RESUMEN
Acinetobacter baumannii is a poorly understood bacterium capable of life-threatening infections in hospitals. Few antibiotics remain effective against this highly resistant pathogen. Development of rationally designed antimicrobials that can target A. baumannii requires improved knowledge of the proteins that carry out essential processes allowing growth of the organism. Unfortunately, studying essential genes has been challenging using traditional techniques, which usually require time-consuming recombination-based genetic manipulations. Here, we performed saturating mutagenesis with dual transposon systems to identify essential genes in A. baumannii, and we developed a CRISPR interference (CRISPRi) system for facile analysis of these genes. We show that the CRISPRi system enables efficient transcriptional silencing in A. baumannii. Using these tools, we confirmed the essentiality of the novel cell division protein AdvA and discovered a previously uncharacterized AraC family transcription factor (ACX60_RS03245) that is necessary for growth. In addition, we show that capsule biosynthesis is a conditionally essential process, with mutations in late-acting steps causing toxicity in strain ATCC 17978 that can be bypassed by blocking early-acting steps or activating the BfmRS stress response. These results open new avenues for analysis of essential pathways in A. baumannii. IMPORTANCE New approaches are urgently needed to control A. baumannii, one of the most drug-resistant pathogens known. To facilitate the development of novel targets that allow inhibition of the pathogen, we performed a large-scale identification of genes whose products the bacterium needs for growth. We also developed a CRISPR-based gene knockdown tool that operates efficiently in A. baumannii, allowing rapid analysis of these essential genes. We used these methods to define multiple processes vital to the bacterium, including a previously uncharacterized gene regulatory factor and export of a protective polymeric capsule. These tools will enhance our ability to investigate processes critical for the essential biology of this challenging hospital-acquired pathogen.
Asunto(s)
Acinetobacter baumannii/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Elementos Transponibles de ADN/fisiología , Cápsulas Bacterianas , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Elementos Transponibles de ADN/genética , Regulación Bacteriana de la Expresión Génica , Técnicas de Silenciamiento del Gen , MutagénesisRESUMEN
A unique, protective cell envelope contributes to the broad drug resistance of the nosocomial pathogen Acinetobacter baumannii. Here we use transposon insertion sequencing to identify A. baumannii mutants displaying altered susceptibility to a panel of diverse antibiotics. By examining mutants with antibiotic susceptibility profiles that parallel mutations in characterized genes, we infer the function of multiple uncharacterized envelope proteins, some of which have roles in cell division or cell elongation. Remarkably, mutations affecting a predicted cell wall hydrolase lead to alterations in lipooligosaccharide synthesis. In addition, the analysis of altered susceptibility signatures and antibiotic-induced morphology patterns allows us to predict drug synergies; for example, certain beta-lactams appear to work cooperatively due to their preferential targeting of specific cell wall assembly machineries. Our results indicate that the pathogen may be effectively inhibited by the combined targeting of multiple pathways critical for envelope growth.
Asunto(s)
Infecciones por Acinetobacter/tratamiento farmacológico , Acinetobacter baumannii/genética , Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Infección Hospitalaria/tratamiento farmacológico , Farmacorresistencia Bacteriana Múltiple/genética , Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/efectos de los fármacos , Antibacterianos/uso terapéutico , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Pared Celular/efectos de los fármacos , Pared Celular/genética , Pared Celular/metabolismo , Infección Hospitalaria/microbiología , Análisis Mutacional de ADN , Elementos Transponibles de ADN/genética , ADN Bacteriano/genética , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Pruebas de Sensibilidad Microbiana , MutaciónRESUMEN
A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-20098-z.