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Essential Gene Analysis in Acinetobacter baumannii by High-Density Transposon Mutagenesis and CRISPR Interference.

ABSTRACT: 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.

SUBMITTER: Bai J

PROVIDER: S-EPMC8316057 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The emergence of multidrug-resistant Gram-negative bacteria underscores the need to define genetic vulnerabilities that can be therapeutically exploited. The Gram-negative pathogen, Acinetobacter baumannii, is considered an urgent threat due to its propensity to evade antibiotic treatments. Essential cellular processes are the target of existing antibiotics and a likely source of new vulnerabilities. Although A. baumannii essential genes have been identified by transposon sequencing, they have not been prioritized by sensitivity to knockdown or antibiotics. Here, we take a systems biology approach to comprehensively characterize A. baumannii essential genes using CRISPR interference (CRISPRi). We show that certain essential genes and pathways are acutely sensitive to knockdown, providing a set of vulnerable targets for future therapeutic investigation. Screening our CRISPRi library against last-resort antibiotics uncovered genes and pathways that modulate beta-lactam sensitivity, an unexpected link between NADH dehydrogenase activity and growth inhibition by polymyxins, and anticorrelated phenotypes that may explain synergy between polymyxins and rifamycins. Our study demonstrates the power of systematic genetic approaches to identify vulnerabilities in Gram-negative pathogens and uncovers antibiotic-essential gene interactions that better inform combination therapies.IMPORTANCEAcinetobacter baumannii is a hospital-acquired pathogen that is resistant to many common antibiotic treatments. To combat resistant A. baumannii infections, we need to identify promising therapeutic targets and effective antibiotic combinations. In this study, we comprehensively characterize the genes and pathways that are critical for A. baumannii viability. We show that genes involved in aerobic metabolism are central to A. baumannii physiology and may represent appealing drug targets. We also find antibiotic-gene interactions that may impact the efficacy of carbapenems, rifamycins, and polymyxins, providing a new window into how these antibiotics function in mono- and combination therapies. Our studies offer a useful approach for characterizing interactions between drugs and essential genes in pathogens to inform future therapies.

Project description:Copper is widely used as antimicrobial in agriculture and medicine. Copper tolerance mechanisms of pathogenic bacteria have been proven to be required for both copper tolerance and survival during bacterial infections. Here, we determined both copper-tolerant phenotype and genotype in A. baumannii originated from clinical and environmental samples. Using copper susceptibility testing, copper-tolerant A. baumannii could be found in both clinical and environmental isolates. Genotypic study revealed that representative copper-related genes of the cluster A (cueR), B (pcoAB), and D (oprC) were detected in all isolates, while copRS of cluster C was detected in only copper-tolerant A. baumannii isolates. Moreover, we found that copper-tolerant phenotype was associated with amikacin resistance, while the presence of copRS was statistically associated with blaNDM-1. We chose the A. baumannii strain AB003 as a representative of copper-tolerant isolate to characterize the effect of copper treatment on external morphology as well as on genes responsible for copper tolerance. The morphological features and survival of A. baumannii AB003 were affected by its exposure to copper, while whole-genome sequencing and analysis showed that it carried fourteen copper-related genes located on four clusters, and cluster C of AB003 was found to be embedded on genomic island G08. Transcriptional analysis of fourteen copper-related genes identified in AB003 revealed that copper treatment induced the expressions of genes of clusters A, B, and D at the micromolar level, while genes of cluster C were over-expressed at the millimolar levels of copper. This study showed that both clinical and environmental A. baumannii isolates have the ability to tolerate copper and carried numerous copper tolerance determinants including intrinsic copper tolerance (clusters A, B, and D) and acquired copper tolerance (cluster C) that could respond to copper toxicity. Our evidence suggests that we need to reconsider the use of copper in hospitals and other medical environments to prevent the selection and spread of copper-tolerant organisms.

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Essential Gene Analysis in Acinetobacter baumannii by High-Density Transposon Mutagenesis and CRISPR Interference.

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