RESUMEN
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.
Asunto(s)
Brucella abortus/genética , Daño del ADN/genética , Interacciones Huésped-Patógeno/genética , Macrófagos/metabolismo , Estrés Fisiológico/genética , Alquilación , Animales , Brucella abortus/metabolismo , Brucelosis , Metilación de ADN/genética , Reparación del ADN/genética , Ratones , Células RAW 264.7 , Vacuolas/metabolismoRESUMEN
Brucella abortus 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 Brucella abortus 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-Tn5 insertion sites in the genome allowed identification of 491 essential coding sequences and essential segments in the B. abortus 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 virB 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 B. abortus to proliferate inside RAW 264.7 macrophages.
Asunto(s)
Brucella abortus/crecimiento & desarrollo , Brucella abortus/genética , Elementos Transponibles de ADN , Genes Bacterianos , Genes Esenciales , Macrófagos/microbiología , Mutagénesis Insercional , Animales , Mapeo Cromosómico , Medios de Cultivo/química , Redes y Vías Metabólicas/genética , Ratones , Células RAW 264.7 , Análisis de Secuencia de ADN , Factores de Virulencia/genéticaRESUMEN
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.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Proteínas Bacterianas/genética , Brucella abortus/genética , Ciclo Celular/genética , División Celular/genética , Regulación Bacteriana de la Expresión Génica , Factores de Transcripción/genética , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Sitios de Unión , Brucella abortus/patogenicidad , Bovinos , Replicación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Retículo Endoplásmico/microbiología , Mutación , Fosforilación , Filogenia , Regiones Promotoras Genéticas , Regulón , Factores de Transcripción/metabolismoRESUMEN
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.
