Meeting report ASOBIOTICS 2024: an interdisciplinary symposium on antisense-based programmable RNA antibiotics.

The international symposium ASOBIOTICS 2024 brought together scientists across disciplines to discuss the challenges of advancing antibacterial antisense oligomers (ASOs) from basic research to clinical application. Hosted by the Helmholtz Institute for RNA-based Infection Research (HIRI) in Wurzburg, Germany, on September 12-13th, 2024, the event featured presentations covering major milestones and current challenges of this antimicrobial technology and its applications against pathogens, commensals, and bacterial viruses. General design principles and modification of ASOs based on peptide nucleic acid (PNA) or phosphorodiamidate-morpholino-oligomer (PMO) chemistry, promising cellular RNA targets, new delivery technologies, as well as putative resistance mechanisms were discussed.

Phage proteins target and co-opt host ribosomes immediately upon infection.

Bacteriophages must seize control of the host gene expression machinery to replicate. To bypass bacterial anti-phage defence systems, this host takeover occurs immediately upon infection. A general understanding of phage mechanisms for immediate targeting of host transcription and translation processes is lacking.

A comparative analysis of peptide-delivered antisense antibiotics using diverse 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 upon delivery with a KFF-peptide carrier into Our systematic analysis of PNA, PMO, phosphorothioate (PTO)-modified DNA, 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.

A MATQ-seq-Based Protocol for Single-Cell RNA-seq in Bacteria.

Microbes exhibit an extraordinary capacity to adapt their physiology to different environments using phenotypic heterogeneity. However, the majority of gene regulation studies are conducted in bulk reflecting only averaged gene expression levels across millions of cells. Bacterial single-cell RNA-seq (scRNA-seq) can overcome this by enabling whole transcriptome and unbiased analysis of microbes at the single-cell level.

Global profiling of the RNA and protein complexes of Escherichia coli by size exclusion chromatography followed by RNA sequencing and mass spectrometry (SEC-seq).

New methods for the global identification of RNA-protein interactions have led to greater recognition of the abundance and importance of RNA-binding proteins (RBPs) in bacteria. Here, we expand this tool kit by developing SEC-seq, a method based on a similar concept as the established Grad-seq approach. In Grad-seq, cellular RNA and protein complexes of a bacterium of interest are separated in a glycerol gradient, followed by high-throughput RNA-sequencing and mass spectrometry analyses of individual gradient fractions.

An unusual mode of baseline translation adjusts cellular protein synthesis capacity to metabolic needs.

In all domains of life, mechanisms exist that adjust translational capacity to nutrient restriction and other growth constraints. The mammalian target of rapamycin (mTOR) regulates the synthesis of ribosomal proteins and translation factors in mammalian cells via phosphorylation of the La-related protein 1 (LARP1). In the present model of starvation-induced translational silencing, LARP1 targets mRNAs carrying a 5' terminal oligopyrimidine (5'TOP) motif to shift these into subpolysomal ribonucleoprotein particles.

Global RNA interactome of Salmonella discovers a 5' UTR sponge for the MicF small RNA that connects membrane permeability to transport capacity.

The envelope of Gram-negative bacteria is a vital barrier that must balance protection and nutrient uptake. Small RNAs are crucial regulators of the envelope composition and function. Here, using RIL-seq to capture the Hfq-mediated RNA-RNA interactome in Salmonella enterica, we discover envelope-related riboregulators, including OppX.

RNA landscape of the emerging cancer-associated microbe Fusobacterium nucleatum.

Fusobacterium nucleatum, long known as a constituent of the oral microflora, has recently garnered renewed attention for its association with several different human cancers. The growing interest in this emerging cancer-associated bacterium contrasts with a paucity of knowledge about its basic gene expression features and physiological responses. As fusobacteria lack all established small RNA-associated proteins, post-transcriptional networks in these bacteria are also unknown.

Analysis of the RNA and Protein Complexome by Grad-seq.

The complexome of a cell is the entirety of its complexes. Complexome capture studies have mostly focused on protein-protein interactions, which has left a gap in our knowledge of the global interactions of RNAs. To overcome these limitations, we recently introduced gradient profiling by sequencing (Grad-seq), which analyzes in a high-throughput fashion soluble cellular complexes after their separation in a glycerol gradient by fraction-wise RNA-seq and mass spectrometry.

Cross-species RNA-seq for deciphering host-microbe interactions.

The human body is constantly exposed to microorganisms, which entails manifold interactions between human cells and diverse commensal or pathogenic bacteria. The cellular states of the interacting cells are decisive for the outcome of these encounters such as whether bacterial virulence programmes and host defence or tolerance mechanisms are induced. This Review summarizes how next-generation RNA sequencing (RNA-seq) has become a primary technology to study host-microbe interactions with high resolution, improving our understanding of the physiological consequences and the mechanisms at play.

A Grad-seq View of RNA and Protein Complexes in Pseudomonas aeruginosa under Standard and Bacteriophage Predation Conditions.

The Gram-negative rod-shaped bacterium is not only a major cause of nosocomial infections but also serves as a model species of bacterial RNA biology. While its transcriptome architecture and posttranscriptional regulation through the RNA-binding proteins Hfq, RsmA, and RsmN have been studied in detail, global information about stable RNA-protein complexes in this human pathogen is currently lacking. Here, we implement gradient profiling by sequencing (Grad-seq) in exponentially growing cells to comprehensively predict RNA and protein complexes, based on glycerol gradient sedimentation profiles of >73% of all transcripts and ∼40% of all proteins.

MAPS integrates regulation of actin-targeting effector SteC into the virulence control network of Salmonella small RNA PinT.

A full understanding of the contribution of small RNAs (sRNAs) to bacterial virulence demands knowledge of their target suites under infection-relevant conditions. Here, we take an integrative approach to capturing targets of the Hfq-associated sRNA PinT, a known post-transcriptional timer of the two major virulence programs of Salmonella enterica. Using MS2 affinity purification and RNA sequencing (MAPS), we identify PinT ligands in bacteria under in vitro conditions mimicking specific stages of the infection cycle and in bacteria growing inside macrophages.

Single-cell RNA-sequencing reports growth-condition-specific global transcriptomes of individual bacteria.

Bacteria respond to changes in their environment with specific transcriptional programmes, but even within genetically identical populations these programmes are not homogenously expressed. Such transcriptional heterogeneity between individual bacteria allows genetically clonal communities to develop a complex array of phenotypes, examples of which include persisters that resist antibiotic treatment and metabolically specialized cells that emerge under nutrient-limiting conditions. Fluorescent reporter constructs have played a pivotal role in deciphering heterogeneous gene expression within bacterial populations but have been limited to recording the activity of single genes in a few genetically tractable model species, whereas the vast majority of bacteria remain difficult to engineer and/or even to cultivate.

The minimal meningococcal ProQ protein has an intrinsic capacity for structure-based global RNA recognition.

FinO-domain proteins are a widespread family of bacterial RNA-binding proteins with regulatory functions. Their target spectrum ranges from a single RNA pair, in the case of plasmid-encoded FinO, to global RNA regulons, as with enterobacterial ProQ. To assess whether the FinO domain itself is intrinsically selective or promiscuous, we determine in vivo targets of Neisseria meningitidis, which consists of solely a FinO domain.

An Advanced Human Intestinal Coculture Model Reveals Compartmentalized Host and Pathogen Strategies during Infection.

A major obstacle in infection biology is the limited ability to recapitulate human disease trajectories in traditional cell culture and animal models, which impedes the translation of basic research into clinics. Here, we introduce a three-dimensional (3D) intestinal tissue model to study human enteric infections at a level of detail that is not achieved by conventional two-dimensional monocultures. Our model comprises epithelial and endothelial layers, a primary intestinal collagen scaffold, and immune cells.

An RNA Surprise in Bacterial Effector Mechanisms.

Bacterial pathogens secrete effector proteins to manipulate host signaling proteins and cellular structures. In this issue of Cell Host & Microbe, Pagliuso et al. (2019) propose an effector mechanism in Listeria monocytogenes whereby an RNA-binding protein associates with bacterial RNA that stimulates RIG-I (retinoic acid inducible gene I)-based innate immunity in the host cytosol.

The Major RNA-Binding Protein ProQ Impacts Virulence Gene Expression in Salmonella enterica Serovar Typhimurium.

FinO domain proteins such as ProQ of the model pathogen have emerged as a new class of major RNA-binding proteins in bacteria. ProQ has been shown to target hundreds of transcripts, including mRNAs from many virulence regions, but its role, if any, in bacterial pathogenesis has not been studied. Here, using a Dual RNA-seq approach to profile ProQ-dependent gene expression changes as infects human cells, we reveal dysregulation of bacterial motility, chemotaxis, and virulence genes which is accompanied by altered MAPK (mitogen-activated protein kinase) signaling in the host.

RNA-binding proteins in bacteria.

RNA-binding proteins (RBPs) are central to most if not all cellular processes, dictating the fate of virtually all RNA molecules in the cell. Starting with pioneering work on ribosomal proteins, studies of bacterial RBPs have paved the way for molecular studies of RNA-protein interactions. Work over the years has identified major RBPs that act on cellular transcripts at the various stages of bacterial gene expression and that enable their integration into post-transcriptional networks that also comprise small non-coding RNAs.

Global Maps of ProQ Binding In Vivo Reveal Target Recognition via RNA Structure and Stability Control at mRNA 3' Ends.

The conserved RNA-binding protein ProQ has emerged as the centerpiece of a previously unknown third large network of post-transcriptional control in enterobacteria. Here, we have used in vivo UV crosslinking and RNA sequencing (CLIP-seq) to map hundreds of ProQ binding sites in Salmonella enterica and Escherichia coli. Our analysis of these binding sites, many of which are conserved, suggests that ProQ recognizes its cellular targets through RNA structural motifs found in small RNAs (sRNAs) and at the 3' end of mRNAs.

Discovery of new RNA classes and global RNA-binding proteins.

The identification of new RNA functions and the functional annotation of transcripts in genomes represent exciting yet challenging endeavours of modern biology. Crucial insights into the biological roles of RNA molecules can be gained from the identification of the proteins with which they form specific complexes. Modern interactome techniques permit to profile RNA-protein interactions in a genome-wide manner and identify new RNA classes associated with globally acting RNA-binding proteins.

A systematic analysis of the RNA-targeting potential of secreted bacterial effector proteins.

Many pathogenic bacteria utilize specialized secretion systems to deliver proteins called effectors into eukaryotic cells for manipulation of host pathways. The vast majority of known effector targets are host proteins, whereas a potential targeting of host nucleic acids remains little explored. There is only one family of effectors known to target DNA directly, and effectors binding host RNA are unknown.

RNA target profiles direct the discovery of virulence functions for the cold-shock proteins CspC and CspE.

The functions of many bacterial RNA-binding proteins remain obscure because of a lack of knowledge of their cellular ligands. Although well-studied cold-shock protein A (CspA) family members are induced and function at low temperature, others are highly expressed in infection-relevant conditions. Here, we have profiled transcripts bound in vivo by the CspA family members of serovar Typhimurium to link the constitutively expressed CspC and CspE proteins with virulence pathways.

Molecular mechanism of mRNA repression in by a ProQ-dependent small RNA.

Research into post-transcriptional control of mRNAs by small noncoding RNAs (sRNAs) in the model bacteria and has mainly focused on sRNAs that associate with the RNA chaperone Hfq. However, the recent discovery of the protein ProQ as a common binding partner that stabilizes a distinct large class of structured sRNAs suggests that additional RNA regulons exist in these organisms. The cellular functions and molecular mechanisms of these new ProQ-dependent sRNAs are largely unknown.

New RNA-seq approaches for the study of bacterial pathogens.

Understanding how bacteria cause disease requires knowledge of which genes are expressed and how they are regulated during infection. While RNA-seq is now a routine method for gene expression analysis in bacterial pathogens, the past years have also witnessed a surge of novel RNA-seq based approaches going beyond standard mRNA profiling. These include variations of the technique to capture post-transcriptional networks controlled by small RNAs and to discover associated RNA-binding proteins in the pathogen itself.