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.
Brucella abortus is a facultative intracellular pathogen causing a severe zoonotic disease worldwide. The two-component regulatory system (TCS) BvrR/BvrS of B. abortus is conserved in members of the Alphaproteobacteria class. It is related to the expression of genes required for host interaction and intracellular survival. Here we report that bvrR and bvrS are part of an operon composed of 16 genes encoding functions related to nitrogen metabolism, DNA repair and recombination, cell cycle arrest, and stress response. Synteny of this genomic region within close Alphaproteobacteria members suggests a conserved role in coordinating the expression of carbon and nitrogen metabolic pathways. In addition, we performed a ChIP-Seq analysis after exposure of bacteria to conditions that mimic the intracellular environment. Genes encoding enzymes at metabolic crossroads of the pentose phosphate shunt, gluconeogenesis, cell envelope homeostasis, nucleotide synthesis, cell division, and virulence are BvrR/BvrS direct targets. A 14 bp DNA BvrR binding motif was found and investigated in selected gene targets such as virB1, bvrR, pckA, omp25, and tamA. Understanding gene expression regulation is essential to elucidate how Brucella orchestrates a physiological response leading to a furtive pathogenic strategy.
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.
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