The pathogen Brucella suis resides and multiplies within a phagocytic vacuole of its host cell, the macrophage. The resulting complex relationship has been investigated by the analysis of the set of genes required for virulence, which we call intramacrophagic virulome. Ten thousand two hundred and seventy-two miniTn5 mutants of B. suis constitutively expressing gfp were screened by fluorescence microscopy for lack of intracellular multiplication in human macrophages. One hundred thirty-one such mutants affected in 59 different genes could be isolated, and a function was ascribed to 53 of them. We identified genes involved in (i) global adaptation to the intracellular environment, (ii) amino acid, and (iii) nucleotide synthesis, (iv) sugar metabolism, (v) oxidoreduction, (vi) nitrogen metabolism, (vii) regulation, (viii) disulphide bond formation, and (ix) lipopolysaccharide biosynthesis. Results led to the conclusion that the replicative compartment of B. suis is poor in nutrients and characterized by low oxygen tension, and that nitrate may be used for anaerobic respiration. Intramacrophagic virulome analysis hence allowed the description of the nature of the replicative vacuole of the pathogen in the macrophage and extended our understanding of the niche in which B. suis resides. We propose calling this specific compartment ''brucellosome.'' I nteractions between microorganisms and their hosts extend from acute infections to persistent infectious diseases or symbiosis. This type of interaction can result from a million years of coevolution and coadaptation of the two organisms. Thus, the biology of the interaction can be read, at least partially, in the genome of the microorganism. In the specific case of pathogenic bacteria, deciphering of the genes involved in the interaction and analysis of their functions will shed light on the environment encountered by the parasite in the host and will contribute to the understanding of the complex relationship between two organisms. As a name for the whole set of genes required for virulence, i.e., involved in the invasion of the host by the bacteria and their adaptation to the environment provided by this host, we propose virulome. In this study, we will perform a thorough analysis of the intramacrophagic virulome of Brucella suis.Brucella spp. is an ␣ proteobacteriaceae that induces a persistent disease in some mammals, resulting in abortion. In humans, initial septicemia may be followed by a subacute or a chronic infection (1). Brucella spp. is phyletically related as well to plant symbionts such as rhizobiaceae, as to rickettsiae, which generate an acute infectious disease (2). In terms of virulence, brucellae occupy an intermediate position where the adaptation results in a mild disease that allows them to persist in mammal hosts. It is usually considered that, for a facultative intracellular bacterium such as Brucella spp., which multiplies in trophoblasts or macrophages (3), one of the challenges is to rapidly adapt to the intracellular settings but also to resis...

A type IV secretion system similar to the VirB system of the phytopathogen Agrobacterium tumefaciens is essential for the intracellular survival and multiplication of the mammalian pathogen Brucella. Reverse transcriptase-PCR showed that the 12 genes encoding the Brucella suis VirB system form an operon. Semiquantitative measurements of virB mRNA levels by slot blotting showed that transcription of the virB operon, but not the flanking genes, is regulated by environmental factors in vitro. Flow cytometry used to measure green fluorescent protein expression from the virB promoter confirmed the data from slot blots. Fluorescence-activated cell sorter analysis and fluorescence microscopy showed that the virB promoter is induced in macrophages within 3 h after infection. Induction only occurred once the bacteria were inside the cells, and phagosome acidification was shown to be the major signal inducing intracellular expression. Because phagosome acidification is essential for the intracellular multiplication of Brucella, we suggest that it is the signal that triggers the secretion of unknown effector molecules. These effector molecules play a role in the remodeling of the phagosome to create the unique intracellular compartment in which Brucella replicates.

During the complex interaction between an infectious agent and a host organism, the pathogen can interfere with the host cell's programmed death to its own benefit. Induction or prevention of host cell apoptosis appears to be a critical step for determining the infection outcome. Members of the gram-negative bacterial genus Brucella are intracellular pathogens which preferentially invade monocytic cells and develop within these cells. We investigated the effect of Brucella suis infection on apoptosis of human monocytic phagocytes. The present study provides evidence that Brucella infection inhibited spontaneously occurring apoptosis in human monocytes. Prevention of monocyte apoptosis was not mediated by Brucella lipopolysaccharide and required bacterial survival within infected cells. Both invaded and noninvaded cells were protected, indicating that soluble mediators released during infection were involved in the phenomenon. Analysis of Brucella-infected monocytes revealed specific overexpression of the A1 gene, a member of the bcl-2 family implicated in the survival of hematopoietic cells. Brucella infection also rendered macrophage-like cells resistant to Fas ligand-or gamma interferon-induced apoptosis, suggesting that Brucella infection protected host cells from several cytotoxic processes occurring at different steps of the immune response. The present data clearly show that Brucella suis modulated the monocyte/macrophage's apoptotic response to the advantage of the pathogen, thus preventing host cell elimination. This might represent a strategy for Brucella development in infected hosts.In recent years, it has become obvious that programmed death (or apoptosis) of cells of the monocytic lineage may be relevant for local immune responses against microorganisms (26, 31). Several bacterial organisms, such as Shigella flexneri (47), Legionella pneumophilia (35), Yersinia enterocolitica (41), Bordetella pertussis (20), Actinobacillus actinomycetemcomitans (18), Listeria monocytogenes (40), and Salmonella enterica serovar Typhimurium (27), promote the destruction of monocytic phagocytes by apoptosis, thus circumventing the first line of defense of the immune system. Surviving bacteria infect neighboring cells and disseminate to other tissues, often epithelial cells. Recently, it was reported that some intracellular bacteria that preferentially infect monocytic phagocytes have a totally opposite strategy and protect their host against apoptosis. Mycobacterium tuberculosis, which was reported to promote alveolar macrophage apoptosis (19,22), and Mycobacterium bovis were shown to inhibit spontaneously occurring apoptosis in human monocytes (9, 24), possibly by inducing tumor necrosis factor alpha (TNF-␣) production. Furthermore, HeLa cells infected with the obligate intracellular bacteria chlamydiae are resistant to apoptosis triggered by exogeneous stimuli (12), and Rickettsia rickettsii prevents the programmed cell death of endothelial cells (7). Molloy et al. showed that apoptosis of BCG-infected macrophages ki...

Brucella species are gram-negative, facultative intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment inside professional and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both cell types. However, the molecular mechanisms and the microbial factors involved are poorly understood. Smooth lipopolysaccharide (LPS) of Brucella has been reported to be an important virulence factor, although its precise role in pathogenesis is not yet clear. In this study, we show that the LPS O side chain is involved in inhibition of the early fusion between Brucella suis-containing phagosomes and lysosomes in murine macrophages. In contrast, the phagosomes containing rough mutants, which fail to express the O antigen, rapidly fuse with lysosomes. In addition, we show that rough mutants do not enter host cells by using lipid rafts, contrary to smooth strains. Thus, we propose that the LPS O chain might be a major factor that governs the early behavior of bacteria inside macrophages.Brucella species are gram-negative, facultative intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment inside professional (5,14,17,28) and nonprofessional (9, 25, 26) phagocytic cells. Usually, phagosomes mature gradually into phagolysosomes which are capable of degrading proteins by lysosomal enzymes. In the case of

SummaryPhysiological adaptation of intracellular bacteria is critical for timely interaction with eukaryotic host cells. One mechanism of adaptation, the stringent response, is induced by nutrient stress via its effector molecule (p)ppGpp, synthesized by the action of RelA/SpoT homologues. The intracellular pathogen Brucella spp., causative agent of brucellosis, possesses a gene homologous to relA / spoT , named rsh , encoding a (p)ppGpp synthetase as confirmed by heterologous complementation of a relA mutant of Sinorhizobium meliloti . The Rsh deletion mutants in Brucella suis and Brucella melitensis were characterized by altered morphology, and by reduced survival under starvation conditions and in cellular and murine models of infection. Most interestingly, we evidenced that expression of virB , encoding the type IV secretion system, a major virulence factor of Brucella , was Rsh-dependent. All mutant phenotypes, including lack of VirB proteins, were complemented with the rsh gene of Brucella . In addition, RelA of S. meliloti functionally replaced Brucella Rsh, describing the capacity of a gene from a plant symbiont to restore virulence in a mammalian pathogen. We therefore concluded that in the intramacrophagic environment encountered by Brucella , Rsh might participate in the adaptation of the pathogen to low-nutrient environments, and indirectly in the VirB-mediated formation of the final replicative niche.

A beta-carbonic anhydrase (CA, EC 4.2.1.1) from the bacterial pathogen Brucella suis, bsCA 1, has been cloned, purified, and characterized kinetically. bsCA 1 has appreciable activity as catalyst for the hydration of CO(2) to bicarbonate, with a k(cat) of 6.4 x 10(5) s(-1) and k(cat)/K(m) of 3.9 x 10(7) M(-1).s(-1). A panel of 38 sulfonamides and one sulfamate have been investigated for inhibition of this new beta-CA. All types of activities have been detected, with K(I)s in the range of 17 nM to 5.87 microM. The best bsCA 1 inhibitors were ethoxzolamide (17 nM), celecoxib (18 nM), dorzolamide (21 nM), valdecoxib, and sulpiride (19 nM). Whether bsCA 1 inhibitors may have application in the fight against brucellosis, an endemic disease and the major bacterial zoonosis, producing debilitating infection in humans and animals, warrants further studies.

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In experimental cellular and murine infections, B. microti exhibited a high pathogenic potential, compared with other Brucella species.