María P. Jiménez de Bagüés

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

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.

Requirement of norD for Brucella suis Virulence in a Murine Model of In Vitro and In Vivo Infection

ABSTRACT 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 New Species Brucella microti Replicates in Macrophages and Causes Death in Murine Models of Infection

BACKGROUND The recent isolation of Brucella microti from the common vole, the red fox, and the soil raises the possibility of an eventual reemergence of brucellosis in Europe. In this work, the pathogenic potential of this new Brucella species in both in vitro and in vivo models of infection was analyzed. METHODS The ability of B. microti (as compared to that of the closely related species Brucella suis) to replicate in human macrophages and in human and murine macrophage-like cells was determined. The behavior of B. microti and B. suis was evaluated in vivo in murine models of infection with Balb/c, CD1, and C57BL/6 mice. RESULTS B. microti showed an enhanced capacity for intramacrophagic replication compared with that of B. suis. Surprisingly, and in contrast to other species of Brucella, 10(5) colony-forming units of B. microti killed 82% of Balb/c mice within 7 days. Infection of spleen and liver with B. microti peaked at day 3, compared with B. suis infection, which peaked at day 7. Sublethal doses of B. microti induced good protection against a subsequent challenge with lethal doses. CONCLUSIONS In experimental cellular and murine infections, B. microti exhibited a high pathogenic potential, compared with other Brucella species.

Differential use of the two high-oxygen-affinity terminal oxidases of Brucella suis for in vitro and intramacrophagic multiplication.

ABSTRACT 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.

Different Roles of the Two High-Oxygen-Affinity Terminal Oxidases of Brucella suis: Cytochrome c Oxidase, but Not Ubiquinol Oxidase, Is Required for Persistence in Mice

ABSTRACT The survival of Brucella suis mutant strains in mice demonstrated different roles of the two high-oxygen-affinity terminal oxidases. The cbb3-type cytochrome c oxidase was essential for chronic infection in oxygen-deficient organs. Lack of the cytochrome bd ubiquinol oxidase led to hypervirulence of bacteria, which could rely on nitrite accumulation inhibiting the inducible nitric oxide synthase of the host.

Elucidating sources and roles of Granzymes A and B during bacterial infection and sepsis

During bacterial sepsis, proinflammatory cytokines contribute to multiorgan failure and death in a process regulated in part by cytolytic cell granzymes. When challenged with a sublethal dose of the identified mouse pathogen Brucella microti, wild-type (WT) and granzyme A (gzmA)(-/-) mice eliminate the organism from liver and spleen in 2 or 3 weeks, whereas the bacteria persist in mice lacking perforin or granzyme B as well as in mice depleted of Tc cells. In comparison, after a fatal challenge, only gzmA(-/-) mice exhibit increased survival, which correlated with reduced proinflammatory cytokines. Depletion of natural killer (NK) cells protects WT mice from sepsis without influencing bacterial clearance and the transfer of WT, but not gzmA(-/-) NK, cells into gzmA(-/-) recipients restores the susceptibility to sepsis. Therefore, infection-related pathology, but not bacterial clearance, appears to require gzmA, suggesting the protease may be a therapeutic target for the prevention of bacterial sepsis without affecting immune control of the pathogen.

Regulation of the mitogen-activated protein kinases by Brucella spp. expressing a smooth and rough phenotype : relationship to pathogen invasiveness

ABSTRACT By comparing smooth wild-type Brucella spp. to their rough mutants, we show that the LPS O chain restricted the activation of the ERK1/2 and p38 mitogen-activated protein kinase (MAPK) pathways, thus preventing the synthesis of immune mediators that regulate host defense. We conclude that the MAPKs are a target for immune intervention by virulent smooth Brucella.

Analysis of the Behavior of eryC Mutants of Brucella suis Attenuated in Macrophages

ABSTRACT The facultatively intracellular pathogen Brucella, characterized by its capacity to replicate in professional and non professional phagocytes, also causes abortion in ruminants. This property has been linked to the presence of erythritol in the placenta, as brucellae preferentially utilize erythritol. The ery operon encodes enzymes involved in erythritol metabolism, and a link with virulence has since been discussed. Allelic exchange mutants in eryC of Brucella suis were erythritol sensitive in vitro with a MIC of 1 to 5 mM of erythritol. Their multiplication in macrophage-like cells was 50- to 90-fold reduced, but complementation of the mutant restored wild-type levels of intracellular multiplication and the capacity to use erythritol as a sole carbon source. In vivo, the eryC mutant colonized the spleens of infected BALB/c mice to a significantly lower extent than the wild type and the complemented strain. Interestingly, eryC mutants that were in addition spontaneously erythritol tolerant nevertheless exhibited wild-type-like intramacrophagic and intramurine replication. We concluded from our results that erythritol was not an essential carbon source for the pathogen in the macrophage host cell but that the inactivation of the eryC gene significantly reduced the intramacrophagic and intramurine fitness of B. suis.

The glutamic acid decarboxylase system of the new species Brucella microti contributes to its acid resistance and to oral infection of mice

BACKGROUND Genome analysis indicated that the new species Brucella microti possesses a potentially functional glutamate decarboxylase (GAD) system involved in extreme acid resistance in several foodborne bacteria. The contribution of this system in adaptation of B. microti to an acidic environment, including the intracellular vacuole and stomach, was investigated. RESULTS B. microti was GAD positive and able to export its product, γ-aminobutyrate, to the extracellular medium. The resistance of B. microti to acid stress (pH 2.5) was glutamate dependent. Mutants affected in the GAD system lost this resistance, demonstrating its direct involvement in survival under these conditions. The reciprocal heterologous complementation of mutants with the GAD systems of Escherichia coli or B. microti confirmed conserved functions in both bacterial species. A gad mutant was not attenuated during infection of macrophages, where Brucella resides in an acidified vacuole at a pH of 4-4.5 during the early phase of macrophage infection, but GAD contributed to the survival of B. microti in a murine model following oral infection. CONCLUSIONS This work provides first evidence that the GAD system might play an essential role in the resistance of an environment-borne, pathogenic Brucella species to extreme acid shock and during passage through the host stomach following oral infection.

RegA, the Regulator of the Two-Component System RegB/RegA of Brucella suis, Is a Controller of Both Oxidative Respiration and Denitrification Required for Chronic Infection in Mice

ABSTRACT Adaptation to oxygen deficiency is essential for virulence and persistence of Brucella inside the host. The flexibility of this bacterium with respect to oxygen depletion is remarkable, since Brucella suis can use an oxygen-dependent transcriptional regulator of the FnrN family, two high-oxygen-affinity terminal oxidases, and a complete denitrification pathway to resist various conditions of oxygen deficiency. Moreover, our previous results suggested that oxidative respiration and denitrification can be simultaneously used by B. suis under microaerobiosis. The requirement of a functional cytochrome bd ubiquinol oxidase for nitrite reductase expression evidenced the linkage of these two pathways, and the central role of the two-component system RegB/RegA in the coordinated control of both respiratory systems was demonstrated. We propose a scheme for global regulation of B. suis respiratory pathways by the transcriptional regulator RegA, which postulates a role for the cytochrome bd ubiquinol oxidase in redox signal transmission to the histidine sensor kinase RegB. More importantly, RegA was found to be essential for B. suis persistence in vivo within oxygen-limited target organs. It is conceivable that RegA acts as a controller of numerous systems involved in the establishment of the persistent state, characteristic of chronic infections by Brucella.