Biology subjects

Myers, G. S. A.

Publications and source records attributed to Myers, G. S. A..

7 recordsLinked to original sources

bioRxiv2026

Interrogating the Escherichia coli epitranscriptome via CRISPR interference and Nanopore native RNA sequencing

Epitranscriptomics has recently gained significant momentum due to technological advances and translational applications, however, studies on bacterial RNA modifications remain limited. Bacterial RNA remains notoriously prone to degradation and methodologies to investigate the epitranscriptome are challenging. Prior research has shown RNA modifications modulate antimicrobial resistance, virulence and pathogenicity. This research employed CRISPR interference to knock down five known Escherichia coli rRNA modification genes (rlmF, rlmJ, rluD, rsmF and rsmG) in three E. coli strains. These isolates underwent growth curves, proteome analysis and native RNA sequencing CRISPRi adequately silenced the majority of RNA modification genes in E. coli (>80% reduction). Significant growth delays were associated with rlmF, rsmF and rsmG repression. Unique protein pathways corresponding with RNA modification loss were found for rlmJ (TreB, XylF), rluD (CysH, HycB, PutP, TrpB), rsmF (EvgA) and rsmG (OppC). Known rRNA modification sites for rluD ({Psi}) and rsmG (m7G) were detected from analysis of nanopore electrical signal, however, only a weak signal was apparent for m6A (rlmF, rlmJ) and m5C (rsmF) modifications. The inhibition of rRNA modifications resulted in mRNA modification changes including for genes ompC, cspC, dbhA, dbhB and secY. Our work provides an approach for unravelling the epitranscriptome of E. coli and gain insight into its functional role.

microbiology↗

bioRxiv2025

Co-isolation of genetically distinct Burkholderia pseudomallei strains from a single patient in North Queensland

While investigating colony morphology variation (CMV) within clinical isolates of Burkholderia pseudomallei (Bp) isolated from a single infection, we identified two distinct strains (based on their multi-locus sequence type) from Bp TSV292. Simultaneous co-infection is rare, previously reported in only 2 of 133 cases. Here, we present the first comprehensive multi-omics characterization of co-infecting Bp strains isolated from a single infection at a hospital in Townsville, Australia in 2018. This finding impacts the design of future diagnosis and treatment of Bp. ImportanceMelioidosis, a severe disease caused by the bacterium Burkholderia pseudomallei, is rarely caused by more than one strain at the same time. In this study, we examined two bacterial strains isolated at once from a patient treated for melioidosis at a hospital in northern Queensland (Australia). By comparing their genomes, proteins, and physical traits, we found important strain differences that may affect how they adapt and survive during infection. Considering strain diversity when studying how this pathogen causes disease is essential, as it directly impacts future diagnosis and treatment.

microbiology↗

bioRxiv2025

Profiling of Burkholderia pseudomallei variants derived from Queensland clinical isolates

Burkholderia pseudomallei (Bp), an environmental bacterium and opportunistic pathogen endemic to tropical regions, is highly adaptive and thrives in diverse environments, from soil to human hosts. Bacterial adaptation is critical for survival, virulence modulation, and persistence during infection and can manifest as colony morphotype variation (CMV). While Bp adaptation has been well studied, CMV remains poorly understood. Here, we characterized five clinical Bp isolates exhibiting heterogeneous populations with rough and smooth colony morphologies. We used phenotypic assays, whole-genome sequencing, and proteomics to investigate the molecular pathways impacted by CMV - by comparing smooth and rough morphotypes. While phenotypic differences in protease activity, haemolysis, mucoid, iron uptake and antibiotic sensitivity --including to antimicrobial agents commonly used to treat infections--were rare, these traits alone could not distinguish morphotypes or group of isolates. Genomic comparisons revealed either no differences or limited isolate-specific mutations, which does not explain the overall difference in phenotypes. In contrast, proteomic analysis uncovered consistent shifts in protein abundance related to virulence, including quorum sensing, DNA methylation, and secretion systems. Rough variants showed higher expression of EPS-associated proteins, the BpsI3/R3 quorum sensing system, and the global regulator ScmR, whereas smooth variants upregulated type III/VI secretion and siderophore biosynthesis pathways. These findings suggest that CMV is driven by phase variation and regulatory mechanisms rather than punctual genomic modifications. Our study underscores the limitations of phenotype or genome-based classification alone in the context of CMV and highlights the value of integrated multi-omics approaches to uncover CMV-associated biomarkers, with potential applications in diagnostics and the development of targeted therapies against persistent and drug-resistant Bp infections.

microbiology↗

bioRxiv2020

Dual RNA-Seq analysis of in vitro infection multiplicity in Chlamydia-infected epithelial cells

Dual RNA-seq experiments examining viral and bacterial pathogens are increasing, but vary considerably in their experimental designs, such as infection rates and RNA depletion methods. Here, we have applied dual RNA-seq to Chlamydia trachomatis infected epithelial cells to examine transcriptomic responses from both organisms. We compared two time points post infection (1 and 24 hours), three multiplicity of infection (MOI) ratios (0.1, 1 and 10) and two RNA depletion methods (rRNA and polyA). Capture of bacterial-specific RNA were greatest when combining rRNA and polyA depletion, and when using a higher MOI. However, under these conditions, host RNA capture was negatively impacted. Although it is tempting to use high infection rates, the implications on host cell survival, the potential reduced length of infection cycles and real world applicability should be considered. This data highlights the delicate nature of balancing host-pathogen RNA capture and will assist future transcriptomic-based studies to achieve more specific and relevant infection-related biological insights.

systems biology↗

bioRxiv2019

Early transcriptional landscapes of Chlamydia trachomatis-infected epithelial cells at single cell resolution

Chlamydia are Gram-negative obligate intracellular bacterial pathogens responsible for a variety of disease in humans and animals worldwide. C. trachomatis causes trachoma (infectious blindness) in disadvantaged populations, and is the most common bacterial sexually transmitted infection in humans, causing reproductive tract disease. Antibiotic therapy successfully treats diagnosed chlamydial infections, however asymptomatic infections are common. High-throughput transcriptomic approaches have explored chlamydial gene expression and infected host cell gene expression. However, these were performed on large cell populations, averaging gene expression profiles across all cells sampled and potentially obscuring biologically relevant subsets of cells. We generated a pilot dataset, applying single cell RNA-Seq (scRNA-Seq) to C. trachomatis infected and mock-infected epithelial cells to assess the utility of single cell approaches to identify early host cell biomarkers of chlamydial infection. 264 time-matched C. trachomatis-infected and mock-infected HEp-2 cells were collected and subjected to scRNA-Seq. After quality control, 200 cells were retained for analysis. Two distinct clusters distinguished 3-hour cells from 6- and 12-hours. Pseudotime analysis identified a possible infection-specific cellular trajectory for Chlamydia-infected cells, while differential expression analyses found temporal expression of metallothioneins and genes involved with cell cycle regulation, innate immune responses, cytoskeletal components, lipid biosynthesis and cellular stress. Changes to the host cell transcriptome at early times of C. trachomatis infection are readily discernible by scRNA-Seq, supporting the utility of single cell approaches to identify host cell biomarkers of chlamydial infection, and to further deconvolute the complex host response to infection.

systems biology↗

bioRxiv2019

Chromatin accessibility dynamics of Chlamydia-infected epithelial cells

Chlamydia are Gram-negative, obligate intracellular bacterial pathogens responsible for a broad spectrum of human and animal diseases. In humans, Chlamydia trachomatis is the most prevalent bacterial sexually transmitted infection worldwide and is the causative agent of trachoma (infectious blindness) in disadvantaged populations. Over the course of its developmental cycle, Chlamydia extensively remodels its intracellular niche and parasitises the host cell for nutrients, with substantial resulting changes to the host cell transcriptome and proteome. However, little information is available on the impact of chlamydial infection on the host cell epigenome and global gene regulation. Regions of open eukaryotic chromatin correspond to nucleosome-depleted regions, which in turn are associated with regulatory functions and transcription factor binding. We applied Formaldehyde-Assisted Isolation of Regulatory Elements enrichment followed by sequencing (FAIRE-Seq) to generate temporal chromatin maps of C. trachomatis-infected human epithelial cells in vitro over the chlamydial developmental cycle. We detected both conserved and distinct temporal changes to genome-wide chromatin accessibility associated with C. trachomatis infection. The observed differentially accessible chromatin regions, including several Clusters of Open Regulatory Elements (COREs) and temporally-enriched sets of transcription factors, may help shape the host cell response to infection. These regions and motifs were linked to genomic features and genes associated with immune responses, re-direction of host cell nutrients, intracellular signaling, cell-cell adhesion, extracellular matrix, metabolism and apoptosis. This work provides another perspective to the complex response to chlamydial infection, and will inform further studies of transcriptional regulation and the epigenome in Chlamydia-infected human cells and tissues

systems biology↗

bioRxiv2017

Dual RNA sequencing (dRNA-Seq) of bacteria and their host cells

Bacterial pathogens subvert host cells by manipulating cellular pathways for survival and replication; in turn, host cells respond to the invading pathogen through cascading changes in gene expression. Deciphering these complex temporal and spatial dynamics to identify novel bacterial virulence factors or host response pathways is crucial for improved diagnostics and therapeutics. Dual RNA sequencing (dRNA-Seq) has recently been developed to simultaneously capture host and bacterial transcriptomes from an infected cell. This approach builds on the high sensitivity and resolution of RNA-Seq technology and is applicable to any bacteria that interact with eukaryotic cells, encompassing parasitic, commensal or mutualistic lifestyles. We pioneered dRNA-Seq to simultaneously capture prokaryotic and eukaryotic expression profiles of cells infected with bacteria, using in vitro Chlamydia-infected epithelial cells as proof of principle. Here we provide a detailed laboratory and bioinformatics protocol for dRNA-seq that is readily adaptable to any host-bacteria system of interest.

genomics↗