Cytotoxicity in Macrophages Infected with Rough Brucella Mutants Is Type IV Secretion System Dependent

Abstract

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...

Quorum-Sensing and BvrR/BvrS Regulation, the Type IV Secretion System, Cyclic Glucans, and BacA in the Virulence of Brucella ovis: Similarities to and Differences from Smooth Brucellae

“…In agreement with the observations reported for S Brucella strains (16,17,42,44), the T4SS is also essential for the multiplication and survival of R virulent B. ovis strain PA in mice and in J774.A1 macrophages (Fig. 4 and 5).…”

“…However, these results differ from those published for an R mutant (⌬manBA) of S B. melitensis 16M. The intracellular counts of that mutant decreased over time in J774.A1 murine macrophages, whereas the double mutant 16M ⌬manBA ⌬virB2 showed an unexpected resistance to killing by macrophages in the absence of T4SS that was coupled to a loss of the cytotoxic effect of the single ⌬manBA mutant (44). In contrast, the cytotoxic effect for macrophages-which has also been observed in R mutants derived from S B. abortus (43)-was not evidenced with B. ovis PA or the mutant strains obtained in this work (data not shown).…”

“…Strains of the genus Brucella may be smooth (S) or rough (R), depending on the presence or absence, respectively, of O-polysaccharide (O-PS) chains in the LPS. O-PS masks other components of the bacterial surface (6), and since R mutant (M) strains derived from S strains are impaired in the interaction with phagocytes and are attenuated in animal models of infection (22,40,44), it is considered a key virulence factor in S Brucella species (such as B.…”

“…Strains of the genus Brucella may be smooth (S) or rough (R), depending on the presence or absence, respectively, of O-polysaccharide (O-PS) chains in the LPS. O-PS masks other components of the bacterial surface (6), and since R mutant (M) strains derived from S strains are impaired in the interaction with phagocytes and are attenuated in animal models of infection (22,40,44), it is considered a key virulence factor in S Brucella species (such as B. melitensis, B. abortus, and B. suis).Nevertheless, B. ovis is a naturally R Brucella species that is virulent in its natural host and in laboratory animal models (9,33,51). This ability of B. ovis to infect the host despite the absence of O-PS in the OM, together with its tropism for the male genital tract, is indicative of virulence mechanisms differing, at least in part, from those employed by S Brucella species, which are able to colonize the placenta and typically induce abortions.…”

Mechanism of Asp24 Upregulation in Brucella abortus Rough Mutant with a Disrupted O-Antigen Export System and Effect of Asp24 in Bacterial Intracellular Survival

“…However, the mechanisms behind attenuated infection by the rough mutant have not been deciphered completely. In a previous study, artificial Brucella rough mutants were found to be cytopathic for macrophages and dependent on the type 4 secretion system (T4SS) (36,37), but the effectors delivered by the T4SS are still unknown.…”