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INFECTION AND IMMUNITY,0019-9567/01/$04.0010 DOI: 10.1128/IAI.69.7.4528–4535.2001

July 2001, p. 4528–4535 Vol. 69, No. 7

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Brucella abortus Cyclic b-1,2-Glucan Mutants Have Reduced Virulencein Mice and Are Defective in Intracellular Replication in HeLa Cells

GABRIEL BRIONES,1,2 NORA INON DE IANNINO,1 MARA ROSET,1 ANA VIGLIOCCO,2

PATRICIA SILVA PAULO,2 AND RODOLFO A. UGALDE1*

Instituto de Investigaciones Biotecnologicas, Instituto Tecnologico de Chascomus (IIB-INTECH), Consejo de InvestigacionesCientıficas y Tecnicas, Universidad Nacional de General San Martın (CONICET-UNSAM),1

and Comision Nacional de Energıa Atomica, Division Agropecuaria,Centro Atomico Ezeiza,2 Buenos Aires, Argentina

Received 27 September 2000/Returned for modification 9 January 2001/Accepted 22 March 2001

Null cyclic b-1,2-glucan synthetase mutants (cgs mutants) were obtained from Brucella abortus virulentstrain 2308 and from B. abortus attenuated vaccinal strain S19. Both mutants show greater sensitivity tosurfactants like deoxycholic acid, sodium dodecyl sulfate, and Zwittergent than the parental strains, suggestingcell surface alterations. Although not to the same extent, both mutants display reduced virulence in mice anddefective intracellular multiplication in HeLa cells. The B. abortus S19 cgs mutant was completely cleared fromthe spleens of mice after 4 weeks, while the 2308 mutant showed a 1.5-log reduction of the number of brucellaeisolated from the spleens after 12 weeks. These results suggest that cyclic b-1,2-glucan plays an important rolein the residual virulence of the attenuated B. abortus S19 strain. Although the cgs mutant was cleared from thespleens earlier than the wild-type parental strain (B. abortus S19) and produced less inflammatory response,its ability to confer protection against the virulent strain B. abortus 2308 was fully retained. Equivalent levelsof induction of spleen gamma interferon mRNA and anti-lipopolysaccharide (LPS) of immunoglobulin G2a(IgG2a) subtype antibodies were observed in mice injected with B. abortus S19 or the cgs mutant. However, thetiter of anti-LPS antibodies of the IgG1 subtype induced by the cgs mutant was lower than that observed withthe parental S19 strain, thus suggesting that the cgs mutant induces a relatively exclusive Th1 response.

Brucella abortus is an intracellular pathogen that causesabortion in bovines and can infect humans. Abortion in cattleis the consequence of the tropism that the bacterium has forthe placenta of pregnant animals, in which it multiplies intra-cellularly (10). Brucellosis in humans is primarily a disease ofthe reticuloendothelial system, in which the bacteria multiplyinside the phagocytic cell; the intermittent release of bacteriafrom the cells into the bloodstream causes undulant fever (17,29). Brucellosis does not spread among humans; consequently,eradication of the disease from the natural reservoirs, cattle,pigs, sheep, goats, and other susceptible animals, will lead toelimination of human infection. In regions with high preva-lence of the disease, the only way of controlling and eventuallyeradicating this zoonosis is by vaccination of all susceptiblehosts and elimination of infected animals.

Vaccination represents an important tool for the control ofbovine brucellosis. One of the most used vaccines is the atten-uated strain B. abortus S19 obtained spontaneously from thevirulent strain B. abortus 2308 (24, 25, 26, 29). Live attenuatedB. abortus S19 has served for many years as an effective vaccineto prevent brucellosis in cattle (8, 18). The genetic defect thatleads to attenuation of this strain has not yet been defined.B. abortus S19 has lost some essential unknown mechanism ofvirulence. Despite this fact, the vaccinal strain conserves somedegree of virulence, being pathogenic for humans (37), and

produces abortion and persistent infection in adult vaccinatedcattle. Vaccination with B. abortus S19 is used only for sexuallyimmature animals (25, 26). Brucella, Agrobacterium, and Rhi-zobium belong, according to 16S rRNA sequences, to the a-2subgroup of the Proteobacteria (16), and comparative studies ofthe virulence genes of the plant pathogen Agrobacterium andthe endosymbiotic Rhizobium might give us new insights onBrucella virulence factors. The Brucella two-component regu-latory system (30) is highly similar to the two-component reg-ulatory system ChvG-ChvI of Agrobacterium tumefaciens (5)and ExoS-ChvI of Rhizobium meliloti (6). These two-compo-nent regulatory genes are equivalent to Salmonella PhoP-PhoQ (31) and Bordetella bronchiseptica BvgA-BvgS systems(32). In all these bacteria, the two-component sensory systemsare involved in controlling virulence or, in the case of Rhizo-bium, in nodule invasion. B. abortus bvrS bvrR mutants displayreduced invasiveness and virulence (22, 30).

A Brucella virB operon highly homologous to the A. tume-faciens virB operon was identified in Brucella suis (20) and inB. abortus (28). A B. abortus virB10 mutant lost the ability tomultiply in HeLa cells and was not recovered from the spleensof infected BALB/c mice (28). The same results were obtainedwith a B. suis virB10 mutant (20), thus demonstrating that inBrucella, as in Agrobacterium, the virB operon is involved invirulence.

In a recent report, a highly conserved B. abortus homologueof the R. meliloti bacA gene, which encodes a putative cyto-plasmic membrane transport protein required for symbiosis,was identified (14). The B. abortus bacA mutant shows de-

* Corresponding author. Mailing address: Instituto de Investiga-ciones Biotecnologicas, UNSAM, P.O. Box 30 (1650) San Martın,Pcia. de Buenos Aires, Argentina. Phone: (54-11) 4580-7285. Fax:(54-11) 4752-9639. E-mail: [email protected].

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creased survival in macrophages and reduced virulence inBALB/c mouse infection (14).

Brucella, like Agrobacterium and Rhizobium, produces cyclicb-1,2-glucans (34). chvB in A. tumefaciens and ndvB in R.meliloti were identified as the genes coding for the cyclic b-1,2-glucan synthetase (12). We recently reported that in Brucellathe biosynthesis of cyclic b-1,2-glucan proceeds by the samemechanism as in Rhizobium and Agrobacterium (4). The cyclicglucan synthetase (Cgs) acts as an intermediate during thesynthesis of the cyclic b-1,2-glucan (12). So far, cyclic b-1,2-glucan has been described only for bacteria that interact withplants as either pathogens or endosymbionts. This glucan isrequired for effective nodule invasion in symbiotic nitrogen-fixing R. meliloti and for crown gall tumor induction in A.tumefaciens (3). Agrobacterium cyclic b-1,2-glucan mutantshave several altered cell surface properties including loss ofmotility due to a defective assembly of flagella and increasedsensitivity to certain antibiotics and detergents (3).

The B. abortus S19 gene that codes for a cyclic b-1,2-glucansynthetase has previously been identified and sequenced (12).Brucella cgs, Agrobacterium chvB, and Rhizobium ndvB areinterchangeable genes. Agrobacterium or Rhizobium cyclicb-1,2-glucan mutants can be complemented by the Brucella cgsgenes, indicating that their functions are highly conserved (11,12). A preliminary characterization of B. abortus S19 cgs mu-tants showed that they had reduced survival in BALB/c mousespleen tissues, thus suggesting that this glucan might be avirulence factor (12). In this study, we examined the virulenceof B. abortus cgs mutants in mice and their intracellular repli-cation in HeLa cells. The protection induced in mice by aB. abortus S19 cgs mutant against a challenge with the virulentstrain B. abortus 2308 was also evaluated.

MATERIALS AND METHODS

Bacterial strains and growth conditions. Bacterial strains and plasmids used inthis study are listed in Table 1. Brucella strains were grown in Brucella agar (BA)(Difco Laboratories, Detroit, Mich.). Escherichia coli strains were grown in Luriabroth. Fuchsin, sodium dodecyl sulfate (SDS), Triton X-100, Zwittergent 316,and deoxycholic acid (DOC) sensitivity tests were carried out as previouslydescribed (1, 30). The absence of smooth-to-rough dissociation was checked bytesting the sensitivity to smooth-specific phages (Tb, Wb, and Iz) (1).

HeLa cell culture and infection assay. HeLa cells were grown at 37°C in 5%CO2 atmosphere in minimal essential medium (Gibco, Paisley, Scotland) sup-plemented with 5 mM glutamine and 5% fetal calf serum. Infection of cells withdifferent Brucella strains was performed as previously described (22, 28).

Construction of B. abortus b-1,2-glucan synthetase mutant and geneticcomplementation. Construction of B. abortus strains was carried out by genereplacement of the wild-type cgs gene with a Tn3-HoHo 1 mutated gene (12)(strain BAI129). Confirmation of transposon position was carried out by PCRand Southern blot hybridization. For genetic complementation of cgs mutants,plasmid pCD523 containing the A. tumefaciens chvB gene (9) or plasmid pBA19containing the B. abortus cgs gene (12), were introduced in strains BAI129 orBvI129 by biparental mating using E. coli S17.1 as the donor strain (7, 12).Complemented B. abortus cgs mutants are described in Table 1.

Pathogenicity in mice. Nine-week-old female BALB/c mice were injectedintraperitoneally with 0.2 ml of a suspension containing the appropriate numberof viable brucellae. Stock cultures were grown for 48 h on BA plates, and cellswere suspended in sterile 0.15 M NaCl and adjusted turbidimetrically to theselected concentration. The exact bacterial concentration was calculated retro-spectively by viable counts. At selected times postinfection, groups of five micewere bled by cardiac puncture and sera were pooled and held frozen at 220°Cuntil use. Mice were killed by cervical dislocation. Spleens were homogenized in5 ml of 0.15 M NaCl, serially diluted, and plated by triplicate on BA plates withthe appropriate antibiotic (15).

Vaccination and evaluation of protection after challenging with virulent strain2308. Nine-week-old BALB/c mice were vaccinated with 104 CFU of B. abortusS19 wild type or B. abortus S19 BAI129 cgs mutant (12). At 8 weeks postvacci-nation, no bacteria were isolated from the spleens of animals infected with S19or BAI129 and animals were challenged intraperitoneally with different doses ofB. abortus virulent strain 2308. One week after challenge, five animals pertreatment were killed the numbers of viable Brucella recovered from the spleensand the weights of the spleens were determined as described (15). In order todistinguish strain S19 from strain 2308, counts were carried out on BA plates with0.01% erythritol as previously described (15).

Semiquantitation of IFN-g and IL-4 mRNA in spleens of vaccinated mice.BALB/c mice injected with 104 CFU of B. abortus S19 or the B. abortus S19 cgsmutant were sacrificed after 4 or 8 days, and total RNA was extracted fromspleens with TRIzol reagent (GIBCO BRI, Gaithersburg, Md.) as previouslydescribed (21). Concentration and purity of extracted RNA were determined bydetermining the A260 and the A260/A280 ratio. Five micrograms of RNA werereverse transcribed using a commercial kit (Superscript II RT, GIBCO BRL)with a 12- to 18-mer deoxy-T oligonucleotide primer. Quantitation of reverse-transcribed mRNA by PCR was carried out as described previously (21). PlasmidpMus (kindly provided by D. Shire and F. Pitossi) harboring the same primersequences for the amplification of several murine cytokines and some house-keeping mRNAs was used as a competitive fragment. The primer sequences usedwere as follows: for b2-microglobulin sense, TGACCGGCTTGTATGCTATC;for b2-microglobulin antisense, CAGTGTGAGCCAGGATATAG; for gammainterferon (IFN-g) sense, GCTCTGAGACAATGAACGCT; for IFN-g anti-sense, AAAGAGATAATCTGGCTCTGC; for interleukin-4 (IL-4) sense, TCGGCATTTTGAACGAGGTC; and for IL-4 antisense, GAAAAGCCCGAAAGAGTCTC. The expected sizes of the PCR products were 222 bp for b2-microglobulin, 227 bp for IFN-g, and 216 bp for IL-4. The concentration ofb2-microglobulin in each sample was determined to check reverse-transcription(RT) efficiency and the accuracy of the determination of RNA concentration.For semiquantitation of IL-4 and IFN-g mRNA, 5 3 104 molecules of pMuswere coamplified in each PCR in the presence of 3 mCi of [a-32P]dCTP. Theseparation of amplicons was accomplished on agarose gel (1.2%) in Tris boratebuffer in the presence of ethidium bromide. The bands of the gel were inspectedat 365 nm and excised, and radioactivity was counted in a liquid scintillator. Foreach amplification, the ratio of counts per minute of cellular amplicon to countsper minute of standard amplicon was calculated. Each individual sample wasamplified by PCR at least twice to exclude casual errors. Spleen RNA sampleswere obtained from three mice subjected to the same treatment and analyzedseparately.

TABLE 1. Bacteria and plasmids

Strain orplasmid Phenotypea and/or genotype Reference

or source

StrainsB. abortus 2308 Virulent, field isolated; wild type,

Nalr, erythritolr25

B. abortus S19 Vaccine strain, Nalr, erythritols;naturally occurring derivative ofB. abortus 2308

25

B. abortus RB51 Vaccine strain, stable rough mutant 27BAI129 B. abortus S19 cgs mutant; Tn3-

HoHo 1::chromosome; Ampr12

BvI129 B. abortus 2308 cgs mutant; Tn3-HoHo 1::chromosome; Ampr

This study

BAI129(pBA19) B. abortus S19 cgs mutant withcosmid pBA19; Ampr Tcr

This study

BvI129(pBA19) B. abortus 2308 cgs mutant withcosmid pBA19; Ampr Tcr

This study

BAI129(pCD523) B. abortus S19 cgs mutant withcosmid; pCD523; Ampr Tcr

11

BvI129(pCD523) B. abortus 2308 cgs mutant withcosmid; pCD523; Ampr Tcr

This study

PlasmidspBA19 pVK102 containing B. abortus S19

cgs gene; Tcr12

pCD523 pLAFR1 containing A. tumefacienschvB gene; Tcr

9

a Nalr, nalidixic acid resistance; Ampr, ampicillin resistance; Tcr, tetracyclineresistance.

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Determination of antibody titers by ELISA and KELA. Antibody titers againstB. abortus lipopolysaccharide (LPS) were measured in an indirect, computer-assisted kinetics-based enzyme-linked assay (KELA), as described by Jimenez deBagues et al. and Winter et al. (13, 36). The indirect enzyme-linked immunosor-bent assay (ELISA) to measure antibodies against Brucella LPS in the sera ofmice was performed as described by Nielsen et al. (19) with some modification(7).

TLC of cyclic b-1,2-glucan and PAGE of membrane proteins. Cyclic b(1-2)glucans were extracted by the ethanol method (70% ethanol for 1 h at 37°C) fromcell pellets of the different strains. Ethanolic extracts were concentrated andsubmitted to thin-layer chromatography (TLC) as described previously (4). SDS-polyacrylamide gel electrophoresis (PAGE) of membrane proteins and fluoro-graphy were performed as previously described (4, 12).

Western blotting. SDS-PAGE and Western blot analysis of LPS O antigenwere performed as described by Comerci et al. (7). Whole-cell lysates weresolubilized in Laemmli buffer at 100°C, electrophoresed by SDS–12% PAGE andtransferred to nitrocellulose filters. The filters were reacted with M84 anti-Ochain monoclonal antibody (19) (kindly provided by K. Nielsen) diluted 1:5,000and incubated with peroxidase-conjugated goat anti-mouse immunoglobulin(Sigma Chemicals Co., St. Louis, Mo.) diluted 1:10,000. Peroxidase activity wasdetected by the ECL Western blotting kit from Amersham Pharmacia Biotech.

Statistical analysis. Differences between the means of experimental and con-trol groups were analyzed using the Student t test. Differences were consideredsignificant at P values of ,0.05.

RESULTS

Characterization of B. abortus cgs mutants. The B. abortus2308 cgs mutant (strain BvI129) and the B. abortus S19 cgs mutant(strain BAI129) were obtained by targeted insertional mu-tagenesis as described in Materials and Methods. Both mutantswere complemented with plasmid pBA19 containing theB. abortus cgs gene or with plasmid pCD523 containing the A.tumefaciens chvB gene. Some of the phenotypic characteristicsof the mutants and complemented strains are shown in Table2. B. abortus cgs mutants do not form cyclic b-1,2-glucan, andthe synthesis was restored by plasmid pBA19 (Fig. 1 and Table2). Complementation of b-1,2-glucan synthesis of cgs mutantswas also achieved with plasmid pCD523, which contains theAgrobacterium cyclic b-1,2-glucan synthetase gene (11), thusindicating that Agrobacterium Cgs is also active in the Brucellabackground. Brucella cgs mutants did not grow in media con-taining DOC, SDS, Zwittergent, or fuchsin (Table 2), suggest-ing that the lack of the Cgs membrane protein or the inabilityto produce cyclic b-1,2-glucan determines a major membranedefect that affects susceptibility to these compounds. All these

inhibitions were relieved when the mutants were comple-mented with Agrobacterium chvB or Brucella cgs genes.

The O antigen transfer to the outer membrane of the cgsmutants was demonstrated by studying sensitivity to three dif-ferent lytic phages that are known to recognize the O chain ofthe Brucella LPS (phages Tb, Wb, and Iz) and resistance to onephage that recognizes rough strains (phage Rc) (1). The pres-ence of the O chain in cgs mutants was confirmed by Westernblot analysis using monoclonal antibody against the BrucellaLPS O antigen (Fig. 2) (19).

TABLE 2. Phenotypic characterization of B. abortus strains

Characteristic

Result for B. abortus strain:

S19 BAI129 BAI129(pBA19)a 2308 BvI129 BvI129

(pBA19)a

b (1-2) Glucanb 1 2 1 1 2 1316-kDa proteinc 1 2 1 1 2 1Fuchsind 1 2 1 1 2 1DOCd 1 2 1 1 2 1Zwittergentd 1 2 1 1 2 1SDSd 1 2 1 1 2 1

a Strains BAI129 and BvI129 were complemented with plasmid pBA19.b The presence of b(1-2)glucan was determined by TLC of 70% ethanol

extract.c A 316-kDa protein was detected by SDS-PAGE as described in Materials and

Methods.d Sensitivity to DOC (0.4 mg/ml), Zwittergent (20 mg/ml), SDS (20 mg/ml), and

fuchsin (20 mg/ml) assays were carried out as previously described (1).

FIG. 1. TLC of cyclic b-1,2-glucans accumulated by differentB. abortus strains. Cyclic b-1,2-glucans were extracted and submitted toTLC as described in Materials and Methods. Lanes: 1, B. abortus 2308;2, B. abortus 2308 cgs mutant (BvI129 strain); 3, B. abortus strainBvI129 complemented with plasmid pBA19; 4, B. abortus S19; 5,B. abortus S19 cgs mutant (BAI129 strain); 6, B. abortus strain BAI129complemented with plasmid pBA19. p and pp, migration of chargedand neutral B. abortus cyclic b-1,2-glucans, respectively.

FIG. 2. Western blot of different B. abortus strains. Whole-cell ly-sates of different B. abortus strains were electrophoresed and trans-ferred, and LPS O antigen was detected as described in Materials andMethods. Lanes: 1, B. abortus 2308 (wild-type strain); 2, B. abortus2308 BvI129 (cgs mutant); 3, B. abortus S19 (vaccinal strain); 4, B. abor-tus S19 BAI129 (cgs mutant); 5, B. abortus RB51.

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B. abortus cgs mutants have reduced virulence in mice. Therecovery of B. abortus 2308 and B. abortus S19 cgs mutants(strains BvI129 and BAI129) from the spleens of mice wasstudied as described in Materials and Methods. Twelve weekspostinfection, the persistence of the B. abortus 2308 cgs mutant(strain BvI129) in the spleens of mice inoculated with 104 CFUper mouse was reduced by 1.5 logs (P , 0.01) compared to theparental wild-type strain (Fig. 3A), while the B. abortus 2308-infected mice remained relatively at the same value at 4 and 12weeks.

It can be seen in Fig. 3B that at the same infection dose noB. abortus S19 cgs mutant was recovered from the spleens after4 weeks. These results indicate that the persistence of B. abor-tus in mouse spleens is reduced by the cgs mutation, althoughthe effect was more drastic in the attenuated B. abortus S19strain. The time of clearance of B. abortus S19 cgs mutantdepends on the doses (Table 3); even when the initial dose wasas high as 7 3 108 CFU/animal, the recovery from the spleen

after 4 weeks was reduced by 3 logs compared to the parentalwild-type strain (Table 3), thus suggesting that this gene mightcode for a function that is required, but not sufficient, forvirulence.

Complementation of B. abortus cgs mutants with plasmidpBA19 or pCD523 restored to wild-type levels the numbers ofBrucella organisms isolated from spleens 4 weeks postinfection(Table 3), thus suggesting that Cgs is responsible for persis-tence in the spleen and that the Agrobacterium gene is correctlyexpressed in the Brucella background.

Induction of splenomegaly. As shown in Table 3, B. abortusS19 induces a dose-dependent transient splenomegaly as aconsequence of inflammatory response. At all the tested doses,B. abortus S19 cgs mutant induced a reduced response com-pared to that of the wild-type strain. Complementation of cgsmutant with plasmids pBA19 or pCD523 restored the induc-tion of splenomegaly (Table 3). The pathogenic B. abortusstrain 2308 at infection doses of 104 CFU per animal induced

FIG. 3. Brucella persistence in spleens of mice inoculated with different strains of B. abortus. Mice were inoculated intraperitoneally asdescribed in Materials and Methods with 104 CFU of B. abortus. (A) Shaded bars, B. abortus 2308 (wild-type strain); open bars, B. abortus 2308BvI129 (cgs mutant). (B) Shaded bars, B. abortus S19 (vaccinal strain); open bars, B. abortus S19 BAI129 cgs mutant. At different timespostinfection (2, 4, or 12 weeks) five mice per group were killed and their spleens were removed. The numbers of CFU in spleen tissues weredetermined as indicated in Materials and Methods.

TABLE 3. Recovery of B. abortus S19 and B. abortus S19 cgs mutant from the spleens of BALB/c micea

Dose(CFU)

Time(wks)

Log CFU/spleen inoculated with: Wt (mg) of spleen inoculated with:

S19 BAI129 BAI129(pBA19) S19 BAI129 BAI129(pBA19)

7 3 108 2 7.04 6 0.04 5.56 6 0.19 7.11 6 0.08 993 6 21 668 6 13 1,140 6 444 5.98 6 0.06 3.19 6 0.01 5.00 6 0.07 409 6 13 189 6 8 392 6 32

5 3 106 2 6.88 6 0.05 5.70 6 0.09 NDb 750 6 33 131 6 7 ND4 6.98 6 0.04 1.70 6 0.01 ND 564 6 12 104 6 7 ND

103 2 6.2 6 0.02 1.9 6 0.02 ND 102 6 11 90 6 10 ND4 5.1 6 0.05 0 ND 115 6 12 87 6 9 ND

a Mice inoculated with 7 3 108 CFU of B. abortus cgs mutant complemented with plasmids pBA19 or pCD523 showed values not statistically different from thoseobserved with the wild-type parental strain. Mice were inoculated intraperitoneally with different doses of B. abortus S19 or B. abortus BAI129 cgs mutant. At differenttimes postinfection, five mice were killed and their spleens were processed as described in Materials and Methods. The mean spleen weight for noninfected mice was90 6 10 mg. Bacterial doses were calculated retrospectively. Results are means 6 standard errors of values obtained in triplicate.

b ND, not determined.

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a threefold increase in spleen weight 2 weeks postinfection(225 6 25 mg [mean 6 standard deviation]) which persistedafter 12 weeks (249 6 60 mg). Although the numbers of bac-teria recovered from spleens of mice infected with wild-typestrains and from those infected with cgs mutant strains werenot significantly different, no induction of splenomegaly wasobserved with the B. abortus 2308 cgs mutant at 2 weeks postin-fection (77 6 30 mg). At 12 weeks postinfection the spleno-megaly increased twofold (170 6 27).

These results suggest a correlation between the presence ofcyclic b-1,2-glucan and/or Cgs protein and the induction ofspleen inflammatory response in mice.

HeLa cells infection. B. abortus is able to infect and multiplyinside HeLa cells. The infection process has two phases,invasion and replication. The invasion phase (0 to 8 h post-infection) is a period during which Brucella enters into thecells but does not multiply (28). During the replication phase,Brucella organisms reach the rough endoplasmic reticulumand start to replicate, causing no cytopathological cell signs(22).

It is shown in Fig. 4 that 4 h postinfection the same numbersof intracellular bacteria were recovered from cells inoculatedwith B. abortus 2308, B. abortus S19, and their respective cgsmutants. These results indicate that the absence of cyclic glu-can does not affect cell invasion. After 4 h, virulent B. abortusstrain 2308 started multiplying exponentially, reaching 1.7 3107 CFU at 48 h (Fig. 4). The B. abortus 2308 cgs mutant (strainBvI129) displayed a lower rate of intracellular replication thanthe wild-type parental strain, reaching 9 3 105 CFU at 48 h

(Fig. 4). The attenuated B. abortus strain S19 has an intracel-lular rate of replication similar to that observed with theB. abortus 2308 cgs mutant BvI129, reaching a similar number(3.5 3 105 CFU) at 48 h (Fig. 4). On the other hand, theB. abortus S19 cgs mutant strain BAI129 multiplies at a verylow rate, reaching 1.2 3 104 CFU at 48 h (Fig. 4).

The intracellular replication of BvI129 and BAI129 cgs mu-tants was restored by plasmid pBA19 (Fig. 4). These resultssuggest that the cyclic glucan and/or the Cgs protein is requiredfor normal B. abortus intracellular replication in both thepathogenic 2308 strain and the attenuated S19 strain.

B. abortus BAI129 as vaccine. One of the drawbacks of thevaccine strain B. abortus S19 is that in cattle, mice, and humansit displays some degree of virulence, causing abortion in preg-nant cows (10). In order to determine if the less pathogenicB. abortus BAI129 (cgs mutant) remains immunogenic, exper-iments of protection in mice were carried out. The B. abortusS19 cgs mutant strain BAI129 protected mice against a chal-lenge with the virulent strain B. abortus 2308 to the same extentas the wild-type B. abortus S19 strain (Table 4). This indicatesthat this mutant, although having reduced virulence and im-paired ability to multiply intracellularly in HeLa cells, com-pletely retained the ability to protect mice. As shown in Table4, protection experiments with B. abortus S19 or B. abortusBAI129 were carried out and the animals were challenged withdifferent doses of the wild-type pathogenic strain. No signifi-cant differences were observed between the B. abortus S19 wildtype and the cgs mutant at any dose.

Quantitation of IFN-g and IL-4 mRNA in spleens of in-fected mice. BALB/c mice were infected with 104 CFU ofB. abortus S19 or the B. abortus S19 cgs mutant and at differentpostinfection times spleens were removed and total RNA wasextracted. Quantitation of IFN-g and IL-4 mRNA was carriedout by RT-PCR as described in Materials and Methods. At 4

FIG. 4. Intracellular replication of different strains of B. abortus.HeLa cells were inoculated with 5 3 107 CFU of different strains ofB. abortus. After 2 h of incubation at 37°C, cells were washed andstreptomycin and gentamicin were added as described in Materials andMethods. Numbers of CFU were determined at the indicated times.Each determination is the average of two independent experimentscarried out in duplicate. In all cases, the standard error for each pointwas less than 5%. Symbols: v——v, B. abortus 2308; v----v, B. abor-tus BvI129 (cgs mutant strain); j——j, B. abortus S19; V....V;B. abortus BAI129 (cgs mutant strain); Œ——Œ, B. abortus BvI129(pBA19); ‚——‚, B. abortus BAI129(pBA19).

TABLE 4. Protection against B. abortus 2308 provided toBALB/c mice after vaccination with B. abortus S19

or the B. abortus BAI129 cgs mutanta

Challengedose

(CFU)

Vaccinationstrainb

Log CFU/spleen1 wk p.i.c (mean

6 SD)

Spleen wt (g)(mean 6

SD)Protectiond

109 None 8.60 6 0.04 NDe 0.00S19 6.44 6 0.03 0.51 6 0.02 2.16BAI129 6.79 6 0.03 0.44 6 0.03 1.91

107 None 6.27 6 0.10 0.34 6 0.03 0.00S19 3.52 6 0.01 0.19 6 0.05 3.05BAI129 2.99 6 0.56 0.14 6 0.01 3.29

105 None 6.55 6 0.05 0.20 6 0.04 0.00S19 0 f 0.18 6 0.04 6.55BAI129 0 f 0.13 6 0.04 6.55

a Mice were inoculated intraperitoneally with 104 CFU of B. abortus S19 orB. abortus BAI129. One month postvaccination, mice were challenged withdifferent doses of B. abortus 2308. Eight week later, the animals were killed andtheir spleens were removed and weighed. Spleens were processed as described inMaterials and Methods.

b Vaccination doses were 104 CFU.c p.i., postinfectiond Protection 5 log CFU of control non-vaccinated mice 2 log CFU of vacci-

nated mice (15).e ND, not determined.f Detection limit was 102 CFU.

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days postinfection B. abortus S19 or the B. abortus S19 cgsmutant induced in the spleen an accumulation of IFN-gmRNA that persisted after 8 days (Fig. 5). No induction ofIL-4 mRNA was detected at any time with the mutant or theparental S19 strain (data not shown).

Patterns of immunoglobulins elicited in mice by B. abortusS19 and B. abortus BAI129 cgs mutant. The Th1 cytokineIFN-g promotes switching to IgG2a, whereas IL-4, a productof Th2 cells, promotes switching to IgG1 and IgE (25, 37). AnELISA for IgG1 and IgG2a was carried out to estimate theTh1/Th2 ratio of host immune response to B. abortus S19 andthe B. abortus S19 cgs mutant. Figure 6 shows the anti-LPSantibody titers of serum pools obtained from animals infectedwith 7 3 108 CFU of B. abortus S19 and the B. abortus S19 cgsmutant BAI129 (Fig. 6A and B, respectively) at differentpostinfection times. It is observed that BAI129 induced aswitching to IgG2a with low levels of IgG1, thus confirminga good IFN-g response and the absence of IL-4 induction

(see “Quantitation of IFN-g and IL-4 mRNA in spleens ofinfected mice” above) (23, 35).

DISCUSSION

In this study, we showed that the absence of cyclic b-1,2-glucan in B. abortus is associated with a reduction of virulencein mice and defective intracellular multiplication in HeLa cells.

B. abortus cgs mutants display increased sensitivity to surfac-tant compounds. This association between reduction of vir-ulence and sensitivity to surfactants was also observed inB. abortus mutants in the two-component regulatory system(30). With cgs mutants no correlation was observed betweensensitivity to surfactants and virulence. B. abortus 2308 and S19cgs mutants have the same sensitivity to DOC, SDS, and Zwit-tergent. However, in strain 2308 the effect on attenuation ofvirulence was lower than that observed with the attenuatedvaccinal S19 strain, even though this may be the result ofmultiple defects in S19.

Some reports suggest that the presence of cyclic b-1,2-glucanin the bacterial periplasm may stabilize membrane proteinsagainst improper assembly or disassembly (33). For example,Swart et al. (33) showed that chvB mutants of A. tumefaciensproduced an inactive form of the protein rhicadhesin whichresulted in the attachment-minus phenotype of chvB mutantand that the normal phenotype was restored by the addition ofthe purified rhicadhesin from a wild-type strain. Banta et al. (2)showed that the chvB mutant of A. tumefaciens exhibits lowerlevels of the VirB10 protein than does the wild type. VirB10 isa transmembrane protein that is part of the type IV secretionsystem required for T-DNA delivery into plant cells; it hasbeen recently demonstrated that this type IV secretion systemis conserved in B. abortus and that virB10 null mutants areavirulent (28). Rhizobium and Agrobacterium cgs mutants arenonmotile due to a defective assembly of the flagella, a processthat is known to take place in the periplasmic space (34). Allthese observations suggest that the absence of cyclic glucanand/or Cgs inner membrane may be important for virulence.On the other hand, it cannot be excluded that the Cgs 316-kDa

FIG. 5. Quantitation of IFN-g in spleen of infected mice. BALB/cmice were infected with 104 CFU of B. abortus S19 (shaded bars) orB. abortus cgs mutant (open bars). At different times postinfection,spleens were removed and total RNA was extracted and analyzedseparately. (A) Quantitation of IFN-g mRNA was carried out byRT-PCR as described in Materials and Methods. Error bars indicatestandard deviations. (B) Agarose gel of the amplicon products fromthree mice at each time point. After the gel dried, radioactivity wasdetected by autoradiograph.

FIG. 6. Pattern of immunoglobulins elicited by B. abortus S19 orthe B. abortus BAI129 cgs mutant. KELA results for pooled sera fromfive mice inoculated with 7 3 108 CFU of B. abortus S19 (A) orB. abortus BAI129 (B) obtained at 1, 2, or 4 weeks postinfection areshown. IgG1 plus IgG2a give total KELA units.

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inner membrane protein may have a direct effect on virulenceby itself.

Our results demonstrate that the residual virulence ofB. abortus S19 depends on the presence of cyclic b-1,2-glucan,since BAI129 (cgs S19 mutant) was strongly attenuated in miceand displayed no intracellular multiplication in HeLa cells.

Montaraz and Winter (15) have shown that the number ofCFU recovered from spleens of mice infected with B. abortusS19 peaked at 2 weeks postinfection with doses of 3.8 3 104 or3.8 3 105 CFU. The behavior of the B. abortus S19 cgs mutant(strain BAI129) showed a different pattern of growth with asharp declination of spleen counts after 2 weeks at all testeddoses, thus indicating the inability of the mutant to replicate inthe mice.

Despite the fact that the B. abortus S19 cgs mutant wascleared from the infected mice faster than the parental strain,due to reduction of virulence, our results showed that themutant protected the mice against a challenge with the patho-genic B. abortus strain 2308 to the same extent as the parentalvaccinal S19 strain.

In a recent study, Power et al. (23) proposed that the dose ofthe Mycobacterium bovis BCG vaccine is crucial in determiningthe Th1/Th2 nature of the immune response. They demon-strated that relatively low doses lead to an almost exclusivecell-mediated Th1 response, while higher doses induce a mixedTh1/Th2 response. We show that mice inoculated with theB. abortus S19 cgs mutant present equivalent levels of IFN-gmRNA as well as lower titers of anti-LPS antibodies of IgG1subtype than those observed in mice vaccinated with the pa-rental S19 strain, suggesting an almost exclusively Th1 re-sponse. These results may be explained by the limited replica-tion of the mutant in the spleen.

The decreased virulence with retention of the capacity toconfer immunity suggests that exploring the cgs S19 mutant asa potential improvement of the B. abortus S19 vaccine might beworthwhile.

ACKNOWLEDGMENTS

We thank J. J. Cazzulo, A. C. C. Frasch, D. Comerci, and J. Ugaldefor helpful comments, and Fabio Fraga, Ernesta Bissi, and Juan Be-nitez for technical assistance.

This work was supported by grants from the Ministerio de Cultura yEducacion, Republica Argentina, to the Instituto de InvestigacionesBiotecnologicas de la Universidad Nacional de General San Martın(Agencia Nacional de Promocion Cientıfica y Tecnologica PICT 97-00080-01768 and PICT 99-06565). N.I.D.I. and R.A.U. are membersof the Research Career of CONICET. M.R. is a fellow of the ConsejoNacional de Investigaciones Cientıficas y Tecnicas CONICET. G.B.,A.V., and P.S.P. are members of CNEA.

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Editor: V. J. DiRita

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