Microbiology & Immunology - Research Publications

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    A highly conserved gene locus in endofungal bacteria codes for the biosynthesis of symbiosis-specific cyclopeptides
    Niehs, SP ; Scherlach, K ; Dose, B ; Uzum, Z ; Stinear, TP ; Pidot, SJ ; Hertweck, C ; Yooseph, S (OXFORD UNIV PRESS, 2022-09)
    The tight association of the pathogenic fungus Rhizopus microsporus and its toxin-producing, bacterial endosymbionts (Mycetohabitans spp.) is distributed worldwide and has significance for agriculture, food production, and human health. Intriguingly, the endofungal bacteria are essential for the propagation of the fungal host. Yet, little is known about chemical mediators fostering the symbiosis, and universal metabolites that support the mutualistic relationship have remained elusive. Here, we describe the discovery of a complex of specialized metabolites produced by endofungal bacteria under symbiotic conditions. Through full genome sequencing and comparative genomics of eight endofungal symbiont strains from geographically distant regions, we discovered a conserved gene locus (hab) for a nonribosomal peptide synthetase as a unifying trait. Bioinformatics analyses, targeted gene deletions, and chemical profiling uncovered unprecedented depsipeptides (habitasporins) whose structures were fully elucidated. Computational network analysis and labeling experiments granted insight into the biosynthesis of their nonproteinogenic building blocks (pipecolic acid and β-phenylalanine). Deletion of the hab gene locus was shown to impair the ability of the bacteria to enter their fungal host. Our study unveils a common principle of the endosymbiotic lifestyle of Mycetohabitans species and expands the repertoire of characterized chemical mediators of a globally occurring mutualistic association.
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    Environmental risk factors associated with the presence of Mycobacterium ulcerans in Victoria, Australia
    Blasdell, KR ; McNamara, B ; O'Brien, DP ; Tachedjian, M ; Boyd, V ; Dunn, M ; Mee, PT ; Clayton, S ; Gaburro, J ; Smith, I ; Gibney, KB ; Tay, EL ; Hobbs, EC ; Waidyatillake, N ; Lynch, SE ; Stinear, TP ; Athan, E ; Becker, D (PUBLIC LIBRARY SCIENCE, 2022-09-13)
    In recent years reported cases of Buruli ulcer, caused by Mycobacterium ulcerans, have increased substantially in Victoria, Australia, with the epidemic also expanding geographically. To develop an understanding of how M. ulcerans circulates in the environment and transmits to humans we analyzed environmental samples collected from 115 properties of recent Buruli ulcer cases and from 115 postcode-matched control properties, for the presence of M. ulcerans. Environmental factors associated with increased odds of M. ulcerans presence at a property included certain native plant species and native vegetation in general, more alkaline soil, lower altitude, the presence of common ringtail possums (Pseudocheirus peregrinus) and overhead powerlines. However, only overhead powerlines and the absence of the native plant Melaleuca lanceolata were associated with Buruli ulcer case properties. Samples positive for M. ulcerans were more likely to be found at case properties and were associated with detections of M. ulcerans in ringtail possum feces, supporting the hypothesis that M. ulcerans is zoonotic, with ringtail possums the strongest reservoir host candidate. However, the disparity in environmental risk factors associated with M. ulcerans positive properties versus case properties indicates the involvement of human behavior or the influence of other environmental factors in disease acquisition that requires further study.
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    RNase III-CLASH of multi-drug resistant Staphylococcus aureus reveals a regulatory mRNA 3′UTR required for intermediate vancomycin resistance (vol 13, 3558, 2022)
    Mediati, DG ; Wong, JL ; Gao, W ; McKellar, S ; Pang, CNI ; Wu, S ; Wu, W ; Sy, B ; Monk, IR ; Biazik, JM ; Wilkins, MR ; Howden, BP ; Stinear, TP ; Granneman, S ; Tree, JJ (NATURE PORTFOLIO, 2022-09-27)
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    Barriers to genetic manipulation of Enterococci: Current Approaches and Future Directions
    Krause, AL ; Stinear, TP ; Monk, IR (OXFORD UNIV PRESS, 2022-11-02)
    Enterococcus faecalis and Enterococcus faecium are Gram-positive commensal gut bacteria that can also cause fatal infections. To study clinically relevant multi-drug resistant E. faecalis and E. faecium strains, methods are needed to overcome physical (thick cell wall) and enzymatic barriers that limit the transfer of foreign DNA and thus prevent facile genetic manipulation. Enzymatic barriers to DNA uptake identified in E. faecalis and E. faecium include type I, II and IV restriction modification systems and CRISPR-Cas. This review examines E. faecalis and E. faecium DNA defence systems and the methods with potential to overcome these barriers. DNA defence system bypass will allow the application of innovative genetic techniques to expedite molecular-level understanding of these important, but somewhat neglected, pathogens.
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    RNase III-CLASH of multi-drug resistant Staphylococcus aureus reveals a regulatory mRNA 3′UTR required for intermediate vancomycin resistance
    Mediati, DG ; Wong, JL ; Gao, W ; McKellar, S ; Pang, CNI ; Wu, S ; Wu, W ; Sy, B ; Monk, IR ; Biazik, JM ; Wilkins, MR ; Howden, BP ; Stinear, TP ; Granneman, S ; Tree, JJ (NATURE PORTFOLIO, 2022-06-22)
    Treatment of methicillin-resistant Staphylococcus aureus infections is dependent on the efficacy of last-line antibiotics including vancomycin. Treatment failure is commonly linked to isolates with intermediate vancomycin resistance (termed VISA). These isolates have accumulated point mutations that collectively reduce vancomycin sensitivity, often by thickening the cell wall. Changes in regulatory small RNA expression have been correlated with antibiotic stress in VISA isolates however the functions of most RNA regulators is unknown. Here we capture RNA-RNA interactions associated with RNase III using CLASH. RNase III-CLASH uncovers hundreds of novel RNA-RNA interactions in vivo allowing functional characterisation of many sRNAs for the first time. Surprisingly, many mRNA-mRNA interactions are recovered and we find that an mRNA encoding a long 3' untranslated region (UTR) (termed vigR 3'UTR) functions as a regulatory 'hub' within the RNA-RNA interaction network. We demonstrate that the vigR 3'UTR promotes expression of folD and the cell wall lytic transglycosylase isaA through direct mRNA-mRNA base-pairing. Deletion of the vigR 3'UTR re-sensitised VISA to glycopeptide treatment and both isaA and vigR 3'UTR deletions impact cell wall thickness. Our results demonstrate the utility of RNase III-CLASH and indicate that S. aureus uses mRNA-mRNA interactions to co-ordinate gene expression more widely than previously appreciated.
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    Niche-specific genome degradation and convergent evolution shaping Staphylococcus aureus adaptation during severe infections
    Giulieri, SG ; Guerillot, R ; Duchene, S ; Hachani, A ; Daniel, D ; Seemann, T ; Davis, JS ; Tong, SYC ; Young, BC ; Wilson, DJ ; Stinear, TP ; Howden, BP (eLIFE SCIENCES PUBL LTD, 2022-06-14)
    During severe infections, Staphylococcus aureus moves from its colonising sites to blood and tissues and is exposed to new selective pressures, thus, potentially driving adaptive evolution. Previous studies have shown the key role of the agr locus in S. aureus pathoadaptation; however, a more comprehensive characterisation of genetic signatures of bacterial adaptation may enable prediction of clinical outcomes and reveal new targets for treatment and prevention of these infections. Here, we measured adaptation using within-host evolution analysis of 2590 S. aureus genomes from 396 independent episodes of infection. By capturing a comprehensive repertoire of single nucleotide and structural genome variations, we found evidence of a distinctive evolutionary pattern within the infecting populations compared to colonising bacteria. These invasive strains had up to 20-fold enrichments for genome degradation signatures and displayed significantly convergent mutations in a distinctive set of genes, linked to antibiotic response and pathogenesis. In addition to agr-mediated adaptation, we identified non-canonical, genome-wide significant loci including sucA-sucB and stp1. The prevalence of adaptive changes increased with infection extent, emphasising the clinical significance of these signatures. These findings provide a high-resolution picture of the molecular changes when S. aureus transitions from colonisation to severe infection and may inform correlation of infection outcomes with adaptation signatures.
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    Staphylococcus aureus specific lung resident memory CD4+ Th1 cells attenuate the severity of influenza virus induced secondary bacterial pneumonia
    Braverman, J ; Monk, IR ; Ge, C ; Westall, GP ; Stinear, TP ; Wakim, LM (ELSEVIER SCIENCE INC, 2022-04)
    Staphylococcus aureus is a major cause of severe pulmonary infections. The evolution of multi-drug resistant strains limits antibiotic treatment options. To date, all candidate vaccines tested have failed, highlighting the need for an increased understanding of the immunological requirements for effective S. aureus immunity. Using an S. aureus strain engineered to express a trackable CD4+ T cell epitope and a murine model of S. aureus pneumonia, we show strategies that lodge Th1 polarised bacterium specific CD4+ tissue resident memory T cells (Trm) in the lung can significantly attenuate the severity of S. aureus pneumonia. This contrasts natural infection of mice that fails to lodge CD4+ Trm cells along the respiratory tract or provide protection against re-infection, despite initially generating Th17 bacterium specific CD4+ T cell responses. Interestingly, lack of CD4+ Trm formation after natural infection in mice appears to be reflected in humans, where the frequency of S. aureus reactive CD4+ Trm cells in lung tissue is also low. Our findings reveal the protective capacity of S. aureus specific respiratory tract CD4+ Th1 polarised Trm cells and highlight the potential for targeting these cells in vaccines that aim to prevent the development of S. aureus pneumonia.
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    Detection of Chimeric Cellular: HIV mRNAs Generated Through Aberrant Splicing in HIV-1 Latently Infected Resting CD4+T Cells
    Lee, MY-H ; Khoury, G ; Olshansky, M ; Sonza, S ; Carter, GP ; McMahon, J ; Stinear, TP ; Turner, SJ ; Lewin, SR ; Purcell, DFJ (FRONTIERS MEDIA SA, 2022-04-28)
    Latent HIV-1 provirus in infected individuals on suppressive therapy does not always remain transcriptionally silent. Both HIV-1 LTR and human gene promoter derived transcriptional events can contribute HIV-1 sequences to the mRNA produced in the cell. In addition, chimeric cellular:HIV mRNA can arise through readthrough transcription and aberrant splicing. Using target enrichment coupled to the Illumina Mi-Seq and PacBio RS II platforms, we show that 3' LTR activation is frequent in latently infected cells from both the CCL19-induced primary cell model of HIV-1 latency as well as ex vivo samples. In both systems of latent HIV-1 infection, we detected several chimeric species that were generated via activation of a cryptic splice donor site in the 5' LTR of HIV-1. Aberrant splicing involving the major HIV-1 splice donor sites, SD1 and SD4 disrupts post-transcriptional processing of the gene in which HIV-1 is integrated. In the primary cell model of HIV-1 latency, Tat-encoding sequences are incorporated into the chimeric mRNA transcripts through the use of SD4. Our study unravels clues to the characteristics of HIV-1 integrants that promote formation of chimeric cellular:HIV mRNA and improves the understanding of the HIV-1 RNA footprint in latently infected cells.
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    Considerations for the Analysis of Bacterial Membrane Vesicles: Methods of Vesicle Production and Quantification Can Influence Biological and Experimental Outcomes
    Bitto, NJ ; Zavan, L ; Johnston, EL ; Stinear, TP ; Hill, AF ; Kaparakis-Liaskos, M ; Edelmann, MJ (AMER SOC MICROBIOLOGY, 2021-12)
    Bacterial membrane vesicles (BMVs) are produced by all bacteria and facilitate a range of functions in host-microbe interactions and pathogenesis. Quantification of BMVs is a critical first step in the analysis of their biological and immunological functions. Historically, BMVs have been quantified by protein assay, which remains the preferred method of BMV quantification. However, recent studies have shown that BMV protein content can vary significantly between bacterial strains, growth conditions, and stages of bacterial growth, suggesting that protein concentration may not correlate directly with BMV quantity. Here, we show that the method used to quantify BMVs can alter experimental outcomes. We compared the enumeration of BMVs using different protein assays and nanoparticle tracking analysis (NTA). We show that different protein assays vary significantly in their quantification of BMVs and that their sensitivity varies when quantifying BMVs produced by different species. Moreover, stimulation of epithelial cells with an equivalent amount of BMV protein quantified using different protein assays resulted in significant differences in interleukin 8 (IL-8) responses. Quantification of Helicobacter pylori, Pseudomonas aeruginosa, and Staphylococcus aureus BMVs by NTA and normalization of BMV cargo to particle number revealed that BMV protein, DNA, and RNA contents were variable between strains and species and throughout bacterial growth. Differences in BMV-mediated activation of Toll-like receptors, NF-κB, and IL-8 responses were observed when stimulations were performed with equivalent BMV particle number but not equivalent protein amount. These findings reveal that the method of BMV quantification can significantly affect experimental outcomes, thereby potentially altering the observed biological functions of BMVs. IMPORTANCE Recent years have seen a surge in interest in the roles of BMVs in host-microbe interactions and interbacterial communication. As a result of such rapid growth in the field, there is a lack of uniformity in BMV enumeration. Here, we reveal that the method used to enumerate BMVs can significantly alter experimental outcomes. Specifically, standardization of BMVs by protein amount reduced the ability to distinguish strain differences in the immunological functions of BMVs. In contrast, species-, strain-, and growth stage-dependent differences in BMV cargo content were evident when BMVs were enumerated by particle number, and this was reflected in differences in their ability to induce immune responses. These findings indicate that parameters critical to BMV function, including bacterial species, strain, growth conditions, and sample purity, should form the basis of standard reporting in BMV studies. This will ultimately bring uniformity to the field to advance our understanding of BMV functions.
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    Long-read RNA sequencing identifies polyadenylation elongation and differential transcript usage of host transcripts during SARS-CoV-2 in vitro infection
    Chang, JJ-Y ; Gleeson, J ; Rawlinson, D ; Pitt, M ; De Paoli-Iseppi, R ; Zhou, C ; Mordant, F ; Londrigan, S ; Clark, M ; Subbarao, K ; Stinear, T ; Coin, LJM ( 2021-12-15)
    Better methods to interrogate host-pathogen interactions during Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections are imperative to help understand and prevent this disease. Here we implemented RNA-sequencing (RNA-seq) combined with the Oxford Nanopore Technologies (ONT) long-reads to measure differential host gene expression, transcript polyadenylation and isoform usage within various epithelial cell lines permissive and non-permissive for SARS-CoV-2 infection. SARS-CoV-2-infected and mock-infected Vero (African green monkey kidney epithelial cells), Calu-3 (human lung adenocarcinoma epithelial cells), Caco-2 (human colorectal adenocarcinoma epithelial cells) and A549 (human lung carcinoma epithelial cells) were analysed over time (0, 2, 24, 48 hours). Differential polyadenylation was found to occur in both infected Calu-3 and Vero cells during a late time point (48 hpi), with Gene Ontology (GO) terms such as viral transcription and translation shown to be significantly enriched in Calu-3 data. Poly(A) tails showed increased lengths in the majority of the differentially polyadenylated transcripts in Calu-3 and Vero cell lines (up to ~136 nt in mean poly(A) length, padj = 0.029). Of these genes, ribosomal protein genes such as RPS4X and RPS6 also showed downregulation in expression levels, suggesting the importance of ribosomal protein genes during infection. Furthermore, differential transcript usage was identified in Caco-2, Calu-3 and Vero cells, including transcripts of genes such as GSDMB and KPNA2 , which have previously been implicated in SARS-CoV-2 infections. Overall, these results highlight the potential role of differential polyadenylation and transcript usage in host immune response or viral manipulation of host mechanisms during infection, and therefore, showcase the value of long-read sequencing in identifying less-explored host responses to disease.