Search

SummaryOrdered collections of precise gene deletions inE. coliand other model microbes have proven to be invaluable resources to study the dispensability of the non-essential genome in different environmental and genetic contexts. The dispensability of canonically essential genes, however, has been largely unstudied in such contexts. Advances in gene editing, in particular CRISPR interference (CRISPRi), have enabled depletion of essential cellular machinery to study the downstream effects on bacterial physiology. Here, we describe the construction of an orderedE. coliCRISPRi collection, designed to knock down the expression of 356 essential genes with the induction of a catalytically inactive Cas9, harbored on the conjugative plasmid pFD152, that also encodes for expression of specific guide RNA scaffolds. This mobile CRISPRi library can be conjugated into other ordered genetic libraries, to assess combined effects of essential gene knockdowns with non-essential gene deletions. As proof of concept, we probed cell envelope synthesis with two crosses. First, we crossed a deletion in the genelppwith the entire CRISPRi library. Similarly, we crossed the CRISPRi knockdown oflolAwith ∼4,000 deletion strains of the Keio collection. These experiments revealed a number of notable genetic interactions for the essential phenotype probed and, in particular, showed supressing interactions for the loci in question.

Bloodstream infections caused by invasive, non-typhoidal salmonellae (iNTS) are a major global health concern. These infections are especially problematic in sub-Saharan Africa, where the sequence type (ST) 313 of invasive non-typhoidal Salmonella Typhimurium (iNTS) is dominant. Unlike S. Typhimurium strains that cause mild gastroenteritis, iNTS strains are resistant to multiple first-line antibiotics and have higher extraintestinal invasiveness, limiting current treatment options. Here, we performed multiple small molecule screens under infection-relevant conditions to reveal chemical sensitivities in ST313 as entry points to drug discovery to combat the clinical burden of iNTS. By screening the invasive ST313 sequence type under host-mimicking conditions, we identified the antimicrobial activity of the nucleoside analog 3’-azido-3’-deoxythymidine, which required bacterial thymidine kinase activity for its antimicrobial activity. In a parallel macrophage-based screening platform, we also identified three host-directed compounds (amodiaquine, berbamine, and indatraline) that significantly restricted intracellular replication of ST313 in macrophages without directly impacting bacterial viability. This work provides evidence that despite elevated invasiveness and multidrug resistance, iNTS S. Typhimurium remains susceptible to unconventional drug discovery approaches.

There is an urgent need to develop new antibiotics for the treatment of infections caused by drug-resistant Gram-negative bacteria. In particular, new and diverse chemical classes of antibiotics are needed, as most antibiotics in clinical development are derivatives of existing drugs. Despite a history of use as antimicrobials, metals and metal-based compounds have largely been overlooked as a source of new chemical matter for antibacterial drug discovery. In this work, we identify several ruthenium complexes, ruthenium red, Ru265, and Ru360’, that possess potent antibacterial activity against both laboratory and clinical isolates of Pseudomonas aeruginosa. Suppressors with increased resistance were sequenced and found to contain mutations in the mechanosensitive ion channel mscS-1 or the colRS two component system. The antibacterial activity of these compounds translated in vivo to Galleria mellonella larvae and mouse infection models. Finally, we identify strong synergy between these compounds and the antibiotic rifampicin, with a dose-sparing combination therapy showing efficacy in both infection models. Our findings provide clear evidence that these ruthenium complexes are effective antibacterial compounds against a critical priority pathogen and show promise for the development of future therapeutics.

Multi-drug resistant bacteria present an urgent threat to modern medicine, creating a desperate need for the discovery of antibiotics with new modes of action. Natural products whose unique highly diverse structures have been shaped by evolution to possess biologically relevant activity are an ideal discovery ground for new antibiotics with new mechanisms of action. In this study we elucidate the mechanism of action of the Gram-positive antibiotic armeniaspirol, a compound for which resistant bacteria could not be selected for. We show that armeniaspirol inhibits the ATP-dependent proteases ClpXP and ClpYQ in biochemical assays and in the Gram-positive bacteria Bacillus subtilis. We then show that this activity dysregulates key proteins involved in the divisome and elongasome including FtsZ, DivIVA, and MreB all of which are known to inhibit cell division when upregulated. Inhibition of ClpXP and ClpYQ leading to dysregulation of the divisome and elongasome represents a new mechanism of action and armeniaspirol is the first known natural product inhibitor of the coveted anti-virulence target ClpP. Thus armeniaspirol is the lead compound for a promising new class of antibiotics with a unique pharmacology and a novel mechanism for combating antimicrobial resistance, making it a highly promising candidate for further development.

Four decades after early in vitro assembly studies demonstrated that ribosome assembly is a controlled process, our understanding of ribosome assembly is still incomplete. Just as structure determination has been so important to understanding ribosome function, so too will it be critical to sorting out the assembly process. Here, we used a viable deletion in the yjeQ gene, a recognized ribosome assembly factor, to isolate and structurally characterize immature 30S subunits assembled in vivo. These small ribosome subunits contained unprocessed 17S rRNA and lacked some late ribosomal proteins. Cryo-electron microscopy reconstructions revealed that the presence of precursor sequences in the rRNA induces a severe distortion in the 3′ minor domain of the subunit involved in the decoding of mRNA and interaction with the large ribosome subunit. These findings suggest that rRNA processing events induce key local conformational changes directing the structure toward the mature assembly. We concluded that rRNA processing, folding, and the entry of tertiary r-proteins are interdependent events in the late stages of 30S subunit assembly. In addition, we demonstrate how studies of emerging assembly factors in ribosome biogenesis can help to elucidate the path of subunit assembly in vivo.