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  • INFECTION AND IMMUNITY,0019-9567/01/$04.0010 DOI: 10.1128/IAI.69.7.45284535.2001

    July 2001, p. 45284535 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



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

    and Comision Nacional de Energa 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 Martn,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.


    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 37C 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

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