Rationally designed pooled CRISPRi-seq uncovers an inhibitor of bacterial peptidyl-tRNA hydrolase.

Bacterial mutant libraries with downregulated antibiotic targets are useful tools for elucidating the mechanisms of action of antibacterial compounds, a pivotal step in antibiotic discovery. However, achieving genomic coverage of antibacterial targets poses a challenge due to the uneven proliferation of knockdown mutants during pooled growth, leading to the unintended loss of important targets. To overcome this issue, we constructed an arrayed essential gene mutant library (EGML) in the antibiotic-resistant bacterium Burkholderia cenocepacia using CRISPR interference (CRISPRi).

Rationally Designed Pooled CRISPRi-Seq Uncovers an Inhibitor of Bacterial Peptidyl-tRNA Hydrolase.

Unlabelled: Pooled knockdown libraries of essential genes are useful tools for elucidating the mechanisms of action of antibacterial compounds, a pivotal step in antibiotic discovery. However, achieving genomic coverage of antibacterial targets poses a challenge due to the uneven proliferation of knockdown mutants during pooled growth, leading to the unintended loss of important targets. To overcome this issue, we describe the construction of CIMPLE ( C RISPR i - m ediated p ooled library of e ssential genes), a rationally designed pooled knockdown library built in a model antibiotic-resistant bacteria, By analyzing growth parameters of clonal knockdown populations of an arrayed CRISPRi library, we predicted strain depletion levels during pooled growth and adjusted mutant relative abundance, approaching genomic coverage of antibacterial targets during antibiotic exposure.

A CRISPR-Cas-associated transposon system for genome editing in complex species.

Unlabelled: Genome editing in non-model bacteria is important to understand gene-to-function links that may differ from those of model microorganisms. Although species of the complex (Bcc) have great biotechnological capacities, the limited genetic tools available to understand and mitigate their pathogenic potential hamper their utilization in industrial applications. To broaden the genetic tools available for Bcc species, we developed RhaCAST, a targeted DNA insertion platform based on a CRISPR-associated transposase driven by a rhamnose-inducible promoter.

Cell envelope structural and functional contributions to antibiotic resistance in .

Antibiotic activity is limited by the physical construction of the Gram-negative cell envelope. Species of the complex (Bcc) are known as intrinsically multidrug-resistant opportunistic pathogens with low permeability cell envelopes. Here, we re-examined a previously performed chemical-genetic screen of barcoded transposon mutants in K56-2, focusing on cell envelope structural and functional processes.

The mechanism of action of auranofin analogs in revealed by chemogenomic profiling.

Drug repurposing efforts led to the discovery of bactericidal activity in auranofin, a gold-containing drug used to treat rheumatoid arthritis. Auranofin kills Gram-positive bacteria by inhibiting thioredoxin reductase, an enzyme that scavenges reactive oxygen species (ROS). Despite the presence of thioredoxin reductase in Gram-negative bacteria, auranofin is not always active against them.

Profiling cell envelope-antibiotic interactions reveals vulnerabilities to β-lactams in a multidrug-resistant bacterium.

The cell envelope of Gram-negative bacteria belonging to the Burkholderia cepacia complex (Bcc) presents unique restrictions to antibiotic penetration. As a consequence, Bcc species are notorious for causing recalcitrant multidrug-resistant infections in immunocompromised individuals. Here, we present the results of a genome-wide screen for cell envelope-associated resistance and susceptibility determinants in a Burkholderia cenocepacia clinical isolate.

Complete Genome Sequence Burkholderia vietnamiensis LMG16232.

We report here the complete annotated closed genome sequence of Burkholderia vietnamiensis LMG16232. There were three contigs, with a combined size of 6,739,172 bp and a GC content of 67%.

The Burkholderia contaminans prevalent phenotypes as possible markers of poor clinical outcomes in chronic lung infection of children with cystic fibrosis.

Burkholderia contaminans, a species of the Burkholderia cepacia complex-prevalent in certain Latin-American and European countries-can cause chronic pulmonary infection in persons with cystic fibrosis. Our aim was to gain insights into long-term lung infections with a focus on correlating how bacterial phenotypic traits in the chronic infection impact on patients' clinical outcome. Genotypic characteristics of 85 B.

ABT-MPNN: an atom-bond transformer-based message-passing neural network for molecular property prediction.

Graph convolutional neural networks (GCNs) have been repeatedly shown to have robust capacities for modeling graph data such as small molecules. Message-passing neural networks (MPNNs), a group of GCN variants that can learn and aggregate local information of molecules through iterative message-passing iterations, have exhibited advancements in molecular modeling and property prediction. Moreover, given the merits of Transformers in multiple artificial intelligence domains, it is desirable to combine the self-attention mechanism with MPNNs for better molecular representation.

A machine learning model trained on a high-throughput antibacterial screen increases the hit rate of drug discovery.

Screening for novel antibacterial compounds in small molecule libraries has a low success rate. We applied machine learning (ML)-based virtual screening for antibacterial activity and evaluated its predictive power by experimental validation. We first binarized 29,537 compounds according to their growth inhibitory activity (hit rate 0.

Deep clustering of small molecules at large-scale via variational autoencoder embedding and K-means.

Background: Converting molecules into computer-interpretable features with rich molecular information is a core problem of data-driven machine learning applications in chemical and drug-related tasks. Generally speaking, there are global and local features to represent a given molecule. As most algorithms have been developed based on one type of feature, a remaining bottleneck is to combine both feature sets for advanced molecule-based machine learning analysis.

Deep learning-driven prediction of drug mechanism of action from large-scale chemical-genetic interaction profiles.

Motivation: Chemical-genetic interaction profiling is a genetic approach that quantifies the susceptibility of a set of mutants depleted in specific gene product(s) to a set of chemical compounds. With the recent advances in artificial intelligence, chemical-genetic interaction profiles (CGIPs) can be leveraged to predict mechanism of action of compounds. This can be achieved by using machine learning, where the data from a CGIP is fed into the machine learning platform along with the chemical descriptors to develop a chemogenetically trained model.

Gradients in gene essentiality reshape antibacterial research.

Essential genes encode the processes that are necessary for life. Until recently, commonly applied binary classifications left no space between essential and non-essential genes. In this review, we frame bacterial gene essentiality in the context of genetic networks.

Identification of putative essential protein domains from high-density transposon insertion sequencing.

A first clue to gene function can be obtained by examining whether a gene is required for life in certain standard conditions, that is, whether a gene is essential. In bacteria, essential genes are usually identified by high-density transposon mutagenesis followed by sequencing of insertion sites (Tn-seq). These studies assign the term "essential" to whole genes rather than the protein domain sequences that encode the essential functions.

New Auranofin Analogs with Antibacterial Properties against Clinical Isolates.

Bacteria of the genus include pathogenic , and the complex (Bcc). These Gram-negative pathogens have intrinsic drug resistance, which makes treatment of infections difficult. Bcc affects individuals with cystic fibrosis (CF) and the species is associated with one of the worst clinical outcomes.

Improved Dynamic Range of a Rhamnose-Inducible Promoter for Gene Expression in spp.

A diverse genetic toolkit is critical for understanding bacterial physiology and genotype-phenotype relationships. Inducible promoter systems are an integral part of this toolkit. In and related species, the l-rhamnose-inducible promoter is among the first choices due to its tight control and the lack of viable alternatives.

Comparative genomics of ST5 and ST30 methicillin-resistant sequential isolates recovered from paediatric patients with cystic fibrosis.

chronic airway infection in patients with cystic fibrosis (CF) allows this pathogen to adapt over time in response to different selection pressures. We have previously shown that the main sequence types related to community-acquired methicillin-resistant (MRSA) infections in Argentina - ST5 and ST30 - are also frequently isolated from the sputum of patients with CF, but in these patients they usually display multi-drug antimicrobial resistance. In this study, we sequenced the genomes of MRSA from four paediatric CF patients with the goal of identifying mutations among sequential isolates, especially those possibly related to antimicrobial resistance and virulence, which might contribute to the adaptation of the pathogen in the airways of patients with CF.

Phenylacetyl Coenzyme A, Not Phenylacetic Acid, Attenuates CepIR-Regulated Virulence in Burkholderia cenocepacia.

During phenylalanine catabolism, phenylacetic acid (PAA) is converted to phenylacetyl coenzyme A (PAA-CoA) by a ligase, PaaK, and then PAA-CoA is epoxidized by a multicomponent monooxygenase, PaaABCDE, before further degradation through the tricarboxylic acid (TCA) cycle. In the opportunistic pathogen , loss of attenuates virulence factor expression, which is under the control of the LuxIR-like quorum sensing (QS) system, CepIR. To further investigate the link between CepIR-regulated virulence and PAA catabolism, we created knockout mutants of the first step of the pathway (PAA-CoA synthesis by PaaK) and characterized them in comparison to a mutant using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and virulence assays.

The Effect of 2-Thiocyanatopyridine Derivative 11026103 on : Resistance Mechanisms and Systemic Impact.

Bacteria of the complex (Bcc) are associated with significant decline of lung functions in cystic fibrosis patients. Bcc infections are virtually impossible to eradicate due to their irresponsiveness to antibiotics. The 2-thiocyanatopyridine derivative 11026103 is a novel, synthetic compound active against .

A Broad-Host-Range CRISPRi Toolkit for Silencing Gene Expression in .

Genetic tools are critical to dissecting the mechanisms governing cellular processes, from fundamental physiology to pathogenesis. Members of the genus have potential for biotechnological applications but can also cause disease in humans with a debilitated immune system. The lack of suitable genetic tools to edit GC-rich genomes has hampered the exploration of useful capacities and the understanding of pathogenic features.

Synthesis and antibiotic activity of novel acylated phloroglucinol compounds against methicillin-resistant Staphylococcus aureus.

The rise in antibiotic resistance among pathogenic microorganisms has created an imbalance in the drugs available for treatment, in part due to the slow development of new antibiotics. Cystic fibrosis (CF) patients are highly susceptible to antibiotic-resistant pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). Phloroglucinols and related polyketide natural products have demonstrated antimicrobial activity against a number of Gram-positive bacteria including S.

A c-di-GMP-Modulating Protein Regulates Swimming Motility of in Response to Arginine and Glutamate.

is an opportunistic bacterium that can thrive in different environments, including the amino acid-rich mucus of the cystic fibrosis (CF) lung. responds to the nutritional conditions that mimic the CF sputum by increasing flagellin expression and swimming motility. Individual amino acids also induce swimming but not flagellin expression.