Different responses of macrophages to smooth and rough Brucella spp.: relationship to virulence - PubMed (original) (raw)

Different responses of macrophages to smooth and rough Brucella spp.: relationship to virulence

María P Jiménez de Bagüés et al. Infect Immun. 2004 Apr.

Abstract

By comparing smooth wild-type Brucella strains to their rough mutants, we show that the lipopolysaccharide (LPS) O side chain of pathogenic Brucella has a dramatic impact on macrophage activation. It favors the development of virulent Brucella by preventing the synthesis of immune mediators, important for host defense. We conclude that this O chain property is firmly linked to Brucella virulence.

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Figures

FIG. 1.

(A) Infection of J774.A1 cells by different smooth and rough strains of Brucella. J774.A1 cells were infected (MOI = 40) with B. suis (▪), B. suis manb (□), B. melitensis 16M (•), B. melitensis B3B2 (▿), or B. melitensis R5 (○), and the intracellular fate of the bacteria was evaluated. The data presented are means ± standard deviations of triplicate plate counts and are representative of three different experiments. (B) Cytokine and iNOS mRNA expression in _Brucella_-infected cells. The gene expression of different cytokines or iNOS was analyzed by RT-PCR performed on mRNAs of J774.A1 cells infected for 5 h and compared to gene expression in control cells (cells) or in cells induced with 100 ng/E. coli LPS/ml. The housekeeping gene coding for β2-microglobulin was used as a standard (31). Cells were infected with B. melitensis 16M, R5, or B3B2 or B. suis 1330 or manb. Data are representative of three different experiments. The mRNAs and cDNA preparations, primers, and method used to compare amplicon intensities have been described elsewhere in detail (28, 31).

FIG. 2.

(A) Intracellular development of B. suis GFP (CFU per well) in macrophages infected with B. suis GFP or coinfected with B. suis GFP and a rough mutant of Brucella. J774.A1 cells (106/well) were infected with B. suis GFP (MOI = 40) (•) or coinfected with B. suis GFP and B. suis manb (○) or B. suis GFP and B. melitensis B3B2 (▿) (MOI = 40 for each bacteria). Infections were performed in triplicate. At different periods of time, the cells were lysed and the number of intracellular B. suis GFP cells was determined by plating the cell lysates on agar plates supplemented with ampicillin and expressed in CFU per well ± standard deviation. At 48 h p.i., in four different experiments, the number of B. suis GFP cells in macrophages infected with only B. suis GFP was significantly higher than the number of B. suis GFP cells in macrophages coinfected with B. suis GFP and B. suis manb (P < 0.05) or with B. suis GFP and B. melitensis B3B2 (P < 0.01). (B) Effect of NO and TNF-α on the development of B. suis in J774.A1 cells coinfected with B. suis and B. melitensis B3B2. J774.A1 cells (106/well) were infected with B. suis GFP (MOI = 40) or coinfected with B. suis GFP and B. melitensis B3B2 (MOI = 40, for each bacteria). After the phagocytosis step (i.e., at the same time as gentamicin), L-NAME (3 mM) or the blocking anti-TNF-αR antibody (5 μg/ml) was added (or not) to the infected cultures. Infections were performed in triplicate. Forty-eight hours later, the intracellular number of B. suis GFP cells was determined for each condition and expressed as CFU per well ± standard deviation. Experiments C and D were performed separately and repeated four times. The numbers of B. suis GFP cells phagocytosed in 30 min were as follows: 1,200 ± 400 (inoculum of B. suis GFP) and 1,500 ± 200 (inoculum of B. suis GFP and B. melitensis B3B2) for experiment C and 2,200 ± 500 (inoculum of B. suis GFP) and 1,900 ± 300 (inoculum of B. suis GFP and B. melitensis B3B2) for experiment D. In four separate experiments, at 48 h p.i., the numbers of intracellular B. suis GFP cells were (i) significantly lower in coinfections than in macrophages infected with only B. suis GFP (P < 0.001 and P < 0.01 for experiments C and D, respectively), (ii) significantly higher in coinfections performed in the presence of L-NAME than in its absence (P < 0.05), and (iii) significantly higher in coinfections performed in the presence of anti-TNF-αR than in its absence (P < 0.01).

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Different responses of macrophages to smooth and rough Brucella spp.: relationship to virulence - PubMed (original) (raw)