Biology subjects

Monk, I. R.

Publications and source records attributed to Monk, I. R..

10 recordsLinked to original sources

bioRxiv2026

Mutations in mfd cause Staphylococcus aureus mucoid hyper-biofilm phenotype in chronic rhinosinusitis

Chronic Rhinosinusitis (CRS) is a common chronic inflammation of the paranasal sinus mucosa. Staphylococcus aureus contributes to its severity through biofilm formation. In this study, we isolated eight sequential methicillin-resistant S. aureus (MRSA) isolates from a patient with severe CRS over a period of 672 days (T1-T8). The isolates were phenotypically and genomically characterised, and the extracellular biofilm proteome analysed. We identified an accumulation of mutations that included the acquisition of an IS21 family insertion sequence inactivating the icaR gene and nucleotide variants in various genes including the transcription repair coupling factor (mfd). The genomic changes were associated with a switch to a mucoid phenotype from T3 onwards (Day 178), with a significant increase in biofilm-forming capacity and the secretion of multiple enterotoxins. Targeted mutagenesis confirmed mfd is a regulator of strain mucoidy with enhanced biofilm and enterotoxin production. These findings support mfd as a target for novel anti-virulence therapies.

microbiology↗

bioRxiv2025

Phosphorylation by the PknB serine/threonine kinase stimulates dimer formation by WalR

WalKR is a two-component system that regulates cell wall homeostasis and other processes in low-GC Gram-positive bacteria. It is essential for viability in Staphylococcus aureus and regulates genes that encode the autolysins and other critical proteins. The sensor kinase WalK phosphorylates WalR at aspartic acid residue 53 (D53) in the receiver domain, stimulating promoter DNA binding. Unlike other response regulators, WalR is thought to have a second kinase, the serine/threonine kinase PknB, which phosphorylates the receiver domain at threonine residue 101. We previously reported that a walR mutation that changed T101 to a methionine conferred low level resistance to vancomycin and greatly increased susceptibility to tunicamycin along with several other phenotypic traits. In this work we demonstrate similarities between pknB null and the WalRT101M mutants that support a regulatory role for PknB-mediated phosphorylation of WalR. Using a combination of in vitro and in vivo approaches, we confirm the specificity of this posttranslational modification and confirm that phosphorylation at both D53 and T101 is important for dimer formation. Importantly, using an in silico approach, we have discovered that phosphorylation at T101 creates an intermolecular hydrogen bond between the phosphate group and E108, that strengthens contacts along the WalR dimerization interface. This is the first time that a clear molecular role has been assigned to this posttranslational modification and it is the strongest molecular evidence to date for a direct functional relationship between PknB and WalR. We propose that, together with primary activation by WalK, PknB serves as a second potentiating input into the WalKR system, stimulating WalR dimer formation, and target DNA binding and tethering this transcriptional event to important extracellular stimuli.

microbiology↗

bioRxiv2025

Regulation of airway fumarate by host and pathogen promotes S. aureus pneumonia

Staphylococcus aureus is a leading cause of healthcare-associated pneumonia, contributing significantly to morbidity and mortality worldwide. As a ubiquitous colonizer of the upper respiratory tract, S. aureus must undergo substantial metabolic adaptation to achieve persistent infection in the distinctive microenvironment of the lung. We observed that fumC, which encodes the enzyme that converts fumarate to malate, is highly conserved with low mutation rates in S. aureus isolates from chronic lung infections. Fumarate, a pro-inflammatory metabolite produced by macrophages during infection, is regulated by the host fumarate hydratase (FH) to limit inflammation. Here, we demonstrate that fumarate, which accumulates in the chronically infected lung, is detrimental to S. aureus, blocking primary metabolic pathways such as glycolysis and oxidative phosphorylation (OXPHOS). This creates a metabolic bottleneck that drives staphylococcal FH (FumC) activity for airway adaptation. FumC not only degrades fumarate but also directs its utilization into critical pathways including the tricarboxylic acid (TCA) cycle, gluconeogenesis and hexosamine synthesis to maintain metabolic fitness and form a protective biofilm. Itaconate, another abundant immunometabolite in the infected airway enhances FumC activity, in synergy with fumarate. In a mouse model of pneumonia, a {Delta}fumC mutant displays significant attenuation compared to its parent and complemented strains, particularly in fumarate- and itaconate-replete conditions. Our findings underscore the pivotal role of immunometabolites in promoting S. aureus pulmonary adaptation.

microbiology↗

bioRxiv2025

Transposon-directed insertion-site sequencing (TraDIS) analysis of Enterococcus faecium using nanopore sequencing and a WebAssembly analysis platform

Vancomycin-resistant Enterococcus faecium (VREfm) are healthcare-associated opportunistic pathogens of global significance. Genetic tools are needed to understand the molecular basis for VREfm clinically relevant phenotypes, such as persistence within the human gut or antimicrobial resistance. Here, we present a transposon-directed insertion-site sequencing (TraDIS) platform optimised for E. faecium. We engineered a transposon delivery plasmid, pIMTA(tetM), that can generate high density transposon mutant libraries, combined with Oxford Nanopore Technology amplicon sequencing to map the transposon insertion sites. We have also customised a bioinformatic analysis suite that includes a WebAssembly-powered visualisation tool called Diana, for TraDIS data exploration and analysis (https://diana.cpg.org.au/). To demonstrate performance of our platform, we assessed the impact of vancomycin exposure on a library of 48,458 unique transposon mutants. As expected, we could confirm the importance of the vanB operon for VREfm vancomycin resistance. Our end-to-end platform for running TraDIS experiments in VREfm will permit accessible, genome-scale, forward genetic screens to probe molecular mechanisms of persistence and pathogenesis. ImportanceThere are limited genetic tools specifically developed and optimized for function in Enterococcus faecium. Here, we addressed this gap through the development of a transposon-directed insertion-site sequencing (TraDIS) platform with a plasmid we engineered to specifically function in E. faecium. The application of nanopore sequencing, with a highly accessible sequence data processing and bioinformatic analysis pipeline streamlines and simplifies the methodology. These developments will allow the functional genomic analysis of important traits involved in the pathobiology of this understudied bacterium. The approach and tools we have described here are likely applicable to other Gram-positive bacteria.

microbiology↗

bioRxiv2025

Are reads required? High-precision variant calling from bacterial genome assemblies

1.Accurate nucleotide variant calling is essential in microbial genomics, particularly for outbreak tracking and phylogenetics. This study evaluates variant calls derived from genome assemblies compared to traditional read-based variant-calling methods, using seven closely related Staphylococcus aureus isolates sequenced on Illumina and Oxford Nanopore Technologies platforms. By benchmarking multiple assembly and variant-calling pipelines against a ground truth dataset, we found that read-based methods consistently achieved high accuracy. Assembly-based approaches performed well in some cases but were highly dependent on assembly quality, as errors in the assembly led to false-positive variant calls. These findings underscore the need for improved assembly techniques before the potential benefits of assembly-based variant calling - such as reduced computational requirements and simpler data management - can be realised. 2. Impact statementVariant calling is foundational to microbial genomics, yet traditional workflows rely heavily on sequencing reads, which for a typical bacterial genome can be hundreds of megabytes. In contrast, genome assemblies are far smaller - usually just a few megabytes - making them significantly easier to manage. If accurate variant calls could be made directly from assemblies, this would reduce computational demands and, in some cases, may even eliminate the need to retain raw sequencing reads. This study addresses the key question of whether variant calling from assemblies is accurate enough to replace read-based methods. Our findings show that while assembly-based variant calling can achieve high accuracy, this is only possible with error-free assemblies. Since most assemblies contain errors, assembly-based variant-calling approaches should currently be used with caution. Nevertheless, as sequencing and assembly technologies continue to advance, improved assembly accuracy may make assembly-based variant calling a viable alternative, reducing data complexity and storage demands while streamlining microbial genomic analyses. 3. Data summarySupplementary methods, data, figures and tables are available at github.com/rrwick/Are-reads-required which is also archived on Zenodo (10.5281/zenodo.14868870). Reads and assemblies are available via BioProject PRJNA1193226.

bioinformatics↗

bioRxiv2024

A marine sponge-associated mycobacterium closely related to Mycobacterium tuberculosis

Reconstructing the evolutionary origins of Mycobacterium tuberculosis, the causative agent of human tuberculosis, has helped identify bacterial factors that have led to the tubercle bacillus becoming such a formidable human pathogen. Here we report the discovery and detailed characterization of an exceedingly slow growing mycobacterium that is closely related to M. tuberculosis for which we have proposed the species name Mycobacterium spongiae sp. nov., (strain ID: FSD4b-SM). The bacterium was isolated from a marine sponge, taken from the waters of the Great Barrier Reef in Queensland, Australia. Comparative genomics revealed that, after the opportunistic human pathogen Mycobacterium decipiens, M. spongiae is the most closely related species to the M. tuberculosis complex reported to date, with 80% shared average nucleotide identity and extensive conservation of key M. tuberculosis virulence factors, including intact ESX secretion systems and associated effectors. Proteomic and lipidomic analyses showed that these conserved systems are functional in FSD4b-SM, but that it also produces cell wall lipids not previously reported in mycobacteria. We investigated the virulence potential of FSD4b-SM in mice and found that, while the bacteria persist in lungs for 56 days after intranasal infection, no overt pathology was detected. The similarities with M. tuberculosis, together with its lack of virulence, motivated us to investigate the potential of FSD4b-SM as a vaccine strain and as a genetic donor of the ESX-1 genetic locus to improve BCG immunogenicity. However, neither of these approaches resulted in superior protection against M. tuberculosis challenge compared to BCG vaccination alone. The discovery of M. spongiae adds to our understanding of the emergence of the M. tuberculosis complex and it will be another useful resource to refine our understanding of the factors that shaped the evolution and pathogenesis of M. tuberculosis.

microbiology↗

bioRxiv2023

The WalKR two-component regulator coordinates cell wall homeostasis with DNA replication in Staphylococcus aureus

Among the 16 two-component systems (TCSs) in the opportunistic human pathogen Staphylococcus aureus, only WalKR is essential. Like orthologous systems in other Bacillota, S. aureus WalKR controls autolysins involved in peptidoglycan remodelling and is therefore intimately involved in cell division. However, despite the importance of WalKR in S. aureus, the basis for its essentiality is not understood and the regulon poorly defined. Here, we defined a consensus WalR DNA-binding motif and the direct WalKR regulon by using functional genomics, including ChIP-seq, with a panel of isogenic walKR mutants that had a spectrum of altered activities. Consistent with prior findings, the direct regulon includes multiple autolysin genes. However, this work also revealed that WalR directly regulates at least five essential genes involved in lipoteichoic acid synthesis (ltaS); translation (rplK); DNA compaction (hup); initiation of DNA replication (dnaA, hup); and purine nucleotide metabolism (prs). Thus, WalKR in S. aureus serves as a polyfunctional regulator that contributes to fundamental control over critical cell processes by co-ordinately linking cell wall homeostasis with purine biosynthesis, protein biosynthesis, and DNA replication. Collectively, our findings address the essentiality of this locus and highlight the importance of WalKR as a bona fide target for novel anti-staphylococcal therapeutics.

microbiology↗

bioRxiv2020

Validation of a Single-step, Single-tube Reverse Transcription-Loop-Mediated Isothermal Amplification Assay for Rapid Detection of SARS-CoV-2 RNA

2.IntroductionThe SARS-CoV-2 pandemic of 2020 has resulted in unparalleled requirements for RNA extraction kits and enzymes required for virus detection, leading to global shortages. This has necessitated the exploration of alternative diagnostic options to alleviate supply chain issues. AimTo establish and validate a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SARS-CoV-2 from nasopharyngeal swabs. MethodologyWe used a commercial RT-LAMP mastermix from OptiGene Ltd in combination with a primer set designed to detect the CDC N1 region of the SARS-CoV-2 nucleocapsid (N) gene. A single-tube, single-step fluorescence assay was implemented whereby as little as 1 L of universal transport medium (UTM) directly from a nasopharyngeal swab could be used as template, bypassing the requirement for RNA purification. Amplification and detection could be conducted in any thermocycler capable of holding 65{degrees}C for 30 minutes and measure fluorescence in the FAM channel at one-minute intervals. ResultsAssay evaluation by assessment of 157 clinical specimens previously screened by E-gene RT-qPCR revealed assay sensitivity and specificity of 87% and 100%, respectively. Results were fast, with an average time-to-positive (Tp) for 93 clinical samples of 14 minutes (SD {+/-}7 minutes). Using dilutions of SARS-CoV-2 virus spiked into UTM, we also evaluated assay performance against FDA guidelines for implementation of emergency-use diagnostics and established a limit-of-detection of 54 Tissue Culture Infectious Dose 50 per ml (TCID50 mL-1), with satisfactory assay sensitivity and specificity. A comparison of 20 clinical specimens between four laboratories showed excellent interlaboratory concordance; performing equally well on three different, commonly used thermocyclers, pointing to the robustness of the assay. ConclusionWith a simplified workflow, N1-STOP-LAMP is a powerful, scalable option for specific and rapid detection of SARS-CoV-2 and an additional resource in the diagnostic armamentarium against COVID-19. 3. Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

microbiology↗

bioRxiv2019

Functional genomics reveals extensive diversity in Staphylococcus epidermidis restriction modification systems compared to Staphylococcus aureus.

Staphylococcus epidermidis is a significant opportunistic pathogen of humans. Molecular studies in this species have been hampered by the presence of restriction-modification (RM) systems that limit introduction of foreign DNA. Here we establish the complete genomes and methylomes for seven clinically significant, genetically diverse S. epidermidis isolates and perform the first systematic genomic analyses of the type I RM systems within both S. epidermidis and Staphylococcus aureus. Our analyses revealed marked differences in the gene arrangement, chromosomal location and movement of type I RM systems between the two species. Unlike S. aureus, S. epidermidis type I RM systems demonstrate extensive diversity even within a single genetic lineage. This is contrary to current assumptions and has important implications for approaching the genetic manipulation of S. epidermidis. Using Escherichia coli plasmid artificial modification (PAM) to express S. epidermidis hsdMS, we readily overcame restriction barriers in S. epidermidis, and achieved transformation efficiencies equivalent to those of modification deficient mutants. With these functional experiments we demonstrate how genomic data can be used to predict both the functionality of type I RM systems and the potential for a strain to be transformation proficient. We outline an efficient approach for the genetic manipulation of S. epidermidis from diverse genetic backgrounds, including those that have hitherto been intractable. Additionally, we identified S. epidermidis BPH0736, a naturally restriction defective, clinically significant, multidrug-resistant ST2 isolate as an ideal candidate for molecular studies.\n\nImportanceStaphylococcus epidermidis is a major cause of hospital-acquired infections, especially those related to implanted medical devices. Understanding how S. epidermidis causes disease and devising ways to combat these infections has been hindered by an inability to genetically manipulate \"hospital-adapted\" strains that cause clinical disease. Here we provide the first comprehensive analyses of the mechanisms whereby S. epidermidis resists the uptake of foreign DNA and demonstrate that these are distinct from those described for S. aureus. Until now it had been assumed that these are the same. Using these insights, we demonstrate an efficient approach for the genetic manipulation of S. epidermidis to enable the study of clinically relevant isolates for the first time.

microbiology↗

bioRxiv2018

Zinc-binding to the cytoplasmic PAS domain regulates the essential WalK histidine kinase of Staphylococcus aureus

WalKR (YycFG) is the only essential two-component regulator in the human pathogen Staphylococcus aureus. WalKR regulates peptidoglycan synthesis, but this function alone appears not to explain its essentiality. To understand WalKR function we investigated a suppressor mutant that arose when WalKR activity was impaired; a single histidine to tryptophan substitution (H271Y) in the cytoplasmic Per-Arnt-Sim (PASCYT) domain of the histidine kinase WalK. Introduction of the WalKH271Y mutation into wild-type S. aureus activated the WalKR regulon. Structural analyses of the WalK PASCYT domain revealed a hitherto unknown metal binding site, in which a zinc ion (Zn2+) was tetrahedrally-coordinated by four amino acid residues including H271. The WallkH271Y mutation abrogated metal binding, increasing WalK kinase activity and WalR phosphorylation. Thus, Zn2+-binding negatively regulates WalKR activity. Identification of a metal ligand sensed by the WalKR system substantially expands our understanding of this critical S. aureus regulon.

microbiology↗