Lethality of Brucella microti in a murine model of infection depends on the wbkE gene involved in O-polysaccharide synthesis.

Brucella microti was isolated a decade ago from wildlife and soil in Europe. Compared to the classical Brucella species, it exhibits atypical virulence properties such as increased growth in human and murine macrophages and lethality in experimentally infected mice. A spontaneous rough (R) mutant strain, derived from the smooth reference strain CCM4915T, showed increased macrophage colonization and was non-lethal in murine infections. Whole-genome sequencing and construction of an isogenic mutant of B. microti and Brucella suis 1330 revealed that the R-phenotype was due to a deletion in a single gene, namely wbkE (BMI_I539), encoding a putative glycosyltransferase involved in lipopolysaccharide (LPS) O-polysaccharide biosynthesis. Complementation of the R-strains with the wbkE gene restored the smooth phenotype and the ability of B. microti to kill infected mice. LPS with an intact O-polysaccharide is therefore essential for lethal B. microti infections in the murine model, demonstrating its importance in pathogenesis.

RegA Plays a Key Role in Oxygen-Dependent Establishment of Persistence and in Isocitrate Lyase Activity, a Critical Determinant of In vivo Brucella suis Pathogenicity.

For aerobic human pathogens, adaptation to hypoxia is a critical factor for the establishment of persistent infections, as oxygen availability is low inside the host. The two-component system RegB/A of Brucella suis plays a central role in the control of respiratory systems adapted to oxygen deficiency, and in persistence in vivo. Using an original "in vitro model of persistence" consisting in gradual oxygen depletion, we compared transcriptomes and proteomes of wild-type and ΔregA strains to identify the RegA-regulon potentially involved in the set-up of persistence. Consecutive to oxygen consumption resulting in growth arrest, 12% of the genes in B. suis were potentially controlled directly or indirectly by RegA, among which numerous transcriptional regulators were up-regulated. In contrast, genes or proteins involved in envelope biogenesis and in cellular division were repressed, suggesting a possible role for RegA in the set-up of a non-proliferative persistence state. Importantly, the greatest number of the RegA-repressed genes and proteins, including aceA encoding the functional IsoCitrate Lyase (ICL), were involved in energy production. A potential consequence of this RegA impact may be the slowing-down of the central metabolism as B. suis progressively enters into persistence. Moreover, ICL is an essential determinant of pathogenesis and long-term interactions with the host, as demonstrated by the strict dependence of B. suis on ICL activity for multiplication and persistence during in vivo infection. RegA regulates gene or protein expression of all functional groups, which is why RegA is a key regulator of B. suis in adaptation to oxygen depletion. This function may contribute to the constraint of bacterial growth, typical of chronic infection. Oxygen-dependent activation of two-component systems that control persistence regulons, shared by several aerobic human pathogens, has not been studied in Brucella sp. before. This work therefore contributes significantly to the unraveling of persistence mechanisms in this important zoonotic pathogen.

Toll-Like Receptors 2 and 4 Cooperate in the Control of the Emerging Pathogen Brucella microti.

Toll-like receptors (TLRs) recognize pathogen-derived molecules and play a critical role during the host innate and adaptive immune response. Brucella spp. are intracellular gram-negative bacteria including several virulent species, which cause a chronic zoonotic infection in a wide range of mammalian hosts known as brucellosis. A new Brucella species, Brucella microti, was recently isolated from wild rodents and found to be highly pathogenic in mice. Using this species-specific model, it was previously found that CD8+ T cells are required to control this infection. In order to find out the role of TLR-mediated responses in the control of this pathogen, the course of infection of B. microti was analyzed over 3 weeks in wild-type (WT) and TLR knock out (KO) mice including TLR2-/-, TLR4-/-, TLR9-/-, TLR2×4-/- and TLR2×4×9-/-. WT and single TLR2, TLR4 and TLR9 KO mice similarly control infection in liver and spleen. In contrast, bacterial clearance was delayed in TLR2×4-/- and TLR2×4×9-/- mice at 7 and 14 days post-infection. This defect correlated with impaired maturation and pro-inflammatory cytokine production in B. microti-infected dendritic cells from TLR2×4-/- and TLR2×4×9-/- mice. Finally, it was found that Tc cells from TLR2×4-/- and TLR2×4×9-/- mice showed reduced ability to inhibit growth of B. microti in macrophages, suggesting the involvement of TLR2 and 4 in the generation of specific Tc cells. Our findings indicate that TLR2 and TLR4 are required to control B. microti infection in mice and that this effect could be related to its participation in the maturation of dendritic cells and the generation of specific CD8+ Tc cells.

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.

The new strains Brucella inopinata BO1 and Brucella species 83-210 behave biologically like classic infectious Brucella species and cause death in murine models of infection.

BACKGROUND: Recently, novel atypical Brucella strains isolated from humans and wild rodents have been reported. They are phenotypically close to Ochrobactrum species but belong to the genus Brucella, based on genetic relatedness, although genetic diversity is higher among the atypical Brucella strains than between the classic species. They were classified within or close to the novel species Brucella inopinata. However, with the exception of Brucella microti, the virulence of these novel strains has not been investigated in experimental models of infection. METHODS: The type species B. inopinata strain BO1 (isolated from a human) and Brucella species strain 83-210 (isolated from a wild Australian rodent) were investigated. A classic infectious Brucella reference strain, B. suis 1330, was also used. BALB/c, C57BL/6, and CD1 mice models and C57BL/6 mouse bone-marrow-derived macrophages (BMDMs) were used as infection models. RESULTS: Strains BO1 and 83-210 behaved similarly to reference strain 1330 in all mouse infection models: there were similar growth curves in spleens and livers of mice and similar intracellular replication rates in BMDMs. However, unlike strain 1330, strains BO1 and 83-210 showed lethality in the 3 mouse models. CONCLUSIONS: The novel atypical Brucella strains of this study behave like classic intracellular Brucella pathogens. In addition, they cause death in murine models of infection, as previously published for B. microti, another recently described environmental and wildlife species.

Course of infection with the emergent pathogen Brucella microti in immunocompromised mice.

A new Brucella species, Brucella microti, has been isolated from wild rodents and found to be pathogenic in mice. The biological relevance of this new mouse pathogen is clear, as it allows us to study Brucella infection in a species-specific model. The course of infection in wild-type (wt) and immunodeficient mice that lack B (Jh), T and B (SCID), or T, B, and NK (SCID.Beige) cells was analyzed over 3 weeks. wt mice completely cleared bacteria from the liver and spleen after that time. However, SCID mice showed a much higher bacterial load in the spleen and liver than wt and Jh mice after 1 week and maintained the same level during the next 2 weeks. All mice tested survived for the 3 weeks. In contrast, the bacterial levels in mice that lacked NK cell activity progressively increased and these mice succumbed to infection after 16 to 18 days. Histopathology analysis of infected mice showed extensive areas of necrotic tissue and thrombosis in liver after 1 week in all infected SCID.Beige mice but were not seen in either SCID or wt animals. These processes were dramatically increased after 21 days, corresponding with the death of SCID.Beige animals. Our results indicate that T and/or B cells are required for the control of infection with the mouse pathogen Brucella microti in liver and spleen but that NK cells are crucial for survival in the absence of B and T cells. In addition, they suggest that controlled granuloma formation is critical to clear this type of infection in wt mice.

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.

Analysis of the behavior of eryC mutants of Brucella suis attenuated in macrophages.

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.

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

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.

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

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.