Complete genome sequences of clinical Pandoraea fibrosis isolates

Pandoraea fibrosis is a newly identified Gram-negative bacterial species that was isolated from the respiratory tract of an Australian cystic fibrosis patient. The complete assembled genome sequences of two consecutive isolates (second isolate collected 11 months after antibiotic treatment) from the same individual are presented here

Fluorescent macrolide probes – synthesis and use in evaluation of bacterial resistance

This is the final version. Available on open access from the Royal Society of Chemistry via the DOI in this recordThe emerging crisis of antibiotic resistance requires a multi-pronged approach in order to avert the onset of a post-antibiotic age. Studies of antibiotic uptake and localisation in live cells may inform the design of improved drugs and help develop a better understanding of bacterial resistance and persistence. To facilitate this research, we have synthesised fluorescent derivatives of the macrolide antibiotic erythromycin. These analogues exhibit a similar spectrum of antibiotic activity to the parent drug and are capable of labelling both Gram-positive and -negative bacteria for microscopy. The probes localise intracellularly, with uptake in Gram-negative bacteria dependent on the level of efflux pump activity. A plate-based assay established to quantify bacterial labelling and localisation demonstrated that the probes were taken up by both susceptible and resistant bacteria. Significant intra-strain and -species differences were observed in these preliminary studies. In order to examine uptake in real-time, the probe was used in single-cell microfluidic microscopy, revealing previously unseen heterogeneity of uptake in populations of susceptible bacteria. These studies illustrate the potential of fluorescent macrolide probes to characterise and explore drug uptake and efflux in bacteria.Australian Postgraduate AwardInstitute for Molecular Biosciences Research Advancement AwardMedical Research Council (MRC)Gordon and Betty and Gordon Moore FoundationCampus France the Programme Hubert Curien FASIC 2018Wellcome TrustNHMR

Correction: Metal complexes as a promising source for new antibiotics

Correction for ‘Metal complexes as a promising source for new antibiotics’ by Angelo Frei et al., Chem. Sci., 2020, 11, 2627–2639

Synthesis of vancomycin fluorescent probes that retain antimicrobial activity, identify Gram-positive bacteria, and detect Gram-negative outer membrane damage

This is the final version. Available from Nature Research via the DOI in this record. All relevant data are available in this article, its Supplementary Information and Supplementary Data files (the source data behind the graphs in the paper is contained in Supplementary Data 2), except for original image files, which are available from the corresponding author upon reasonable request.Antimicrobial resistance is an urgent threat to human health, and new antibacterial drugs are desperately needed, as are research tools to aid in their discovery and development. Vancomycin is a glycopeptide antibiotic that is widely used for the treatment of Gram-positive infections, such as life-threatening systemic diseases caused by methicillin-resistant Staphylococcus aureus (MRSA). Here we demonstrate that modification of vancomycin by introduction of an azide substituent provides a versatile intermediate that can undergo copper-catalysed azide-alkyne cycloaddition (CuAAC) reaction with various alkynes to readily prepare vancomycin fluorescent probes. We describe the facile synthesis of three probes that retain similar antibacterial profiles to the parent vancomycin antibiotic. We demonstrate the versatility of these probes for the detection and visualisation of Gram-positive bacteria by a range of methods, including plate reader quantification, flow cytometry analysis, high-resolution microscopy imaging, and single cell microfluidics analysis. In parallel, we demonstrate their utility in measuring outer-membrane permeabilisation of Gram-negative bacteria. The probes are useful tools that may facilitate detection of infections and development of new antibiotics.Biotechnology & Biological Sciences Research Council (BBSRC)Engineering and Physical Sciences Research Council (EPSRC)Wellcome TrustMedical Research Council (MRC)Gordon and Betty Moore Foundation Marine Microbiology InitiativeNHMRCNHMRCNHMRCThe Royal SocietyChina Scholarship Council (CSC)University of QueenslandInstitute for Molecular BiosciencesQUEXGW

The Induction of APC with a Distinct Tolerogenic Phenotype via Contact-Dependent STAT3 Activation

BACKGROUND: Activation of the signal transducer and activator of transcription 3 (STAT3) within antigen presenting cells (APCs) is linked to abnormal APCs differentiation and function. We have previously shown that STAT3 is activated within APC by a novel contact-dependent mechanism, which plays a key role in mediating the immunomodulatory effects of hMSC. In order to better understand the underlying mechanisms that control APC maturation in a contact dependent manner, we extended our observation to tumor cells. Tumors were shown to secrete a variety of tumor-derived factors that activate STAT3 within infiltrating APCs. We now tested whether tumor cells can activate STAT3 within APC using the contact-dependent mechanism, in addition to soluble factors, and compared these two STAT3 activating pathways. PRINCIPAL FINDINGS: We demonstrate that in addition to tumor-derived secreted factors tumor cells activate STAT3 by a mechanism that is based on cell-cell interaction. We further demonstrate that these two STAT3 activating mechanisms differ in their JAK usage and their susceptibility to JSI-124 inhibition thereby representing two distinct pathways. Significantly, although both pathways activate STAT3, they modulate DCs maturation in a different manner that results in disparate phenotypic outcomes. Whereas the soluble-dependent pathway results in an immature phenotype, the contact-dependent pathway results in an apparently mature phenotype. Albeit their mature-like phenotype these latter cells express the tolerogenic markers ILT3 and ILT4 and possess T cell inhibitory activity. SIGNIFICANCE: This data suggests that, in at least certain cellular microenvironments, cell:cell interactions represent a novel way to activate STAT3 signaling, uncouple APC activation events and consequently regulate immunity and tolerance. Significantly, we have now demonstrated that this contact-dependent signaling pathway differs from that mediated by soluble factors and cytokines, inducing disparate phenotypic outcome, suggesting these two mechanisms have different and possibly complementary biological functions

Cucurbitacin IIa: a novel class of anti-cancer drug inducing non-reversible actin aggregation and inhibiting survivin independent of JAK2/STAT3 phosphorylation

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Dendritic Cells as Danger-Recognizing Biosensors

Dendritic cells (DCs) are antigen presenting cells that are characterized by a potent capacity to initiate immune responses. DCs comprise several subsets with distinct phenotypes. After sensing any danger(s) to the host via their innate immune receptors such as Toll-like receptors, DCs become mature and subsequently present antigens to CD4+ T cells. Since DCs possess the intrinsic capacity to polarize CD4+ helper cells, it is critical to understand the immunological roles of DCs for clinical applications. Here, we review the different DC subsets, their danger-sensing receptors and immunological functions. Furthermore, the cytokine reporter mouse model for studying DC activation is introduced

Cucurbitacin I Inhibits Cell Motility by Indirectly Interfering with Actin Dynamics

Cucurbitacins are plant natural products that inhibit activation of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway by an unknown mechanism. They are also known to cause changes in the organization of the actin cytoskeleton. actin depolymerization experiments, cucurbitacin I had no effect on the rate of actin filament disassembly at the nanomolar concentrations that inhibit cell migration. At elevated concentrations, the depolymerization rate was also unaffected, although there was a delay in the initiation of depolymerization. Therefore, cucurbitacin I targets some factor involved in cellular actin dynamics other than actin itself. Two candidate proteins that play roles in actin depolymerization are the actin-severing proteins cofilin and gelsolin. Cucurbitacin I possesses electrophilic reactivity that may lead to chemical modification of its target protein, as suggested by structure-activity relationship data. However, mass spectrometry revealed no evidence for modification of purified cofilin or gelsolin by cucurbitacin I.Cucurbitacin I results in accumulation of actin filaments in cells by a unique indirect mechanism. Furthermore, the proximal target of cucurbitacin I relevant to cell migration is unlikely to be the same one involved in activation of the JAK2/STAT3 pathway