Acinetobacter baumannii poses a substantial global health threat, causing severe multi-drug-resistant infections in hospitalized patients. Circulating clinical isolates present remarkable diversity, with a proportion capable of establishing a transient intracellular niche suitable for persistence, multiplication, and spread. Yet, it remains unknown which bacterial factors mediate the formation and maintenance of this niche, especially within non-phagocytic cells, nor what host responses are elicited. This work demonstrates that the invasive A. baumannii ABC141 strain does not secrete ammonia in endothelial cells as previously shown for other A. baumannii strains multiplying within macrophages but resides in an acidic vacuole devoid of active lysosomal degradative enzymes. This compartment mediates bacterial egress and infection of neighboring cells, promoting dissemination. Using a Dual-RNAseq approach, we mapped the host and bacterial gene expression during the replicative stage of the infection. An atypical hypoxia cell response was observed without significant induction of the HIF1 pathway, with no metabolic shift or disturbance of mitochondria. Surprisingly, ABC141 efficiently grew in hypoxic conditions in culture and within host cells. In addition, we found a bacterial signature reflective of an adaptation to a nutrient-deprived environment. Our work also highlights a differential role for ABC141 secretion systems, with the T1SS assisting intracellular multiplication and the T2SS required for host cell invasion, implicating for the first time the T2SS in the intracellular lifecycle of invasive ABC141 in endothelial cells.

DNA methylation is a regulator of bacterial gene expression and adaptation, influencing traits such as virulence and antimicrobial resistance. The dynamic nature of DNA methylation enables rapid responses to changing environments and is a source of heterogeneity in bacterial populations. However, condition-dependent DNA methylation and consequences for transcriptional output remain poorly understood. We applied Oxford Nanopore sequencing to profile DNA methylation during exponential growth and late stationary phase of Salmonella enterica serovar Typhimurium and integrated these data with transcriptomic analyses. We found that each DNA methyltransferase (MTases) exhibits a distinct activity pattern across growth stages, which could not be explained by transcriptional levels of the corresponding enzymes. As predicted, DNA methylation patterns determined by regulatory MTases were dynamic across growth conditions whereas methylation patterns of MTases belonging to R-M systems were comparatively stable. We identified growth stage-specific methylation patterns for all studied MTases and correlations between methylation states and gene expression patterns. Together, these findings chart DNA methylation networks in the epigenetic regulation of bacterial physiology. ### Competing Interest Statement The authors have declared no competing interest. European Union, https://ror.org/019w4f821, Marie Skłodowska-Curie grant agreement No. 896441 University of Regina Fir cluster of the Digital Research Alliance of Canada

The yeast Swi-Snf co-activator was the first chromatin remodelling complex discovered which can disrupt promoter nucleosomes to enable transcription. The Tup1-Cyc8 complex was the first global co-repressor identified which can position nucleosomes across promoters to block transcription. However, the global extent of their antagonistic regulation of gene transcription in yeast is unknown. We have identified the genes repressed by Tup1-Cyc8 and activated by Swi-Snf in glucose-grown cells which include inactive and actively transcribed genes. We show that Swi-Snf and Tup1-Cyc8 are present at both active and inactive gene promoters, where SNF2 is required for full Tup1-Cyc8 occupancy. The influence of Swi-Snf and Tup1-Cyc8 upon nucleosome positioning is specific to each co-regulated gene and extends over the available upstream intergenic region. We propose Tup1-Cyc8 and Swi-Snf are constitutively present at co-regulated gene promoters where their interplay influences the entire intergenic upstream chromatin environment of target genes and transcription.

Age-related macular degeneration (AMD) is the leading cause of irreversible central blindness and can result in pathological neovascularization. Using a “human-first” approach, we identify immunotherapy as a disease modifier in models of neovascular AMD (nAMD). Plasma cytokine analysis in a large population cohort reveals an imbalance of lymphocytic cytokines associated with severity of AMD, leading to discovery of a skewed peripheral natural killer (NK) cell phenotype in individuals with AMD. Peripheral NK cells are rapidly activated in nAMD models, and single-cell RNA sequencing demonstrates expansion of activated cytolytic NK cells within neovascular lesions during resolution. NK cells localize to neovessels in human AMD donor eyes; however, they exhibit markers of terminal differentiation and quiescence. Adoptive transfer of pre-activated NK cells reduces neovascularization and restores barrier integrity. Our data identify a distinct, functionally altered NK cell phenotype in nAMD and suggests harnessing NK cells represents an immunotherapeutic alternative for the treatment of nAMD.

Acinetobacter baumannii is associated with severe hospital- acquired, multi- drug- resistant infections worldwide, causing significant mortality and morbidity in intensive care patients or those under prolonged hospitalization. Multiple studies have recently shown that a proportion of circulating clinical isolates establish a transient multiplication niche inside phagocytic and non- phagocytic eukaryotic cells. We have previously demonstrated that the A. baumannii ABC141 strain invades human endothelial and epithelial cells, where it efficiently multiplies without induction of cytotoxicity. Here, we show that ABC141 adhesion, invasion and intracellular multiplication depend on the growth stage, being most efficient in the exponential growth phase. To define the gene expression signature most favourable to an intracellular lifestyle, a transcriptomic comparison was carried out between exponentially grown ABC141 and cultures in the stationary phase. Although most of the pathways identified reflected growth- related metabolic changes, we observed an up- regulation of the twin- arginine translocation (Tat) export system. Analysis of a mutant strain lacking the tatABC operon revealed that this export system is required only for adhesion to host cells, but not for invasion or intracellular multiplication. These data highlight a new role for the Tat export pathway in A. baumannii pathogenesis.

Post-transcriptional regulation can be mediated by small regulatory RNAs (sRNAs) in bacteria, which can act by base-pairing to a target mRNA. The discovery and mechanistic validation of base-pairing sRNAs in multidrug-resistant Acinetobacter baumannii has been hampered by the lack of genetic tools to assess RNA-RNA interactions. Here, we created two compatible plasmids for A. baumannii, which addresses this need. The newly designed plasmids validated the known Aar sRNA-carO mRNA and a new interaction of sRNA44 and the mRNA of the biofilm-associated protein Bap. The new plasmid system should accelerate the mechanistic characterization of sRNAs in A. baumannii.

Streptococcus pneumoniae (Sp) is an opportunistic pathogen that colonizes the mucosal surfaces of the human upper respiratory tract. While transcriptomic studies of Sp have become more common, most have focused on laboratory-adapted strains such as D39 or TIGR4. These strains, though widely used in research, may not fully capture the biology of clinical isolates, particularly the hypervirulent serotype 1 (S1). S1 is clinically significant due to its association with invasive disease, epidemic outbreaks and a distinct global distribution, particularly in regions with a high pneumococcal disease burden. Unlike many other serotypes, S1 is frequently linked to hypervirulence and a propensity for rapid spread, making it a high-priority target for understanding the molecular mechanisms underpinning pneumococcal pathogenesis. In this study, we conducted a comprehensive in vitro transcriptomic analyses of Sp S1 strains, positioning this work as a valuable resource for the pneumococcal research community. Using a straightforward approach, we cultured three distinct S1 strains - ST306, ST217 and ST615, representing European, African and South American S1 lineages, respectively - in Brain Heart Infusion medium and compared transcriptomic profiles during exponential growth to those of the well-characterized laboratory-adapted D39 strain. Our analysis revealed significant differential expression of 292 genes in all three S1 isolates compared to D39. Among these, 151 genes had higher expression, including those involved in competence pathways and purine metabolism, while 141 genes exhibited lower expression, particularly those linked to lactose metabolism and iron/amino acid transport. These findings underscore the distinct molecular features of S1 strains, which likely contribute to the unique pathogenic properties of this serotype. The identification of the distinct transcriptional signatures of hypervirulent S1 strains paves the way for future efforts to design targeted therapeutics against pneumococcal S1 infections.

The 26th annual Bioinformatics Open Source Conference (BOSC 2025, open-bio.org/events/bosc-2025) brought its community-driven focus on open-source bioinformatics and open science to the 2025 conference on Intelligent Systems for Molecular Biology and the European Conference on Computational Biology (ISMB/ECCB 2025). Since its launch in 2000, BOSC has been the premier annual meeting covering open-source bioinformatics and open science. Framed by two keynote addresses and a thought-provoking panel discussion, the two-day conference included sessions dedicated to open data, analytic tools and pipelines, workflow platforms, knowledge representation, and the application of AI/ML. The first keynote talk was delivered by Christine Orengo: “Working together to develop, promote and protect our data resources: Lessons learnt developing CATH and TED.” A joint session with the Bio-Ontologies and Knowledge Representation (BOKR) track the second day of BOSC started with a keynote talk by Chris Mungall entitled “Open Knowledge Bases in the Age of Generative AI”. A closing panel on Data Sustainability, moderated by Mónica Muñoz Torres, featured panelists Scott Edmunds, Varsha Khodiyar, Tony Burdett, Nicky Mulder, and Chris Mungall. This year, the CollaborationFest collaborative work event that typically precedes or follows ISMB was incorporated as part of the main conference and organized by BOSC with help from the Function and 3D-SIG tracks.

Salmonella enterica serovar Typhimurium must adapt to rapid environmental shifts, including those encountered upon entry and during replication to survive within macrophages during pathogenesis. Despite extensive RNA-seq-based investigations, questions remain regarding the range, timing and magnitude of response dynamics. Here we constructed a comprehensive GFP-reporter strain library representing 2,901 computationally identified Salmonella promoter regions to study time-resolved Salmonella transcriptional responses. Promoter activity was measured during in vitro growth and during intracellular infection of RAW 264.7 macrophages. Using bulk measurements and single-cell imaging, we uncovered condition-specific transcriptional regulation and population-level heterogeneity in SPI2-related promoter activity. We also discovered previously unidentified transcriptional activity from 234 promoters. These analyses revealed metabolic shifts including requirements for mntS expression to support manganese homeostasis and expression of Entner-Doudoroff pathway-associated genes to support growth within macrophages. Our library and datasets, made available through the online tool SalComKinetics, provide resources for systems-level interrogation of Salmonella transcriptional dynamics.

Circulating tumour cells (CTCs) are rare yet crucial biomarkers with significant prognostic potential across different cancer types. However, their role in high-grade serous ovarian cancer (HSGC) is not well defined. To capture the full spectrum of CTCs found in HGSC, we employed an EpCAM independent enrichment technique in patients with advanced HGSC and investigated the prognostic value and molecular signatures of these rare cells. CTC enumeration was performed in 43 newly diagnosed patients with HGSC using Parsortix® CTC enrichment and benchmarked against a metastatic breast cancer (MBC) cohort for which the device is FDA approved. CTCs were also isolated from the ovarian vein of patients with HGSC during primary cytoreductive surgery. CTCs were assessed as prognostic markers in patients with HGSC. FACS single cell sorting and scRNAseq was performed on CTCs isolated from the ovarian vein. CTCs isolated using Parsortix® enrichment in HGSC ranged between 1-22 cells/7.5 ml blood. Concordance was seen between Parsortix® enrichment and CellSearch® enumeration in patients with MBC (R2 = 0.8786). CTC clusters were isolated from the ovarian vein (P = 0.0195) and were cloaked in platelets/immune cells. Detection of CTCs in patients with HGSC was predictive of a poorer progression free survival (P = 0.0183). Patients with CTCs were found to have increased serum levels of CD73 (P = 0.0311). scRNAseq of CTCs isolated from the ovarian vein identified enrichment in genes associated with immune signalling. Peripheral CTCs isolated from patients with HGSC were predictors of a poor prognosis. The ovarian vein was found to be a rich source of disseminating CTC clusters in HGSC. Further studies are warranted to investigate the utility of CTCs as markers of neoadjuvant chemotherapy response as well as for longitudinal monitoring. Molecular analysis of CTCs in HGSCs reveals a potential role of the immune system in CTC-mediated haematogenous metastasis.

Acinetobacter baumannii is a major opportunistic pathogen capable of natural transformation, a process driven by type IV pili (T4P) that facilitates horizontal gene transfer and accelerates the spread of antimicrobial resistance. While the transcriptional regulation of T4P is increasingly understood, post-transcriptional mechanisms controlling pilus assembly remain unexplored. Here, we identify and characterise a small RNA, Arp (Acinetobacter repressor of pilin), as a post-transcriptional repressor of T4P-mediated functions in A. baumannii. In a previous Hi-GRIL-seq experiment, we detected specific ligation events between Arp and the ribosome binding site of the pilA mRNA, encoding the major pilin subunit PilA. In-line probing and translational reporter assays revealed that Arp represses pilA translation by sequestering the Shine-Dalgarno sequence and the first 17 codons of the mRNA. Overexpression of Arp significantly impairs DNA uptake and twitching motility, two hallmark T4P-dependent phenotypes. Together, our findings identify a native A. baumannii sRNA that modulates natural competence by targeting pilin synthesis, revealing a new regulatory layer that could be exploited to disrupt horizontal gene transfer in multidrug-resistant strains.

Heterozygous missense mutations in EZH2 cause Weaver syndrome (WS), a developmental disorder characterized by intellectual disability and overgrowth. EZH2 encodes the enzymatic subunit of Polycomb repressive complex 2 (PRC2), which mediates monomethylation, dimethylation, and trimethylation of histone H3 lysine 27 (H3K27me1/2/3). Most WS-associated EZH2 variants lack functional characterization but are presumed loss-of-function. However, the lack of early truncating mutations in EZH2 led us to hypothesize a dominant-negative mechanism for WS, which was supported by our structural analysis of all known WS-associated EZH2 variants. We isogenically modeled 10 representative variants in embryonic stem cells and showed that they reduce global H3K27me2/3 with concomitant increases in H3K27ac and chromatin decompaction. Notably, the pattern of H3K27me2/3 reductions indicated dominant-negative interference on PRC2 activity even when WS variants were expressed at low levels. RNA-seq identified weakly Polycomb-bound genes that lose canonical PRC1 (cPRC1) occupancy and become derepressed, including several phenotypically relevant growth control genes. Comparative analysis of a gain-of-function EZH2 variant causing growth restriction revealed reciprocal chromatin and transcriptional changes compared with WS-associated variants. Taken together, our findings support a model in which EZH2 variants associated with opposing developmental growth syndromes affect not only H3K27me3 but also intergenic H3K27me2, chromatin architecture, and cPRC1 recruitment.

The 25th annual Bioinformatics Open Source Conference (BOSC 2024, open-bio.org/events/bosc-2024) was part of the 2024 conference on Intelligent Systems for Molecular Biology (ISMB 2024). Launched in 2000 and held yearly since, BOSC is the premier meeting covering open-source bioinformatics and open science. ISMB 2024 was held in Montréal, Canada, with an online participation option. A total of nearly 2000 people attended; about 200 people participated in BOSC sessions. Over the course of two days, BOSC covered a wide range of topics in open science and open source bioinformatics, including Data Analysis, Open Data, Visualization, Developer Tools and Libraries, Standards and Frameworks for Open Science, and Open AI/ML. Mélanie Courtot delivered an impactful first keynote with a perspective on how “The Data Shows We Need Better Data”. The second keynote speaker, Andrew Su, discussed “Open Data, Knowledge Graphs, and Large Language Models.” BOSC ended with a panel, “Open Source AI/ML: A Game Changer for Bioinformatics?,” in which Lawrence Hunter and Thomas Hervé Mboa Nkoudou joined BOSC’s keynote speakers as panelists. Immediately following BOSC, the CollaborationFest was held at Montréal’s University of Québec campus. First launched in 2010, CoFest is a collaborative work event held yearly around BOSC. This year’s CoFest included 42 participants who worked together on 10 projects.

Salmonella enterica is comprised of genetically distinct 'serovars' that together provide an intriguing model for exploring the genetic basis of pathogen evolution. Although the genomes of numerous Salmonella isolates with broad variations in host range and human disease manifestations have been sequenced, the functional links between genetic and phenotypic differences among these serovars remain poorly understood. Here, we conduct high-throughput functional genomics on both generalist (Typhimurium) and human-restricted (Typhi and Paratyphi A) Salmonella at unprecedented scale in the study of this enteric pathogen. Using a comprehensive systems biology approach, we identify gene networks with serovar-specific fitness effects across 25 host-associated stresses encountered at key stages of human infection. By experimentally perturbing these networks, we characterize previously undescribed pseudogenes in human-adapted Salmonella. Overall, this work highlights specific vulnerabilities encoded within human-restricted Salmonella that are linked to the degradation of their genomes, shedding light into the evolution of this enteric pathogen.

The transcriptome from a Saccharomyces cerevisiae tup1 deletion mutant was one of the first comprehensive yeast transcriptomes published. Subsequent transcriptomes from tup1 and cyc8 mutants firmly established the Tup1-Cyc8 complex as predominantly acting as a repressor of gene transcription. However, transcriptomes from tup1/cyc8 gene deletion or conditional mutants would all have been influenced by the striking flocculation phenotypes that these mutants display. In this study, we have separated the impact of flocculation from the transcriptome in a cyc8 conditional mutant to reveal those genes (i) subject solely to Cyc8p-dependent regulation, (ii) regulated by flocculation only and (iii) regulated by Cyc8p and further influenced by flocculation. We reveal a more accurate list of Cyc8p-regulated genes that includes newly identified Cyc8p-regulated genes that were masked by the flocculation phenotype and excludes genes which were indirectly influenced by flocculation and not regulated by Cyc8p. Furthermore, we show evidence that flocculation exerts a complex and potentially dynamic influence upon global gene transcription. These data should be of interest to future studies into the mechanism of action of the Tup1-Cyc8 complex and to studies involved in understanding the development of flocculation and its impact upon cell function.

Invasive non-typhoidal Salmonella (iNTS) disease is a serious bloodstream infection that targets immune-compromised individuals, and causes significant mortality in sub-Saharan Africa. Salmonella enterica serovar Typhimurium ST313 causes the majority of iNTS in Malawi. We performed an intensive comparative genomic analysis of 608 S. Typhimurium ST313 isolates dating between 1996 and 2018 from Blantyre, Malawi. We discovered that following the arrival of the well-characterized S. Typhimurium ST313 lineage 2 in 1999, two multidrug-resistant variants emerged in Malawi in 2006 and 2008, designated sublineages 2.2 and 2.3, respectively. The majority of S. Typhimurium isolates from human bloodstream infections in Malawi now belong to sublineages 2.2 or 2.3. To understand the emergence of the prevalent ST313 sublineage 2.2, we studied two representative strains, D23580 (lineage 2) and D37712 (sublineage 2.2). The chromosome of ST313 lineage 2 and sublineage 2.2 only differed by 29 SNPs/small indels and a 3 kb deletion of a Gifsy-2 prophage region including the sseI pseudogene. Lineage 2 and sublineage 2.2 had distinctive plasmid profiles. The transcriptome was investigated in 15 infection-relevant in vitro conditions and within macrophages. During growth in physiological conditions that do not usually trigger S. Typhimurium SPI2 gene expression, the SPI2 genes of D37712 were transcriptionally active. We identified down-regulation of flagellar genes in D37712 compared with D23580. Following phenotypic confirmation of transcriptomic differences, we discovered that sublineage 2.2 had increased fitness compared with lineage 2 during mixed growth in minimal media. We speculate that this competitive advantage is contributing to the emergence of sublineage 2.2 in Malawi.

ABSTRACT Acinetobacter baumannii is an opportunistic Gram-negative pathogen that infects critically ill patients. The emergence of antimicrobial resistant A. baumannii has exacerbated the need to functionally characterise environmental adaptation, antibiotic resistance and pathogenicity of this organism and their genetic regulators to inform intervention strategies. Critical to rapid adaptation to changing environments in bacteria are small regulatory RNAs (sRNAs), however, the role that sRNAs play in the biology of A. baumannii is poorly understood. To assess the regulatory function of sRNAs and to uncover their RNA interaction partners in A. baumannii , we employed an RNA proximity ligation and sequencing method (Hi-GRIL-seq) in three different environmental conditions. We found that 40 sRNA candidates were ligated to sRNA-RNA chimeric sequencing reads, suggesting that sRNA-mediated gene regulation is pervasive in A. baumannii and that sRNAs act as direct regulators of mRNA molecules through antisense base-pairing. In-depth characterisation uncovered the sRNA Aar to be a post-transcriptional regulator of four mRNA targets including that of the outer membrane protein CarO and the siderophore receptor BfnH. We show that Aar initiates base-pairing with these mRNA molecules using a conserved seed region of nine nucleotides, sequestering the ribosome binding sites and inhibiting translation. Aar is differentially expressed in response to multiple stress stimuli suggesting a role in fine-tuning translation of the Aar-target molecules in A. baumannii under hostile conditions. Together, our study provides mechanistic insights into sRNA-mediated gene expression control in A. baumannii and represents a valuable resource for future RNA-centric research endeavours in this ESKAPE pathogen.