Publications by authors named "Xavier De Bolle"

65 Publications

Brucellosis in wildlife in Africa: a systematic review and meta-analysis.

Sci Rep 2021 Mar 16;11(1):5960. Epub 2021 Mar 16.

Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Pretoria, South Africa.

This study aimed to consolidate current knowledge of wildlife brucellosis in Africa and to analyse available predictors of infection. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were followed. Information on species, test used, test results, area, rainfall, livestock and wildlife contact and year of study were extracted. This systematic review revealed 42 prevalence studies, nine disease control articles and six articles on epidemiology. Brucella abortus, Brucella melitensis, Brucella inopinata and Brucella suis were reported in wildlife. The prevalence studies revealed serological evidence of brucellosis in buffalo, antelope (positive in 14/28 species), carnivores (4/12) and other species (7/20) over the last five decades. Buffalo populations were more likely to be infected and had a higher seroprevalence than other species; the pooled seroprevalence was 13.7% (95% CI 10.3-17.3%) in buffalo, 7.1% (95% CI 1.1-15.5%) in carnivores and 2.1% (95% CI 0.1-4.9%) in antelope. Wildlife in high rainfall areas (≥ 800 mm) were more likely to be infected, and infected populations showed higher seroprevalence in high rainfall areas and in studies published after 2000. Domestic animal contact was associated with increased seroprevalence in antelope and carnivore species, but not in buffalo, supporting the hypothesis that buffalo may be a reservoir species.
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http://dx.doi.org/10.1038/s41598-021-85441-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7966391PMC
March 2021

β-Barrels covalently link peptidoglycan and the outer membrane in the α-proteobacterium Brucella abortus.

Nat Microbiol 2021 01 2;6(1):27-33. Epub 2020 Nov 2.

Research Unit in Microorganisms Biology (URBM), Department of Biology, Namur Research Institute for Life Sciences (NARILIS), Namur, Belgium.

Gram-negative bacteria are surrounded by a cell envelope that comprises an outer membrane (OM) and an inner membrane that, together, delimit the periplasmic space, which contains the peptidoglycan (PG) sacculus. Covalent anchoring of the OM to the PG is crucial for envelope integrity in Escherichia coli. When the OM is not attached to the PG, the OM forms blebs and detaches from the cell. The Braun lipoprotein Lpp covalently attaches OM to the PG but is present in only a small number of γ-proteobacteria; the mechanism of OM-PG attachment in other species is unclear. Here, we report that the OM is attached to PG by covalent cross-links between the N termini of integral OM β-barrel-shaped proteins (OMPs) and the peptide stems of PG in the α-proteobacteria Brucella abortus and Agrobacterium tumefaciens. Cross-linking is catalysed by L,D-transpeptidases and attached OMPs have a conserved alanyl-aspartyl motif at their N terminus. Mutation of the aspartate in this motif prevents OMP cross-linking and results in OM membrane instability. The alanyl-aspartyl motif is conserved in OMPs from Rhizobiales; it is therefore feasible that OMP-PG cross-links are widespread in α-proteobacteria.
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http://dx.doi.org/10.1038/s41564-020-00799-3DOI Listing
January 2021

Convergent evolution of zoonotic species toward the selective use of the pentose phosphate pathway.

Proc Natl Acad Sci U S A 2020 10 5;117(42):26374-26381. Epub 2020 Oct 5.

Research Unit in Biology of Microorganisms, Narilis, University of Namur, B-5000 Namur, Belgium;

Mechanistic understanding of the factors that govern host tropism remains incompletely understood for most pathogens. species, which are capable of infecting a wide range of hosts, offer a useful avenue to address this question. We hypothesized that metabolic fine-tuning to intrahost niches is likely an underappreciated axis underlying pathogens' ability to infect new hosts and tropism. In this work, we compared the central metabolism of seven species by stable isotopic labeling and genetics. We identified two functionally distinct groups, one overlapping with the classical zoonotic species of domestic livestock that exclusively use the pentose phosphate pathway (PPP) for hexose catabolism, whereas species from the second group use mostly the Entner-Doudoroff pathway (EDP). We demonstrated that the metabolic dichotomy among emerged after the acquisition of two independent EDP-inactivating mutations in all classical zoonotic species. We then examined the pathogenicity of key metabolic mutants in mice and confirmed that this trait is tied to virulence. Altogether, our data are consistent with the hypothesis that the PPP has been incrementally selected over the EDP in parallel to adaptation to domestic livestock.
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http://dx.doi.org/10.1073/pnas.2008939117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7584911PMC
October 2020

Intracellular Growth and Cell Cycle Progression are Dependent on (p)ppGpp Synthetase/Hydrolase in .

Pathogens 2020 Jul 14;9(7). Epub 2020 Jul 14.

Unité de Recherche en Biologie des Microorganismes (URBM), University of Namur, 61 rue de Bruxelles, 5000 Namur, Belgium.

is a pathogenic bacterium able to proliferate inside host cells. During the first steps of its trafficking, it is able to block the progression of its cell cycle, remaining at the G1 stage for several hours, before it reaches its replication niche. We hypothesized that starvation mediated by guanosine tetra- or penta-phosphate, (p)ppGpp, could be involved in the cell cycle arrest. Rsh is the (p)ppGpp synthetase/hydrolase. A ∆ mutant is unable to grow in minimal medium, it is unable to survive in stationary phase in rich medium and it is unable to proliferate inside RAW 264.7 macrophages. A strain producing the heterologous constitutive (p)ppGpp hydrolase Mesh1b is also unable to proliferate inside these macrophages. Altogether, these data suggest that (p)ppGpp is necessary to allow to adapt to its intracellular growth conditions. The deletion of , proposed to mediate a part of the effect of (p)ppGpp on transcription, does not affect growth in culture or inside macrophages. Expression of a gene coding for a constitutively active (p)ppGpp synthetase slows down growth in rich medium and inside macrophages. Using an mCherry-ParB fusion able to bind to the replication origin of the main chromosome of , we observed that expression of the constitutive (p)ppGpp synthetase gene generates an accumulation of bacteria at the G1 phase. We thus propose that (p)ppGpp accumulation could be one of the factors contributing to the G1 arrest observed for in RAW 264.7 macrophages.
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http://dx.doi.org/10.3390/pathogens9070571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7400157PMC
July 2020

Shedding of Brucella melitensis happens through milk macrophages in the murine model of infection.

Sci Rep 2020 06 10;10(1):9421. Epub 2020 Jun 10.

German Federal Institute for Risk Assessment, Diedersdorfer Weg 1, 12277, Berlin, Germany.

Although shedding of zoonotic brucellae in milk has been demonstrated in natural hosts, these data are still missing for the standard murine infection model. We therefore analysed shedding kinetics and the niche of B. melitensis in murine milk. Pregnant Balb/cByJ mice were intraperitoneally infected with 10 CFU of the 16 M reference strain, a 16 M mCherry mutant or a human isolate. Milk was collected over the course of lactation, and subjected to culture and immunofluorescence assays. Bacteria were also quantified in spleen and mammary glands of maternal mice and in spleen of the litter. The shedding of the three strains did not differ significantly (p = 0.301), ranging from log 1.5 to 4.04 CFU/ml. A total of 73% of the mice excreted B. melitensis into the milk with peak values at mid-lactation; up to 30 bacteria/cell were found in macrophages and neutrophils. While the bacterial counts in the spleen of lactating females confirmed a well-established infection, only 50% of the pups harboured brucellae in their spleen, including the spleen of an uninfected pup fed by an infected foster mother. In conclusion, the murine model of infection may contribute to a better understanding of the zoonotic transmission of brucellosis.
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http://dx.doi.org/10.1038/s41598-020-65760-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7287137PMC
June 2020

Occurrence and repair of alkylating stress in the intracellular pathogen Brucella abortus.

Nat Commun 2019 10 24;10(1):4847. Epub 2019 Oct 24.

URBM, Narilis, University of Namur, Namur, Belgium.

It is assumed that intracellular pathogenic bacteria have to cope with DNA alkylating stress within host cells. Here we use single-cell reporter systems to show that the pathogen Brucella abortus does encounter alkylating stress during the first hours of macrophage infection. Genes encoding direct repair and base-excision repair pathways are required by B. abortus to face this stress in vitro and in a mouse infection model. Among these genes, ogt is found to be under the control of the conserved cell-cycle transcription factor GcrA. Our results highlight that the control of DNA repair in B. abortus displays distinct features that are not present in model organisms such as Escherichia coli.
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http://dx.doi.org/10.1038/s41467-019-12516-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6813329PMC
October 2019

Route of Infection Strongly Impacts the Host-Pathogen Relationship.

Front Immunol 2019 11;10:1589. Epub 2019 Jul 11.

Unité de Recherche en Biologie des Microorganismes, Laboratoire d'Immunologie et de Microbiologie, NARILIS, Université de Namur, Namur, Belgium.

Live attenuated vaccines play a key role in the control of many human and animal pathogens. Their rational development is usually helped by identification of the reservoir of infection, the lymphoid subpopulations associated with protective immunity as well as the virulence genes involved in pathogen persistence. Here, we compared the course of infection in C57BL/6 mice infected via intraperitoneal (i.p.), intranasal (i.n.) and intradermal (i.d.) route and demonstrated that the route of infection strongly impacts all of these parameters. Following i.p. and i.n. infection, most infected cells observed in the spleen or lung were F4/80 myeloid cells. In striking contrast, infected Ly6G neutrophils and CD140a fibroblasts were also observed in the skin after i.d. infection. The operon encoding for the type IV secretion system is considered essential to deflecting vacuolar trafficking in phagocytic cells and allows to multiply and persist. Unexpectedly, the Δ strain, which does not persist in the lung after i.n. infection, persists longer in skin tissues than the wild strain after i.d. infection. While the CD4 T cell-mediated Th1 response is indispensable to controlling the challenge in the i.p. model, it is dispensable for the control of in the i.d. and i.n. models. Similarly, B cells are indispensable in the i.p. and i.d. models but dispensable in the i.n. model. γδ T cells appear able to compensate for the absence of αβ T cells in the i.d. model but not in the other models. Taken together, our results demonstrate the crucial importance of the route of infection for the host pathogen relationship.
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http://dx.doi.org/10.3389/fimmu.2019.01589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6637429PMC
October 2020

Localized incorporation of outer membrane components in the pathogen .

EMBO J 2019 03 11;38(5). Epub 2019 Jan 11.

Research Unit in Biology of Microorganisms (URBM), Narilis University of Namur (UNamur), Namur, Belgium

The zoonotic pathogen 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.
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http://dx.doi.org/10.15252/embj.2018100323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6396147PMC
March 2019

Transposon Sequencing of Brucella abortus Uncovers Essential Genes for Growth and Inside Macrophages.

Infect Immun 2018 08 23;86(8). Epub 2018 Jul 23.

Research Unit in Microorganisms Biology (URBM), Narilis, University of Namur, Namur, Belgium

is a class III zoonotic bacterial pathogen able to survive and replicate inside host cells, including macrophages. Here we report a multidimensional transposon sequencing analysis to identify genes essential for growth in rich medium and replication in RAW 264.7 macrophages. The construction of a dense transposon mutant library and mapping of 929,769 unique mini-Tn insertion sites in the genome allowed identification of 491 essential coding sequences and essential segments in the genome. Chromosome II carries a lower proportion (5%) of essential genes than chromosome I (19%), supporting the hypothesis of a recent acquisition of a megaplasmid as the origin of chromosome II. Temporally resolved transposon sequencing analysis as a function of macrophage infection stages identified 79 genes with a specific attenuation phenotype in macrophages, at either 2, 5, or 24 h postinfection, and 86 genes for which the attenuated mutant phenotype correlated with a growth defect on plates. We identified 48 genes required for intracellular growth, including the operon, encoding the type IV secretion system, which supports the validity of the screen. The remaining genes encode amino acid and pyrimidine biosynthesis, electron transfer systems, transcriptional regulators, and transporters. In particular, we report the need of an intact pyrimidine nucleotide biosynthesis pathway in order for to proliferate inside RAW 264.7 macrophages.
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http://dx.doi.org/10.1128/IAI.00312-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6056880PMC
August 2018

PBP2b plays a key role in both peripheral growth and septum positioning in Lactococcus lactis.

PLoS One 2018 23;13(5):e0198014. Epub 2018 May 23.

Institut des Sciences de la Vie (ISV), Université catholique de Louvain (UCL), Louvain-la-Neuve, Belgium.

Lactococcus lactis is an ovoid bacterium that forms filaments during planktonic and biofilm lifestyles by uncoupling cell division from cell elongation. In this work, we investigate the role of the leading peptidoglycan synthase PBP2b that is dedicated to cell elongation in ovococci. We show that the localization of a fluorescent derivative of PBP2b remains associated to the septal region and superimposed with structural changes of FtsZ during both vegetative growth and filamentation indicating that PBP2b remains intimately associated to the division machinery during the whole cell cycle. In addition, we show that PBP2b-negative cells of L. lactis are not only defective in peripheral growth; they are also affected in septum positioning. This septation defect does not simply result from the absence of the protein in the cell growth machinery since it is also observed when PBP2b-deficient cells are complemented by a catalytically inactive variant of PBP2b. Finally, we show that round cells resulting from β-lactam treatment are not altered in septation, suggesting that shape elongation as such is not a major determinant for selection of the division site. Altogether, we propose that the specific PBP2b transpeptidase activity at the septum plays an important role for tagging future division sites during L. lactis cell cycle.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0198014PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5965867PMC
November 2018

Learning from the master: targets and functions of the CtrA response regulator in Brucella abortus and other alpha-proteobacteria.

FEMS Microbiol Rev 2018 07;42(4):500-513

URBM-Biology, Université de Namur, Unité de recherche en biologie moléculaire, Belgium.

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.
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http://dx.doi.org/10.1093/femsre/fuy019DOI Listing
July 2018

Mitochondrial fragmentation affects neither the sensitivity to TNFα-induced apoptosis of Brucella-infected cells nor the intracellular replication of the bacteria.

Sci Rep 2018 03 26;8(1):5173. Epub 2018 Mar 26.

Laboratory of Biochemistry and Cell Biology (URBC, Unité de Recherche en Biologie Cellulaire)-NARILIS (Namur Research Institute for Life Sciences), University of Namur, Rue de Bruxelles 61, 5000, Namur, Belgium.

Mitochondria are complex organelles that participate in many cellular functions, ranging from ATP production to immune responses against viruses and bacteria. This integration of a plethora of functions within a single organelle makes mitochondria a very attractive target to manipulate for intracellular pathogens. We characterised the crosstalk that exists between Brucella abortus, the causative agent of brucellosis, and the mitochondria of infected cells. Brucella replicates in a compartment derived from the endoplasmic reticulum (ER) and modulates ER functionality by activating the unfolded protein response. However, the impact of Brucella on the mitochondrial population of infected cells still requires a systematic study. We observed physical contacts between Brucella containing vacuoles and mitochondria. We also found that B. abortus replication is independent of mitochondrial oxidative phosphorylation and that mitochondrial reactive oxygen species do not participate to the control of B. abortus infection in vitro. We demonstrated that B. abortus and B. melitensis induce a drastic mitochondrial fragmentation at 48 hours post-infection in different cell types, including myeloid and non-myeloid cells. This fragmentation is DRP1-independent and might be caused by a deficit of mitochondrial fusion. However, mitochondrial fragmentation does not change neither Brucella replication efficiency, nor the susceptibility of infected cells to TNFα-induced apoptosis.
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http://dx.doi.org/10.1038/s41598-018-23483-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5979954PMC
March 2018

OXA-427, a new plasmid-borne carbapenem-hydrolysing class D β-lactamase in Enterobacteriaceae.

J Antimicrob Chemother 2017 09;72(9):2469-2477

Laboratory of Clinical Microbiology, Belgian National Reference Center for Monitoring Antimicrobial Resistance in Gram-negative Bacteria, CHU UCL Namur, Yvoir, Belgium.

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.
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http://dx.doi.org/10.1093/jac/dkx184DOI Listing
September 2017

Erythritol Availability in Bovine, Murine and Human Models Highlights a Potential Role for the Host Aldose Reductase during Infection.

Front Microbiol 2017 13;8:1088. Epub 2017 Jun 13.

Research Unit in Biology of Microorganisms, Department of Veterinary Medicine, University of NamurNamur, Belgium.

Erythritol is the preferential carbon source for most brucellae, a group of facultative intracellular bacteria that cause a worldwide zoonosis. Since this polyol is abundant in genital organs of ruminants and swine, it is widely accepted that erythritol accounts at least in part for the characteristic genital tropism of brucellae. Nevertheless, proof of erythritol availability and essentiality during intracellular multiplication has remained elusive. To investigate this relationship, we compared Δ (erythritol-sensitive and thus predicted to be attenuated if erythritol is present), Δ (erythritol-tolerant but showing reduced growth if erythritol is a crucial nutrient) and wild type in various infection models. This reporting system indicated that erythritol was available but not required for multiplication in bovine trophoblasts. However, mice and humans have been considered to lack erythritol, and we found that it was available but not required for multiplication in human and murine trophoblastic and macrophage-like cells, and in mouse spleen and conceptus (fetus, placenta and envelopes). Using this animal model, we found that infected cells and tissues contained aldose reductase, an enzyme that can account for the production of erythritol from pentose cycle precursors.
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http://dx.doi.org/10.3389/fmicb.2017.01088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5468441PMC
June 2017

Genital Tropism: What's on the Menu.

Front Microbiol 2017 28;8:506. Epub 2017 Mar 28.

Facultad de Medicina, Departamento de Microbiología y Parasitología, Edificio de Investigación, Instituto de Salud Tropical e Instituto de Investigación Sanitaria de Navarra, Universidad de Navarra Pamplona, Spain.

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http://dx.doi.org/10.3389/fmicb.2017.00506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5368252PMC
March 2017

CtrA controls cell division and outer membrane composition of the pathogen Brucella abortus.

Mol Microbiol 2017 03 10;103(5):780-797. Epub 2017 Jan 10.

Microorganisms Biology Research Unit (URBM), Narilis, University of Namur, Namur, Belgium.

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.
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http://dx.doi.org/10.1111/mmi.13589DOI Listing
March 2017

Strain 2308 Wisconsin Genome: Importance of the Definition of Reference Strains.

Front Microbiol 2016 29;7:1557. Epub 2016 Sep 29.

Programa de Investigación en Enfermedades Tropicales, Escuela de Medicina Veterinaria, Universidad Nacional de Costa RicaHeredia, Costa Rica; Centro de Investigación en Enfermedades Tropicales, Facultad de Microbiología, Universidad de Costa RicaSan José, Costa Rica.

Brucellosis is a bacterial infectious disease affecting a wide range of mammals and a neglected zoonosis caused by species of the genetically homogenous genus . As in most studies on bacterial diseases, research in brucellosis is carried out by using reference strains as canonical models to understand the mechanisms underlying host pathogen interactions. We performed whole genome sequencing analysis of the reference strain 2308 routinely used in our laboratory, including manual curated annotation accessible as an editable version through a link at https://en.wikipedia.org/wiki/Brucella#Genomics. Comparison of this genome with two publically available 2308 genomes showed significant differences, particularly indels related to insertional elements, suggesting variability related to the transposition of these elements within the same strain. Considering the outcome of high resolution genomic techniques in the bacteriology field, the conventional concept of strain definition needs to be revised.
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http://dx.doi.org/10.3389/fmicb.2016.01557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5041503PMC
September 2016

Phosphotransferase-dependent accumulation of (p)ppGpp in response to glutamine deprivation in Caulobacter crescentus.

Nat Commun 2016 04 25;7:11423. Epub 2016 Apr 25.

Bacterial Cell cycle and Development (BCcD), URBM, University of Namur, 61 Rue de Bruxelles, Namur 5000, Belgium.

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.
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http://dx.doi.org/10.1038/ncomms11423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4848567PMC
April 2016

Metabolic control of cell division in α-proteobacteria by a NAD-dependent glutamate dehydrogenase.

Commun Integr Biol 2016 Jan-Feb;9(1):e1125052. Epub 2016 Jan 5.

Bacterial Cell cycle & Development (BCcD), URBM, University of Namur , Namur, Belgium.

Prior to initiate energy-consuming processes, such as DNA replication or cell division, cells need to evaluate their metabolic status. We have recently identified and characterized a new connection between metabolism and cell division in the α-proteobacterium Caulobacter crescentus. We showed that an NAD-dependent glutamate dehydrogenase (GdhZ) coordinates growth with cell division according to its enzymatic activity. Here we report the conserved role of GdhZ in controlling cell division in another α-proteobacterium, the facultative intracellular pathogen Brucella abortus. We also discuss the importance of amino acids as a main carbon source for α-proteobacteria.
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http://dx.doi.org/10.1080/19420889.2015.1125052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4802739PMC
April 2016

Brucella abortus Cell Cycle and Infection Are Coordinated.

Trends Microbiol 2015 Dec 20;23(12):812-821. Epub 2015 Oct 20.

University of Namur, 61 rue de Bruxelles, 5000 Namur, Belgium.

Brucellae are facultative intracellular pathogens. The recent development of methods and genetically engineered strains allowed the description of cell-cycle progression of Brucella abortus, including unipolar growth and the ordered initiation of chromosomal replication. B. abortus cell-cycle progression is coordinated with intracellular trafficking in the endosomal compartments. Bacteria are first blocked at the G1 stage, growth and chromosome replication being resumed shortly before reaching the intracellular proliferation compartment. The control mechanisms of cell cycle are similar to those reported for the bacterium Caulobacter crescentus, and they are crucial for survival in the host cell. The development of single-cell analyses could also be applied to other bacterial pathogens to investigate their cell-cycle progression during infection.
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http://dx.doi.org/10.1016/j.tim.2015.09.007DOI Listing
December 2015

A NAD-dependent glutamate dehydrogenase coordinates metabolism with cell division in Caulobacter crescentus.

EMBO J 2015 Jul 7;34(13):1786-800. Epub 2015 May 7.

Bacterial Cell Cycle & Development (BCcD), URBM, University of Namur, Namur, Belgium

Coupling cell cycle with nutrient availability is a crucial process for all living cells. But how bacteria control cell division according to metabolic supplies remains poorly understood. Here, we describe a molecular mechanism that coordinates central metabolism with cell division in the α-proteobacterium Caulobacter crescentus. This mechanism involves the NAD-dependent glutamate dehydrogenase GdhZ and the oxidoreductase-like KidO. While enzymatically active GdhZ directly interferes with FtsZ polymerization by stimulating its GTPase activity, KidO bound to NADH destabilizes lateral interactions between FtsZ protofilaments. Both GdhZ and KidO share the same regulatory network to concomitantly stimulate the rapid disassembly of the Z-ring, necessary for the subsequent release of progeny cells. Thus, this mechanism illustrates how proteins initially dedicated to metabolism coordinate cell cycle progression with nutrient availability.
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http://dx.doi.org/10.15252/embj.201490730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4516431PMC
July 2015

On the link between cell cycle and infection of the Alphaproteobacterium .

Microb Cell 2014 Sep 29;1(10):346-348. Epub 2014 Sep 29.

URBM, UNamur, Belgium.

Bacteria of the genus are responsible for brucellosis, a worldwide zoonosis. These bacteria are known to have a peculiar intracellular trafficking, with a first long and non-proliferative endosomal stage and a second proliferation stage, often associated with its localization of the bacteria in the endoplasmic reticulum (ER). However, the status of the bacterial cell cycle during the non-proliferative phase was still unknown. In a recent study [Nat. Communic. 5:4366], we followed the cell cycle of in culture and inside the host cells. In culture, initiates the replication of its large chromosome before the small chromosome. The origin and terminator regions of these two chromosomes display distinct localization and dynamics within . In HeLa cells and RAW264.7 macrophages, the bacteria in G1 (i.e. before the initiation of chromosomes replication) are preferentially found during the endosomal stage of the infection. During this period, growth is also arrested. The cell cycle arrest and resume during the trafficking in host cell suggest that like the model Alphaproteobacterium , these bacteria are able to block their cell cycle at the G1 phase when starvation is sensed.
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http://dx.doi.org/10.15698/mic2014.10.171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5349176PMC
September 2014

Replication of Brucella abortus and Brucella melitensis in fibroblasts does not require Atg5-dependent macroautophagy.

BMC Microbiol 2014 Sep 2;14:223. Epub 2014 Sep 2.

Background: Several intracellular bacterial pathogens have evolved subtle strategies to subvert vesicular trafficking pathways of their host cells to avoid killing and to replicate inside the cells. Brucellae are Gram-negative facultative intracellular bacteria that are responsible for brucellosis, a worldwide extended chronic zoonosis. Following invasion, Brucella abortus is found in a vacuole that interacts first with various endosomal compartments and then with endoplasmic reticulum sub-compartments. Brucella establishes its replication niche in ER-derived vesicles. In the past, it has been proposed that B. abortus passed through the macroautophagy pathway before reaching its niche of replication. However, recent experiments provided evidence that the classical macroautophagy pathway was not involved in the intracellular trafficking and the replication of B. abortus in bone marrow-derived macrophages and in HeLa cells. In contrast, another study showed that macroautophagy favoured the survival and the replication of Brucella melitensis in infected RAW264.7 macrophages. This raises the possibility that B. abortus and B. melitensis followed different intracellular pathways before replicating. In the present work, we have addressed this issue by comparing the replication rate of B. abortus and B. melitensis in embryonic fibroblasts derived from wild-type and Atg5-/- mice, Atg5 being a core component of the canonical macroautophagic pathway.

Results: Our results indicate that both B. abortus S2308 and B. melitensis 16M strains are able to invade and replicate in Atg5-deficient fibroblasts, suggesting that the canonical Atg5-dependent macroautophagic pathway is dispensable for Brucella replication. The number of viable bacteria was even slightly higher in Atg5-/- fibroblasts than in wild-type fibroblasts. This increase could be due to a more efficient uptake or to a better survival rate of bacteria before the beginning of the replication in Atg5-deficient cells as compared to wild-type cells. Moreover, our data show that the infection with B. abortus or with B. melitensis does not stimulate neither the conversion of LC3-I to LC3-II nor the membrane recruitment of LC3 onto the BCV.

Conclusion: Our study suggests that like Brucella abortus, Brucella melitensis does not subvert the canonical macroautophagy to reach its replicative niche or to stimulate its replication.
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http://dx.doi.org/10.1186/s12866-014-0223-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4159544PMC
September 2014

G1-arrested newborn cells are the predominant infectious form of the pathogen Brucella abortus.

Nat Commun 2014 Jul 9;5:4366. Epub 2014 Jul 9.

Microorganisms biology research unit (URBM), University of Namur (UNamur), Namur, Belgium.

Several intracellular pathogens, such as Brucella abortus, display a biphasic infection process starting with a non-proliferative stage of unclear nature. Here, we study the cell cycle of B. abortus at the single-cell level, in culture and during infection of HeLa cells and macrophages. The localization of segregation and replication loci of the two bacterial chromosomes indicates that, immediately after being engulfed by host-cell endocytic vacuoles, most bacterial cells are newborn. These bacterial cells do not initiate DNA replication for the next 4 to 6 h, indicating a G1 arrest. Moreover, growth is completely stopped during that time, reflecting a global cell cycle block. Growth and DNA replication resume later, although bacteria still reside within endosomal-like compartments. We hypothesize that the predominance of G1-arrested bacteria in the infectious population, and the bacterial cell cycle arrest following internalization, may constitute a widespread strategy among intracellular pathogens to colonize new proliferation niches.
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http://dx.doi.org/10.1038/ncomms5366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4104442PMC
July 2014

Quorum sensing and self-quorum quenching in the intracellular pathogen Brucellamelitensis.

PLoS One 2013 11;8(12):e82514. Epub 2013 Dec 11.

URBM, Department of Biology, University of Namur, Namur, Belgium.

Brucella quorum sensing has been described as an important regulatory system controlling crucial virulence determinants such as the VirB type IV secretion system and the flagellar genes. However, the basis of quorum sensing, namely the production of autoinducers in Brucella has been questioned. Here, we report data obtained from the use of a genetic tool allowing the in situ detection of long-chain N-acyl-homoserine lactones (AHL) activity at single bacterium level in Brucella melitensis. These data are consistent with an intrinsic production of AHL by B. melitensis in low concentration both during in vitro growth and macrophage infection. Moreover, we identified a protein, named AibP, which is homologous to the AHL-acylases of various bacterial species. In vitro and during infection, expression of aibP coincided with a decrease in endogenous AHL activity within B. melitensis, suggesting that AibP could efficiently impair AHL accumulation. Furthermore, we showed that deletion of aibP in B. melitensis resulted in enhanced virB genes expression and VirB8 production as well as in a reduced flagellar genes expression and production of FlgE (hook protein) and FliC (flagellin) in vitro. Altogether, these results suggest that AHL-dependent quorum sensing and AHL-quorum quenching coexist in Brucella, at least to regulate its virulence.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0082514PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3859601PMC
October 2014

Insights into the function of YciM, a heat shock membrane protein required to maintain envelope integrity in Escherichia coli.

J Bacteriol 2014 Jan 1;196(2):300-9. Epub 2013 Nov 1.

de Duve Institute, Université Catholique de Louvain, Brussels, Belgium.

The cell envelope of Gram-negative bacteria is an essential organelle that is important for cell shape and protection from toxic compounds. Proteins involved in envelope biogenesis are therefore attractive targets for the design of new antibacterial agents. In a search for new envelope assembly factors, we screened a collection of Escherichia coli deletion mutants for sensitivity to detergents and hydrophobic antibiotics, a phenotype indicative of defects in the cell envelope. Strains lacking yciM were among the most sensitive strains of the mutant collection. Further characterization of yciM mutants revealed that they display a thermosensitive growth defect on low-osmolarity medium and that they have a significantly altered cell morphology. At elevated temperatures, yciM mutants form bulges containing cytoplasmic material and subsequently lyse. We also discovered that yciM genetically interacts with envC, a gene encoding a regulator of the activity of peptidoglycan amidases. Altogether, these results indicate that YciM is required for envelope integrity. Biochemical characterization of the protein showed that YciM is anchored to the inner membrane via its N terminus, the rest of the protein being exposed to the cytoplasm. Two CXXC motifs are present at the C terminus of YciM and serve to coordinate a redox-sensitive iron center of the rubredoxin type. Both the N-terminal membrane anchor and the C-terminal iron center of YciM are important for function.
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http://dx.doi.org/10.1128/JB.00921-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911248PMC
January 2014

The Brucella pathogens are polarized bacteria.

Microbes Infect 2013 Dec 18;15(14-15):998-1004. Epub 2013 Oct 18.

Global Health Institute, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Station 19, 1015 Lausanne, Switzerland.

Brucella pathogens are responsible for brucellosis, a worldwide zoonosis. They are facultative intracellular pathogens characterized by their asymmetric division and their unipolar growth. This growth modality generates poles with specialized functions (through polar recruitment of polar adhesins or of cell cycle regulators) and progeny cells with potentially different fates.
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http://dx.doi.org/10.1016/j.micinf.2013.10.008DOI Listing
December 2013

BtaE, an adhesin that belongs to the trimeric autotransporter family, is required for full virulence and defines a specific adhesive pole of Brucella suis.

Infect Immun 2013 Mar 14;81(3):996-1007. Epub 2013 Jan 14.

Fundación Instituto Leloir, IIBBA CONICET, Buenos Aires, Argentina.

Brucella is responsible for brucellosis, one of the most common zoonoses worldwide that causes important economic losses in several countries. Increasing evidence indicates that adhesion of Brucella spp. to host cells is an important step to establish infection. We have previously shown that the BmaC unipolar monomeric autotransporter mediates the binding of Brucella suis to host cells through cell-associated fibronectin. Our genome analysis shows that the B. suis genome encodes several additional potential adhesins. In this work, we characterized a predicted trimeric autotransporter that we named BtaE. By expressing btaE in a nonadherent Escherichia coli strain and by phenotypic characterization of a B. suis ΔbtaE mutant, we showed that BtaE is involved in the binding of B. suis to hyaluronic acid. The B. suis ΔbtaE mutant exhibited a reduction in the adhesion to HeLa and A549 epithelial cells compared with the wild-type strain, and it was outcompeted by the wild-type strain in the binding to HeLa cells. The knockout btaE mutant showed an attenuated phenotype in the mouse model, indicating that BtaE is required for full virulence. BtaE was immunodetected on the bacterial surface at one cell pole. Using old and new pole markers, we observed that both the BmaC and BtaE adhesins are consistently associated with the new cell pole, suggesting that, in Brucella, the new pole is functionally differentiated for adhesion. This is consistent with the inherent polarization of this bacterium, and its role in the invasion process.
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http://dx.doi.org/10.1128/IAI.01241-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3584859PMC
March 2013

Innate immune recognition of flagellin limits systemic persistence of Brucella.

Cell Microbiol 2013 Jun 7;15(6):942-960. Epub 2013 Jan 7.

Department of Medical Microbiology & Immunology, University of California, Davis, CA, USA.

Brucella are facultative intracellular bacteria that cause chronic infections by limiting innate immune recognition. It is currently unknown whether Brucella FliC flagellin, the monomeric subunit of flagellar filament, is sensed by the host during infection. Here, we used two mutants of Brucella melitensis, either lacking or overexpressing flagellin, to show that FliC hinders bacterial replication in vivo. The use of cells and mice genetically deficient for different components of inflammasomes suggested that FliC was a target of the cytosolic innate immune receptor NLRC4 in vivo but not in macrophages in vitro where the response to FliC was nevertheless dependent on the cytosolic adaptor ASC, therefore suggesting a new pathway of cytosolic flagellin sensing. However, our work also suggested that the lack of TLR5 activity of Brucella flagellin and the regulation of its synthesis and/or delivery into host cells are both part of the stealthy strategy of Brucella towards the innate immune system. Nevertheless, as a flagellin-deficient mutant of B. melitensis wasfound to cause histologically demonstrable injuries in the spleen of infected mice, we suggested that recognition of FliC plays a role in the immunological stand-off between Brucella and its host, which is characterized by a persistent infection with limited inflammatory pathology.
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http://dx.doi.org/10.1111/cmi.12088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4026035PMC
June 2013

Small GTPases and Brucella entry into the endoplasmic reticulum.

Biochem Soc Trans 2012 Dec;40(6):1348-52

URBM, NARILIS, University of Namur (FUNDP), Namur, Belgium.

A key determinant for intracellular pathogenic bacteria to ensure their virulence within host cells is their ability to bypass the endocytic pathway and to reach a safe niche of replication. In the case of Brucella, the bacterium targets the ER (endoplasmic reticulum) to create a replicating niche called the BCV (Brucella-containing vacuole). The ER is a suitable strategic place for pathogenic Brucella. Indeed, bacteria can be hidden from host cell defences to persist within the host, and they can take advantage of the membrane reservoir delivered by the ER to replicate. Interaction with the ER leads to the presence on the BCV of the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and the small GTPase Rab2 known to be located on secretory vesicles that traffic between the ER and the Golgi apparatus. GAPDH and the small GTPase Rab2 controls Brucella replication at late times post-infection. A specific interaction between the human small GTPase Rab2 and a Brucella spp. protein named RicA was identified. Altered kinetics of intracellular trafficking and faster proliferation of the Brucella abortus ΔricA mutant was observed compared with the wild-type strain. RicA is the first reported effector with a proposed function for B. abortus.
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http://dx.doi.org/10.1042/BST20120156DOI Listing
December 2012
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