Biology subjects

Carter, G. P.

Publications and source records attributed to Carter, G. P..

13 recordsLinked to original sources

bioRxiv2026

Regulation and Characterization of the Cex system of enteric pathogens: a pathway for the lipidation and secretion of noncanoncial bacterial lipoproteins

CexE is an outer membrane lipoprotein of enterotoxigenic Escherichia coli (ETEC); one of the most prevalent etiological agents of diarrheal disease. Homologs of CexE are present in enteroaggregative E. coli, Citrobacter rodentium, and other enteropathogens. We have previously shown that expression of CexE is dependent upon Rns, the master virulence regulator of ETEC and a member of the AraC/XylS superfamily of transcription factors. In this study we report that the expression of cexD and cexPABC, the genes required for lipidation and delivery of CexE to the outer leaflet of the outer membrane, are also Rns-dependent. Although cexE and cexPABC are arranged in the same orientation, they are separated by an intergenic region sufficiently large to accommodate a promoter and transcription factor binding sites. Nevertheless, expression of the four gene cluster does not originate within the intergenic region. Rather, cexPABC are expressed from the Rns-dependent cexE promoter. The remaining gene of the system, cexD, is monocistronic and expressed from its own Rns-dependent promoter. Mutagenesis of a predicted Rns binding site upstream of cexD abolished Rns binding to a cexD promoter fragment in vitro and activation of the promoter in vivo. CexD is a novel bacterial acyltransferase. Unlike canonical bacterial acyltransferases that lipidate amino-terminal cysteines, CexD attaches a lipid to the amino-terminal glycine of CexE. Lipidation must occur in the periplasm because topological mapping revealed that CexD has six transmembrane domains with both its amino-terminus and enzymatic domain in the periplasm. We further show that CexD is required for CexE localization to the outer leaflet of the outer membrane and that a cexD mutant of C. rodentium is attenuated in a murine model. These findings further define the Rns virulence regulon, the enzymology of a noncanonical acyltranferase and its contributions to pathogenicity.

microbiology↗

bioRxiv2026

Viral Microglia Reprogramming Clears Oligomeric Neurotoxic Debris

Owing to pivotal roles in CNS debris clearance and homeostasis, microglia are central targets for the therapy of neurodegenerative diseases. Intricate proximity to neurons, the inherent danger of neuroimmune toxicity, and intrinsically high plasticity and adaptability, impose high hurdles on microglia modulation. Attenuated viruses are being tested extensively against CNS malignancies (i.e., cancer virotherapy); yet, aside from viral vector-mediated payload delivery, virotherapy for non-neoplastic CNS disease remains unexplored. Here we report disseminated targeting of microglia with the highly attenuated polio:rhinovirus chimera, PVSRIPO, that culminated in profound, durable microglia reprogramming. This phenotype, rooted in extended cytoplasmic viral (v)RNA replication, was non-cytopathogenic and did not yield virus progeny or dissemination. vRNA replication in microglia triggered selective interferon (IFN) regulatory factor (IRF) 3/IRF7 transcriptional programs in the relative absence of NF{kappa}B-driven proinflammatory cytokine responses and elicited robust phagocytosis of both tumor cells and amyloid-beta. Targeting of microglia with PVSRIPO mediated immunotherapy in a mouse glioma model and the clearance of oligomeric amyloid-beta deposits in an injectable model of neurotoxic amyloid accumulation. This work identifies attenuated virotherapy as an approach to safely and effectively invigorate microglia function in immune surveillance and neurotoxic debris clearance.

neuroscience↗

bioRxiv2025

Uncovering bacterial pseudaminylation with pan-specific antibody tools

Pseudaminic acids (Pse) are a family of carbohydrates found within bacterial lipopolysaccharides, capsular polysaccharides and glycoproteins that are critical for the virulence of human pathogens. However, a dearth of effective tools for detecting and enriching Pse has restricted study to only the most abundant Pse-containing glycoconjugates. Here, we devise a synthesis of - and {beta}-O-pseudaminylated glycopeptides to generate pan-specific monoclonal antibodies (mAbs) that recognise - and {beta}-configured Pse and its C8 epimer (8ePse) presented within glycans or directly linked to polypeptide backbones. Structural characterisation reveals the molecular basis of Pse recognition across a range of diverse chemical contexts. Using these mAbs, we establish a glycoproteomic platform to provide unprecedented depth in mapping the Pse glycome of Helicobacter pylori, Campylobacter jejuni, and Acinetobacter baumannii strains. Finally, we demonstrate that the mAbs recognise diverse capsule types in multidrug-resistant Acinetobacter baumannii and enhance phagocytosis to eliminate infections in mice.

biochemistry↗

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↗

bioRxiv2024

Enteropathogenic Providencia alcalifaciens: A subgroup of P. alcalifaciens that causes diarrhea.

Even though Providencia alcalifaciens is considered as a normal flora of the large intestine, there are reports of it causing diarrhea. In a previous study, a strain, 2939/90 obtained as a pure stool culture from a dead diarrheal patient was shown to cause invasion and actin condensation in mammalian cells, and diarrhea in a rabbit model. In a subsequent study, four TnphoA mutants of 2939/90 produced negligible invasion and actin condensation in mammalian cells. In the present study, the parent strain was sequenced by short-read and long-read sequencing, and the mutants by short read sequencing. In all four mutants, a TnphoA insertion was detected in the type three secretion system (T3SS) locus present on the largest of four plasmids (p2939_90_1) and not in a seemingly independent, functional T3SS locus on the chromosome. A survey of 52 genomes of P. alcalifaciens available in the public database identified the chromosomal T3SS locus in all strains, including both P. alcalifaciens genomic clades that we have classified as (group A) and (group B); a highly related gene layout and gene synteny flanking the locus suggested that these chromosomal loci are orthologous. There is a low sequence similarity between the chromosomal and plasmid-borne T3SS; a survey of plasmid T3SS showed its presence in only 21 of 52 genomes and mostly in group A genomes. Group A included several isolates from an outbreak of haemorrhagic diarrhea in dogs. Using prediction software (EffectiveDB), we detected several known and unknown effectors flanking the plasmid T3SS locus. The observation that TnphoA insertion only in the plasmid T3SS locus affected the invasion phenotype suggested that this locus is critical for causation of diarrhea. This leads us to conclude that a subgroup of P. alcalifaciens that possesses this plasmid-borne T3SS locus (in the case of strain, 2939/90) can cause diarrheal disease. We name this subgroup as enteropathogenic P. alcalifaciens (EPA). EPA should be included in future studies of etiology of diarrhea. A unique sequence that may be present in the T3SS locus in the plasmid may be investigated as a marker in a simple molecular test for diagnosis of EPA.

microbiology↗

bioRxiv2023

Inter-species gene flow drives ongoing evolution of Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis

Streptococcus dysgalactiae subsp. equisimilis (SDSE) is an emerging cause of human infection with invasive disease incidence and clinical manifestations comparable to the closely related species, Streptococcus pyogenes. Through systematic genomic analyses of 501 disseminated SDSE strains, we demonstrate extensive overlap between the genomes of SDSE and S. pyogenes. More than 75% of core genes are shared between the two species with one third demonstrating evidence of cross-species recombination. Twenty-five percent of mobile genetic element (MGE) clusters and 16 of 55 SDSE MGE insertion regions were found across species. Assessing potential cross-protection from leading S. pyogenes vaccine candidates on SDSE, 12/34 preclinical vaccine antigen genes were shown to be present in >99% of isolates of both species. Relevant to possible vaccine evasion, six vaccine candidate genes demonstrated evidence of inter-species recombination. These findings demonstrate previously unappreciated levels of genomic overlap between these closely related pathogens with implications for streptococcal pathobiology, disease surveillance and prevention.

microbiology↗

bioRxiv2019

Microbe-metabolite associations linked to the rebounding murine gut microbiome post-colonization with vancomycin resistant Enterococcus faecium

Vancomycin resistant Enterococcus faecium (VREfm) is an emerging antibiotic resistant pathogen. Strain-level investigations are beginning to reveal the molecular mechanisms used by VREfm to colonize regions of the human bowel. However, the role of commensal bacteria during VREfm colonization, in particular following antibiotic treatment, remains largely unknown. We employed amplicon 16S rRNA gene sequencing and metabolomics in a murine model system to try and investigate functional roles of the gut microbiome during VREfm colonization. First-order taxonomic shifts between Bacteroidetes and Tenerricutes within the gut microbial community composition were detected both in response to pretreatment using ceftriaxone, and to subsequent VREfm challenge. Using neural networking approaches to find co-occurrence profiles of bacteria and metabolites, we detected key metabolome features associated with butyric acid during and after VREfm colonization. These metabolite features were associated with Bacteroides, indicative of a transition towards a pre-antibiotic naive microbiome. This study shows the impacts of antibiotics on the gut ecosystem, and the progression of the microbiome in response to colonisation with VREfm. Our results offer insights towards identifying potential non-antibiotic alternatives to eliminate VREfm through metabolic re-engineering to preferentially select for Bacteroides. ImportanceThis study demonstrates the importance and power of linking bacterial composition profiling with metabolomics to find the interactions between commensal gut bacteria and a specific pathogen. Knowledge from this research will inform gut microbiome engineering strategies, with the aim of translating observations from animal models to human-relevant therapeutic applications.

microbiology↗

bioRxiv2019

Complete microbial genomes for public health in Australia and Southwest Pacific

Complete genomes of microbial pathogens are essential for the phylogenomic analyses that increasingly underpin core public health lab activities. Here, we present complete genomes of pathogen strains of regional importance to the Southwest Pacific and Australia. These enrich the catalogue of globally available complete genomes for public health while providing valuable strains to regional public health labs.\n\nAnnouncementWhole-genome sequence (WGS) data is increasingly important in public health microbiology (1-4). The data can be used to replicate many of the basic bacterial sub-typing approaches, as well as support epidemiological investigations, such as surveillance and outbreak investigation (5-7). The appeal of WGS data comes from the promise of a single workflow to process all microbial pathogens that can provide easily portable data that promotes deeper integration of surveillance and investigation efforts across jurisdictions. This promise is leading to a concerted effort to move microbial public health to a primarily genome-based workflow at numerous jurisdictions (8-10), including Australia (11).

genomics↗

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

Remodelling of pSK1 Family Plasmids and Enhanced Chlorhexidine Tolerance in Methicillin-Resistant Staphylococcus aureus

Staphylococcus aureus is a significant human pathogen whose evolution and adaptation has been shaped in part by mobile genetic elements (MGEs), facilitating global spread of extensive antimicrobial resistance. However, our understanding of the evolutionary dynamics surrounding MGEs is incomplete, in particular how changes in the structure of multidrug-resistant (MDR) plasmids may influence important staphylococcal phenotypes. Here, we undertook a population-and functional-genomics study of 212 methicillin-resistant S. aureus (MRSA) ST239 isolates collected over 32 years to explore the evolution of the pSK1 family of MDR plasmids, illustrating how these plasmids have co-evolved with and contributed to the successful adaptation of this persistent MRSA lineage. Using complete genomes and temporal phylogenomics we reconstructed the evolution of the pSK1 family lineage from its emergence in the late 1970s, with multiple structural variants arising. Plasmid maintenance and stability was linked to IS256- and IS257-mediated chromosomal integration and disruption of plasmid replication machinery. Overlaying genomic comparisons with phenotypic susceptibility data for gentamicin and chlorhexidine, it appeared that pSK1 has contributed to enhanced resistance in ST239 MRSA through two mechanisms: (i) acquisition of plasmid-borne resistance mechanisms increasing rates of gentamicin resistance and reduced chlorhexidine susceptibility, and (ii) changes in plasmid configuration linked with further enhancement of chlorhexidine tolerance. While the exact mechanism of enhanced tolerance remains elusive, this research has uncovered a clear evolutionary response of ST239 MRSA to chlorhexidine, one which may contribute to the ongoing persistence and adaptation of this lineage within healthcare institutions.

evolutionary biology↗

bioRxiv2018

The changing landscape of VREfm in Victoria, Australia: a state-wide genomic snapshot

Vancomycin-resistant Enterococcus faecium (VREfm) represent a major source of nosocomial infection worldwide. In Australia, the vanB genotype is dominant; however there has been a recent increase in the predominantly plasmid-encoded vanA genotype, prompting investigation into the genomic epidemiology of VREfm in this context.\n\nMaterials and MethodsA cross-sectional study of VREfm in Victoria, Australia (Nov.10th - Dec.9th, 2015). A total of 321 VREfm isolates (from 286 patients) were collected and whole-genome sequenced with Illumina NextSeq. Single nucleotide polymorphisms (SNPs) were used to assess relatedness. Multi-locus sequence types (STs), and genes associated with resistance and virulence were identified. The vanA-harbouring plasmid from an isolate from each ST was assembled using long-read data.\n\nResultsvanA-VREfm comprised 17.8% of isolates. ST203, ST80 and a pstS(-) clade, ST1421, predominated (30.5%, 30.5% and 37.2% of vanA-VREfm, respectively). Most vanB-VREfm were ST796 (77.7%). vanA-VREfm isolates were closely-related within hospitals vs. between them (core SNPs 10 [interquartile range 1-357] vs. 356 [179-416] respectively), suggesting discrete introductions of vanA-VREfm, with subsequent intra-hospital transmission. In contrast, vanB-VREfm had similar core SNP distributions within vs. between hospitals, due to widespread dissemination of ST796. Overall, vanA-harbouring plasmids differed across STs, and with exception of ST78 and ST796, Tn1546 transposons also varied.\n\nConclusionsvanA-VREfm in Victoria is associated with multiple STs, and is not solely mediated by a single shared plasmid/Tn1546 transposon; clonal transmission appears to play an important role, predominantly within, rather than between, hospitals. In contrast, vanB-VREfm appears to be well-established and widespread across Victorian healthcare institutions.

genomics↗

bioRxiv2017

Genome reconstruction and characterisation of extensively drug-resistant bacterial pathogens through direct metagenomic sequencing of human faeces

Whole-genome sequencing of microbial pathogens is revolutionising modern approaches to outbreaks of infectious diseases and is reliant upon organism culture. Culture-independent methods have shown promise in identifying pathogens, but high level reconstruction of microbial genomes from microbiologically complex samples for more in-depth analyses remains a challenge. Here, using metagenomic sequencing of a human faecal sample and analysis by tetranucleotide frequency profiling projected onto emergent self-organising maps, we were able to reconstruct the underlying populations of two extensively-drug resistant pathogens, Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae and vancomycin-resistant Enterococcus faecium. From these genomes, we were able to ascertain molecular typing results, such as MLST, and identify highly discriminatory mutations in the metagenome to distinguish closely related strains. These proof-of-principle results demonstrate the utility of clinical sample metagenomics to recover sequences of important drug-resistant bacteria and application of the approach in outbreak investigations, independent of the need to culture the organisms.

genomics↗

bioRxiv2017

A target-specific assay for rapid and quantitative detection of Mycobacterium chimaera DNA in environmental and clinical specimens

Mycobacterium chimaera is an opportunistic environmental mycobacterium, belonging to the Mycobacterium intracellulare complex. Although most commonly associated with pulmonary disease, there has been growing awareness of invasive M. chimaera infections following cardiac surgery. Investigations suggest world-wide spread of a specific M. chimaera clone, associated with contaminated hospital heater-cooler units used during the surgery. Given the global dissemination of this clone, its potential to cause invasive disease, and the laboriousness of current culture-based diagnostic methods, there is a pressing need to develop rapid and accurate diagnostic assays, specific for M. chimaera. Here, we assessed 354 mycobacterial genome sequences and confirmed that M. chimaera is a phylogenetically coherent group. In silico comparisons indicated six DNA regions present only in M. chimaera. We targeted one of these regions and developed a TaqMan qPCR assay for M. chimaera with a detection limit of 10 CFU in whole blood. In vitro screening against DNA extracted from 40 other mycobacteria and 22 bacterial species from 21 diverse genera confirmed in silico predicted specificity for M. chimaera. Screening 33 water samples from heater cooler units with this assay highlighted the increased sensitivity of PCR compared to culture, with 15 of 23 culture negative samples positive by M. chimaera qPCR. We have thus developed a robust molecular assay that can be readily and rapidly deployed to screen clinical and environmental specimens for M. chimaera.

microbiology↗