Scalable approaches for generating, validating and incorporating data from high-throughput functional assays to improve clinical variant classification.

As the adoption and scope of genetic testing continue to expand, interpreting the clinical significance of DNA sequence variants at scale remains a formidable challenge, with a high proportion classified as variants of uncertain significance (VUSs). Genetic testing laboratories have historically relied, in part, on functional data from academic literature to support variant classification. High-throughput functional assays or multiplex assays of variant effect (MAVEs), designed to assess the effects of DNA variants on protein stability and function, represent an important and increasingly available source of evidence for variant classification, but their potential is just beginning to be realized in clinical lab settings. Here, we describe a framework for generating, validating and incorporating data from MAVEs into a semi-quantitative variant classification method applied to clinical genetic testing. Using single-cell gene expression measurements, cellular evidence models were built to assess the effects of DNA variation in 44 genes of clinical interest. This framework was also applied to models for an additional 22 genes with previously published MAVE datasets. In total, modeling data was incorporated from 24 genes into our variant classification method. These data contributed evidence for classifying 4043 observed variants in over 57,000 individuals. Genetic testing laboratories are uniquely positioned to generate, analyze, validate, and incorporate evidence from high-throughput functional data and ultimately enable the use of these data to provide definitive clinical variant classifications for more patients.

Lipopolysaccharide transport regulates bacterial sensitivity to a cell wall-degrading intermicrobial toxin.

Gram-negative bacteria can antagonize neighboring microbes using a type VI secretion system (T6SS) to deliver toxins that target different essential cellular features. Despite the conserved nature of these targets, T6SS potency can vary across recipient species. To understand the functional basis of intrinsic T6SS susceptibility, we screened for essential Escherichia coli (Eco) genes that affect its survival when antagonized by a cell wall-degrading T6SS toxin from Pseudomonas aeruginosa, Tae1. We revealed genes associated with both the cell wall and a separate layer of the cell envelope, lipopolysaccharide, that modulate Tae1 toxicity in vivo. Disruption of genes in early lipopolysaccharide biosynthesis provided Eco with novel resistance to Tae1, despite significant cell wall degradation. These data suggest that Tae1 toxicity is determined not only by direct substrate damage, but also by indirect cell envelope homeostasis activities. We also found that Tae1-resistant Eco exhibited reduced cell wall synthesis and overall slowed growth, suggesting that reactive cell envelope maintenance pathways could promote, not prevent, self-lysis. Together, our study reveals the complex functional underpinnings of susceptibility to Tae1 and T6SS which regulate the impact of toxin-substrate interactions in vivo.

Lipopolysaccharide integrity primes bacterial sensitivity to a cell wall-degrading intermicrobial toxin.

Gram-negative bacteria can antagonize neighboring microbes using a type VI secretion system (T6SS) to deliver toxins that target different essential cellular features. Despite the conserved nature of these targets, T6SS potency can vary across recipient species. To understand the molecular basis of intrinsic T6SS susceptibility, we screened for essential Escherichia coli genes that affect its survival when antagonized by a cell wall-degrading T6SS toxin from Pseudomonas aeruginosa , Tae1. We revealed genes associated with both the cell wall and a separate layer of the cell envelope, surface lipopolysaccharide, that modulate Tae1 toxicity in vivo . Disruption of lipopolysaccharide synthesis provided Escherichia coli (Eco) with novel resistance to Tae1, despite significant cell wall degradation. These data suggest that Tae1 toxicity is determined not only by direct substrate damage, but also by indirect cell envelope homeostasis activities. We also found that Tae1-resistant Eco exhibited reduced cell wall synthesis and overall slowed growth, suggesting that reactive cell envelope maintenance pathways could promote, not prevent, self-lysis. Together, our study highlights the consequences of co-regulating essential pathways on recipient fitness during interbacterial competition, and how antibacterial toxins leverage cellular vulnerabilities that are both direct and indirect to their specific targets in vivo .

Ceragenins and Antimicrobial Peptides Kill Bacteria through Distinct Mechanisms.

Ceragenins are a family of synthetic amphipathic molecules designed to mimic the properties of naturally occurring cationic antimicrobial peptides (CAMPs). Although ceragenins have potent antimicrobial activity, whether their mode of action is similar to that of CAMPs has remained elusive. Here, we reported the results of a comparative study of the bacterial responses to two well-studied CAMPs, LL37 and colistin, and two ceragenins with related structures, CSA13 and CSA131. Using transcriptomic and proteomic analyses, we found that Escherichia coli responded similarly to both CAMPs and ceragenins by inducing a Cpx envelope stress response. However, whereas E. coli exposed to CAMPs increased expression of genes involved in colanic acid biosynthesis, bacteria exposed to ceragenins specifically modulated functions related to phosphate transport, indicating distinct mechanisms of action between these two classes of molecules. Although traditional genetic approaches failed to identify genes that confer high-level resistance to ceragenins, using a Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) approach we identified E. coli essential genes that when knocked down modify sensitivity to these molecules. Comparison of the essential gene-antibiotic interactions for each of the CAMPs and ceragenins identified both overlapping and distinct dependencies for their antimicrobial activities. Overall, this study indicated that, while some bacterial responses to ceragenins overlap those induced by naturally occurring CAMPs, these synthetic molecules target the bacterial envelope using a distinctive mode of action. IMPORTANCE The development of novel antibiotics is essential because the current arsenal of antimicrobials will soon be ineffective due to the widespread occurrence of antibiotic resistance. The development of naturally occurring cationic antimicrobial peptides (CAMPs) for therapeutics to combat antibiotic resistance has been hampered by high production costs and protease sensitivity, among other factors. The ceragenins are a family of synthetic CAMP mimics that kill a broad spectrum of bacterial species but are less expensive to produce, resistant to proteolytic degradation, and seemingly resistant to the development of high-level resistance. Determining how ceragenins function may identify new essential biological pathways of bacteria that are less prone to the development of resistance and will further our understanding of the design principles for maximizing the effects of synthetic CAMPs.

Morphological and Transcriptional Responses to CRISPRi Knockdown of Essential Genes in Escherichia coli.

CRISPR interference (CRISPRi) has facilitated the study of essential genes in diverse organisms using both high-throughput and targeted approaches. Despite the promise of this technique, no comprehensive arrayed CRISPRi library targeting essential genes exists for the model bacterium Escherichia coli, or for any Gram-negative species. Here, we built and characterized such a library. Each of the ∼500 strains in our E. coli library contains an inducible, chromosomally integrated single guide RNA (sgRNA) targeting an essential (or selected nonessential) gene and can be mated with a pseudo-Hfr donor strain carrying a dcas9 cassette to create a CRISPRi knockdown strain. Using this system, we built an arrayed library of CRISPRi strains and performed population and single-cell growth and morphology measurements as well as targeted follow-up experiments. These studies found that inhibiting translation causes an extended lag phase, identified new modulators of cell morphology, and revealed that the morphogene mreB is subject to transcriptional feedback regulation, which is critical for the maintenance of morphology. Our findings highlight canonical and noncanonical roles for essential genes in numerous aspects of cellular homeostasis. IMPORTANCE Essential genes make up only ∼5 to 10% of the genetic complement in most organisms but occupy much of their protein synthesis and account for almost all antibiotic targets. Despite the importance of essential genes, their intractability has, until recently, hampered efforts to study them. CRISPRi has facilitated the study of essential genes by allowing inducible and titratable depletion. However, all large-scale CRISPRi studies in Gram-negative bacteria thus far have used plasmids to express CRISPRi components and have been constructed in pools, limiting their utility for targeted assays and complicating the determination of antibiotic effects. Here, we use a modular method to construct an arrayed library of chromosomally integrated CRISPRi strains targeting the essential genes of the model bacterium Escherichia coli. This library enables targeted studies of essential gene depletions and high-throughput determination of antibiotic targets and facilitates studies targeting the outer membrane, an essential component that serves as the major barrier to antibiotics.

Bacterial CRISPR screens for gene function.

In this review we describe the application of CRISPR tools for functional genomics screens in bacteria, with a focus on the use of interference (CRISPRi) approaches. We review recent developments in CRISPRi titration, which has enabled essential gene functional screens, and genome-scale pooled CRISPRi screens. We summarize progress toward enabling CRISPRi screens in non-model and pathogenic bacteria, including the development of new dCas9 variants. Taking into account the current state of the field, we provide a forward-looking analysis of CRISPRi strategies for determining gene function in bacteria.

Mismatch-CRISPRi Reveals the Co-varying Expression-Fitness Relationships of Essential Genes in Escherichia coli and Bacillus subtilis.

Essential genes are the hubs of cellular networks, but lack of high-throughput methods for titrating gene expression has limited our understanding of the fitness landscapes against which their expression levels are optimized. We developed a modified CRISPRi system leveraging the predictable reduction in efficacy of imperfectly matched sgRNAs to generate defined levels of CRISPRi activity and demonstrated its broad applicability. Using libraries of mismatched sgRNAs predicted to span the full range of knockdown levels, we characterized the expression-fitness relationships of most essential genes in Escherichia coli and Bacillus subtilis. We find that these relationships vary widely from linear to bimodal but are similar within pathways. Notably, despite ∼2 billion years of evolutionary separation between E. coli and B. subtilis, most essential homologs have similar expression-fitness relationships with rare but informative differences. Thus, the expression levels of essential genes may reflect homeostatic or evolutionary constraints shared between the two organisms.

Modulating Pathogenesis with Mobile-CRISPRi.

Conditionally essential (CE) genes are required by pathogenic bacteria to establish and maintain infections. CE genes encode virulence factors, such as secretion systems and effector proteins, as well as biosynthetic enzymes that produce metabolites not found in the host environment. Due to their outsized importance in pathogenesis, CE gene products are attractive targets for the next generation of antimicrobials. However, the precise manipulation of CE gene expression in the context of infection is technically challenging, limiting our ability to understand the roles of CE genes in pathogenesis and accordingly design effective inhibitors. We previously developed a suite of CRISPR interference-based gene knockdown tools that are transferred by conjugation and stably integrate into bacterial genomes that we call Mobile-CRISPRi. Here, we show the efficacy of Mobile-CRISPRi in controlling CE gene expression in an animal infection model. We optimize Mobile-CRISPRi in Pseudomonas aeruginosa for use in a murine model of pneumonia by tuning the expression of CRISPRi components to avoid nonspecific toxicity. As a proof of principle, we demonstrate that knock down of a CE gene encoding the type III secretion system (T3SS) activator ExsA blocks effector protein secretion in culture and attenuates virulence in mice. We anticipate that Mobile-CRISPRi will be a valuable tool to probe the function of CE genes across many bacterial species and pathogenesis models.IMPORTANCE Antibiotic resistance is a growing threat to global health. To optimize the use of our existing antibiotics and identify new targets for future inhibitors, understanding the fundamental drivers of bacterial growth in the context of the host immune response is paramount. Historically, these genetic drivers have been difficult to manipulate precisely, as they are requisite for pathogen survival. Here, we provide the first application of Mobile-CRISPRi to study conditionally essential virulence genes in mouse models of lung infection through partial gene perturbation. We envision the use of Mobile-CRISPRi in future pathogenesis models and antibiotic target discovery efforts.

Enabling genetic analysis of diverse bacteria with Mobile-CRISPRi.

The vast majority of bacteria, including human pathogens and microbiome species, lack genetic tools needed to systematically associate genes with phenotypes. This is the major impediment to understanding the fundamental contributions of genes and gene networks to bacterial physiology and human health. Clustered regularly interspaced short palindromic repeats interference (CRISPRi), a versatile method of blocking gene expression using a catalytically inactive Cas9 protein (dCas9) and programmable single guide RNAs, has emerged as a powerful genetic tool to dissect the functions of essential and non-essential genes in species ranging from bacteria to humans1-6. However, the difficulty of establishing effective CRISPRi systems across bacteria is a major barrier to its widespread use to dissect bacterial gene function. Here, we establish 'Mobile-CRISPRi', a suite of CRISPRi systems that combines modularity, stable genomic integration and ease of transfer to diverse bacteria by conjugation. Focusing predominantly on human pathogens associated with antibiotic resistance, we demonstrate the efficacy of Mobile-CRISPRi in gammaproteobacteria and Bacillales Firmicutes at the individual gene scale, by examining drug-gene synergies, and at the library scale, by systematically phenotyping conditionally essential genes involved in amino acid biosynthesis. Mobile-CRISPRi enables genetic dissection of non-model bacteria, facilitating analyses of microbiome function, antibiotic resistances and sensitivities, and comprehensive screens for host-microorganism interactions.

Ribosomal mutations promote the evolution of antibiotic resistance in a multidrug environment.

Antibiotic resistance arising via chromosomal mutations is typically specific to a particular antibiotic or class of antibiotics. We have identified mutations in genes encoding ribosomal components in Mycobacterium smegmatis that confer resistance to several structurally and mechanistically unrelated classes of antibiotics and enhance survival following heat shock and membrane stress. These mutations affect ribosome assembly and cause large-scale transcriptomic and proteomic changes, including the downregulation of the catalase KatG, an activating enzyme required for isoniazid sensitivity, and upregulation of WhiB7, a transcription factor involved in innate antibiotic resistance. Importantly, while these ribosomal mutations have a fitness cost in antibiotic-free medium, in a multidrug environment they promote the evolution of high-level, target-based resistance. Further, suppressor mutations can then be easily acquired to restore wild-type growth. Thus, ribosomal mutations can serve as stepping-stones in an evolutionary path leading to the emergence of high-level, multidrug resistance.

Inhibition of CRISPR-Cas9 with Bacteriophage Proteins.

Bacterial CRISPR-Cas systems utilize sequence-specific RNA-guided nucleases to defend against bacteriophage infection. As a countermeasure, numerous phages are known that produce proteins to block the function of class 1 CRISPR-Cas systems. However, currently no proteins are known to inhibit the widely used class 2 CRISPR-Cas9 system. To find these inhibitors, we searched cas9-containing bacterial genomes for the co-existence of a CRISPR spacer and its target, a potential indicator for CRISPR inhibition. This analysis led to the discovery of four unique type II-A CRISPR-Cas9 inhibitor proteins encoded by Listeria monocytogenes prophages. More than half of L. monocytogenes strains with cas9 contain at least one prophage-encoded inhibitor, suggesting widespread CRISPR-Cas9 inactivation. Two of these inhibitors also blocked the widely used Streptococcus pyogenes Cas9 when assayed in Escherichia coli and human cells. These natural Cas9-specific "anti-CRISPRs" present tools that can be used to regulate the genome engineering activities of CRISPR-Cas9.

Baeyer-Villiger Monooxygenases EthA and MymA Are Required for Activation of Replicating and Non-replicating Mycobacterium tuberculosis Inhibitors.

Successful treatment of Mycobacterium tuberculosis infection typically requires a complex regimen administered over at least 6 months. Interestingly, many of the antibiotics used to treat M. tuberculosis are prodrugs that require intracellular activation. Here, we describe three small molecules, active against both replicating and non-replicating M. tuberculosis, that require activation by Baeyer-Villiger monooxygenases (BVMOs). Two molecules require BVMO EthA (Rv3854c) for activation and the third molecule requires the BVMO MymA (Rv3083). While EthA is known to activate the antitubercular drug ethionamide, this is the first description of MymA as an activating enzyme of a prodrug. Furthermore, we found that MymA also plays a role in activating ethionamide, with loss of MymA function resulting in ethionamide-resistant M. tuberculosis. These findings suggest overlap in function and specificity of the BVMOs in M. tuberculosis.

Bacterial CRISPR: accomplishments and prospects.

In this review we briefly describe the development of CRISPR tools for genome editing and control of transcription in bacteria. We focus on the Type II CRISPR/Cas9 system, provide specific examples for use of the system, and highlight the advantages and disadvantages of CRISPR versus other techniques. We suggest potential strategies for combining CRISPR tools with high-throughput approaches to elucidate gene function in bacteria.

Identification of host-targeted small molecules that restrict intracellular Mycobacterium tuberculosis growth.

Mycobacterium tuberculosis remains a significant threat to global health. Macrophages are the host cell for M. tuberculosis infection, and although bacteria are able to replicate intracellularly under certain conditions, it is also clear that macrophages are capable of killing M. tuberculosis if appropriately activated. The outcome of infection is determined at least in part by the host-pathogen interaction within the macrophage; however, we lack a complete understanding of which host pathways are critical for bacterial survival and replication. To add to our understanding of the molecular processes involved in intracellular infection, we performed a chemical screen using a high-content microscopic assay to identify small molecules that restrict mycobacterial growth in macrophages by targeting host functions and pathways. The identified host-targeted inhibitors restrict bacterial growth exclusively in the context of macrophage infection and predominantly fall into five categories: G-protein coupled receptor modulators, ion channel inhibitors, membrane transport proteins, anti-inflammatories, and kinase modulators. We found that fluoxetine, a selective serotonin reuptake inhibitor, enhances secretion of pro-inflammatory cytokine TNF-α and induces autophagy in infected macrophages, and gefitinib, an inhibitor of the Epidermal Growth Factor Receptor (EGFR), also activates autophagy and restricts growth. We demonstrate that during infection signaling through EGFR activates a p38 MAPK signaling pathway that prevents macrophages from effectively responding to infection. Inhibition of this pathway using gefitinib during in vivo infection reduces growth of M. tuberculosis in the lungs of infected mice. Our results support the concept that screening for inhibitors using intracellular models results in the identification of tool compounds for probing pathways during in vivo infection and may also result in the identification of new anti-tuberculosis agents that work by modulating host pathways. Given the existing experience with some of our identified compounds for other therapeutic indications, further clinically-directed study of these compounds is merited.

Identification of novel inhibitors of nonreplicating Mycobacterium tuberculosis using a carbon starvation model.

During Mycobacterium tuberculosis infection, a population of bacteria is thought to exist in a nonreplicating state, refractory to antibiotics, which may contribute to the need for prolonged antibiotic therapy. The identification of inhibitors of the nonreplicating state provides tools that can be used to probe this hypothesis and the physiology of this state. The development of such inhibitors also has the potential to shorten the duration of antibiotic therapy required. Here we describe the development of a novel nonreplicating assay amenable to high-throughput chemical screening coupled with secondary assays that use carbon starvation as the in vitro model. Together these assays identify compounds with activity against replicating and nonreplicating M. tuberculosis as well as compounds that inhibit the transition from nonreplicating to replicating stages of growth. Using these assays we successfully screened over 300,000 compounds and identified 786 inhibitors of nonreplicating M. tuberculosis In order to understand the relationship among different nonreplicating models, we tested 52 of these molecules in a hypoxia model, and four different chemical scaffolds in a stochastic persister model, and a streptomycin-dependent model. We found that compounds display varying levels of activity in different models for the nonreplicating state, suggesting important differences in bacterial physiology between models. Therefore, chemical tools identified in this assay may be useful for determining the relevance of different nonreplicating in vitro models to in vivo M. tuberculosis infection. Given our current limited understanding, molecules that are active across multiple models may represent more promising candidates for further development.