Brucella spp. can establish themselves and cause disease in humans and animals. The mechanisms by which Brucella spp. evade the antibacterial defenses of their host, however, remain largely unknown. We have previously reported that live brucellae failed to induce tumor necrosis factor alpha (TNF-␣) production upon human macrophage infection. This inhibition is associated with a nonidentified protein that is released into culture medium. Outer membrane proteins (OMPs) of gram-negative bacteria have been shown to modulate macrophage functions, including cytokine production. Thus, we have analyzed the effects of two major OMPs (Omp25 and Omp31) of Brucella suis 1330 (wild-type [WT] B. suis) on TNF-␣ production. For this purpose, omp25 and omp31 null mutants of B. suis (⌬omp25 B. suis and ⌬omp31 B. suis, respectively) were constructed and analyzed for the ability to activate human macrophages to secrete TNF-␣. We showed that, in contrast to WT B. suis or ⌬omp31 B. suis, ⌬omp25 B. suis induced TNF-␣ production when phagocytosed by human macrophages. The complementation of ⌬omp25 B. suis with WT omp25 (⌬omp25-omp25 B. suis mutant) significantly reversed this effect: ⌬omp25-omp25 B. suis-infected macrophages secreted significantly less TNF-␣ than did macrophages infected with the ⌬omp25 B. suis mutant. Furthermore, pretreatment of WT B. suis with an anti-Omp25 monoclonal antibody directed against an epitope exposed at the surface of the bacteria resulted in substancial TNF-␣ production during macrophage infection. These observations demonstrated that Omp25 of B. suis is involved in the negative regulation of TNF-␣ production upon infection of human macrophages.Members of the genus Brucella are gram-negative, facultatively intracellular bacteria that can induce chronic infections in humans. Following invasion of the reticuloendothelial system, the bacteria develop intracellularly within mononuclear phagocytes. Chronic infection generally results in the fixation of infected macrophages at specific locations within the body (spleen, brain, heart, bones), and the human disease is characterized by undulant fever, endocarditis, arthritis, and osteomyelitis (42). Brucellae are also pathogenic for animals, but the pathophysiology of the human infection differs in many respects from the illness induced in animals. In domestic ruminants, infection results mainly in abortion in females and orchitis in males (15) whereas in mice, infection resembles septicemia and does not become truly chronic (18). These observations therefore suggest a species-specific interaction of Brucella organisms with the immune systems of their different hosts. To survive and multiply within the host, one of the major strategies of pathogens is to affect the expression of cytokines, which is necessary for the normal protective function of the immune response (26).In previous papers (6, 7) we have reported that brucellae can adopt the following strategy. (i) In human monocytic phagocytes (but not in mouse macrophages), Brucella spp. impair the production o...

A mutant of Brucella suis bearing a Tn5 insertion in norD, the last gene of the operon norEFCBQD, encoding nitric oxide reductase, was unable to survive under anaerobic denitrifying conditions. The norD strain exhibited attenuated multiplication within nitric oxide-producing murine macrophages and rapid elimination in mice, hence demonstrating that norD is essential for Brucella virulence.The gram-negative intracellular pathogen Brucella sp. is the causative agent of brucellosis, which is transmissible to humans from domestic animal species that are infected with B. abortus, B. suis, or B. melitensis.Multiplication inside macrophages allows Brucella to be carried throughout the host organism and to colonize specific organs. The pathogenicity of brucellae and chronicity are based on the ability of the pathogen to adapt to the environmental conditions that it encounters in its replicative niche (18) and to avoid the killing mechanisms within macrophages (3,8,10,14).Intensive studies were performed at the genetic level to investigate the factors that are essential for the adaptation of Brucella to the intracellular conditions (5). Analysis of the intramacrophagic virulome (17) confirmed that the type IV secretion system encoded by virB (19) is, to date, the main virulence factor of B. suis. It also revealed that the B. suis replicative niche is characterized by low levels of nutrients and oxygen. The latter parameter was previously observed in the phagosomes of stimulated macrophages (13). Complete genome sequences (6,12,20) have revealed that Brucella possesses all of the genes that are necessary for a complete denitrification pathway resulting in the reduction of nitrate to nitrogen. Genes encoding the four reductases Nar (nitrate reductase), Nir (nitrite reductase), Nor (nitric oxide reductase), and Nos (nitrous oxide reductase) constitute a "denitrification island" in B. suis which shares numerous similarities with that of Sinorhizobium meliloti (21); both organisms belong to the ␣-subclass of the proteobacteria. The respiratory system could allow Brucella to survive under very low oxygen tension, using nitrogen oxides as terminal electron acceptors. Bacteria may also take advantage of denitrification to cope with nitric oxide (NO) production in the macrophages during the innate response against infection. NO production by infected macrophages is a major defense system in control of Brucella infection in mice (9, 16) and possibly, although more controversial, in human infection, as revealed by the use of human macrophages transfected with inducible NO synthase (7). Despite the low levels of NO that were released by human macrophages, the nitric oxide reductase of Neisseria meningitidis was found to confer intracellular resistance to NO and allowed its utilization, resulting in the optimal survival of this bacterium in nasopharyngeal mucosa (22). In fact, during an infection of murine macrophages producing high levels of NO, B. abortus displayed increased late survival (23). The authors of that work suggested th...

Expression of the high-oxygen-affinity cytochrome cbb3 and cytochrome bd ubiquinol oxidases of Brucella suis was studied in vitro and in the intramacrophagic niche, which was previously proposed to be oxygen limited. The cytochrome cbb3 oxidase was exclusively expressed in vitro, whereas the cytochrome bd oxidase was preferentially used inside macrophages and contributed to intracellular bacterial replication.Brucellosis, an anthropozoonosis encountered worldwide, is caused by the gram-negative intracellular pathogens Brucella spp., which utilize macrophages to multiply inside a specific niche (12) and to spread throughout the organism. Low levels of nutriments and oxygen, as revealed by the analysis of the intramacrophagic virulome (11), are major features of the Brucella suis replicative niche. Phagosomes of stimulated macrophages are known to have oxygen concentrations which are lower than those found in the extracellular environment (9). Furthermore, granulomatous structures generated by the immune system during localized infection within livers, spleens, or brains of patients are characterized by oxygen deficiency (2,20,21).Pathogenicity of brucellae and chronicity are due to the ability of the pathogen to adapt to the environmental conditions encountered in its replicative niche. To perform this task, Brucella has to modify its gene expression profile to rapidly adapt to the intracellular conditions. To this end, the bacteria induce a set of virulence genes, the main one being virB, encoding a type IV secretion system (17). Expression of genes involved in adaptation to oxygen-limited conditions appeared to be crucial for intramacrophagic survival of Brucella. A previous study identified a cydB mutant of Brucella abortus lacking the cytochrome bd oxidase with high affinity for oxygen as being highly attenuated in the mouse model of infection (6). Complete genome sequences (5,8,18) have revealed that Brucella possessed the locus ccoNOQP, potentially encoding another high-oxygen-affinity oxidase, the cytochrome cbb3-type terminal oxidase. We also identified a putative transcription regulator of the FixK/Fnr family in B. suis. The present study was undertaken to investigate (i) expression of the two operons encoding the cytochrome bd and cytochrome cbb3-type terminal oxidases in vitro as well as in bacteria obtained from infected cells and (ii) their respective roles in intracellular multiplication.