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Cyclic-di-GMP plays crucial role in the cell cycle regulation of the α-Proteobacterium Caulobacter crescentus. Here we investigated its role in the α-Proteobacterium Brucella abortus, a zoonotic intracellular pathogen. Surprisingly, deletion of all predicted cyclic-di-GMP synthesizing or degrading enzymes did not drastically impair the growth of B. abortus, nor its ability to grow inside cell lines. As other Rhizobiales, B. abortus displays unipolar growth from the new cell pole generated by cell division. We found that the phosphodiesterase PdeA, the ortholog of the essential polar growth factor RgsP of the Rhizobiale Sinorhizobium meliloti, is required for rod shape integrity but is not essential for B. abortus growth. Indeed, the radius of the pole is increased by 31 ± 1.7% in a ΔpdeA mutant, generating a coccoid morphology. A mutation in the cyclic-di-GMP phosphodiesterase catalytic site of PdeA does not generate the coccoid morphology and the ΔpdeA mutant kept the ability to recruit markers of new and old poles. However, the presence of PdeA is required in an intra-nasal mouse model of infection. In conclusion, we propose that PdeA contributes to bacterial morphology and virulence in B. abortus, but it is not crucial for polarity and asymmetric growth., (© 2021 John Wiley & Sons Ltd.)

The zoonotic pathogen Brucella abortus is part of the Rhizobiales, which are alpha-proteobacteria displaying unipolar growth. Here, we show that this bacterium exhibits heterogeneity in its outer membrane composition, with clusters of rough lipopolysaccharide co-localizing with the essential outer membrane porin Omp2b, which is proposed to allow facilitated diffusion of solutes through the porin. We also show that the major outer membrane protein Omp25 and peptidoglycan are incorporated at the new pole and the division site, the expected growth sites. Interestingly, lipopolysaccharide is also inserted at the same growth sites. The absence of long-range diffusion of main components of the outer membrane could explain the apparent immobility of the Omp2b clusters, as well as unipolar and mid-cell localizations of newly incorporated outer membrane proteins and lipopolysaccharide. Unipolar growth and limited mobility of surface structures also suggest that new surface variants could arise in a few generations without the need of diluting pre-existing surface antigens., (© 2019 The Authors.)

The α-proteobacteria are a fascinating group of free-living, symbiotic and pathogenic organisms, including the Brucella genus, which is responsible for a worldwide zoonosis. One common feature of α-proteobacteria is the presence of a conserved response regulator called CtrA, first described in the model bacterium Caulobacter crescentus, where it controls gene expression at different stages of the cell cycle. Here, we focus on Brucella abortus and other intracellular α-proteobacteria in order to better assess the potential role of CtrA in the infectious context. Comparative genomic analyses of the CtrA control pathway revealed the conservation of specific modules, as well as the acquisition of new factors during evolution. The comparison of CtrA regulons also suggests that specific clades of α-proteobacteria acquired distinct functions under its control, depending on the essentiality of the transcription factor. Other CtrA-controlled functions, for instance motility and DNA repair, are proposed to be more ancestral. Altogether, these analyses provide an interesting example of the plasticity of a regulation network, subject to the constraints of inherent imperatives such as cell division and the adaptations to diversified environmental niches.

Objectives: To describe a novel plasmid-borne class D carbapenemase (CHDL) named OXA-427 identified in several Enterobacteriaceae clinical isolates from nine patients in one Belgian hospital., Methods: OXA-427-producing isolates were analysed by an electrochemical imipenem hydrolysis method (BYG Carba test), Carba NP test, conventional phenotypic assays and by molecular methods (PCR, whole sequencing of the OXA-427-encoding plasmid and cloning). The antimicrobial resistance profile of OXA-427 was analysed by expression of the cloned gene in Escherichia coli DH10B and J53., Results: Eleven OXA-427-producing Enterobacteriaceae isolates of various species were identified from clinical specimens of nine patients between March 2012 and June 2014. OXA-427 shares only 22%-29% amino acid identity with OXA-48-like enzymes and other acquired CHDL (e.g. OXA-23, -24/40 and -58 of Acinetobacter spp.). Conversely, it appeared closely related to the chromosomal class D β-lactamase of Aeromonas media, Aeromonas hydrophila and Aeromonas sobria (99%, 89% and 77% of identity, respectively). When expressed in E. coli, OXA-427 hydrolysed imipenem and conferred resistance to extended-spectrum cephalosporins (mostly ceftazidime), penicillins including temocillin, and reduced susceptibility to carbapenems. The blaOXA-427 gene was located in a 45 kb resistance island on a 177 kb IncA/C plasmid., Conclusions: OXA-427 is a novel CHDL most closely related to chromosomal class D β-lactamase of A. media WS. It confers resistance to penicillins, ceftazidime and aztreonam and in some instances to carbapenems. OXA-427, which is not detectable by classical molecular tests, caused a protracted outbreak in one university hospital over a 2 year period., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Brucella abortus is a pathogen infecting cattle, able to survive, traffic, and proliferate inside host cells. It belongs to the Alphaproteobacteria, a phylogenetic group comprising bacteria with free living, symbiotic, and pathogenic lifestyles. An essential regulator of cell cycle progression named CtrA was described in the model bacterium Caulobacter crescentus. This regulator is conserved in many alphaproteobacteria, but the evolution of its regulon remains elusive. Here we identified promoters that are CtrA targets using ChIP-seq and we found that CtrA binds to promoters of genes involved in cell cycle progression, in addition to numerous genes encoding outer membrane components involved in export of membrane proteins and synthesis of lipopolysaccharide. Analysis of a conditional B. abortus ctrA loss of function mutant confirmed that CtrA controls cell division. Impairment of cell division generates elongated and branched morphologies, that are also detectable inside HeLa cells. Surprisingly, abnormal bacteria are able to traffic to the endoplasmic reticulum, the usual replication niche of B. abortus in host cells. We also found that CtrA depletion affected outer membrane composition, in particular the abundance and spatial distribution of Omp25. Control of the B. abortus envelope composition by CtrA indicates the plasticity of the CtrA regulon along evolution., (© 2016 John Wiley & Sons Ltd.)

The alarmone (p)ppGpp is commonly used by bacteria to quickly respond to nutrient starvation. Although (p)ppGpp synthetases such as SpoT have been extensively studied, little is known about the molecular mechanisms stimulating alarmone synthesis upon starvation. Here, we describe an essential role of the nitrogen-related phosphotransferase system (PTS(Ntr)) in controlling (p)ppGpp accumulation in Caulobacter crescentus. We show that cells sense nitrogen starvation by way of detecting glutamine deprivation using the first enzyme (EI(Ntr)) of PTS(Ntr). Decreasing intracellular glutamine concentration triggers phosphorylation of EI(Ntr) and its downstream components HPr and EIIA(Ntr). Once phosphorylated, both HPr∼P and EIIA(Ntr)∼P stimulate (p)ppGpp accumulation by modulating SpoT activities. This burst of second messenger primarily impacts the non-replicative phase of the cell cycle by extending the G1 phase. This work highlights a new role for bacterial PTS systems in stimulating (p)ppGpp accumulation in response to metabolic cues and in controlling cell cycle progression and cell growth.

Brucella spp. and Sinorhizobium meliloti are alphaproteobacteria that share not only an intracellular lifestyle in their respective hosts, but also a crucial requirement for cell envelope components and their timely regulation for a successful infectious cycle. Here, we report the characterization of Brucella melitensis mucR, which encodes a zinc finger transcriptional regulator that has previously been shown to be involved in cellular and mouse infections at early time points. MucR modulates the surface properties of the bacteria and their resistance to environmental stresses (i.e., oxidative stress, cationic peptide, and detergents). We show that B. melitensis mucR is a functional orthologue of S. meliloti mucR, because it was able to restore the production of succinoglycan in an S. meliloti mucR mutant, as detected by calcofluor staining. Similar to S. meliloti MucR, B. melitensis MucR also represses its own transcription and flagellar gene expression via the flagellar master regulator ftcR. More surprisingly, we demonstrate that MucR regulates a lipid A core modification in B. melitensis. These changes could account for the attenuated virulence of a mucR mutant. These data reinforce the idea that there is a common conserved circuitry between plant symbionts and animal pathogens that regulates the relationship they have with their hosts.

Bacterial adaptation to environmental conditions is essential to ensure maximal fitness in the face of several stresses. In this context, two-component systems (TCSs) represent a predominant signal transduction mechanism, allowing an appropriate response to be mounted when a stimulus is sensed. As facultative intracellular pathogens, Brucella spp. face various environmental conditions, and an adequate response is required for a successful infection process. Recently, bioinformatic analysis of Brucella genomes predicted a set of 15 bona fide TCS pairs, among which some have been previously investigated. In this report, we characterized a new TCS locus called prlS/R, for probable proline sensor-regulator. It encodes a hybrid histidine kinase (PrlS) with an unusual Na(+)/solute symporter N-terminal domain and a transcriptional regulator (belonging to the LuxR family) (PrlR). In vitro, Brucella spp. with a functional PrlR/S system form bacterial aggregates, which seems to be an adaptive response to a hypersaline environment, while a prlS/R mutant does not. We identified ionic strength as a possible signal sensed by this TCS. Finally, this work correlates the absence of a functional PrlR/S system with the lack of hypersaline-induced aggregation in particular marine Brucella spp.

Bacterial differentiation is often associated with the asymmetric localization of regulatory proteins, such as histidine kinases. PdhS is an essential and polarly localized histidine kinase in the pathogenic alphaproteobacterium Brucella abortus. After cell division, PdhS is asymmetrically segregated between the two sibling cells, highlighting a differentiation event. However, the function(s) of PdhS in the B. abortus cell cycle remains unknown. We used an original approach, the pentapeptide scanning mutagenesis method, to generate a thermosensitive allele of pdhS. We report that a B. abortus strain carrying this pdhS allele displays growth arrest and an altered DivK-yellow fluorescent protein (YFP) polar localization at the restrictive temperature. Moreover, the production of a nonphosphorylatable PdhS protein or truncated PdhS proteins leads to dominant-negative effects by generating morphological defects consistent with the inhibition of cell division. In addition, we have used a domain mapping approach combined with yeast two-hybrid and fluorescence microscopy methods to better characterize the unusual PdhS sensory domain. We have identified a fragment of the PdhS sensory domain required for protein-protein interaction (amino acids [aa] 210 to 434), a fragment sufficient for polar localization (aa 1 to 434), and a fragment (aa 527 to 661) whose production in B. abortus correlates with the generation of cell shape alterations. The data support a model in which PdhS acts as an essential regulator of cell cycle progression in B. abortus and contribute to a better understanding of the differentiation program inherited by the two sibling cells.

Members of the genus Brucella are facultative intracellular pathogenic bacteria able to control maturation of their vacuoles. In several cell types, Brucella is able to reach a proliferation compartment derived from the endoplasmic reticulum (ER). Since ER exit site (ERES) functions are required for Brucella proliferation, we performed a yeast two-hybrid screen between human ERES-associated proteins and the predicted brucella proteome. This screening led to the identification of CstA, a conserved protein that specifically interacts with Sec24A, a component of the ERES. We found that a tagged CstA is secreted in Brucella abortus culture medium. This secretion is independent of the type IV secretion system VirB and the flagellum, suggesting that CstA is secreted through another system. We also discovered that a B. abortus cstA mutant is impaired for its association with the Sec23 ERES marker. The B. abortus cstA mutant displayed peculiar trafficking, with reduced association with LAMP1 and Calnexin 12 h post-infection in HeLa cells. However, its intracellular proliferation kinetics was not affected. The data reported here suggest that CstA could be directly or indirectly involved in the control of B. abortus intracellular trafficking in HeLa cells.

Background: Protein-protein interactions are at the basis of many cellular processes, and they are also involved in the interaction between pathogens and their host(s). Many intracellular pathogenic bacteria translocate proteins called effectors into the cytoplasm of the infected host cell, and these effectors can interact with one or several host protein(s). An effector named RicA was recently reported in Brucella abortus to specifically interact with human Rab2 and to affect intracellular trafficking of this pathogen., Results: In order to identify regions of the RicA protein involved in the interaction with Rab2, RicA was subjected to extensive random mutagenesis using error prone polymerase chain reaction. The resulting allele library was selected by the yeast two-hybrid assay for Rab2-interacting clones that were isolated and sequenced, following the "absence of interference" approach. A tridimensional model of RicA structure was used to position the substitutions that did not affect RicA-Rab2 interaction, giving a "negative image" of the putative interaction region. Since RicA is a bacterial conserved protein, RicA homologs were also tested against Rab2 in a yeast two-hybrid assay, and the C. crescentus homolog of RicA was found to interact with human Rab2. Analysis of the RicA structural model suggested that regions involved in the folding of the "beta helix" or an exposed loop with the IGFP sequence could also be involved in the interaction with Rab2. Extensive mutagenesis of the IGFP loop suggested that loss of interaction with Rab2 was correlated with insolubility of the mutated RicA, showing that "absence of interference" approach also generates surfaces that could be necessary for folding., Conclusion: Extensive analysis of substitutions in RicA unveiled two structural elements on the surface of RicA, the most exposed β-sheet and the IGFP loop, which could be involved in the interaction with Rab2 and protein folding. Our analysis of mutants in the IGFP loop suggests that, at least for some mono-domain proteins such as RicA, protein interaction analysis using allele libraries could be complicated by the dual effect of many substitutions affecting both folding and protein-protein interaction.

Brucella melitensis is a gram-negative bacteria known to cause brucellosis and to produce severe infections in humans. Whilst brucella's outer membrane proteins have been extensively studied due to their potential role as antigens or virulence factors, their function is still poorly understood at the structural level, as the 3D structure of Brucella β-barrel membrane proteins are still unknown. In this context, the B. melitensis trimeric Omp2a porin has been overexpressed and refolded in n-dodecyl-β-d-maltopyranoside. We here show that this refolding process is insensitive to urea but is temperature- and ionic strength-dependent. Reassembled species were characterized by fluorescence, size-exclusion chromatography and circular dichroism. A refolding mechanism is proposed, suggesting that Omp2a first refolds under a monomeric form and then self-associates into a trimeric state. This first complete in vitro refolding of a membrane protein from B. melitensis shall eventually lead to functional and 3D structure determination., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Brucella abortus is the etiological agent of a worldwide zoonosis called brucellosis. This alpha-proteobacterium is dividing asymmetrically, and PdhS, an essential histidine kinase, was reported to be an old pole marker., Results: We were interested to identify functions that could be recruited to bacterial poles. The Brucella ORFeome, a collection of cloned predicted coding sequences, was placed in fusion with yellow fluorescent protein (YFP) coding sequence and screened for polar localizations in B. abortus. We report that AidB-YFP was systematically localized to the new poles and at constrictions sites in B. abortus, either in culture or inside infected HeLa cells or RAW264.7 macrophages. AidB is an acyl-CoA dehydrogenase (ACAD) homolog, similar to E. coli AidB, an enzyme putatively involved in destroying alkylating agents. Accordingly, a B. abortus aidB mutant is more sensitive than the wild-type strain to the lethality induced by methanesulphonic acid ethyl ester (EMS). The exposure to EMS led to a very low frequency of constriction events, suggesting that cell cycle is blocked during alkylation damage. The localization of AidB-YFP at the new poles and at constriction sites seems to be specific for this ACAD homolog since two other ACAD homologs fused to YFP did not show specific localization. The overexpression of aidB, but not the two other ACAD coding sequences, leads to multiple morphological defects., Conclusions: Data reported here suggest that AidB is a marker of new poles and constriction sites, that could be considered as sites of preparation of new poles in the sibling cells originating from cell division. The possible role of AidB in the generation or the function of new poles needs further investigation.

Bacteria of the Brucella genus are facultative intracellular class III pathogens. These bacteria are able to control the intracellular trafficking of their vacuole, presumably by the use of yet unknown translocated effectors. To identify such effectors, we used a high-throughput yeast two-hybrid screen to identify interactions between putative human phagosomal proteins and predicted Brucella spp. proteins. We identified a specific interaction between the human small GTPase Rab2 and a Brucella spp. protein named RicA. This interaction was confirmed by GST-pull-down with the GDP-bound form of Rab2. A TEM-β-lactamase-RicA fusion was translocated from Brucella abortus to RAW264.7 macrophages during infection. This translocation was not detectable in a strain deleted for the virB operon, coding for the type IV secretion system. However, RicA secretion in a bacteriological culture was still observed in a ΔvirB mutant. In HeLa cells, a ΔricA mutant recruits less GTP-locked myc-Rab2 on its Brucella-containing vacuoles, compared with the wild-type strain. We observed altered kinetics of intracellular trafficking and faster proliferation of the B. abortusΔricA mutant in HeLa cells, compared with the wild-type control. Altogether, the data reported here suggest RicA as the first reported effector with a proposed function for B. abortus., (© 2011 Blackwell Publishing Ltd.)

The genome of Brucella melitensis contains genes coding for the sigma factors RpoD, RpoN, RpoH1, RpoH2, RpoE1 and RpoE2. Previously published data show that B. melitensis is flagellated and that an rpoE1 mutant overexpresses the flagellar protein FlgE. In this study, we demonstrate that mutation of rpoE1 causes an overexpression of the flagellar genes fliF, flgE, fliC, flaF and flbT, correlating with the production of a longer filament and thereby demonstrating that RpoE1 acts as a flagellar repressor. Moreover, mutation of rpoE1 increases the promoter activity of the flagellar master regulator ftcR, suggesting that RpoE1 acts upstream of ftcR. Together, these data show that RpoE1 represses the flagellar synthesis and filament length in B. melitensis.

Background: Inhibition of apoptosis is one of the mechanisms selected by numerous intracellular pathogenic bacteria to control their host cell. Brucellae, which are the causative agent of a worldwide zoonosis, prevent apoptosis of infected cells, probably to support survival of their replication niche., Methodology/principal Findings: In order to identify Brucella melitensis anti-apoptotic effector candidates, we performed a genome-wide functional screening in yeast. The B. melitensis ORFeome was screened to identify inhibitors of Bax-induced cell death in S. cerevisiae. B. melitensis porin Omp2b, here shown to be essential, prevents Bax lethal effect in yeast, unlike its close paralog Omp2a. Our results based on Omp2b size variants characterization suggest that signal peptide processing is required for Omp2b effect in yeast., Conclusion/significance: We report here the first application to a bacterial genome-wide library of coding sequences of this "yeast-rescue" screening strategy, previously used to highlight several new apoptosis regulators. Our work provides B. melitensis proteins that are candidates for an anti-apoptotic function, and can be tested in mammalian cells in the future. Hypotheses on possible molecular mechanisms of Bax inhibition by the B. melitensis porin Omp2b are discussed.

Background: When heterologous recombinant proteins are produced in Escherichia coli, they often precipitate to form insoluble aggregates of unfolded polypeptides called inclusion bodies. These structures are associated with chaperones like IbpA. However, there are reported cases of "non-classical" inclusion bodies in which proteins are soluble, folded and active., Results: We report that the Brucella abortus PdhS histidine kinase fused to the mCherry fluorescent protein forms intermediate aggregates resembling "non-classical" inclusion bodies when overproduced in E. coli, before forming "classical" inclusion bodies. The intermediate aggregates of PdhS-mCherry are characterized by the solubility of PdhS-mCherry, its ability to specifically recruit known partners fused to YFP, suggesting that PdhS is folded in these conditions, and the quick elimination (in less than 10 min) of these structures when bacterial cells are placed on fresh rich medium. Moreover, soluble PdhS-mCherry foci do not systematically colocalize with IpbA-YFP, a marker of inclusion bodies. Instead, time-lapse experiments show that IbpA-YFP exhibits rapid pole-to-pole shuttling, until it partially colocalizes with PdhS-mCherry aggregates., Conclusion: The data reported here suggest that, in E. coli, recombinant proteins like PdhS-mCherry may transit through a soluble and folded state, resembling previously reported "non-classical" inclusion bodies, before forming "classical" inclusion bodies. The dynamic localization of IbpA-YFP foci suggests that the IbpA chaperone could scan the E. coli cell to find its substrates.

Background: In many bacteria, the phosphotransferase system (PTS) is a key player in the regulation of the assimilation of alternative carbon sources notably through catabolic repression. The intracellular pathogens Brucella spp. possess four PTS proteins (EINtr, NPr, EIIANtr and an EIIA of the mannose family) but no PTS permease suggesting that this PTS might serve only regulatory functions., Methodology/principal Findings: In vitro biochemical analyses and in vivo detection of two forms of EIIANtr (phosphorylated or not) established that the four PTS proteins of Brucella melitensis form a functional phosphorelay. Moreover, in vitro the protein kinase HprK/P phosphorylates NPr on a conserved serine residue, providing an additional level of regulation to the B. melitensis PTS. This kinase activity was inhibited by inorganic phosphate and stimulated by fructose-1,6 bisphosphate. The genes encoding HprK/P, an EIIAMan-like protein and NPr are clustered in a locus conserved among α-proteobacteria and also contain the genes for the crucial two-component system BvrR-BvrS. RT-PCR revealed a transcriptional link between these genes suggesting an interaction between PTS and BvrR-BvrS. Mutations leading to the inactivation of EINtr or NPr significantly lowered the synthesis of VirB proteins, which form a type IV secretion system. These two mutants also exhibit a small colony phenotype on solid media. Finally, interaction partners of PTS proteins were identified using a yeast two hybrid screen against the whole B. melitensis ORFeome. Both NPr and HprK/P were shown to interact with an inorganic pyrophosphatase and the EIIAMan-like protein with the E1 component (SucA) of 2-oxoglutarate dehydrogenase., Conclusions/significance: The B. melitensis can transfer the phosphoryl group from PEP to the EIIAs and a link between the PTS and the virulence of this organism could be established. Based on the protein interaction data a preliminary model is proposed in which this regulatory PTS coordinates also C and N metabolism.

Many pathogenic bacteria use a regulatory process termed quorum sensing (QS) to produce and detect small diffusible molecules to synchronize gene expression within a population. In Gram-negative bacteria, the detection of, and response to, these molecules depends on transcriptional regulators belonging to the LuxR family. Such a system has been discovered in the intracellular pathogen Brucella melitensis, a Gram-negative bacterium responsible for brucellosis, a worldwide zoonosis that remains a serious public health concern in countries were the disease is endemic. Genes encoding two LuxR-type regulators, VjbR and BabR, have been identified in the genome of B. melitensis 16 M. A DeltavjbR mutant is highly attenuated in all experimental models of infection tested, suggesting a crucial role for QS in the virulence of Brucella. At present, no function has been attributed to BabR. The experiments described in this report indicate that 5% of the genes in the B. melitensis 16 M genome are regulated by VjbR and/or BabR, suggesting that QS is a global regulatory system in this bacterium. The overlap between BabR and VjbR targets suggest a cross-talk between these two regulators. Our results also demonstrate that VjbR and BabR regulate many genes and/or proteins involved in stress response, metabolism, and virulence, including those potentially involved in the adaptation of Brucella to the oxidative, pH, and nutritional stresses encountered within the host. These findings highlight the involvement of QS as a major regulatory system in Brucella and lead us to suggest that this regulatory system could participate in the spatial and sequential adaptation of Brucella strains to the host environment.

Many organisms use polar localization of signalling proteins to control developmental events in response to completion of asymmetric cell division. Asymmetric division was recently reported for Brucella abortus, a class III facultative intracellular pathogen generating two sibling cells of slightly different size. Here we characterize PdhS, a cytoplasmic histidine kinase essential for B. abortus viability and homologous to the asymmetrically distributed PleC and DivJ histidine kinases from Caulobacter crescentus. PdhS is localized at the old pole of the large cell, and after division and growth, the small cell acquires PdhS at its old pole. PdhS may therefore be considered as a differentiation marker as it labels the old pole of the large cell. Moreover, PdhS colocalizes with its paired response regulator DivK. Finally, PdhS is able to localize at one pole in other alpha-proteobacteria, suggesting that a polar structure associating PdhS with one pole is conserved in these bacteria. We propose that a differentiation event takes place after the completion of cytokinesis in asymmetrically dividing alpha-proteobacteria. Altogether, these data suggest that prokaryotic differentiation may be much more widespread than expected.

The flagellar regulon of Brucella melitensis 16M contains 31 genes clustered in three loci on the small chromosome. These genes encode a polar sheathed flagellum that is transiently expressed during vegetative growth and required for persistent infection in a mouse model. By following the expression of three flagellar genes (fliF, flgE, and fliC, corresponding to the MS ring, hook, and filament monomer, respectively), we identified a new regulator gene, ftcR (flagellar two-component regulator). Inactivation of ftcR led to a decrease in flagellar gene expression and to impaired Brucella virulence. FtcR has a two-component response regulator domain as well a DNA binding domain and is encoded in the first flagellar locus of B. melitensis. Both the ftcR sequence and its genomic context are conserved in other related alpha-proteobacteria. During vegetative growth in rich medium, ftcR expression showed a peak during the early exponential phase that paralleled fliF gene expression. VjbR, a quorum-sensing regulator of the LuxR family, was previously found to control fliF and flgE gene expression. Here, we provide some new elements suggesting that the effect of VjbR on these flagellar genes is mediated by FtcR. We found that ftcR expression is partially under the control of VjbR and that the expression in trans of ftcR in a vjbR mutant restored the production of the hook protein (FlgE). Finally, FtcR binds directly to the upstream region of the fliF gene. Therefore, our data support the role of FtcR as a flagellar master regulator in B. melitensis and perhaps in other related alpha-proteobacteria.

Physiological 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.

We identified two regulators of denitrification genes in Brucella melitensis 16M: NarR, which regulates the nitrate reductase (nar) operon, and NnrA, which is involved in the expression of the last three reductases of the denitrification pathway (nirK, norB, and nosZ). NnrA is required for virulence in mice and for intracellular resistance to nitric oxide.

Both a type IV secretion system and a flagellum have been described in Brucella melitensis. These two multimolecular surface appendages share several features. Their expression in bacteriological medium is growth curve dependent, both are induced intracellularly and are required for full virulence in a mouse model of infection. Here we report the identification of VjbR, a quorum sensing-related transcriptional regulator. A vjbR mutant has a downregulated expression of both virB operon and flagellar genes either during vegetative growth or during intracellular infection. In a cellular model, the vacuoles containing the vjbR mutant or a virB mutant are decorated with the same markers at similar times post infection. The vjbR mutant is also strongly attenuated in a mouse model of infection. As C(12)-homoserine lactone pheromone is known to be involved in virB repression, we postulated that VjbR is mediating this effect. In agreement with this hypothesis, we observed that, as virB operon, flagellar genes are controlled by the pheromone. All together these data support a model in which VjbR acts as a major regulator of virulence factors in Brucella.

The bacteria of the Brucella genus are responsible for a worldwide zoonosis called brucellosis. They belong to the alpha-proteobacteria group, as many other bacteria that live in close association with a eukaryotic host. Importantly, the Brucellae are mainly intracellular pathogens, and the molecular mechanisms of their virulence are still poorly understood. Using the complete genome sequence of Brucella melitensis, we generated a database of protein-coding open reading frames (ORFs) and constructed an ORFeome library of 3091 Gateway Entry clones, each containing a defined ORF. This first version of the Brucella ORFeome (v1.1) provides the coding sequences in a user-friendly format amenable to high-throughput functional genomic and proteomic experiments, as the ORFs are conveniently transferable from the Entry clones to various Expression vectors by recombinational cloning. The cloning of the Brucella ORFeome v1.1 should help to provide a better understanding of the molecular mechanisms of virulence, including the identification of bacterial protein-protein interactions, but also interactions between bacterial effectors and their host's targets.

Brucella spp. are gram-negative intracellular facultative pathogens that are known to produce 2,3-dihydroxybenzoic acid (DHBA), a catechol siderophore that is essential for full virulence in the natural host. The mechanism of DHBA entry into Brucella and other gram-negative bacteria is poorly understood. Using mini-Tn5Kmcat mutagenesis, we created a transposon library of Brucella melitensis 16M and isolated 32 mutants with a defect in iron acquisition or assimilation. Three of these transposon mutants are deficient in utilization of DHBA. Analysis of these three mutants indicated that the ExbB, DstC, and DugA proteins are required for optimal assimilation of DHBA and/or citrate. ExbB is part of the Ton complex, and DstC is a permease homologue of an iron(III) ABC transporter; in gram-negative bacteria these two complexes are involved in the uptake of iron through the outer and inner membranes, respectively. DugA is a new partner in iron utilization that exhibits homology with the bacterial conserved GTPase YchF. Based on this homology, DugA could have a putative regulatory function in iron assimilation in Brucella. None of the three mutants was attenuated in cellular models or in the mouse model of infection, which is consistent with the previous suggestion that DHBA utilization is not required in these models.

The release of an increasing number of complete bacterial genomic sequences allows the evolutionary analysis of processes such as regulatory networks. CtrA is a response regulator of the OmpR subfamily, belonging to a complex regulatory network in the dimorphic bacterium Caulobacter crescentus. It coordinates the cell cycle with an asymmetric division, which is part of the adaptation of Caulobacter to poor-nutrient environments. CtrA is only found in alpha-proteobacteria, a group of bacteria encompassing genera with very distinct lifestyles, including host-associated bacteria. Analyses of CtrA regulatory networks and morphological examinations of some alpha-proteobacteria are presented. Our observations suggest that the core of the CtrA regulation network is conserved and that alpha-proteobacteria divide asymmetrically. We propose that the two daughter cells might be differentiated bacteria, each one displaying specific functions.

Brucella melitensis is a gram-negative alpha2-proteobacterium responsible for abortion in goats and for Malta fever in humans. This facultative intracellular pathogen invades and survives within both professional and nonprofessional phagocytes. A dichloromethane extract of spent culture supernatant from B. melitensis induces bioluminescence in an Escherichia coli acyl-homoserine lactone (acyl-HSL) biosensor strain based upon the activity of the LasR protein of Pseudomonas aeruginosa. HPLC fractionation of the extract, followed by mass spectrometry, identified the major active molecule as N-dodecanoylhomoserine lactone (C12-HSL). This is the first report of the production of an acyl-HSL by an intracellular pathogen. The addition of synthetic C12-HSL to an early log phase culture of either B. melitensis or Brucella suis 1330 reduces the transcription of the virB operon, which contains virulence genes known to be required for intracellular survival. This mimics events seen during the stationary phase of growth and suggests that quorum sensing may play a role in the control of virulence in Brucella.

Brucella is a facultative intracellular parasite that causes brucellosis in animals and humans. The protective immune response against Brucella involves both humoral and cell-mediated immunity. In previous studies, we demonstrated that the T-dominant Brucella antigens bacterioferritin (BFR) and P39 administered either as CpG adjuvant recombinant proteins or as naked-DNA plasmids induced a specific Th1-biased immune response in mice. In order to improve the protection conferred by the BFR and P39 vaccines and to evaluate the additive role of antilipopolysaccharide (anti-LPS) antibodies, we used live attenuated Yersinia enterocolitica serotypes O:3 and O:9 as delivery vectors for naked-DNA plasmids encoding these BFR and P39 antigens. Following two intragastric immunizations in BALB/c mice, the Yersinia vectors harboring a DNA vaccine encoding BFR or P39 induced antigen-specific serum immunoglobulin and Th1-type responses (both lymphocyte proliferation and gamma interferon production) among splenocytes. Moreover, as expected, antibodies recognizing Brucella abortus 544 lipopolysaccharide were detected in O:9-immunized mice but not in O:3-treated animals. Animals immunized with O:9 organisms carrying pCI or with O:9 organisms alone were found to be significantly resistant to infection by B. abortus 544. Our data demonstrated that pCI plasmids encoding BFR or P39 and delivered with live attenuated strains of Yersinia O:3 or O:9 can trigger Th1-type responses. The fact than only O:9 vectors induced a highly significant protective immunity against B. abortus 544 infection pointed out the crucial role of anti-LPS antibodies in protection. The best protection was conferred by a serotype O:9 strain carrying pCIP39, confirming the importance of the P39 T-cell antigen in this mechanism.

In this study, we evaluated the ability of DNA vaccines encoding the bacterioferritin (BFR) or P39 proteins of Brucella spp. to induce cellular and humoral immune responses and to protect BALB/c mice against a challenge with B. abortus 544. We constructed eukaryotic expression vectors called pCIBFR and pCIP39, encoding BFR or P39 antigens, respectively, and we verified that these proteins were produced after transfection of COS-7 cells. PCIBFR or pCIP39 was injected intramuscularly three times, at 3-week intervals. pCIP39 induced higher antibody responses than did the DNA vector encoding BFR. Both vectors elicited a T-cell-proliferative response and also induced a strong gamma interferon production upon restimulation with either the specific antigens or Brucella extract. In this report, we also demonstrate that animals immunized with these plasmids elicited a strong and long-lived memory immune response which persisted at least 3 months after the third vaccination. Furthermore, pCIBFR and pCIP39 induced a typical T-helper 1-dominated immune response in mice, as determined by cytokine or immunoglobulin G isotype analysis. The pCIP39 delivered by intramuscular injection (but not the pCIBFR or control vectors) induced a moderate protection in BALB/c mice challenged with B. abortus 544 compared to that observed in positive control mice vaccinated with S19.

The P39 and the bacterioferrin (BFR) antigens of Brucella melitensis 16M were previously identified as T dominant antigens able to induce both delayed-type hypersensivity in sensitized guinea pigs and in vitro gamma interferon (IFN-gamma) production by peripheral blood mononuclear cells from infected cattle. Here, we analyzed the potential for these antigens to function as a subunitary vaccine against Brucella abortus infection in BALB/c mice, and we characterized the humoral and cellular immune responses induced. Mice were injected with each of the recombinant proteins alone or adjuvanted with either CpG oligodeoxynucleotides (CpG ODN) or non-CpG ODN. Mice immunized with the recombinant antigens with CpG ODN were the only group demonstrating both significant IFN-gamma production and T-cell proliferation in response to either Brucella extract or to the respective antigen. The same conclusion holds true for the antibody response, which was only demonstrated in mice immunized with recombinant antigens mixed with CpG ODN. The antibody titers (both immunoglobulin G1 [IgG1] and IgG2a) induced by P39 immunization were higher than the titers induced by BFR (only IgG2a). Using a B. abortus 544 challenge, the level of protection was analyzed and compared to the protection conferred by one immunization with the vaccine strain B19. Immunization with P39 and CpG ODN gave a level of protection comparable to the one conferred by B19 at 4 weeks postchallenge, and the mice were still significantly protected at 8 weeks postchallenge, although to a lesser extent than the B19-vaccinated group. Intriguingly, no protection was detected after BFR vaccination. All other groups did not demonstrate any protection.

Omp2a and Omp2b are highly homologous porins present in the outer membrane of the bacteria from the genus Brucella, a facultative intracellular pathogen. The genes coding for these proteins are closely linked in the Brucella genome and oriented in opposite directions. In this work, we present the cloning, purification, and characterization of four Omp2b size variants found in various Brucella species, and we compare their antigenic and functional properties to the Omp2a and Omp2b porins of Brucella melitensis reference strain 16M. The variation of the Omp2a and Omp2b porin sequences among the various strains of the genus Brucella seems to result mostly from multiple gene conversions between the two highly homologous genes. As shown in this study, this phenomenon has led to the creation of natural Omp2a and Omp2b chimeric proteins in Omp2b porin size variants. The comparison by liposome swelling assay of the porins sugar permeability suggested a possible functional differences between Omp2a and Omp2b, with Omp2a showing a more efficient pore in sugar diffusion. The sequence variability in the Omp2b size variants was located in the predicted external loops of the porin. Several epitopes recognized by anti-Omp2b monoclonal antibodies were mapped by comparison of the Omp2b size variants antigenicity, and two of them were located in the most exposed surface loops. However, since variations are mostly driven by simple exchanges of conserved motifs between the two genes (except for an Omp2b version from an atypical strain of Brucella suis biovar 3), the porin variability does not result in major antigenic variability of the Brucella surface that could help the bacteria during the reinfection of a host. Porin variation in Brucella seems to result mainly in porin conductivity modifications.

Brucella melitensis 16M is a Gram-negative alpha2-proteobacterium responsible for abortion in goats and for Malta fever in humans. This facultative intracellular pathogen invades into and survives within both professional and non-professional phagocytes. Signature-tagged mutagenesis (STM) was used to identify genes required for the in vivo pathogenesis of Brucella. A library of transposon mutants was screened in a murine infection model. Out of 672 mutants screened, 20 were not recovered after a 5 day passage in BALB/c mice. The attenuation of 18 mutants was confirmed using an in vivo competition assay against the wild-type strain. The 18 mutants were characterized further for their ability to replicate in murine macrophages and in HeLa cells. The sequences disrupted by the transposon in the mutants have homology to genes coding for proteins of different functional classes: transport, amino acid and DNA metabolism, transcriptional regulation, peptidoglycan synthesis, a chaperone-like protein and proteins of unknown function. The mutants selected in this study provide new insights into the molecular basis of Brucella virulence.

A methodology is proposed to solve a difficult modeling problem related to the recently sequenced P39 protein. This sequence shares no similarity with any known 3D structure, but a fold is proposed by several threading tools. The difficulty in aligning the target sequence on one of the proposed template structures is overcome by combining the results of several available prediction methods and by refining a rational consensus between them. In silico validation of the obtained model and a preliminary cross-check with experimental features allow us to state that this borderline prediction is at least reasonable. This model raises relevant hypotheses on the main structural features of the protein and allows the design of site-directed mutations. Knowing the genetic context of the P39 reading frame, we are now able to suggest a function for the P39 protein: it would act as a periplasmic substrate-binding protein.