Previous studies suggest that smooth Brucella organisms inhibit macrophage apoptosis. In contrast, necrotic cell death of macrophages infected with rough Brucella organisms in vitro has been reported, which may in part explain the failure of some rough organisms to thrive. To characterize these potential macrophage killing mechanisms, J774.A1 murine macrophages were infected with Brucella abortus S2308-derived rough mutant CA180. Electron microscopic analysis and polyethylene glycol protection assays revealed that the cells were killed as a result of necrosis and oncosis. This killing was shown to be unaffected by treatment with carbenicillin, an inhibitor of bacterial cell wall biosynthesis and, indirectly, replication. In contrast, chloramphenicol treatment of macrophages infected at multiplicities of infection exceeding 10,000 prevented cell death, despite internalization of large numbers of bacteria. Similarly, heat-killed and gentamicin-killed CA180 did not induce cytopathic effects in the macrophage. These results suggested that killing of infected host cells requires active bacterial protein synthesis. Cytochalasin D treatment revealed that internalization of the bacteria was necessary to initiate killing. Transwell experiments demonstrated that cell death is not mediated by a diffusible product, including tumor necrosis factor alpha and nitric oxide, but does require direct contact between host and pathogen. Furthermore, macrophages preinfected with B. abortus S2308 or pretreated with B. abortus O polysaccharide did not prevent rough CA180-induced cell death. In conclusion, Brucella rough mutant infection induces necrotic and oncotic macrophage cell death that requires bacterial protein synthesis and direct interaction of bacteria with the target cells.

Smooth Brucella spp. inhibit macrophage apoptosis, whereas rough Brucella mutants induce macrophage oncotic and necrotic cell death. However, the mechanisms and genes responsible for Brucella cytotoxicity have not been identified. In the current study, a random mutagenesis approach was used to create a mutant bank consisting of 11,354 mutants by mariner transposon mutagenesis using Brucella melitensis rough mutant 16M⌬ manBA as the parental strain. Subsequent screening identified 56 mutants (0.49% of the mutant bank) that failed to cause macrophage cell death (release of 10% or less of the lactate dehydrogenase). The absence of cytotoxicity during infection with these mutants was independent of demonstrable defects in in vitro bacterial growth or uptake and survival in macrophages. Interrupted genes in 51 mutants were identified by DNA sequence analysis, and the mutations included interruptions in virB encoding the type IV secretion system (T4SS) (n ‫؍‬ 36) and in vjbR encoding a LuxR-like regulatory element previously shown to be required for virB expression (n ‫؍‬ 3), as well as additional mutations (n ‫؍‬ 12), one of which also has predicted roles in virB expression. These results suggest that the T4SS is associated with Brucella cytotoxicity in macrophages. To verify this, deletion mutants were constructed in B. melitensis 16M by removing genes encoding phosphomannomutase/phosphomannoisomerase (⌬manBA) and the T4SS (⌬virB). As predicted, deletion of virB from 16M⌬manBA and 16M resulted in a complete loss of cytotoxicity in rough strains, as well as the low level cytotoxicity observed with smooth strains at extreme multiplicities of infection (>1,000). Taken together, these results demonstrate that Brucella cytotoxicity in macrophages is T4SS dependent.Brucella spp., the causative agents of brucellosis, are facultative intracellular bacteria with a broad host range. These bacteria can survive and multiply in macrophages and trophoblasts, causing abortion in a variety of animals and undulant fever in humans. The economic losses caused by brucellosis in livestock industries can have dramatic consequences for agriculture and public health, especially in developing countries. Because of the public health concerns and the absence of safe and efficacious human vaccines, Brucella melitensis, B. abortus, and B. suis have been classified as category B agents on the CDC biodefense list (31).To survive in hostile environments, intracellular bacteria have developed various strategies or virulence factors to evade elimination from the host. A number of bacteria, including Brucella, inhibit host cell apoptosis (13,17,19,41,43), which presumably enhances bacterial survival in the host. Inactivation of defense systems is another strategy used by bacteria to survive in their hosts (37). Cytotoxicity of Brucella in macrophages was originally described more than 40 years ago (14, 15). Our recent studies revealed that this cytotoxicity is macrophage specific and resembles oncosis and necrosis, not apoptosis (32, 33). The mechani...

Background: Random gene inactivation used to identify cellular functions associated with virulence and survival of Brucella spp has relied heavily upon the use of the transposon Tn5 that integrates at G/C base pairs. Transposons of the mariner family do not require species-specific host factors for efficient transposition, integrate nonspecifically at T/A base pairs, and, at a minimum, provide an alternative approach for gene discovery. In this study, plasmid vector pSC189, containing both the hyperactive transposase C9 and transposon terminal inverted repeats flanking a kanamycin resistance gene, were used to deliver Himar1 transposable element into the B. melitensis genome. Conjugation was performed efficiently and rapidly in less than one generation in order to minimize the formation of siblings while assuring the highest level of genome coverage.

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Brucella melitensis is a facultative intracellular pathogen that mainly resides within macrophages. The mechanisms employed by Brucella to adapt to harsh intracellular environments and survive within host macrophages are not clearly understood. Here, we constructed a cspA gene deletion mutant, NIΔcspA, that did not exhibit any discernible growth defect at a normal culture temperature (37 °C) or at a low temperature (15 °C). However, expression of the cspA gene in Brucella was induced by cold, acidic, and oxidative conditions, as determined via quantitative reverse transcription PCR. Unlike its parental strain, B. melitensis NI, the NIΔcspA mutant showed an increased sensitivity to acidic and H2 O2 stresses, especially during the mid-log-phase, and these stress conditions would presumably be encountered by bacteria during intracellular infections. Moreover, macrophage and mouse infection assays indicated that the NIΔcspA mutant fails to replicate in cultured J774.A1 murine macrophages and is rapidly cleared from the spleens of experimentally infected BALB/c mice. These findings suggest that the Brucella cspA gene makes an essential contribution to virulence in vitro and in vivo, most likely by allowing brucellae to adapt appropriately to the harsh environmental conditions encountered within host macrophages.