Publications

Despite decades of investigation into bacterial pathogens, the conditions met by intracellular bacteria are still unclear. These conditions can include access to nutrients, such as amino acids, and exposure to toxic compounds, like copper. To investigate the ability of Brucella abortus, a facultative intracellular pathogen responsible for a major zoonosis, to cope with copper, we performed a Tn-seq analysis to identify copper-sensitive mutants. Unexpectedly, we realized that classical copper resistance systems (involving CopA and CueO homologs) do not appear to be robustly needed, while histidine and purine biosynthesis pathways are crucial to cope with copper. We show that hisA, hisB, hisC and hisD mutants are auxotrophic for histidine and sensitive to copper. This suggests that the reported attenuation of his mutants in macrophages could be based on auxotrophy or/and copper sensitivity. Therefore, we generated suppressor strains with a restored resistance to copper for hisC, but still auxotrophs for histidine. Our data suggest that this suppression is due to the overproduction of a homolog of OppA, a periplasmic oligopeptide binding protein. Analysis of these suppressors shows that the absence of histidine biosynthesis capacity, and not copper sensitivity, is required for optimal growth of B. abortus in macrophages.

The cell envelope of gram-negative bacteria is composed of an inner and an outer membrane. In Escherichia coli, several pathways mediate phospholipid transport between the two membranes, including the Mla (i.e., maintenance of lipid asymmetry) and Pqi (i.e., paraquat inducible) systems. Here, we identify and characterise, in the intracellular pathogen Brucella abortus, a complex named Mpc, which exhibits homology to both Mla and Pqi components. Mpc is required for bacterial growth under envelope stress conditions, and for survival within macrophages during the early stages of infection. Analyses of protein-protein interactions and structural predictions suggest that the Mpc complex bridges the two membranes of the bacterial cell envelope. Absence of this system results in altered lipid composition of the outer membrane vesicles, indicating that Mpc plays a role in lipid transport between the membranes. Our sequence comparisons reveal that Mpc is conserved across numerous species of Hyphomicrobiales. The discovery of this novel lipid-trafficking system expands our understanding of the diversity and evolution of lipid-transport mechanisms in diderm bacteria.

The facultative intracellular pathogen Brucella abortus interacts with several organelles of the host cell to reach its replicative niche inside the endoplasmic reticulum. However, little is known about the interplay between the intracellular bacteria and the host cell mitochondria. Here, we showed that B. abortus triggers substantive mitochondrial network fragmentation, accompanied by mitophagy and the formation of mitochondrial Brucella- containing vacuoles during the late steps of cellular infection. Bru- cella-induced expression of the mitophagy receptor BNIP3L is essential for these events and relies on the iron-dependent stabili- sation of the hypoxia-inducible factor 1α. Functionally, BNIP3L- mediated mitophagy appears to be advantageous for bacterial exit from the host cell as BNIP3L depletion drastically reduces the num- ber of reinfection events. Altogether, these findings highlight the intricate link between Brucella trafficking and the mitochondria during host cell infection.

Lipopolysaccharide is essential for most Gram-negative bacteria as it is a main component of the outer membrane. In the pathogen Brucella abortus, smooth lipopolysaccharide containing the O-antigen is required for virulence. Being part of the Rhizobiales, Brucella spp. display unipolar growth and lipopolysaccharide was shown to be incorporated at the active growth sites, i.e. the new pole and the division site. By localizing proteins involved in the lipopolysaccharide transport across the cell envelope, from the inner to the outer membrane, we show that the lipopolysaccharide incorporation sites are determined by the inner membrane complex of the lipopolysaccharide transport system. Moreover, we identify the main O-antigen ligase of Brucella spp. involved in smooth lipopolysaccharide synthesis. Altogether, our data highlight a layer of spatiotemporal organization of the lipopolysaccharide biosynthesis pathway and identify an original class of bifunctional O-antigen ligases.

The pathogenic bacterium Brucella abortus invades and multiplies inside host cells. To grow inside host cells, B. abortus requires a functional histidine biosynthesis pathway. Here, we show that a B. abortus histidine auxotroph mutant also displays an unexpected chaining phenotype. The intensity of this phenotype varies according to the culture medium and is exacerbated inside host cells. Chains of bacteria consist of contiguous peptidoglycan, and likely result from the defective cleavage of peptidoglycan at septa. Genetic suppression of the chaining phenotype unearthed two essential genes with a role in B. abortus cell division: dipM and cdlP. Loss of function of dipM and cdlP generates swelling at the division site. While DipM is strictly localized at the division site, CdlP is localized at the growth pole and the division site. Altogether, the unexpected chaining phenotype of a hisB mutant allowed the discovery of new crucial actors in cell division in B. abortus.

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.

Gram negative bacteria are surrounded by a cell envelope that comprises an outer membrane (OM) and an inner membrane (IM) which together delimit the periplasmic space, which contains peptidoglycan (PG). Covalent anchoring of the OM to the peptidoglycan is crucial for envelope integrity in Escherichia coli. When OM is not attached to PG the OM forms blebs and detaches from the cell. The Braun lipoprotein (Lpp) covalently attaches OM to PG but is only present in a small number of gamma-proteobacteria, which leaves the mechanism of OM-PG attachment in other species undetermined. We report that the OM is attached to the peptidoglycan by covalent crosslinks between the N-termini of integral OM beta-barrel-shaped proteins (OMPs) and the peptide stems of PG in the alpha-proteobacteria Brucella abortus and Agrobacterium tumefaciens. Crosslinking is catalyzed 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 crosslinking and results in OM membrane instability. The alanyl-aspartyl motif  is conserved in OMPs from Rhizobiales, which means that it is feasible that OMP-PG crosslinks are widespread in alpha-proteobacteria

Brucella abortus 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 B. abortus ∆rsh 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 B. abortus to adapt to its intracellular growth conditions. The deletion of dksA, proposed to mediate a part of the effect of (p)ppGpp on transcription, does not affect B. abortus 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 B. abortus, 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 B. abortus in RAW 264.7 macrophages.

Over the last decades, many in vitro studies led to a better understanding of the causes and consequences of alkylating stress on DNA, as well as to the repair mechanisms selected by prokaryotic cells to face it. It has been proposed that pathogenic bacteria have to cope with alkylating stress inside host cells, but this assumption has never been demonstrated. Here, using single cell reporter systems, we show that the class III pathogen Brucella abortus does encounter alkylating stress during the first hours of macrophages infection. Direct repair and base excision repair pathways were identified to be needed by B. abortus to face this stress in vitro and in a mice infection model. In addition, B. abortus relies on distinct strategies than Escherichia coli to control DNA repair gene expression, since we found that some of these genes are under the control of the conserved cell-cycle transcription factor GcrA.

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.

Trends Microbiol. (12):812-21.

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 6h, 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.

Immun. 81, 996-1007

Microbes and Infection 15, 998-1004

J. Bacteriol. 194, 5305-5314.

Microbiology 158, 2610-2618

BMC Biochemistry 13, 16

Cell. Microbiol. 13, 1044-1058

BMC Microbiol. 11, 257.

Microbiology 157, 1263-1268.

J. Bacteriol. 192, 3235-3239.

PLoS ONE 5, e13274.

BMC Microbiology 10, 248.

J. Proteome Res. 9, 3200-3217.

PLoS ONE 5, e12679.

Microb. Pathog. 45, 134-141.

EMBO J. 26, 1444-1455.

J. Bacteriol. 189, 6035-6047.

J. Bacteriol. 188, 1615-1619.

J. Bacteriol. 188, 7707-7710

Cell. Microbiol. 8, 1791-1802.

J. Bacteriol. 189, 131-141.

Infect. Immun. 73, 5578-5586.

Cell. Microbiol. 7, 1151-1161.

Genome Res. 14, 2201-2206.

Trends Microbiol. 12, 361-365.

Infect. Immun. 72, 5783-5790.

Infect. Immun. 71, 7053-7060.

Mol. Microbiol. 43, 945-960. IF: 6.3

Bioinformatics 18, 1250-1256. IF: 3.4

Proc Natl. Acad. Sci. USA 109, 1697-1701