹ ¹ rfbA

rfbBrfbGrfbF

100%

III þ ¹ ¹ Candidate rfaL 54% to RfaL of S. typhimuriumIV þ p ¹ rfbC 100%V þ p ¹ rfaX 37% to N-terminal 24 amino acids of RfaY of E. coli

a. DNA sequences of insertion sites of TnphoA were obtained as described in the Experimental procedures. The homology search was performedwith the IntelliGenetics SuiteTM program.b. ‘p’ means formation of pin-point plaques (< 0.2 mm diameter as compared to wild type 1–1.5 mm diameter). ‘¹’ indicates no plaques.c. Temperature regulation of gene expression, determined by measuring the PhoA fusion phosphatase activity of bacterial cultures grown at 378Cvs. 308C; ‘þ’ indicates higher PhoA activity at 378C than 308C and ‘¹’ indicates no difference.

Shigella flexneri lipopolysaccharide mutants 781

Because alkaline phosphatase activity expressed fromTnphoA fusions requires a functional leader sequence,the presence of PhoA activity from pME2 further indicatedthat CldpHS-2 was located at the bacterial membrane, withat least a portion being exposed to the periplasmic spaceor cell surface. CldpHS-2 was suspected of being a cyto-plasmic membrane protein, because it has amino acidhomology to two cytoplasmic membrane proteins, FepE(Ozenberger et al., 1987) and Rol (Morona et al., 1995),and all three proteins have similar hydropathy profiles.This was confirmed by fractionating SA100(pME2) cellsand analysing the cytoplasmic membrane, outer mem-brane and cytosol fractions by immunoblotting with anti-PhoA antiserum. The CldpHS-2–PhoA fusion was detectedin the cytoplasmic membrane fraction (data not shown).

PhoA fusion activity and RNA dot-blot hybridizationwere also used to study the regulation of cldpHS-2 expres-sion. The results indicated that cldpHS-2 was not regulatedby the environmental conditions tested (including iron con-centration, temperature, pH, nutrients, and the intracellu-lar environment; data not shown).

In order to study the role of cldpHS -2 in S. flexneri viru-lence, SA100 was cured of pHS-2 as described in theExperimental procedures. The resulting strain, SA514,was transformed with the recombinant plasmid pMA9, inwhich a cam gene interrupts the cldpHS-2 ORF, or withpMN4, in which the cam gene is inserted downstream ofcldpHS-2, leaving the ORF intact (Fig. 2).

Because cldpHS-2 has homology to the cld/rol genefamily and was proposed to be an O-antigen chain-lengthregulator (Stevenson et al., 1995), it was of interest tocompare mutants defective in this plasmid gene and inthe chromosomal rol with each other and with other poten-tial LPS mutants. The rol mutation (rol ::km) from the S.flexneri serotype Y strain RMA585 (Morona et al., 1995)was introduced into SA100 (wild-type serotype 2a) andSA514 (SA100DpHS-2) by P1 transduction, to generatea single mutant SA100rol and a double mutant SA514rol(SA100rol ::kmDpHS-2).

LPS analysis of the mutants

The sequence data suggested that some of the mutantswould have alterations in LPS structure. Therefore, theLPS profiles of these mutants were analysed by SDS–PAGE. The gels were silver-stained to allow visualizationof total LPS (Fig. 3A), and O-antigens were specificallydetected with S. flexneri group B antiserum in immuno-blots (Fig. 3, B and C). In the silver-stained gel (Fig. 3A),the bottom band (fastest migrating) represents LPS mol-ecules that contain cores but lack any attached O-antigenrepeating units, while the upper bands represent LPSmolecules with increasing numbers of O-antigen repeatingunits attached. In the full-range immunoblot (Fig. 3B), thelowest bands detected with the antiserum represent LPSwith a single O-antigen repeating unit attached. Both thesilver-stained gel and the immunoblot showed that in thewild-type strain most of the LPS had 11–17 O-antigenrepeating units (Mode A) (Fig. 3. A and B, lane 1). Thisis the so-called ‘modal distribution pattern’ observed byothers (Morona et al., 1995; Stevenson et al., 1995). Inthe immunoblots, Mode B indicates the longer O-antigenchains that are probably the 90–100-repeating-unit modeobserved by Stevenson et al. (Stevenson et al., 1995)(Fig. 3, B and C). Panel C (Fig. 3) shows the upper por-tion of an immunoblot from a gel which was overloadedto enhance visualization of the Mode B chains.

The candidate rfaL mutant, SA555-38, produced onlythe LPS core and had no O-antigen attached (Fig. 3A,lane 6 and Fig. 3B, lane 8). This phenotype togetherwith observed sequence homology with Salmonella rfaLare strong evidence that the gene interrupted in this mutantis the S. flexneri O-antigen ligase gene. The rfbA, -B, -F,and -G mutants also produced only LPS cores (data notshown). The rfbC mutant, SA555-96, unlike the other rfb


Fig. 2. pHS-2 map. Sequence was obtained from previous studies(Stieglitz et al., 1989; Stevenson et al., 1995). Only the restrictionsites that were used in this study are shown here. The numbers inparentheses indicate the nucleotide positions using the numberingof Stieglitz et al. (1989). The origin of pHS-2 was inferred from thesequence homology to the origin of ColE1. The HindIII site at 1016was used for the cam insertion in pMA9, while the XbaI site at1966 was the site of the cam insertion in pMN4. Position 986 is thesite of the TnphoA insertion in pME2. The 1.6 kb Sau 3AI fragmentused in the construction of pMB3-4 and the EcoRV–AccI fragmentused to generate the DNA probe for RNA hybridization areindicated in the Figure.


mutants tested, had a single O-antigen repeating unitattached (Fig. 3A, lane 7 and Fig. 3B, lane 9).

The rfaX mutant, SA555-148, produced a distinctivepattern in which its LPS core reproducibly migrated fasterthan the wild-type core and appeared as a fainter smearinstead of a dark band (Fig. 3 A lane 5). In spite of thealtered core, this mutant still had O-antigen chains attached,and the distribution pattern was similar to that of the wildtype; the majority of O-antigens were 11–17 units long(Mode A) and there were a small population of Mode BO-antigen chains (Fig. 3A, lane 5 and Fig. 3B, lane 7).However, the total amount of O-antigen detected by eithersilver-staining or immunoblotting in SA555-148 appearedto be less than that seen in extracts of an equivalent num-ber of wild-type cells (Fig. 3, A and B). Because the muta-tion in rfaX did affect the LPS core, this gene is designatedan rfa gene.

SA514, the mutant that lacks pHS-2, did not have theMode B O-antigen chains (Fig. 3 B and C, lane 2). SA514also appeared to have slightly fewer short O-antigenchains of 1–10 repeating units, but had more of the Mode

A 11–17-repeating-unit chains than SA100 (Fig. 3, A andB). SA514(pMN4), which contains an intact cldpHS-2 gene,had the same LPS O-antigen pattern as SA100 (Fig. 3B),whereas SA514(pMA9), in which cldpHS-2 was interrupted,showed the same pattern as SA514 (Fig. 3B), confirm-ing the role of cldpHS-2 in determining this chain-lengthdistribution.

In the SA100rol mutant, the modal distribution of 11–17repeating-unit O-antigen chains (Mode A) was not observed;instead, the majority of O-antigen chains were shorter andthe number of chains of a given size was inversely propor-tional to the chain length (Fig. 3A, lane 3 and Fig. 3B, lane5), although the Mode B, long O-antigen chains were stillpresent (Fig. 3B, lane 5 and Fig. 3C, lane 3). The doublemutant SA514rol (rol ::kmDpHS-2), however, showed apattern different from either SA514 or SA100rol. Neitherthe Mode A nor the Mode B O-antigen chains were pre-sent, and there appeared to be greater numbers of O-anti-gen chains of 6–30 repeating units than in the SA100rolsingle mutant (Fig. 3, A and C, lane 4 and Fig. 3B, lane6). The silver-stained LPS patterns of SA100rol and


Fig. 3. LPS analysis of representative mutants. LPS samples from equal numbers of bacterial cells were loaded in each lane, and wereseparated on 13% SDS–polyacrylamide gels. The gels were either silver-stained (A) or analysed in immunoblotting with S. flexneri group Bantiserum (B and C). Panel C shows the top portion of an immunoblot in which each lane contained twice the amount in (B). The strains are:SA100, wild type; SA514, SA100DpHS-2; SA514(pMA9), cldpHS-2::cam; SA514(pMN4), CldpHS-2

þ ; SA100rol, SA100rol ::km; SA514rol,SA100rol ::kmDpHS-2; SA555-148, SA100rfaX; SA555-38, SA100rfaL; SA555-96, SA100rfbC; SA555-30, SA100rfbF. ‘Core’ indicates theposition of LPS molecules with only core and no O-antigen attached. Arrows indicating ‘1 unit’ or ‘2 units’ are LPS molecules with one or twoO-antigen repeating units attached. The upper bands represent LPS molecules with an increasing number of O-antigen repeating units.Bracket A: ‘Mode A’, LPS molecules with 11–17 O-antigen repeating units. Bracket B: ‘Mode B’, LPS molecules with long O-antigen chains(probably with 90–100 O-antigen repeating units).


SA514rol were compared to that of RMA585 (the rol mutantof the S. flexneri Y serotype) (Morona et al., 1995), andRMA585 is more like SA514rol than SA100rol (data notshown). The immunoblotting data showed that RMA585O-antigen chain-length distribution was very similar to thatof the SA514rol mutant and did not have the Mode Blong chains (data not shown). The wild-type parent strainof RMA585 did not have the Mode B long O-antigen chains(data not shown), indicating that it may lack cldpHS-2.

Serum sensitivity of LPS mutants

LPS confers resistance to non-specific, complement-medi-ated serum killing in S. typhimurium and E. coli (Rowley,1968; Joiner, 1988; Taylor, 1995), but the role of LPS inprotecting S. flexneri from killing by serum was unknown.While S. flexneri is not associated with bacteraemia, itdoes provoke an inflammatory response, and resistanceto complement could play a role in surviving the localinflammatory reaction. Therefore, the S. flexneri LPSmutants constructed in this study were tested for serumsensitivity by incubating them for 2 h in the presence ofnormal human serum (10% v:v in L broth). The amountof killing by serum was determined and expressed aslog10 kill (Table 2). The killing by serum appeared to becomplement-mediated, since inactivation of complement

by heating at 568C for 15 min prior to bacterial inoculationpermitted survival to the same extent as in L broth alone(data not shown).

The wild-type strain was quite resistant to the killingeffect of serum: during incubation with serum the numberof cells increased, although not as much as in the absenceof serum, yielding a log10 kill ¼ 0.42 for the wild-type con-trol strain (Table 2 and data not shown). The serum sensi-tivities of the mutant strains varied greatly, with the log10

kill spanning more than seven orders of magnitude. All ofthe rfb mutants and the candidate rfaL mutant were killedby serum (log10 kill >6.6, Table 2). The rfaX mutant, whoseLPS profile showed an abnormal core region and a reducedamount of LPS, also was sensitive to serum killing, but wasnot as sensitive as the rfb and rfaL mutants (Table 2).These data indicate that O-antigen was required for serumresistance and that the amount of O-antigen or structureof the core influences serum sensitivity.

The distribution of chain lengths also influenced theability of S. flexneri to survive in serum. The pHS-2-curedstrain, SA514, showed a log10 kill of 1.75 (Table 2). The sen-sitivity was due to loss of the cldpHS-2 gene, because intro-duction of a recombinant plasmid with an intact cldpHS-2

gene (pMN4) restored serum resistance, while pMA9,which has an interrupted cldpHS-2 gene, did not (Table2). In contrast, the chromosomal rol mutation did not


Table 2. Serum sensitivity and tissue-culture cell assays and Sereny test of S. flexneri mutants.

Strain Mutation LPS O-antigen phenotypeaSerumsensitivityb % Invasionc

Plaqueformationd

Serenyteste

SA100 Wild type Normal 0.42 6 0.10 68.48 6 13.13 þ þþ

SA222-24 ipaC::TnphoA NT 0.20 4.70 6 2.1 ¹ ¹

SA555-38 rfaL::TnphoA Absent > 6.6 55.68 6 9.93 ¹ NTSA555-30 rfbF ::TnphoA Absent > 6.6 61.00 6 3.25 ¹ NTSA555-94 rfbA::TnphoA Absent > 6.6 72.65 6 5.16 ¹ NTSA555-85 rfbB ::TnphoA Absent > 6.6 69.15 6 8.27 ¹ NTSA555-144 rfbG::TnphoA Absent > 6.6 65.70 6 12.73 ¹ NTSA555-96 rfbC::TnphoA Only 1 unit > 6.6 73.80 6 3.11 p NTSA555-148 rfaX ::TnphoA Altered LPS core, fewer O-antigen chains,

normal length distribution3.02 6 0.94 56.70 6 14.11 p NT

SA514 SA100DpHS-2 No Mode B (90–100 unit) distribution 1.75 6 0.18 85.10 6 10.12 þ 6SA514(pMA9) cldpHS-2::cam No Mode B distribution 2.25 6 0.39 87.50 6 14.85 þ 6SA514(pMN4) CldpHS-2

þ Normal 0.32 6 0.06 86.00 6 14.14 þ þþ

SA100rol rol ::km No Mode A (11–17 unit) distribution 0.46 6 0.19 31.43 6 6.69 p 6SA514rol rol ::km DpHS-2 No Mode A or Mode B distribution 3.30 6 0.35 68.50 6 2.12 þ 6

a. ‘NT’, not tested. ‘Absent’ indicates no O-antigen but normal LPS cores. ‘Unit’ represents O-antigen repeating unit.b. Serum sensitivity was assayed as described in the Experimental procedures and is expressed as log10 kill ¼ (log10 cfu ml¹1 in the absence ofserum) ¹ (log10 cfu ml¹1 in the presence of serum). High values indicate higher sensitivity to serum killing, while lower values represent resistance.> 6.6 indicates no detectable survivors in the presence of serum. SA222-24 was tested once. Each of the other samples was tested at least threetimes, from which the average and standard deviation were calculated.c. The percentage of HeLa cells infected with three or more bacteria among at least 300 HeLa cells counted per monolayer was calculated, 6standard deviation. Two or more experiments were performed with each strain.d. ‘þ’ indicates formation of clear, round plaques of about 1–1.5 mm diameter on a confluent HeLa cell monolayer. ‘p’ (pin-point) plaques are smallclear areas (< 0.2 mm) surrounded by cells that are infected by bacteria. ‘¹’ indicates that the cell monolayer appeared the same as the negativecontrol (mock infection).e. ‘NT’, not tested. For the strains tested, 3–10 mice were used per sample. The severity of response is based on swelling and keratoconjuncti-vitis: ‘þþ’ indicates a severe response that lasted for > 72 h; ‘¹’ indicates no response; ‘6’ indicates a very weak response that lasted for less than30–40 h.


affect bacterial resistance to serum killing, since SA100rolwas as resistant to serum as the wild-type SA100 (Table2). However, when both rol and cldpHS-2 were mutated,the strain became more sensitive than the cldpHS-2 mutantto serum killing (3.30 log10 kill of SA514rol ) (Table 2).Thus the cldpHS-2-dependent Mode B O-antigens providesubstantial protection against serum killing.

Tissue-culture cell-invasion assays and Sereny testof LPS mutants

To study the role of LPS genes in Shigella invasion andcell-to-cell spread, tissue-culture cell-invasion and plaque-formation assays were carried out (Table 2). The wild-type strain SA100 was invasive and formed large plaquesin HeLa cell monolayers, indicating its ability to multiplyintracellularly and spread to adjacent cells. The negativecontrol, SA222-24, was not invasive because the invasiongene ipaC was interrupted.

All of the rfa and rfb mutants were as invasive as thewild-type strain. The HeLa cells that were infected withthese strains produced fewer, yet obvious, long protru-sions with bacteria at the end, indicating some degree ofintracellular movement of bacteria (data not shown). How-ever, the mutants were defective in cell-to-cell spread asmeasured by plaque assays in which they failed to formwild-type plaques (Table 2). SA555-148 (rfaX ) andSA555-96 (rfbC) formed pin-point plaques, while none ofthe other rfb mutants or the rfaL mutant formed detect-able plaques (Table 2). The results with these well-definedrfb and rfaL mutants are in agreement with previousstudies which showed that uncharacterized rough mutantsof Shigella were invasive but lost the ability to spread toadjacent cells (Okamura and Nakaya, 1977; Okamura etal., 1983).

The HeLa cell invasion assay and the plaqueassay showed that the cldpHS-2 mutants SA514 andSA514(pMA9) were as invasive and formed the samesize of plaques as the wild type (Table 2). This indicatesthat neither the cldpHS-2 gene nor the other genes encodedby pHS-2 were required for cell invasion, intracellularmultiplication, or spread to adjacent cells in tissue culture.The SA100rol mutant, however, was slightly less invasivethan SA100 and SA514, the invasion ratio of SA100rol toSA100 being 0.49 6 0.17 from three experiments (Table 2and data not shown). SA100rol formed plaques that weremuch smaller than those of SA100 and SA514 (Table 2).The HeLa cells that were infected with SA100rol formedthe protrusions indicative of intracellular movement, butthey had many fewer intracellular bacteria per cell (datanot shown). Surprisingly, the double mutant SA514rolwas as invasive as the wild type and formed normal-sizeplaques (Table 2).

The mouse Sereny test was carried out to study further

the roles of cldpHS-2 and rol in virulence (Table 2). Strainswith pHS-2 deleted (SA514) or carrying a disrupted cldpHS-2

gene (SA514(pMA9)) induced a much weaker responsethan that of SA100 (Table 2). Furthermore, the milderkeratoconjunctivitis in mice infected with SA514 andSA514(pMA9) disappeared at between 30 and 40 h, whilethe more severe keratoconjunctivitis in mice infected withSA100 stayed at the same level for more than 72 h (Table2). pMN4 complemented the defect in SA514, andSA514(pMN4) produced a strong positive response lastingfor more than 72 h (Table 2). The SA100rol strain, whichwas defective in intercellular spread, produced a weakand shortened response similar to that of SA514 (Table2). The double mutant SA514rol, which formed normalplaques but was sensitive to serum killing (like SA514),also caused a response that was weak and of short dura-tion (Table 2).

Discussion

A number of studies have pointed to a role for LPS in inva-sion and virulence of S. flexneri. Uncharacterized roughmutants of S. flexneri which lacked O-antigens wereshown to be normal in invasion but deficient in intercellularspread (Okamura and Nakaya, 1977; Okamura et al.,1983), and mutations in galU or rfc abolished S. flexneriplaque-forming ability (Sandlin et al., 1995; 1996). Themutations in some avirulent strains were mapped to rfband rfa regions (Okada et al., 1991a,b; Rajakumar et al.,1994), and transfer of E. coli rfe or rfaL mutations to S.flexneri resulted in mutants which made tiny plaques(Sandlin et al., 1995; 1996). Of these various mutations,galU, rfc, and some rfb were characterized in S. flex-neri. It was found that the polar localization of IcsA onthe S. flexneri surface was changed by galU, rfe, rfaL,and rfc mutations, and thus the host-cell actin polymeriza-tion was affected; this probably caused reduced bacterialintracellular and intercellular movement (Sandlin et al.,1995; 1996). In this study, we isolated and characterizedS. flexneri LPS mutants and determined the roles of var-ious LPS-synthesis genes. Some mutants were still inva-sive but could not spread normally; among these werethose with mutations in either known rfb genes or candi-date rfa genes (including rfaL). This confirms the impor-tance of LPS in Shigella pathogenesis. rfe, rfaL, andrfbA, -B, -G, or -F lack O-antigen side chains but havecomplete cores and thus each would have a longer corethan the galU mutant (this study; Sandlin et al., 1995;1996). Both rfc and rfbC have one O-antigen repeatingunit attached to the core (this study and Sandlin et al.,1996). The cause of the defective intercellular-spreadphenotype in the rfb and rfaL mutants constructed inthis study is probably the same as that for rfc, which isthat the IcsA polar localization is affected (Sandlin et al.,



1996). The rfaL mutant constructed in our study did not formplaques whereas the rfaL mutant constructed by Sandlinet al. was reported to produce tiny plaques (Sandlin etal., 1996). The difference could be a result of differencesin the nature of the mutations or in the assay system.

The observation that the rfbC mutant produced smallplaques in cell monolayers, while rfbA, -B, or -D did notform plaques at all, is consistent with the observationthat interruption of the rfbC gene resulted in less extensivealteration of LPS structure than the rfbA, -B, or -D muta-tions. However, the rfb locus consists of genes in theorder rfbBCADXFG, rfc (Fig. 1B), which were suggestedas being co-transcribed from the promoter upstream ofrfbB (Macpherson et al., 1994; Morona et al., 1994; 1995).If this model were correct, interrupting rfbC with the polarinsertion TnphoA should abolish expression of all thedownstream genes including rfbAD (needed for earlierstages of dTDP-rhamnose synthesis), rfbEFG (rhamnosyltransferases), and rfc. Thus, a polar rfbC mutant shouldhave been at least as defective as any downstream mutant,but our LPS data and plaque assay result showed theopposite, suggesting that there is a promoter betweenrfbC and rfbA. The complexity of the transcription in thisregion was also suggested by the difficulty of mappingthe transcription start point and the possible mRNA secon-dary structure of the intergenic region between galF andrfbB (Macpherson et al., 1994).

The rfaX mutant generated in this study had an alteredLPS core and appeared to have a reduced amount ofO-antigen chains. It is possible that rfaX is involved inattachment of branch sugars to the outer core in S. flex-neri (Fig. 1A). The tissue-culture cell assays showedthat the rfaX mutant was invasive but formed pin-pointplaques. The mechanism for the reduced plaque forma-tion by rfaX is unclear. Whether the defective core orthe reduction in the amount of O-antigen of the rfaXmutant affected the polar localization of IcsA remains tobe determined. Although the rfaX gene has homology toE. coli rfaY (Klena et al., 1992b), it is not possible to com-pare the effects of rfaY mutations in different organisms,because defined rfaY mutants in E. coli or S. typhimuriumhave not been reported and the role of rfaY in LPS coresynthesis was speculative (Schnaitman and Klena, 1993).An uncharacterized mutant of S. flexneri generated in aprevious study, S2687, showed a defective LPS core hav-ing normally attached O-antigen chains (Okada et al.,1991b). The rfaX mutant is probably different from S2687because S2687 had numbers of LPS molecules thatwere similar to those in the wild type (Okada et al., 1991b).

The polar effect of the TnphoA insertion in rfaX alsocould have affected downstream gene expression. In theE. coli and S. typhimurium rfa loci, rfaZ and rfaK arelocated downstream of rfaY (MacLachlan et al., 1991;Klena et al., 1992b). rfaK is thought to be a GlcNAc

transferase gene that is required for LPS core synthesis,and mutations in this gene reduced the size of LPScores and almost abolished O-antigen attachment (Makelaand Stocker, 1984; MacLachlan et al., 1991; Klena et al.,1992a). rfaZ does not have a defined function, but itsmutation in E. coli reduced the size and amount of LPScores without affecting the amount of O-antigens attached(Klena et al., 1992a). Whether analogous genes (rfaZ andrfaK ) are present downstream of rfaX in S. flexneri andexactly which gene loss produced the phenotypes in therfaX mutant strain are questions that require study. Thesequence analysis of the region upstream of rfaX in S.flexneri also revealed a partial ORF with significant homo-logy to the 38-portions of E. coli and S. typhimurium rfaIand rfaJ, which are required for LPS outer core synthesisand are located upstream of rfaY (Schnaitman and Klena,1993). This suggests that the ORF upstream of rfaX alsois an rfa gene.

Additional mutations in this study were in the O-antigenchain-length regulators cldpHS-2 and rol. rol is required forsynthesis of the 11–17 O-antigen-repeating-unit mode(Morona et al., 1995), which is referred to as ‘Mode A’ inthis article. cldpHS-2 is required for synthesizing longO-antigen chains of 90–100 O-antigen repeating units(Stevenson et al., 1995), and is referred to as ‘Mode B’in this article. The tissue-culture assays showed that therol mutant was less invasive than the wild type and formedpin-point-size plaques, whereas the cldpHS-2 mutant wasas invasive as the wild type and normal in the plaqueassay. The finding that the rol mutant was unable toform normal plaques is consistent with recent studies byVan Den Bosch et al. (Van Den Bosch et al., 1997), inwhich it was found that a rol mutant of strain 2457 wasunable to form plaques. It was shown that most rol mutantbacteria produced little or no cell-surface IcsA, and thuswere defective in F-actin tail formation (Van Den Boschet al., 1997). In our study, a rol, cldpHS-2 double mutant(SA514rol ) was also constructed, and, surprisingly, the rolmutation had no effect on invasion and plaque formationin the absence of cldpHS-2. The difference in the O-antigenchain-length distribution pattern between SA100rol andSA514rol is likely to be important to the difference in inter-cellular spread ability shown by the two mutants. Thequestion as to whether production and cell-surface pre-sentation of IcsA were restored in the double mutantSA514rol awaits further study.

The presence of LPS O-antigen side chain is necessaryfor S. flexneri resistance to complement-mediated serumkilling. All rfb mutants and the rfaL mutant were verysensitive to serum killing. Our data with the rfaX mutantfurther showed that either the amount of O-antigen sidechains presented on the cell surface or the completenessof the LPS core, even when there are O-antigen sidechains attached, also influenced serum resistance. The



rfaX mutant was more sensitive to serum killing than wasthe wild type, but less sensitive than rfb and rfaL mutants.Interestingly, while the rol mutant was resistant to serumkilling, the cldpHS-2 mutant was sensitive, although not assensitive as mutants lacking O-antigen side chains. Thedouble mutant of rol and cldpHS-2 showed even greaterserum sensitivity. These data suggest that the modal dis-tribution pattern of O-antigen chains of 11–17 repeatingunits (Mode A) is not required for serum resistance whenthe Mode B long O-antigen chains are present. Whenthe Mode B chains are absent, however, the bacteria ingeneral become more accessible to complement; underthis circumstance, the 11–17-repeating-unit Mode A canprovide some protection against serum killing.

The cldpHS-2 and rol mutants were further assessed inthe mouse Sereny test to determine whether there wasany correlation between serum resistance and ability toprovoke the prolonged keratoconjunctivitis characteristicof virulent wild-type strains. The cldpHS-2 mutants inducedless severe and shorter duration keratoconjunctivitis thanthe wild-type strain. This reduced virulence may reflect theincreased serum sensitivity of these strains. The obser-vations that the rol mutation caused reduced keratocon-junctivitis in Sereny tests (this study and Van Den Boschet al., 1997) are consistent with previous studies by othersshowing that intercellular spreading ability is required forpositive Sereny test responses (Hale, 1991). However, thedouble mutant, SA514rol (SA100rol ::kmDpHS-2), whichformed wild-type plaques, produced a Sereny responseas weak as either of the two single mutants (this work),indicating that restoring intercellular spreading ability aloneis not sufficient to restore a strong inflammatory responsein the Sereny test. These observations suggest that serumresistance is required for keratoconjunctivitis and suggesta role for cldpHS-2 in the induction of the inflammatoryresponse, at least in the mouse Sereny test. In the colonof the natural host, Shigella establishes infection by inva-sion and intercellular spread, and produces inflammatorylesions which may involve complement (Hale, 1991; San-sonetti, 1992). Although Shigella rarely causes bacter-aemia in the host, its ability to survive host killing at thestage of inflammation may be an important part of itsvirulence.

Neither the 90–100-unit O-antigen chain (Mode B) northe cldpHS-2 gene has been observed in organisms otherthan S. flexneri 2a. This may implicate cldpHS-2 in provid-ing a unique self defence advantage for S. flexneri 2astrains. Thus, the strains harbouring the plasmid pHS-2may be more virulent than those without the plasmid.pHS-2 was originally identified in S. flexneri isolates fromindividuals recovering from shigellosis and who developedReiter’s Syndrome, a form of reactive arthritis (Stieglitz etal., 1989). Those patients were all HLA-B27þ (Stieglitz etal., 1989). pHS-2 contains several small ORFs besides

cldpHS-2, including a small ORF of 22 amino acids thatshares an epitope with the human HLA-B27 molecule(Stieglitz et al., 1989). A role for this ORF or CldpHS-2 incausing reactive arthritis remains to be confirmed.

Experimental procedures

Bacterial strains and growth conditions

Bacterial strains, plasmids, and their sources are listed inTable 3. E. coli strains DH5a and TB1 were used for routinecloning (Maniatis et al., 1982). Bacterial strains were grownin L broth for general purposes. M9 minimal medium (Miller,1972) was used to grow bacteria for RNA extraction.

DNA manipulations

E. coli plasmid DNA was isolated by alkaline lysis as describedby Sambrook et al. (Sambrook et al., 1989). S. flexneri chromo-somal DNA was isolated by the method of Marmur (Marmur,


Table 3. Strains and plasmids.

Strain/Plasmid Characteristics Source/Reference

Strain

SA100 S. flexneri wild type,serotype 2a

Payne et al.(1983)

SA514 SA100 cured of pHS-2 This workSA100rol SA100 rol ::km This workSA514rol SA100 rol ::kmDpHS-2 This workSA555-30 SA100 rfbF ::TnphoA This workSA555-38 SA100 rfaL::TnphoA This workSA555-85 SA100 rfbB::TnphoA This workSA555-94 SA100 rfbA::TnphoA This workSA555-96 SA100 rfbC::TnphoA This workSA555-144 SA100 rfbG::TnphoA This workSA555-148 SA100 rfaX ::TnphoA This workSA222-24 SA514(pMA9) ipaC::TnphoA This workSA202 SA100(F8::TnphoA) Headley (1990)RMA585 S. flexneri serotype Y

rol ::kmMorona et al.

(1995)SM10lpir E. coli, pirR6K Taylor et al.

(1989)

Plasmid

F 8::TnphoA PhoA¹, KmR Manoil andBeckwith (1985)

pAT153 Cloning vector, ApR TcR Twigg andSherratt (1980)

pMA9 pHS-2 with cam inserted incldpHS-2, CmR

This work

pMB3-4 pAT153 with pHS-2 origin,ApR

This work

pME2 pHS-2 with TnphoA insertedin cldpHS-2 (cldpHS-2–phoAfusion), KmR

This work

pMN4 pHS-2 with cam inserteddownstream of cldpHS-2,CmR

This work

pNK2884 Tn10 derivative, cam flankedwith two HindIII sites

Kleckner et al.(1991)

pRT733 oriR6K mobþ, TnphoA, ApR

KmRTaylor et al.

(1989)


1961). The method of Kado and Liu (Kado and Liu, 1981) wasused to obtain the large virulence plasmid from Shigella, and amodification (Marko et al., 1982) of the alkaline lysis pro-cedure was used to obtain Shigella small plasmids. pHS-2was separated from the other Shigella plasmids by elutingthe DNA band from a 0.8% agarose gel.

TnphoA mutagenesis

In order to generate insertional mutations in potential mem-brane-protein genes, S. flexneri strain SA514(pMA9), a chlor-amphenicol-resistant (CmR) derivative of SA100 that retainedinvasion and intercellular spread abilities, was mated withSM10lpir (pRT733), which carries TnphoA (Manoil and Beck-with, 1985) on a suicide vector (Taylor et al., 1989). Kana-mycin-resistant (KmR), CmR colonies were selected and thenscreened for PhoAþ and for their ability to bind Congo reddye (Crbþ), a characteristic of invasive Shigella strains(Payne and Finkelstein, 1977). PhoAþ Crbþ colonies werescreened for ampicillin sensitivity (AmpS), indicating loss ofthe suicide vector. These mutants (SA222 series) were testedin a rapid plaque assay on HeLa cell monolayers as describedbelow. Those that did not form plaques or formed smallplaques were further tested for invasion and in the standardplaque assays in order to identify the mutants that were stillinvasive yet could not form wild-type plaques.

To confirm that the loss of plaque-forming ability in themutants was due to a single insertion, Southern hybridiza-tions were performed. Total DNA (chromosome and plas-mids) was isolated and digested with BamHI, which cuts ata single site within TnphoA, then separated on an agarosegel, blotted to nylon membranes and detected with a phoAprobe (Sambrook et al., 1989). Only one hybridizing bandwas detected in each strain, indicating a single insertion ofTnphoA. The TnphoA insertions in each of the rfb and thecandidate rfa mutants (SA222 series) were moved to thewild-type SA100 background by P1 transduction (Miller,1972), yielding the SA555-series strains. The phenotypes oftwo or more independent colonies from each transductionwere confirmed as being the same as the donor strains. P1transduction also was used to move rol ::km from RMA585into SA100 and SA514.

To obtain a cldpHS-2–phoA translational fusion, SA202(SA100(F8::TnphoA)) was grown under conditions that enrichfor PhoAþ fusions. A PhoA¹ colony of SA202 was inoculatedinto a Tris-buffered medium (Simon and Tessman, 1963)lacking KH2PO4 and containing 40 mg ml¹1 XP (5-bromo-4-chloro-3-indolyl phosphate; Sigma Chemical Co.) as thesole source of phosphate, to select for PhoAþ bacteria.phoA fusions to plasmid genes were selected by transformingan E. coli PhoA¹ strain with the pooled plasmid DNA from thebacteria (SA202) that grew and selecting for KmR and PhoAþ

phenotypes. DNA sequence analysis (Sanger et al., 1977)showed that the plasmid pME2 contained phoA insertedin-frame within cldpHS-2. SA100 was transformed with pME2and, after growth in the presence of kanamycin, pME2 replacedthe incompatible plasmid pHS-2.

Construction of recombinant plasmids and strains

To cure SA100 of pHS-2, the incompatible plasmid pMB3-4

was constructed by cloning the 1.6 kb Sau 3AI fragment con-taining the pHS-2 origin of replication (Fig. 2) into the BamHIsite within the tetracycline-resistance gene (tet ) of pAT153.SA100 was transformed with pMB3-4, which eventuallyreplaced pHS-2. To eliminate pMB3-4, this strain was trans-formed with pAT153, and tetracycline-resistant (TcR) colo-nies were selected. After several passages, pMB3-4 wasreplaced by pAT153 to yield strain SA513. SA513 subse-quently was plated on modified Bochner medium (Bochneret al., 1980; Lawlor et al., 1987) to select for tetracycline-sen-sitive (TcS) isolates, indicating loss of pAT153. The absenceof pHS-2 sequences in this strain (SA514) was confirmed byhybridization of plasmid and chromosomal DNA with a pHS-2probe.

The cldpHS-2 insertion plasmid pMA9 was constructed byinserting a 1.6 kb HindIII fragment carrying a chlorampheni-col-resistance gene (cam) from pNK2884 (Kleckner et al.,1991) into the HindIII site within cldpHS-2 (Fig. 2). The plasmidpMN4, which has the intact cldpHS-2 gene, was constructed byinserting the same, but blunt-ended, cam fragment into anopened and blunted XbaI site which is more than 100 basesdownstream of cldpHS-2 (Fig. 2). S. flexneri SA514 was trans-formed with pMA9 or pMN4, to generate SA514(pMA9), thecldpHS-2 insertion mutant, or to generate SA514(pMN4), thewild-type cldpHS-2 strain.

Sequence analysis of the adjacent regions of TnphoAinsertion in SA222 strains

The junction regions of TnphoA insertion in SA222 mutantswere obtained with one of two approaches. One approachwas to ligate BamHI fragments of the total DNA intopACYC184, transform DH5a, and select for KmR transfor-mants. As BamHI cuts within TnphoA only once and cutsdownstream of the kan gene, the BamHI fragment clonedshould carry the upstream junction region. The DNA sequenceupstream of phoA was obtained (Sanger et al., 1977) with theprimer derived from phoA (58-ATATCGCCCTGAGCAG-38).The other approach was inverse polymerase chain reaction(PCR). The total DNA was digested with TaqI and ligated atlow DNA concentration. The PCR reaction was carried outwith two primers derived from phoA sequence: Primer A58-ATATCGCCCTGAGCAG-38, and Primer B 58-CAACCGG-TGTCAAAACC-38. The PCR products were isolated by elut-ing the DNA band from an agarose gel. Sequencingreactions were carried out directly with the PCR fragmentsfrom either the A or B primer using an ABI PrismTM 377DNA sequencer (Perkin-Elmer Co., Applied Biosystem Divi-sion). With this approach, the DNA sequence betweenTnphoA and the first TaqI site upstream of the insertion sitewas obtained.

Sequence homology searches were performed with theIntelliGenetics SuiteTM Program (Release 5.4, IntelliGenetics,Inc.).

RNA isolation and dot-blot hybridization

RNA isolation was performed as described previously (Schmittand Payne, 1988). Twenty micrograms of RNA was denatured,loaded, and hybridized to a cldpHS-2 probe or a control probein a solution containing 50% formamide (Sambrook et al.,



1989). The intensity of the dots on the exposed X-ray filmswas quantified by densitometry as described by L. L. Poulsenand D. M. Ziegler (US Patent No. 5 194 949, 1993).

Phosphatase-activity assay

Bacteria were grown to exponential phase in L broth (in vitrosample) or harvested from infected HeLa cells (intracellularsample). Alkaline phosphatase (PhoA) activity was determinedas previously described (Brickman and Beckwith, 1975).

Tissue-culture cell-invasion and plaque assays

HeLa cell monolayers were used in all experiments andwere cultured in Earle’s minimal essential mediumþ 2 mMglutamineþ 10% fetal calf serum (Life Technologies) in a5% CO2 atmosphere at 378C.

The ability of S. flexneri to invade HeLa cells was deter-mined by the procedure of Hale and Formal (Hale and For-mal, 1981) as follows. HeLa cell monolayers were grown in35-mm diameter plates and infected with 2 ×108 cfu of bac-teria. The total time of incubation of the infected monolayerswas 2 h, with 16 mg gentamicin being added per ml after thefirst hour of incubation. At least 300 HeLa cells from eachplate were observed and those containing 3 or more bacteriaper cell were considered infected. Where indicated, the HeLacell protrusions indicative of bacterial intra- and intercellularmovement were counted, and the numbers of intracellularbacteria per infected HeLa cell were also counted. To isolatethe intracellular bacteria, the HeLa cell monolayers weredetached with trypsin and lysed with 0.5% w/v sodium deoxy-cholate, and the intracellular bacteria were harvested bycentrifugation as described previously (Headley and Payne,1990).

The standard HeLa cell plaque assay was performed asdescribed (Oaks et al., 1985). Confluent HeLa cell mono-layers in 35-mm plates were infected with 102–105 bacteria.After 90 min of incubation, the HeLa cells were overlaid withfresh medium plus 0.45% glucose, 0.5% agarose and 20 mgml¹1 gentamicin, and incubated for 48 h. The rapid plaqueassay was modified (K. Reed, personal communication) fromthe standard plaque assay as follows. A confluent HeLa cellmonolayer in a 100-mm plate was overlaid with 30 ml of themedium containing 0.45% glucose and 0.5% agarose. Afterthe overlay had solidified, a platinum wire was used to toucha S. flexneri colony and was stabbed through the mediumoverlay until gently touching the HeLa cell monolayer. Another20 ml of the same medium with 60 mg ml¹1 gentamicin wasadded on top of the first overlay after inoculation, and theplate was incubated for 48 h. Up to 50 strains could be testedon one plate with this method. Plaques were visualized bystaining with Wright–Giemsa stain (Baxter Scientific Products).

Mouse Sereny test

The mouse Sereny test was performed as described byMurayama et al. (Murayama et al., 1986). S. flexneri cellswere harvested from plates, washed, and resuspended insaline. Inoculum (7 ×107 cfu) was placed in one eye of each4-week-old BALB/c mouse, and sterile saline was inoculatedinto the other eye as a negative control.

Animal protocols were approved by the University of TexasAnimal Care and Use Committee.

Lipopolysaccharide extraction and SDS–PAGE analysis

LPS was extracted from S. flexneri whole-cell lysates asdescribed by elsewhere (Hitchcock and Brown, 1983). LPSpreparations from equal numbers of cells were subjected toSDS–PAGE (13%) and gels were either silver-stained usingthe Silver Stain Plus kit (Bio-Rad Laboratories) or immuno-blotted with S. flexneri group B antiserum (Difco Laboratories)and detected with horseradish peroxidase colour develop-ment reagent (Bio-Rad Laboratories) after incubation withgoat anti-rabbit IgG HRP conjugate (Bio-Rad Laboratories).

Serum sensitivity assay

Human serum was from individuals with no history of Shigellainfection. Shigella strains were diluted from exponential phaseL-broth cultures to an OD650 value of 0.05 into fresh L broth, orinto L broth with 10% serum, and were incubated without aera-tion at 378C for 2 h. Bacterial cultures at the starting point (time0) and after 2 h of incubation in each of the media were dilutedand plated to determine the cfu per ml. Serum sensitivity isexpressed by killing. Log10 kill¼ (log10 cfu ml¹1 after 2 h in theabsence of serum) – (log10 cfu ml¹1 after 2 h in the presenceof serum).

Note added in proof

The mutations in SA555-94 and SA555-96 both lie within therfb operon. However, there is not agreement on the order andidentification of the rfb genes in the cited papers and in Gen-Bank Release 100.0. Thus, the assignments of these muta-tions to rfbA and rfbC may be incorrect. The recent paperby Van Den Bosch et al. (1997) indicates that the geneorder should be rfbBDAC. The mutations in 555-94 and555-96 are in the rfb genes encoding proteins with amino-terminal sequences MNILL and MKTRK, respectively.

Acknowledgements

We gratefully thank these people for their generous help: DrMark McIntosh for contributing ideas, Dr Renato Moronaand Dr Paul Manning for providing the RMA585 (rol ) strain,Dr Robin Sandlin and Dr Anthony Maurelli for assisting withthe LPS analysis, Dr Leodocia Pope and Ms Mary Lozanofor help with the mouse Sereny test, Dr Sara Chenault for pro-viding serum, the DNA core facility of the Institute for Cellularand Molecular Biology for DNA sequencing, and Dr LawrencePoulsen and Dr R. Malcolm Brown, Jr for assisting with den-sitometry. The critical reading of this manuscript by Dr Eliza-beth Wyckoff is also gratefully acknowledged. This work wassupported by Public Health Service Grant AI 16935 from theNational Institutes of Health.

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