Biology subjects

Popella, L.

Publications and source records attributed to Popella, L..

8 recordsLinked to original sources

bioRxiv2026

ASO-mediated mRNA silencing enables functional analysis and selective depletion of the human microbiota Prevotellaceae

The Prevotellaceae family comprises abundant, taxonomically diverse bacteria of the human microbiota that exhibit remarkable intraspecies variability and distinct phenotypes during host-microbe interactions. Yet functional investigations are hampered by the limited genetic tractability of this medically important bacterial family, rendering it understudied. Here, we apply antisense oligomer (ASO) technology to selectively inhibit translation of single or multiple mRNAs across nine Prevotellaceae species. Using ftsZ- and mreB-related phenotypes as morphogenic readouts, we demonstrate that ASOs can function as a tunable system to study essential gene function. Further, we show that ASOs can selectively deplete target species from a synthetic Bacteroidales community. These results establish ASOs as practical tools for functional genomics and community modulation in otherwise genetically intractable anaerobic microbiota members.

microbiology↗

bioRxiv2025

An antisense oligomer conjugate with unpredicted bactericidal activity against Fusobacterium nucleatum

Fusobacteria are commensal members of the oral microbiome that can spread from their primary niche and colonize distal sites in the human body. Their enrichment in colorectal and breast cancer tissue has been associated with tumor growth, metastasis, and chemotherapeutic resistance. The use of non-selective antibiotics to remove fusobacteria impairs tumor progression, but prolonged application risks side effects such as gastrointestinal problems and dysbiosis. Species-specific antisense antibiotics based on peptide nucleic acid (PNA) have shown efficacy in many Gram-negative species, suggesting that antisense PNAs may also enable a tailored depletion of fusobacteria. Here, we have investigated the antibacterial potential of cell-penetrating peptide (CPP)-PNA conjugates targeting the mRNA of putative essential genes in Fusobacterium nucleatum. Unexpectedly, we observed no growth inhibition with any of the target-specific PNAs, but identified a non-targeting control CPP-PNA (FUS79, (RXR)4XB-GACATAATTGT) as a potent growth inhibitor of F. nucleatum. Our data suggest that the CPP and specific sequence features of FUS79 are responsible for its activity, rather than an antisense effect. Interestingly, FUS79 also inhibits the growth of five additional fusobacterial strains but not of F. nucleatum ssp. vincentii (FNV). RNA-seq analysis indicates that FUS79 induces a membrane stress response in a vulnerable F. nucleatum strain but not in FNV. Collectively, our attempt at developing antisense antibiotics for fusobacteria discovers a potent growth inhibitor, whose bactericidal effect appears independent of target-specific mRNA inhibition. IMPORTANCEEnrichment of F. nucleatum at cancer sites is associated with increased tumor growth and metastasis. Antibiotic treatment to remove the bacteria was shown to change the course of cancer progression. Here we explore first steps to establish peptide nucleic acids (PNAs) as specific antisense antibiotics, thereby laying the foundation for further development of antisense technology in fusobacteria. While the CPP-PNA FUS79 was initially designed as a control, we observed that the compound was bactericidal for specific fusobacterial strains. Our results suggest that FUS79 might be able to selectively deplete fusobacterial strains from bacterial communities, offering a new perspective on fusobacterial removal at the tumor site.

microbiology↗

bioRxiv2024

A systematic identification of resistance determinants to antisense antibiotics suggests adaptation strategies dependent on the delivery peptide

The rise of antimicrobial resistance (AMR) among human pathogenic microbes is a serious threat to global health, calling for the development of novel treatment strategies. Antibiotics based on programmable antisense oligomers (asobiotics) offer an attractive solution to the "arms-race", as their specificity can be quickly updated and tailored to target resistant bacteria. In order to understand the genetic architecture of resistance to asobiotics, we employed laboratory evolution assays to identify mutations that decrease susceptibility to antisense peptide nucleic acid (PNA) against four major gram-negative pathogens: Escherichia coli, Klebsiella pneumoniae, Salmonella enterica, and Pseudomonas aeruginosa. We observed that the reduction in susceptibility upon asobiotics treatment was dependent on the specific cell penetrating peptide (CPP) being conjugated to the PNAs, suggesting that reduced uptake is a common adaptation strategy only against the (KFF)3-K CPP. We in fact observed that sbmA was frequently mutated in all tested species when treated with (KFF)3-K conjugated PNAs. We further identified mutations related to translation, peptide transport and cell envelope, which provide new hypotheses on cellular response to CPP-PNAs conjugates. Furthermore, for (RXR)4XB-acpP we observed a modest increase in resistance only when mutations in the PNA binding site were induced, which could easily be bypassed by changing the PNA sequence. These findings indicate that the specific identity of the CPP used plays a key role in determining its robustness against the evolution of resistance, and that laboratory evolution can illuminate the remaining gaps in our knowledge on the mechanisms of action of asobiotics.

microbiology↗

bioRxiv2024

RNA toehold switch-based reporter assay to assess bacterial uptake of antisense oligomers

Antisense oligomers (ASOs) hold promise as antibiotics for selective targeting of bacterial pathogens and as tools for the modulation of gene function in genetically intractable microbes. However, their efficient delivery across the complex bacterial envelope remains a major challenge. There are few methods to assess the efficiency of carrier-mediated ASO uptake by bacteria. Here, we have developed a "switch-on" reporter assay to measure ASO uptake efficiency in a semi-quantitative manner. The assay uses a synthetic RNA toehold switch fused to the mRNA of a fluorescent reporter protein, which is activated in vivo by a peptide nucleic acid (PNA)-based ASO upon delivery into the bacterial cytosol. We have used this assay to screen different cell penetrating peptides (CPPs) as ASO carriers in Escherichia coli and Salmonella enterica and observed up to 60-fold activation, depending on the CPP and bacterial strain used. Our assay shows high dynamic range and sensitivity, which should enable high-throughput screens for bacterial ASO carriers. We also show that the reporter can be used to study routes of PNA uptake, as demonstrated by reduced reporter activity in the absence of the inner membrane protein SbmA. In summary, we present a portable tool for the discovery of species-specific and efficient ASO carriers that will also be useful for a broader investigation of cellular uptake mechanisms of antibacterial ASOs. ImportanceThe rise of antimicrobial resistance presents a major global health challenge. If not addressed, the death toll from resistant infections is expected to rise dramatically in the coming years. As a result, it is essential to explore alternative antimicrobial therapies. One promising approach is to target bacterial mRNAs using antisense oligomers (ASOs) to silence genes involved in essential functions, virulence, or resistance. However, delivering ASOs across bacterial membranes remains a major challenge and effective methods to monitor their uptake are limited. In this study, we develop a reporter assay to facilitate the high-throughput discovery of bacterial ASO carriers. This research paves the way for developing novel precision antisense-based antibacterial therapies.

microbiology↗

bioRxiv2024

Non-genetic messenger RNA silencing reveals essential genes in phage-host interplay

Bacteriophages are the most abundant entities on earth and exhibit vast genetic and phenotypic diversity. Exploitation of this largely unexplored molecular space requires identification and functional characterisation of genes that act at the phage-host interface. To date, this is restricted to few model phage-host systems that are amenable to genetic manipulation. To overcome this limitation, we introduce a non-genetic mRNA targeting approach using exogenous delivery of programmable antisense oligomers (ASOs) to silence genes of both DNA and RNA phages. A systematic knockdown screen of core and accessory genes of the nucleus-forming jumbo phage {Phi}KZ, coupled to RNA-sequencing and microscopy analyses, reveals previously unrecognised proteins that are essential for phage replication and that, upon silencing, elicit distinct phenotypes at the level of the phage and host response. One of these factors is a {Phi}KZ-encoded nuclease that acts at a major decision point during infection, linking the formation of the protective phage nucleus to phage genome amplification. The non-genetic ASO-based gene silencing used here promises to be a versatile tool for molecular discovery in phage biology, will help elucidate defence and anti-defence mechanisms in non-model phage-host pairs, and offers potential for optimising phage therapy and biotechnological procedures.

molecular biology↗

bioRxiv2023

A side-by-side comparison of peptide-delivered antisense antibiotics employing different nucleotide mimics

Antisense oligomer (ASO)-based antibiotics that target mRNAs of essential bacterial genes have great potential for counteracting antimicrobial resistance and for precision microbiome editing. To date, the development of such antisense antibiotics has primarily focused on using phosphorodiamidate morpholino (PMO) and peptide nucleic acid (PNA) backbones, largely ignoring the growing number of chemical modalities that have spurred the success of ASO-based human therapy. Here, we directly compare the activities of seven chemically distinct 10mer ASOs, all designed to target the essential gene acpP upon delivery with a KFF-peptide carrier into Salmonella. Our systematic analysis of PNA, PMO, phosphorothioate-modified DNA (PTO), 2-methylated RNA (RNA-OMe), 2-methoxyethylated RNA (RNA-MOE), 2-fluorinated RNA (RNA-F) and 2-4-locked RNA (LNA) is based on a variety of in vitro and in vivo methods to evaluate ASO uptake, target pairing and inhibition of bacterial growth. Our data show that only PNA and PMO are efficiently delivered by the KFF peptide into Salmonella to inhibit bacterial growth. Nevertheless, the strong target binding affinity and in vitro translational repression activity of LNA and RNA-MOE make them promising modalities for antisense antibiotics that will require the identification of an effective carrier.

microbiology↗

bioRxiv2022

Predicting off-target effects of antisense oligomers targeting bacterial mRNAs with the MASON webserver

Antisense oligomers (ASOs) such as peptide nucleic acids (PNAs), designed to inhibit the translation of essential bacterial genes, have emerged as attractive sequence- and species-specific programmable RNA antibiotics. Yet, potential drawbacks include unwanted side effects caused by their binding to transcripts other than the intended target. To facilitate the design of PNAs with minimal off-target effects, we developed MASON (Make AntiSense Oligomers Now), a webserver for the design of PNAs that target bacterial mRNAs. MASON generates PNA sequences complementary to the translational start site of a bacterial gene of interest and reports critical sequence attributes and potential off-target sites. We based MASONs off-target predictions on experiments in which we treated Salmonella enterica serovar Typhimurium with a series of 10mer PNAs derived from a PNA targeting the essential gene acpP but carrying two serial mismatches. Growth inhibition and RNA-sequencing (RNA-seq) data revealed that PNAs with terminal mismatches are still able to target acpP, suggesting wider off-target effects than anticipated. Comparison of these results to an RNA-seq dataset from uropathogenic Escherichia coli (UPEC) treated with eleven different PNAs confirmed our findings are not unique to Salmonella. We believe that MASONs off-target assessment will improve the design of specific PNAs and other ASOs.

bioinformatics↗

bioRxiv2022

Comprehensive analysis of PNA-based antisense antibiotics targeting various essential genes in uropathogenic Escherichia coli

Antisense peptide nucleic acids (PNAs) that target mRNAs of essential bacterial genes exhibit specific bactericidal effects in several microbial species, but our mechanistic understanding of PNA activity and their target gene spectrum is limited. Here, we present a systematic analysis of PNAs targeting eleven essential genes with varying expression levels in uropathogenic Escherichia coli (UPEC). We demonstrate that UPEC is susceptible to killing by peptide-conjugated PNAs, especially when targeting the widely-used essential gene acpP. Our evaluation yields three additional promising target mRNAs for effective growth inhibition, i.e., dnaB, ftsZ, and rpsH. The analysis also shows that transcript abundance does not predict target vulnerability and that PNA-mediated growth inhibition is not universally associated with target mRNA depletion. Global transcriptomic analyses further reveal PNA sequence-dependent but also -independent responses, including the induction of envelope stress response pathways. Importantly, we show that the growth inhibitory capacity of 9mer PNAs is generally as effective as their 10mer counterparts. Overall, our systematic comparison of a range of PNAs targeting mRNAs of different essential genes in UPEC suggests important features for PNA design, reveals a general bacterial response to PNA conjugates and establishes the feasibility of using PNA antibacterials to combat UPEC.

microbiology↗