Biology subjects

Romano, K. P.

Publications and source records attributed to Romano, K. P..

5 recordsLinked to original sources

bioRxiv2025

An Inducible CRISPRi system for phenotypic analysis of essential genes in Pseudomonas aeruginosa

Precise and tunable genetic tools are essential for high throughput functional genomics. To address this need in the important gram-negative pathogen Pseudomonas aeruginosa, we developed and characterized a tightly regulated CRISPRi system that enables precise and tunable repression of essential genes. The system utilizes a rhamnose-inducible promoter to control both the Streptococcus pasteurianus-derived dCas9 and gene-specific sgRNAs, each encoded on separate plasmids for modularity and efficiency. The combination of tight regulation and high conjugation efficiency facilitated the rapid and facile construction of strains with regulated depletion of 16 essential genes spanning diverse pathways. Comparison of phenotypes across the different genetically depleted strains, including growth rate, susceptibility to antibiotics, and changes in transcriptional programs, revealed novel aspects of gene function or small molecule mechanism of action. Finally, the rhamnose-inducible CRISPRi system supports the generation and stable maintenance of pooled mutant libraries, thereby paving the way for future genome-wide, systematic assessment of individual gene vulnerabilities, which will provide critical insights for target prioritization in antibiotic discovery efforts against this recalcitrant pathogen. IMPORTANCECRISPRi has become an invaluable tool for studying genetics. In particular, the ability to knockdown genes enables the study of essential genes and their role in cell survival. However, a tightly regulated gene knockdown system is required to gain valuable insights into these vulnerable genes by virtue of their essentiality. We report a tightly regulated CRISPRi system to study the biology of essential gene perturbations in Pseudomonas aeruginosa, an important gram-negative pathogen that causes severe infections and is increasingly resistant to current antibiotics. This system enables characterization of both chemical genetic interactions between small molecules and specific gene depletions, and the impact of genetic perturbations on transcriptional networks. Genetic perturbations using CRISPRi can thus further our understanding of basic biology with translation towards future antimicrobial development.

microbiology↗

bioRxiv2024

Perturbation-Specific Transcriptional Mapping facilitates unbiased target elucidation of antibiotics

The rising prevalence of antibiotic resistance threatens human health. While more sophisticated strategies for antibiotic discovery are being developed, target elucidation of new chemical entities remains challenging. In the post-genomic era, expression profiling can play an important role in mechanism-of-action (MOA) prediction by reporting on the cellular response to perturbation. However, the broad application of transcriptomics has yet to fulfill its promise of transforming target elucidation due to challenges in identifying the most relevant, direct responses to target inhibition. We developed an unbiased strategy for MOA prediction, called Perturbation-Specific Transcriptional Mapping (PerSpecTM), in which large-throughput expression profiling of wildtype or hypomorphic mutants, depleted for essential targets, enables a computational strategy to address this challenge. We applied PerSpecTM to perform reference-based MOA prediction based on the principle that similar perturbations, whether chemical or genetic, will elicit similar transcriptional responses. Using this approach, we elucidated the MOAs of three new molecules with activity against Pseudomonas aeruginosa by comparing their expression profiles to those of a reference set of antimicrobial compounds with known MOAs. We also show that transcriptional responses to small molecule inhibition resemble those resulting from genetic depletion of essential targets by CRISPRi by PerSpecTM, demonstrating proof-of-concept that correlations between expression profiles of small molecule and genetic perturbations can facilitate MOA prediction when no chemical entities exist to serve as a reference. Empowered by PerSpecTM, this work lays the foundation for an unbiased, readily scalable, systematic reference-based strategy for MOA elucidation that could transform antibiotic discovery efforts. Significance StatementNew antibiotics are critically needed in the face of increasing antibiotic resistance. However, mechanism-of-action (MOA) elucidation remains challenging and imposes a major bottleneck in antibiotic discovery and development. Building on the principle that molecules with similar MOAs elicit similar transcriptional responses, we have developed a highly scalable strategy for MOA prediction in the important bacterial pathogen Pseudomonas aeruginosa based on correlations between the expression profiles of new molecules and known perturbations, either small molecule inhibition by known antibiotics or transcriptional repression of essential targets by CRISPRi. By rapidly assigning MOAs to three new molecules with anti-pseudomonal activity, we provide proof-of-concept for a rapid, comprehensive, systematic, reference-based approach to MOA prediction with the potential to transform antibiotic discovery efforts.

genomics↗

bioRxiv2024

Multiplexed screen identifies a Pseudomonas aeruginosa-specific small molecule targeting the outer membrane protein OprH and its interaction with LPS

SUMMARYThe surge of antimicrobial resistance threatens efficacy of current antibiotics, particularly against Pseudomonas aeruginosa, a highly resistant gram-negative pathogen. The asymmetric outer membrane (OM) of P. aeruginosa combined with its array of efflux pumps provide a barrier to xenobiotic accumulation, thus making antibiotic discovery challenging. We adapted PROSPECT1, a target-based, whole-cell screening strategy, to discover small molecule probes that kill P. aeruginosa mutants depleted for essential proteins localized at the OM. We identified BRD1401, a small molecule that has specific activity against a P. aeruginosa mutant depleted for the essential lipoprotein, OprL. Genetic and chemical biological studies identified that BRD1401 acts by targeting the OM {beta}-barrel protein OprH to disrupt its interaction with LPS and increase membrane fluidity. Studies with BRD1401 also revealed an interaction between OprL and OprH, directly linking the OM with peptidoglycan. Thus, a whole-cell, multiplexed screen can identify species-specific chemical probes to reveal novel pathogen biology.

microbiology↗

bioRxiv2019

Mutations in pmrB confer cross-resistance to the LptD inhibitor POL7080 and colistin in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a major bacterial pathogen for which there is rising antibiotic resistance. We evaluated the resistance mechanisms of P. aeruginosa against POL7080, a species-specific, first-in-class antibiotic in phase 3 clinical trials targeting the lipopolysaccharide transport protein LptD. We found resistance mutations in the two-component regulator pmrB. Genome-wide transcriptomics and confocal microscopy studies together suggest that POL7080 is vulnerable to the same resistance mechanisms described previously for polymyxins, including colistin, that involve lipid A modifications to mitigate antibiotic cell surface binding.

microbiology↗

bioRxiv2018

Resistance from Afar: Distal Mutation V36M Allosterically Modulates the Active Site to Accentuate Drug Resistance in HCV NS3/4A Protease

Hepatitis C virus rapidly evolves, conferring resistance to direct acting antivirals. While resistance via active site mutations in the viral NS3/4A protease has been well characterized, the mechanism for resistance of non-active site mutations is unclear. R155K and V36M often co-evolve and while R155K alters the electrostatic network at the binding site, V36M is more than 13 [A] away. In this study the mechanism by which V36M confers resistance, in the context of R155K, is elucidated with drug susceptibility assays, crystal structures, and molecular dynamics (MD) simulations for three protease inhibitors: telaprevir, boceprevir and danoprevir. The R155K and R155K/V36M crystal structures differ in the -2 helix and E2 strand near the active site, with alternative conformations at M36 and side chains of active site residues D168 and R123, revealing an allosteric coupling, which persists dynamically in MD simulations, between the distal mutation and the active site. This allosteric modulation validates the network hypothesis and elucidates how distal mutations confer resistance through propagation of conformational changes to the active site.

molecular biology↗