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Journal articleKennedy C, Goya Grocin A, Kovacic T, et al., 2021,
A probe for NLRP3 inflammasome inhibitor MCC950 identifies carbonic anhydrase 2 as a novel target
, ACS Chemical Biology, Vol: 16, Pages: 982-990, ISSN: 1554-8929Inhibition of inflammasome and pyroptotic pathways are promising strategies for clinical treatment of autoimmune and inflammatory disorders. MCC950, a potent inhibitor of the NLR-family inflammasome pyrin domain-containing 3 (NLRP3) protein, has shown encouraging results in animal models for a range of conditions; however, until now, no off-targets have been identified. Herein, we report the design, synthesis, and application of a novel photoaffinity alkyne-tagged probe for MCC950 (IMP2070) which shows direct engagement with NLRP3 and inhibition of inflammasome activation in macrophages. Affinity-based chemical proteomics in live macrophages identified several potential off-targets, including carbonic anhydrase 2 (CA2) as a specific target of IMP2070, and independent cellular thermal proteomic profiling revealed stabilization of CA2 by MCC950. MCC950 displayed noncompetitive inhibition of CA2 activity, confirming carbonic anhydrase as an off-target class for this compound. These data highlight potential biological mechanisms through which MCC950 and derivatives may exhibit off-target effects in preclinical or clinical studies.
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Journal articleWang Z, Wang H, Mulvenna N, et al., 2021,
A Bacteriophage DNA Mimic Protein Employs a Non-specific Strategy to Inhibit the Bacterial RNA Polymerase
, FRONTIERS IN MICROBIOLOGY, Vol: 12, ISSN: 1664-302X- Author Web Link
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- Citations: 4
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Journal articleAltwiley D, Brignoli T, Edwards A, et al., 2021,
A Functional Menadione Biosynthesis Pathway is Required for Capsule Production by <i>Staphylococcus aureus</i>
<jats:title>Abstract</jats:title><jats:p><jats:italic>Staphylococcus aureus</jats:italic> is a major human pathogen that utilises a wide array of pathogenic and immune evasion strategies to cause disease. One immune evasion strategy, common to many bacterial pathogens, is the ability of <jats:italic>S. aureus</jats:italic> to produce a capsule that protects the bacteria from several aspects of the human immune system. To identify novel regulators of capsule production by <jats:italic>S. aureus</jats:italic> we applied a genome wide association study (GWAS) to a collection of 300 bacteraemia isolates that represent the two major MRSA clones in UK and Irish hospitals: CC22 and CC30. One of the loci associated with capsule production, the <jats:italic>menD</jats:italic> gene, encodes an enzyme critical to the biosynthesis of menadione. Mutations in this gene that result in menadione auxotrophy induce the slow growing small-colony variant (SCV) form of <jats:italic>S. aureus</jats:italic> often associated with chronic infections due to their increased resistance to antibiotics and ability to survive inside phagocytes. Utilising such an SCV we functionally verified this association between <jats:italic>menD</jats:italic> and capsule production. Although the clinical isolates with polymorphisms in the <jats:italic>menD</jats:italic> gene in our collections had no apparent growth defects, they were more resistant to gentamicin when compared to those with the wild-type <jats:italic>menD</jats:italic> gene. Our work suggests that menadione plays a critical role in the production of the <jats:italic>S. aureus</jats:italic> capsule, and that amongst clinical isolates polymorphisms exist in the <jats:italic>menD</jats:italic> gene that confer the characteristic increased gentamicin resistance, but not the major growth defect associated with S
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Journal articleMullish BH, Ghani R, McDonald JAK, et al., 2021,
Reply to Woodworth, et al.
, Clin Infect Dis, Vol: 72, Pages: e924-e925 -
Journal articleVivian T, Yi L, Ashleigh C, et al., 2021,
Metabolomics in infectious diseases and drug discovery
, Molecular Omics, Vol: 17, Pages: 376-393, ISSN: 2515-4184Metabolomics has emerged as an invaluable tool that can be used along with genomics, transcriptomics and proteomics to understand host–pathogen interactions at small-molecule levels. Metabolomics has been used to study a variety of infectious diseases and applications. The most common application of metabolomics is for prognostic and diagnostic purposes, specifically the screening of disease-specific biomarkers by either NMR-based or mass spectrometry-based metabolomics. In addition, metabolomics is of great significance for the discovery of druggable metabolic enzymes and/or metabolic regulators through the use of state-of-the-art flux analysis, for example, via the elucidation of metabolic mechanisms. This review discusses the application of metabolomics technologies to biomarker screening, the discovery of drug targets in infectious diseases such as viral, bacterial and parasite infections and immunometabolomics, highlights the challenges associated with accessing metabolite compartmentalization and discusses the available tools for determining local metabolite concentrations.
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Journal articleRaynaud C, Sheppard D, Berry J, et al., 2021,
PilB from Streptococcus sanguinis is a bimodular type IV pilin with a direct role in adhesion
, Proceedings of the National Academy of Sciences of USA, Vol: 118, Pages: 1-10, ISSN: 0027-8424Type IV pili (T4P) are functionally versatile filamentous nanomachines, nearly ubiquitous in prokaryotes. They are predominantly polymers of one major pilin but also contain minor pilins whose functions are often poorly defined and likely to be diverse. Here, we show that the minor pilin PilB from the T4P of Streptococcus sanguinis displays an unusual bimodular three-dimensional structure with a bulky von Willebrand factor A–like (vWA) module “grafted” onto a small pilin module via a short loop. Structural modeling suggests that PilB is only compatible with a localization at the tip of T4P. By performing a detailed functional analysis, we found that 1) the vWA module contains a canonical metal ion–dependent adhesion site, preferentially binding Mg2+ and Mn2+, 2) abolishing metal binding has no impact on the structure of PilB or piliation, 3) metal binding is important for S. sanguinis T4P–mediated twitching motility and adhesion to eukaryotic cells, and 4) the vWA module shows an intrinsic binding ability to several host proteins. These findings reveal an elegant yet simple evolutionary tinkering strategy to increase T4P functional versatility by grafting a functional module onto a pilin for presentation by the filaments. This strategy appears to have been extensively used by bacteria, in which modular pilins are widespread and exhibit an astonishing variety of architectures.
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Journal articleHoward SA, Furniss RCD, Bonini D, et al., 2021,
The breadth and molecular basis of Hcp-driven type six secretion system (T6SS) effector delivery
, mBio, Vol: 12, Pages: 1-19, ISSN: 2150-7511The type VI secretion system (T6SS) is a bacterial nanoscale weapon that delivers toxins into prey ranging from bacteria and fungi to animal hosts. The cytosolic contractile sheath of the system wraps around stacked hexameric rings of Hcp proteins, which form an inner tube. At the tip of this tube is a puncturing device comprising a trimeric VgrG topped by a monomeric PAAR protein. The number of toxins a single system delivers per firing event remains unknown, since effectors can be loaded on diverse sites of the T6SS apparatus, notably the inner tube and the puncturing device. Each VgrG or PAAR can bind one effector, and additional effector cargoes can be carried in the Hcp ring lumen. While many VgrG- and PAAR-bound toxins have been characterized, to date, very few Hcp-bound effectors are known. Here, we used 3 known Pseudomonas aeruginosa Hcp proteins (Hcp1 to -3), each of which associates with one of the three T6SSs in this organism (H1-T6SS, H2-T6SS, and H3-T6SS), to perform in vivo pulldown assays. We confirmed the known interactions of Hcp1 with Tse1 to -4, further copurified a Hcp1-Tse4 complex, and identified potential novel Hcp1-bound effectors. Moreover, we demonstrated that Hcp2 and Hcp3 can shuttle T6SS cargoes toxic to Escherichia coli. Finally, we used a Tse1-Bla chimera to probe the loading strategy for Hcp passengers and found that while large effectors can be loaded onto Hcp, the formed complex jams the system, abrogating T6SS function.
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Journal articleDenny S, Abdolrasouli A, Elamin T, et al., 2021,
A retrospective multicenter analysis of candidaemia among COVID-19 patients during the first UK pandemic wave
, Journal of Infection, Vol: 82, Pages: 276-316, ISSN: 0163-4453 -
Journal articleBakovic J, Yu BYK, Silva D, et al., 2021,
Redox Regulation of the Quorum-sensing Transcription Factor AgrA by Coenzyme A
, ANTIOXIDANTS, Vol: 10- Author Web Link
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- Citations: 4
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Journal articleSteinchen W, Ahmad S, Valentini M, et al., 2021,
Dual role of a (p)ppGpp- and (p)ppApp-degrading enzyme in biofilm formation and interbacterial antagonism
, Molecular Microbiology, Vol: 115, Pages: 1339-1356, ISSN: 0950-382XThe guanosine nucleotide‐based second messengers ppGpp and pppGpp (collectively: (p)ppGpp) enable adaptation of microorganisms to environmental changes and stress conditions. In contrast, the closely related adenosine nucleotides (p)ppApp are involved in type VI secretion system (T6SS)‐mediated killing during bacterial competition. Long RelA‐SpoT Homolog (RSH) enzymes regulate synthesis and degradation of (p)ppGpp (and potentially also (p)ppApp) through their synthetase and hydrolase domains, respectively. Small alarmone hydrolases (SAH) that consist of only a hydrolase domain are found in a variety of bacterial species, including the opportunistic human pathogen Pseudomonas aeruginosa. Here, we present the structure and mechanism of P. aeruginosa SAH showing that the enzyme promiscuously hydrolyses (p)ppGpp and (p)ppApp in a strictly manganese‐dependent manner. While being dispensable for P. aeruginosa growth or swimming, swarming, and twitching motilities, its enzymatic activity is required for biofilm formation. Moreover, (p)ppApp‐degradation by SAH provides protection against the T6SS (p)ppApp synthetase effector Tas1, suggesting that SAH enzymes can also serve as defense proteins during interbacterial competition.
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