Antibiotic hypersensitivity signatures identify targets for attack in theAcinetobacter baumanniicell envelope

Abstract

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

DNA damage response coregulator ddrR affects many cellular pathways and processes in Acinetobacter baumannii 17978

“…In the ddrR mutant, the tol-pal operon had a higher basal expression when compared to WT. Also upregulated was the newly characterized gene advA involved in chromosome segregation and cell division in A. baumannii (Geisinger et al, 2020) and t he lytic transglycosylase rlpA which facilitates daughter cell separation in Pseudomonas aeruginosa (Jorgenson et al, 2014) and Vibrio cholerae (Weaver et al, 2019). This upregulation of rlpA was confirmed with RT-qPCR analyses (Figure 8).…”

Transposon Insertion Sequencing, a Global Measure of Gene Function

“…However, the list of important genes in the presence of cell wall synthesis inhibitors stretches far beyond peptidoglycan synthesis genes and includes many metabolic genes, regulators, transporters, membrane integrity genes, and those involved in energy generation. These results highlight how no single gene or pathway acts just by itself but instead is tightly integrated into a genomic network, interacting with all kinds of other pathways and systems (15,25,27,28,41,42,62,68,95). The effects of most stressors, including antibiotics, thus may start at the stressor's direct target, but because of network interdependencies, the effect of the stress reverberates throughout the organism, and with increasing stress the response becomes more complex, involving more and more genes.…”

Micro‐/Nanorobots in Antimicrobial Applications: Recent Progress, Challenges, and Opportunities

“…Conventional antibiotic therapies are virtually ineffective in treating biofilm‐associated infections, derived not only from the multidrug resistance mechanism of mature biofilms but also from insufficient enrichment of antibiotics in the vicinity of the bacteria in the EPS barrier. [ 135 ] The establishment of channels or pathways in biofilms is a convenient way to resist biofilms because it not only facilitates the penetration of antimicrobial agents but improves the efficacy of antimicrobial agents and reduces cell–cell communication between bacteria as well. In 2019, Quan et al.…”