Microbiology & Immunology - Research Publications
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ItemTissue-associated and vertically transmitted bacterial symbiont in the coral Pocillopora acuta(OXFORD UNIV PRESS, 2024-01-08)Coral microhabitats are colonized by a myriad of microorganisms, including diverse bacteria which are essential for host functioning and survival. However, the location, transmission, and functions of individual bacterial species living inside the coral tissues remain poorly studied. Here, we show that a previously undescribed bacterial symbiont of the coral Pocillopora acuta forms cell-associated microbial aggregates (CAMAs) within the mesenterial filaments. CAMAs were found in both adults and larval offspring, suggesting vertical transmission. In situ laser capture microdissection of CAMAs followed by 16S rRNA gene amplicon sequencing and shotgun metagenomics produced a near complete metagenome-assembled genome. We subsequently cultured the CAMA bacteria from Pocillopora acuta colonies, and sequenced and assembled their genomes. Phylogenetic analyses showed that the CAMA bacteria belong to an undescribed Endozoicomonadaceae genus and species, which we propose to name Candidatus Sororendozoicomonas aggregata gen. nov sp. nov. Metabolic pathway reconstruction from its genome sequence suggests this species can synthesize most amino acids, several B vitamins, and antioxidants, and participate in carbon cycling and prey digestion, which may be beneficial to its coral hosts. This study provides detailed insights into a new member of the widespread Endozoicomonadaceae family, thereby improving our understanding of coral holobiont functioning. Vertically transmitted, tissue-associated bacteria, such as Sororendozoicomonas aggregata may be key candidates for the development of microbiome manipulation approaches with long-term positive effects on the coral host.
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ItemTackling antimicrobial resistance by integrating One Health and the Sustainable Development Goals(Springer Science and Business Media LLC, )Abstract Antimicrobial resistance (AMR) has been identified as a leading threat to global public health. One Health approaches that integrate sectors across human health, animal health, food production and the environment are essential to both addressing the growing threat of AMR and achieving the Sustainable Development Goals.
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ItemMosquitoes provide a transmission route between possums and humans for Buruli ulcer in southeastern Australia(NATURE PORTFOLIO, 2024-02)Buruli ulcer, a chronic subcutaneous infection caused by Mycobacterium ulcerans, is increasing in prevalence in southeastern Australia. Possums are a local wildlife reservoir for M. ulcerans and, although mosquitoes have been implicated in transmission, it remains unclear how humans acquire infection. We conducted extensive field survey analyses of M. ulcerans prevalence among mosquitoes in the Mornington Peninsula region of southeastern Australia. PCR screening of trapped mosquitoes revealed a significant association between M. ulcerans and Aedes notoscriptus. Spatial scanning statistics revealed overlap between clusters of M. ulcerans-positive Ae. notoscriptus, M. ulcerans-positive possum excreta and Buruli ulcer cases, and metabarcoding analyses showed individual mosquitoes had fed on humans and possums. Bacterial genomic analysis confirmed shared single-nucleotide-polymorphism profiles for M. ulcerans detected in mosquitoes, possum excreta and humans. These findings indicate Ae. notoscriptus probably transmit M. ulcerans in southeastern Australia and highlight mosquito control as a Buruli ulcer prevention measure.
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ItemCryo-EM structure of the extracellular domain of murine Thrombopoietin Receptor in complex with Thrombopoietin(NATURE PORTFOLIO, 2024-02-07)Thrombopoietin (Tpo) is the primary regulator of megakaryocyte and platelet numbers and is required for haematopoetic stem cell maintenance. Tpo functions by binding its receptor (TpoR, a homodimeric Class I cytokine receptor) and initiating cell proliferation or differentiation. Here we characterise the murine Tpo:TpoR signalling complex biochemically and structurally, using cryo-electron microscopy. Tpo uses opposing surfaces to recruit two copies of receptor, forming a 1:2 complex. Although it binds to the same, membrane-distal site on both receptor chains, it does so with significantly different affinities and its highly glycosylated C-terminal domain is not required. In one receptor chain, a large insertion, unique to TpoR, forms a partially structured loop that contacts cytokine. Tpo binding induces the juxtaposition of the two receptor chains adjacent to the cell membrane. The therapeutic agent romiplostim also targets the cytokine-binding site and the characterisation presented here supports the future development of improved TpoR agonists.
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ItemIncreasing HbA1c is associated with reduced CD8+ T cell functionality in response to influenza virus in a TCR-dependent manner in individuals with diabetes mellitus(SPRINGER BASEL AG, 2024-12)Diabetes mellitus is on the rise globally and is a known susceptibility factor for severe influenza virus infections. However, the mechanisms by which diabetes increases the severity of an influenza virus infection are yet to be fully defined. Diabetes mellitus is hallmarked by high glucose concentrations in the blood. We hypothesized that these high glucose concentrations affect the functionality of CD8+ T cells, which play a key role eliminating virus-infected cells and have been shown to decrease influenza disease severity. To study the effect of hyperglycemia on CD8+ T cell function, we stimulated peripheral blood mononuclear cells (PBMCs) from donors with and without diabetes with influenza A virus, anti-CD3/anti-CD28-coated beads, PMA and ionomycin (PMA/I), or an influenza viral peptide pool. After stimulation, cells were assessed for functionality [as defined by expression of IFN-γ, TNF-α, macrophage inflammatory protein (MIP)-1β, and lysosomal-associated membrane protein-1 (CD107a)] using flow cytometry. Our results showed that increasing HbA1c correlated with a reduction in TNF-α production by CD8+ T cells in response to influenza stimulation in a TCR-specific manner. This was not associated with any changes to CD8+ T cell subsets. We conclude that hyperglycemia impairs CD8+ T cell function to influenza virus infection, which may be linked with the increased risk of severe influenza in patients with diabetes.
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ItemNon-SARS-CoV-2 respiratory viral detection and whole genome sequencing from COVID-19 rapid antigen test devices: a laboratory evaluation study.(Elsevier BV, 2024-02-07)BACKGROUND: There has been high uptake of rapid antigen test device use for point-of-care COVID-19 diagnosis. Individuals who are symptomatic but test negative on COVID-19 rapid antigen test devices might have a different respiratory viral infection. We aimed to detect and sequence non-SARS-CoV-2 respiratory viruses from rapid antigen test devices, which could assist in the characterisation and surveillance of circulating respiratory viruses in the community. METHODS: We applied archival clinical nose and throat swabs collected between Jan 1, 2015, and Dec 31, 2022, that previously tested positive for a common respiratory virus (adenovirus, influenza, metapneumovirus, parainfluenza, rhinovirus, respiratory syncytial virus [RSV], or seasonal coronavirus; 132 swabs and 140 viral targets) on PCR to two commercially available COVID-19 rapid antigen test devices, the Panbio COVID-19 Ag Rapid Test Device and Roche SARS-CoV-2 Antigen Self-Test. In addition, we collected 31 COVID-19 rapid antigen test devices used to test patients who were symptomatic at The Royal Melbourne Hospital emergency department in Melbourne, Australia. We extracted total nucleic acid from the device paper test strips and assessed viral recovery using multiplex real-time PCR (rtPCR) and capture-based whole genome sequencing. Sequence and genome data were analysed through custom computational pipelines, including subtyping. FINDINGS: Of the 140 respiratory viral targets from archival samples, 89 (64%) and 88 (63%) were positive on rtPCR for the relevant taxa following extraction from Panbio or Roche rapid antigen test devices, respectively. Recovery was variable across taxa: we detected influenza A in nine of 18 samples from Panbio and seven of 18 from Roche devices; parainfluenza in 11 of 20 samples from Panbio and 12 of 20 from Roche devices; human metapneumovirus in 11 of 16 from Panbio and 14 of 16 from Roche devices; seasonal coronavirus in eight of 19 from Panbio and two of 19 from Roche devices; rhinovirus in 24 of 28 from Panbio and 27 of 28 from Roche devices; influenza B in four of 15 in both devices; and RSV in 16 of 18 in both devices. Of the 31 COVID-19 devices collected from The Royal Melbourne Hospital emergency department, 11 tested positive for a respiratory virus on rtPCR, including one device positive for influenza A virus, one positive for RSV, four positive for rhinovirus, and five positive for SARS-CoV-2. Sequences of target respiratory viruses from archival samples were detected in 55 (98·2%) of 56 samples from Panbio and 48 (85·7%) of 56 from Roche rapid antigen test devices. 98 (87·5%) of 112 viral genomes were completely assembled from these data, enabling subtyping for RSV and influenza viruses. All 11 samples collected from the emergency department had viral sequences detected, with near-complete genomes assembled for influenza A and RSV. INTERPRETATION: Non-SARS-CoV-2 respiratory viruses can be detected and sequenced from COVID-19 rapid antigen devices. Recovery of near full-length viral sequences from these devices provides a valuable opportunity to expand genomic surveillance programmes for public health monitoring of circulating respiratory viruses. FUNDING: Australian Government Medical Research Future Fund and Australian National Health and Medical Research Council.
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ItemInterim results from a phase I randomized, placebo-controlled trial of novel SARS-CoV-2 beta variant receptor-binding domain recombinant protein and mRNA vaccines as a 4th dose booster(ELSEVIER, 2023-12)BACKGROUND: SARS-CoV-2 booster vaccination should ideally enhance protection against variants and minimise immune imprinting. This Phase I trial evaluated two vaccines targeting SARS-CoV-2 beta-variant receptor-binding domain (RBD): a recombinant dimeric RBD-human IgG1 Fc-fusion protein, and an mRNA encoding a membrane-anchored RBD. METHODS: 76 healthy adults aged 18-64 y, previously triple vaccinated with licensed SARS-CoV-2 vaccines, were randomised to receive a 4th dose of either an adjuvanted (MF59®, CSL Seqirus) protein vaccine (5, 15 or 45 μg, N = 32), mRNA vaccine (10, 20, or 50 μg, N = 32), or placebo (saline, N = 12) at least 90 days after a 3rd boost vaccination or SARS-CoV-2 infection. Bleeds occurred on days 1 (prior to vaccination), 8, and 29. CLINICALTRIALS: govNCT05272605. FINDINGS: No vaccine-related serious or medically-attended adverse events occurred. The protein vaccine reactogenicity was mild, whereas the mRNA vaccine was moderately reactogenic at higher dose levels. Best anti-RBD antibody responses resulted from the higher doses of each vaccine. A similar pattern was seen with live virus neutralisation and surrogate, and pseudovirus neutralisation assays. Breadth of immune response was demonstrated against BA.5 and more recent omicron subvariants (XBB, XBB.1.5 and BQ.1.1). Binding antibody titres for both vaccines were comparable to those of a licensed bivalent mRNA vaccine. Both vaccines enhanced CD4+ and CD8+ T cell activation. INTERPRETATION: There were no safety concerns and the reactogenicity profile was mild and similar to licensed SARS-CoV-2 vaccines. Both vaccines showed strong immune boosting against beta, ancestral and omicron strains. FUNDING: Australian Government Medical Research Future Fund, and philanthropies Jack Ma Foundation and IFM investors.
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ItemSystemic inflammatory response syndrome triggered by blood-borne pathogens induces prolonged dendritic cell paralysis and immunosuppression(CELL PRESS, 2024-02-27)Blood-borne pathogens can cause systemic inflammatory response syndrome (SIRS) followed by protracted, potentially lethal immunosuppression. The mechanisms responsible for impaired immunity post-SIRS remain unclear. We show that SIRS triggered by pathogen mimics or malaria infection leads to functional paralysis of conventional dendritic cells (cDCs). Paralysis affects several generations of cDCs and impairs immunity for 3-4 weeks. Paralyzed cDCs display distinct transcriptomic and phenotypic signatures and show impaired capacity to capture and present antigens in vivo. They also display altered cytokine production patterns upon stimulation. The paralysis program is not initiated in the bone marrow but during final cDC differentiation in peripheral tissues under the influence of local secondary signals that persist after resolution of SIRS. Vaccination with monoclonal antibodies that target cDC receptors or blockade of transforming growth factor β partially overcomes paralysis and immunosuppression. This work provides insights into the mechanisms of paralysis and describes strategies to restore immunocompetence post-SIRS.
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ItemIon Mobility-Based Enrichment-Free N-Terminomics Analysis Reveals Novel Legumain Substrates in Murine Spleen.(Elsevier BV, 2024-02)Aberrant levels of the asparaginyl endopeptidase legumain have been linked to inflammation, neurodegeneration, and cancer, yet our understanding of this protease is incomplete. Systematic attempts to identify legumain substrates have been previously confined to in vitro studies, which fail to mirror physiological conditions and obscure biologically relevant cleavage events. Using high-field asymmetric waveform ion mobility spectrometry (FAIMS), we developed a streamlined approach for proteome and N-terminome analyses without the need for N-termini enrichment. Compared to unfractionated proteomic analysis, we demonstrate FAIMS fractionation improves N-termini identification by >2.5 fold, resulting in the identification of >2882 unique N-termini from limited sample amounts. In murine spleens, this approach identifies 6366 proteins and 2528 unique N-termini, with 235 cleavage events enriched in WT compared to legumain-deficient spleens. Among these, 119 neo-N-termini arose from asparaginyl endopeptidase activities, representing novel putative physiological legumain substrates. The direct cleavage of selected substrates by legumain was confirmed using in vitro assays, providing support for the existence of physiologically relevant extra-lysosomal legumain activity. Combined, these data shed critical light on the functions of legumain and demonstrate the utility of FAIMS as an accessible method to improve depth and quality of N-terminomics studies.
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ItemWhole genome sequencing of drug-resistant Mycobacterium tuberculosis isolates in Victoria, Australia(ELSEVIER SCI LTD, 2024-01)OBJECTIVES: Whole genome sequencing (WGS) can identify clusters, transmission patterns, and drug resistance mutations. This is important in low-burden settings such as Australia, as it can assist in efficient contact tracing and surveillance. METHODS: We conducted a retrospective cohort study using WGS from 155 genomically defined drug-resistant Mycobacterium tuberculosis (DR-TB) isolates collected between 2018-2021 in Victoria, Australia. Bioinformatic analysis was performed to identify resistance-conferring mutations, lineages, clusters and understand how local sequences compared with international context. RESULTS: Of the 155 sequences, 42% were identified as lineage 2 and 35% as lineage 1; 65.8% (102/155) were isoniazid mono-resistant, 8.4% were multi-drug resistant TB and 5.8% were pre-extensively drug-resistant / extensively drug-resistant TB. The most common mutations were observed in katG and fabG1 genes, especially at Ser315Thr and fabG1 -15 C>T for first-line drugs. Ser450Leu was the most frequent mutation in rpoB gene. Phylogenetic analysis confirmed that Victorian DR-TB were associated with importation events. There was little evidence of local transmission with only five isolate pairs. CONCLUSION: Isoniazid-resistant TB is the commonest DR-TB in Victoria, and the mutation profile is similar to global circulating DR-TB. Most cases are diagnosed among migrants with limited transmission. This study highlights the value of WGS in identification of clusters and resistance-conferring mutations. This information is crucial in supporting disease mitigation and treatment strategies.