Microbiology & Immunology - Research Publications

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    Bioinformatic investigation of discordant sequence data for SARS- CoV-2: insights for robust genomic analysis during pandemic surveillance
    Zufan, SE ; Lau, KA ; Donald, A ; Hoang, T ; Foster, CSP ; Sikazwe, C ; Theis, T ; Rawlinson, WD ; Ballard, SA ; Stinear, TP ; Howden, BP ; Jennison, AV ; Seemann, T (MICROBIOLOGY SOC, 2023-11)
    The COVID-19 pandemic has necessitated the rapid development and implementation of whole-genome sequencing (WGS) and bioinformatic methods for managing the pandemic. However, variability in methods and capabilities between laboratories has posed challenges in ensuring data accuracy. A national working group comprising 18 laboratory scientists and bioinformaticians from Australia and New Zealand was formed to improve data concordance across public health laboratories (PHLs). One effort, presented in this study, sought to understand the impact of the methodology on consensus genome concordance and interpretation. SARS-CoV-2 WGS proficiency testing programme (PTP) data were retrospectively obtained from the 2021 Royal College of Pathologists of Australasia Quality Assurance Programmes (RCPAQAP), which included 11 participating Australian laboratories. The submitted consensus genomes and reads from eight contrived specimens were investigated, focusing on discordant sequence data and findings were presented to the working group to inform best practices. Despite using a variety of laboratory and bioinformatic methods for SARS-CoV-2 WGS, participants largely produced concordant genomes. Two participants returned five discordant sites in a high-Cτ replicate, which could be resolved with reasonable bioinformatic quality thresholds. We noted ten discrepancies in genome assessment that arose from nucleotide heterogeneity at three different sites in three cell-culture-derived control specimens. While these sites were ultimately accurate after considering the participants' bioinformatic parameters, it presented an interesting challenge for developing standards to account for intrahost single nucleotide variation (iSNV). Observed differences had little to no impact on key surveillance metrics, lineage assignment and phylogenetic clustering, while genome coverage <90 % affected both. We recommend PHLs bioinformatically generate two consensus genomes with and without ambiguity thresholds for quality control and downstream analysis, respectively, and adhere to a minimum 90 % genome coverage threshold for inclusion in surveillance interpretations. We also suggest additional PTP assessment criteria, including primer efficiency, detection of iSNVs and minimum genome coverage of 90 %. This study underscores the importance of multidisciplinary national working groups in informing guidelines in real time for bioinformatic quality acceptance criteria. It demonstrates the potential for enhancing public health responses through improved data concordance and quality control in SARS-CoV-2 genomic analysis during pandemic surveillance.
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    Functional potential and evolutionary response to long-term heat selection of bacterial associates of coral photosymbionts
    Maire, J ; Philip, GK ; Livingston, J ; Judd, LM ; Blackall, LL ; van Oppen, MJH ; Wilkins, LGE (AMER SOC MICROBIOLOGY, 2023-12-21)
    Symbiotic microorganisms are crucial for the survival of corals and their resistance to coral bleaching in the face of climate change. However, the impact of microbe-microbe interactions on coral functioning is mostly unknown but could be essential factors for coral adaption to future climates. Here, we investigated interactions between cultured dinoflagellates of the Symbiodiniaceae family, essential photosymbionts of corals, and associated bacteria. By assessing the genomic potential of 49 bacteria, we found that they are likely beneficial for Symbiodiniaceae, through the production of B vitamins and antioxidants. Additionally, bacterial genes involved in host-symbiont interactions, such as secretion systems, accumulated mutations following long-term exposure to heat, suggesting symbiotic interactions may change under climate change. This highlights the importance of microbe-microbe interactions in coral functioning.
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    Kidney organoids reveal redundancy in viral entry pathways during ACE2-dependent SARS-CoV-2 infection
    Vanslambrouck, JM ; Neil, JA ; Rudraraju, R ; Mah, S ; Tan, KS ; Groenewegen, E ; Forbes, TA ; Karavendzas, K ; Elliott, DA ; Porrello, ER ; Subbarao, K ; Little, MH ; Liu, S-L (AMER SOC MICROBIOLOGY, 2024-03-19)
    UNLABELLED: With a high incidence of acute kidney injury among hospitalized COVID-19 patients, considerable attention has been focussed on whether SARS-CoV-2 specifically targets kidney cells to directly impact renal function, or whether renal damage is primarily an indirect outcome. To date, several studies have utilized kidney organoids to understand the pathogenesis of COVID-19, revealing the ability for SARS-CoV-2 to predominantly infect cells of the proximal tubule (PT), with reduced infectivity following administration of soluble ACE2. However, the immaturity of standard human kidney organoids represents a significant hurdle, leaving the preferred SARS-CoV-2 processing pathway, existence of alternate viral receptors, and the effect of common hypertensive medications on the expression of ACE2 in the context of SARS-CoV-2 exposure incompletely understood. Utilizing a novel kidney organoid model with enhanced PT maturity, genetic- and drug-mediated inhibition of viral entry and processing factors confirmed the requirement for ACE2 for SARS-CoV-2 entry but showed that the virus can utilize dual viral spike protein processing pathways downstream of ACE2 receptor binding. These include TMPRSS- and CTSL/CTSB-mediated non-endosomal and endocytic pathways, with TMPRSS10 likely playing a more significant role in the non-endosomal pathway in renal cells than TMPRSS2. Finally, treatment with the antihypertensive ACE inhibitor, lisinopril, showed negligible impact on receptor expression or susceptibility of renal cells to infection. This study represents the first in-depth characterization of viral entry in stem cell-derived human kidney organoids with enhanced PTs, providing deeper insight into the renal implications of the ongoing COVID-19 pandemic. IMPORTANCE: Utilizing a human iPSC-derived kidney organoid model with improved proximal tubule (PT) maturity, we identified the mechanism of SARS-CoV-2 entry in renal cells, confirming ACE2 as the sole receptor and revealing redundancy in downstream cell surface TMPRSS- and endocytic Cathepsin-mediated pathways. In addition, these data address the implications of SARS-CoV-2 exposure in the setting of the commonly prescribed ACE-inhibitor, lisinopril, confirming its negligible impact on infection of kidney cells. Taken together, these results provide valuable insight into the mechanism of viral infection in the human kidney.
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    A CAF01-adjuvanted whole asexual blood-stage liposomal malaria vaccine induces a CD4+ T-cell-dependent strain-transcending protective immunity in rodent models
    Okoth, WA ; Ho, M-F ; Zaman, M ; Cooper, E ; Som, P ; Burgess, M ; Walton, M ; Nevagi, RJ ; Beattie, L ; Murphy, D ; Stanisic, DI ; Good, MF ; Miller, LH (AMER SOC MICROBIOLOGY, 2023-12-19)
    Malaria is a devastating disease that has claimed many lives, especially children <5 years of age in Sub-Saharan Africa, as documented in World Malaria Reports by WHO. Even though vector control and chemoprevention tools have helped with elimination efforts in some, if not all, endemic areas, these efforts have been hampered by serious issues (including drug and insecticide resistance and disruption to social cohesion caused by the COVID-19 pandemic). Development of an effective malaria vaccine is the alternative preventative tool in the fight against malaria. Vaccines save millions of lives each year and have helped in elimination and/or eradication of global diseases. Development of a highly efficacious malaria vaccine that will ensure long-lasting protective immunity will be a "game-changing" prevention strategy to finally eradicate the disease. Such a vaccine will need to counteract the significant obstacles that have been hampering subunit vaccine development to date, including antigenic polymorphism, sub-optimal immunogenicity, and waning vaccine efficacy.
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    High-performance enrichment-based genome sequencing to support the investigation of hepatitis A virus outbreaks
    Zufan, SE ; Mercoulia, K ; Kwong, JC ; Judd, LM ; Howden, BP ; Seemann, T ; Stinear, TP ; Rantsiou, K (AMER SOC MICROBIOLOGY, 2024-01-11)
    This proof-of-concept study introduces a hybrid capture oligo panel for whole-genome sequencing of all six human pathogenic hepatitis A virus (HAV) subgenotypes, exhibiting a higher sensitivity than some conventional genotyping assays. The ability of hybrid capture to enrich multiple targets allows for a single, streamlined workflow, thus facilitating the potential harmonization of molecular surveillance of HAV with other enteric viruses. Even challenging sample matrices can be accommodated, making them suitable for broad implementation in clinical and public health laboratories. This innovative approach has significant implications for enhancing multijurisdictional outbreak investigations as well as our understanding of the global diversity and transmission dynamics of HAV.
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    Antiviral Wolbachia strains associate with Aedes aegypti endoplasmic reticulum membranes and induce lipid droplet formation to restrict dengue virus replication
    Loterio, RK ; Monson, EA ; Templin, R ; de Bruyne, JT ; Flores, HA ; Mackenzie, JM ; Ramm, G ; Helbig, KJ ; Simmons, CP ; Fraser, JE ; Duggal, NK ; Perera, R (AMER SOC MICROBIOLOGY, 2024-02-14)
    Wolbachia are a genus of insect endosymbiotic bacteria which includes strains wMel and wAlbB that are being utilized as a biocontrol tool to reduce the incidence of Aedes aegypti-transmitted viral diseases like dengue. However, the precise mechanisms underpinning the antiviral activity of these Wolbachia strains are not well defined. Here, we generated a panel of Ae. aegypti-derived cell lines infected with antiviral strains wMel and wAlbB or the non-antiviral Wolbachia strain wPip to understand host cell morphological changes specifically induced by antiviral strains. Antiviral strains were frequently found to be entirely wrapped by the host endoplasmic reticulum (ER) membrane, while wPip bacteria clustered separately in the host cell cytoplasm. ER-derived lipid droplets (LDs) increased in volume in wMel- and wAlbB-infected cell lines and mosquito tissues compared to cells infected with wPip or Wolbachia-free controls. Inhibition of fatty acid synthase (required for triacylglycerol biosynthesis) reduced LD formation and significantly restored ER-associated dengue virus replication in cells occupied by wMel. Together, this suggests that antiviral Wolbachia strains may specifically alter the lipid composition of the ER to preclude the establishment of dengue virus (DENV) replication complexes. Defining Wolbachia's antiviral mechanisms will support the application and longevity of this effective biocontrol tool that is already being used at scale.IMPORTANCEAedes aegypti transmits a range of important human pathogenic viruses like dengue. However, infection of Ae. aegypti with the insect endosymbiotic bacterium, Wolbachia, reduces the risk of mosquito to human viral transmission. Wolbachia is being utilized at field sites across more than 13 countries to reduce the incidence of viruses like dengue, but it is not well understood how Wolbachia induces its antiviral effects. To examine this at the subcellular level, we compared how different strains of Wolbachia with varying antiviral strengths associate with and modify host cell structures. Strongly antiviral strains were found to specifically associate with the host endoplasmic reticulum and induce striking impacts on host cell lipid droplets. Inhibiting Wolbachia-induced lipid redistribution partially restored dengue virus replication demonstrating this is a contributing role for Wolbachia's antiviral activity. These findings provide new insights into how antiviral Wolbachia strains associate with and modify Ae. aegypti host cells.
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    Rapid nanopore sequencing and predictive susceptibility testing of positive blood cultures from intensive care patients with sepsis.
    Harris, PNA ; Bauer, MJ ; Lüftinger, L ; Beisken, S ; Forde, BM ; Balch, R ; Cotta, M ; Schlapbach, L ; Raman, S ; Shekar, K ; Kruger, P ; Lipman, J ; Bialasiewicz, S ; Coin, L ; Roberts, JA ; Paterson, DL ; Irwin, AD ; Papp-Wallace, KM (American Society for Microbiology, 2024-02-06)
    We aimed to evaluate the performance of Oxford Nanopore Technologies (ONT) sequencing from positive blood culture (BC) broths for bacterial identification and antimicrobial susceptibility prediction. Patients with suspected sepsis in four intensive care units were prospectively enrolled. Human-depleted DNA was extracted from positive BC broths and sequenced using ONT (MinION). Species abundance was estimated using Kraken2, and a cloud-based system (AREScloud) provided in silico predictive antimicrobial susceptibility testing (AST) from assembled contigs. Results were compared to conventional identification and phenotypic AST. Species-level agreement between conventional methods and AST predicted from sequencing was 94.2% (49/52), increasing to 100% in monomicrobial infections. In 262 high-quality AREScloud AST predictions across 24 samples, categorical agreement (CA) was 89.3%, with major error (ME) and very major error (VME) rates of 10.5% and 12.1%, respectively. Over 90% CA was achieved for some taxa (e.g., Staphylococcus aureus) but was suboptimal for Pseudomonas aeruginosa. In 470 AST predictions across 42 samples, with both high quality and exploratory-only predictions, overall CA, ME, and VME rates were 87.7%, 8.3%, and 28.4%. VME rates were inflated by false susceptibility calls in a small number of species/antibiotic combinations with few representative resistant isolates. Time to reporting from sequencing could be achieved within 8-16 h from BC positivity. Direct sequencing from positive BC broths is feasible and can provide accurate predictive AST for some species. ONT-based approaches may be faster but significant improvements in accuracy are required before it can be considered for clinical use.IMPORTANCESepsis and bloodstream infections carry a high risk of morbidity and mortality. Rapid identification and susceptibility prediction of causative pathogens, using Nanopore sequencing direct from blood cultures, may offer clinical benefit. We assessed this approach in comparison to conventional phenotypic methods and determined the accuracy of species identification and susceptibility prediction from genomic data. While this workflow holds promise, and performed well for some common bacterial species, improvements in sequencing accuracy and more robust predictive algorithms across a diverse range of organisms are required before this can be considered for clinical use. However, results could be achieved in timeframes that are faster than conventional phenotypic methods.
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    The broccoli-derived antioxidant sulforaphane changes the growth of gastrointestinal microbiota, allowing for the production of anti-inflammatory metabolites
    Marshall, SA ; Young, RB ; Lewis, JM ; Rutten, EL ; Gould, J ; Barlow, CK ; Giogha, C ; Marcelino, VR ; Fields, N ; Schittenhelm, RB ; Hartland, EL ; Scott, NE ; Forster, SC ; Gulliver, EL (ELSEVIER, 2023-08)
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    Multi-targeted loss of the antigen presentation molecule MR1 during HSV-1 and HSV-2 infection.
    Samer, C ; McWilliam, HEG ; McSharry, BP ; Velusamy, T ; Burchfield, JG ; Stanton, RJ ; Tscharke, DC ; Rossjohn, J ; Villadangos, JA ; Abendroth, A ; Slobedman, B (Elsevier BV, 2024-02-16)
    The major histocompatibility complex (MHC), Class-I-related (MR1) molecule presents microbiome-synthesized metabolites to Mucosal-associated invariant T (MAIT) cells, present at sites of herpes simplex virus (HSV) infection. During HSV type 1 (HSV-1) infection there is a profound and rapid loss of MR1, in part due to expression of unique short 3 protein. Here we show that virion host shutoff RNase protein downregulates MR1 protein, through loss of MR1 transcripts. Furthermore, a third viral protein, infected cell protein 22, also downregulates MR1, but not classical MHC-I molecules. This occurs early in the MR1 trafficking pathway through proteasomal degradation. Finally, HSV-2 infection results in the loss of MR1 transcripts, and intracellular and surface MR1 protein, comparable to that seen during HSV-1 infection. Thus HSV coordinates a multifaceted attack on the MR1 antigen presentation pathway, potentially protecting infected cells from MAIT cell T cell receptor-mediated detection at sites of primary infection and reactivation.
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    CREB: A multifaceted transcriptional regulator of neural and immune function in CNS tumors
    Dinevska, M ; Widodo, SS ; Cook, L ; Stylli, SS ; Ramsay, RG ; Mantamadiotis, T (ACADEMIC PRESS INC ELSEVIER SCIENCE, 2024-02)
    Cancers of the central nervous system (CNS) are unique with respect to their tumor microenvironment. Such a status is due to immune-privilege and the cellular behaviors within a highly networked, neural-rich milieu. During tumor development in the CNS, neural, immune and cancer cells establish complex cell-to-cell communication networks which mimic physiological functions, including paracrine signaling and synapse-like formations. This crosstalk regulates diverse pathological functions contributing to tumor progression. In the CNS, regulation of physiological and pathological functions relies on various cell signaling and transcription programs. At the core of these events lies the cyclic adenosine monophosphate (cAMP) response element binding protein (CREB), a master transcriptional regulator in the CNS. CREB is a kinase inducible transcription factor which regulates many CNS functions, including neurogenesis, neuronal survival, neuronal activation and long-term memory. Here, we discuss how CREB-regulated mechanisms operating in diverse cell types, which control development and function of the CNS, are co-opted in CNS tumors.