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.

Acinetobacter baumannii is a poorly understood bacterium capable of life-threatening infections in hospitals. Few antibiotics remain effective against this highly resistant pathogen. Developing 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.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-20098-z

19Acinetobacter baumannii is an opportunistic pathogen that is a critical, high-priority target for 20 new antibiotic development. Clearing of A. baumannii requires relatively high doses of 21 antibiotics across the spectrum, primarily due to its protective cell envelope. Many of the 22 synthesis. Moreover, we provide a genetic strategy that uses hypersensitivity signatures to 33 predict drug synergies, allowing the identification of b-lactams that work cooperatively based on 34 the cell wall assembly machineries that they preferentially target. These data reveal multiple 35 pathways critical for envelope growth in A. baumannii that can be targeted in combination 36 strategies for attacking the pathogen. 37 38 39 Despite the utility of these approaches in measuring gene-antibiotic interactions, understanding 62 4 the mechanisms behind the uncovered resistance determinants is limited by difficulties 63 associated with providing accurate gene annotations. A large fraction of genes in any organism 64 lack characterization and have no known or predicted function (referred to as "orphan" or 65 "hypothetical" genes) 13,21,22 . Lack of functional information complicates downstream analyses, 66 and single gene-antibiotic phenotypes can be insufficient to generate hypotheses on function. 67 Moreover, in species divergent from model organisms, functional annotations predicted by 68 sequence homologies are often inaccurate, as the function of sequence orthologs may not be 69 conserved 22,23 . Hypothetical genes lacking annotation and genes with inaccurate annotation due 70 to noncanonical functions are predicted to be particularly problematic with Acinetobacter, which 71 has diverged from other g-proteobacteria and lacks many canonical proteins that function in 72 envelope biogenesis 10,24 . 73 In this paper, we have comprehensively characterized mechanisms of intrinsic defense in 74A. baumannii against multiple antibiotics via transposon sequencing (Tn-seq) and leveraged the 75 diversity of phenotypes generated to address the problem of uncharacterized gene function in this 76 pathogen. By analyzing the patterns of antibiotic hypersusceptibility caused by gene-inactivating 77 mutations across the genome, we uncovered new functions for conserved hypothetical proteins 78 and expanded the roles of annotated enzymes in envelope synthesis. The identified determinants 79 of susceptibility represent novel targets for potentiating current antibiotics against A. baumannii. 80Moreover, the Tn-seq analysis informed a strategy to combine different classes of β-lactam 81 antibiotics for enhanced antimicrobial activity. 82 83 84 5 Results 85Defining intrinsic drug susceptibility determinants in Acinetobacter baumannii. 86

Acinetobacter baumannii is a common hospital opportunistic pathogen with multidrug resistance, including resistance to last‐resort antibiotics. Many antibiotics today target the cell wall system which is critical for viability. Thus, we sought to better understand cell wall biogenesis in A. baumannii with the goal of finding novel therapeutic targets. L,D‐transpeptidases have been shown to participate in cell wall synthesis by forming unusual peptidoglycan crosslinks. A. baumannii has an L,D‐transpeptidase (Ldt1) that is essential for maintaining the characteristic rod‐shape of the bacterium. However, this transpeptidase and other aspects of cell wall synthesis remain poorly defined. We hypothesized that Ldt1 is important for cell elongation, which is mediated by a multiprotein complex known as the Rod system. To test this hypothesis and define genetic and direct‐protein interactions of Ldt1, we used transposon sequencing (Tn‐seq) and bacterial two‐hybrid techniques. Additionally, we used fluorescent D‐amino acid probes and fluorescence microscopy to visualize differences in active cell wall synthesis sites in WT A. baumannii versus ldt1 deletion mutant. The strongest negative Tn‐seq interactions were with cell division genes, consistent with the hypothesis that Ldt1 is important for the complementary Rod system. The results of the two‐hybrid assay did not show strong direct protein‐protein interaction of Ldt1 with canonical rod‐system components; however, using this method, we mapped novel interactions within the Rod system of A. baumannii. For the fluorescence microscopy, staining conditions of the D‐amino acid probe have been optimized. This study demonstrates methods of elucidating interaction networks in A. baumannii. Through increased understanding of Ldt1 and the Rod system interaction networks, novel synergistic therapeutics can be developed to combat resistant A. baumannii infections.

Acinetobacter baumannii is a poorly understood bacterium capable of life-threatening infections in hospitals. Few antibiotics remain effective against this highly resistant pathogen. Developing 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.ImportanceNew 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.