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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 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 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, Pages: 1-10, ISSN: 1664-302XDNA mimicry by proteins is a strategy that employed by some proteins to occupy the binding sites of the DNA-binding proteins and deny further access to these sites by DNA. Such proteins have been found in bacteriophage, eukaryotic virus, prokaryotic, and eukaryotic cells to imitate non-coding functions of DNA. Here, we report another phage protein Gp44 from bacteriophage SPO1 of Bacillus subtilis, employing mimicry as part of unusual strategy to inhibit host RNA polymerase. Consisting of three simple domains, Gp44 contains a DNA binding motif, a flexible DNA mimic domain and a random-coiled domain. Gp44 is able to anchor to host genome and interact bacterial RNA polymerase via the β and β′ subunit, resulting in bacterial growth inhibition. Our findings represent a non-specific strategy that SPO1 phage uses to target different bacterial transcription machinery regardless of the structural variations of RNA polymerases. This feature may have potential applications like generation of genetic engineered phages with Gp44 gene incorporated used in phage therapy to target a range of bacterial hosts.
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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 articleMatthews-Palmer T, Gonzalez-Rodriguez N, Calcraft T, et al., 2021,
Structure of the cytoplasmic domain of SctV (SsaV) from the Salmonella SPI-2 injectisome and implications for a pH sensing mechanism
, Journal of Structural Biology, Vol: 213, ISSN: 1047-8477Bacterial type III secretion systems assemble the axial structures of both injectisomes and flagella. Injectisome type III secretion systems subsequently secrete effector proteins through their hollow needle into a host, requiring co-ordination. In the Salmonella enterica serovar Typhimurium SPI-2 injectisome, this switch is triggered by sensing the neutral pH of the host cytoplasm. Central to specificity switching is a nonameric SctV protein with an N-terminal transmembrane domain and a toroidal C-terminal cytoplasmic domain. A ‘gatekeeper’ complex interacts with the SctV cytoplasmic domain in a pH dependent manner, facilitating translocon secretion while repressing effector secretion through a poorly understood mechanism. To better understand the role of SctV in SPI-2 translocon-effector specificity switching, we purified full-length SctV and determined its toroidal cytoplasmic region’s structure using cryo-EM. Structural comparisons and molecular dynamics simulations revealed that the cytoplasmic torus is stabilized by its core subdomain 3, about which subdomains 2 and 4 hinge, varying the flexible outside cleft implicated in gatekeeper and substrate binding. In light of patterns of surface conservation, deprotonation, and structural motion, the location of previously identified critical residues suggest that gatekeeper binds a cleft buried between neighboring subdomain 4s. Simulations suggest that a local pH change from 5 to 7.2 stabilizes the subdomain 3 hinge and narrows the central aperture of the nonameric torus. Our results are consistent with a model of local pH sensing at SctV, where pH-dependent dynamics of SctV cytoplasmic domain affect binding of gatekeeper complex.
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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 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 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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Journal articleGonzalo X, Broda A, Drobniewski F, et al., 2021,
Performance of lipid fingerprint-based MALDI-ToF for the diagnosis of mycobacterial infections
, Clinical Microbiology and Infection, Vol: 27, Pages: 912.e1-912.e5, ISSN: 1198-743XObjectivesBacterial diagnosis of mycobacteria is often challenging because of the variability of the sensitivity and specificity of the assay used, and it can be expensive to perform accurately. Although matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has become the workhorse of clinical laboratories, the current MALDI methodology (which is based on cytosolic protein profiling) for mycobacteria is still challenging due to the number of steps involved (up to seven) and potential biosafety concerns. Knowing that mycobacteria produce surface-exposed species-specific lipids, we here hypothesized that the detection of those molecules could offer a rapid, reproducible and robust method for mycobacterial identification.MethodsWe evaluated the performance of an alternative methodology based on characterized species-specific lipid profiling of intact bacteria, without any sample preparation, by MALDI MS; it uses MALDI-time-of-flight (ToF) MS combined with a specific matrix (super-2,5-dihydroxybenzoic acid solubilized in an apolar solvent system) to analyse lipids of intact heat-inactivated mycobacteria. Cultured mycobacteria are heat-inactivated and loaded directly onto the MALDI target followed by addition of the matrix. Acquisition of the data is done in both positive and negative ion modes. Blinded studies were performed using 273 mycobacterial strains comprising both the Mycobacterium tuberculosis (Mtb) complex and non-tuberculous mycobacteria (NTMs) subcultured in Middlebrook 7H9 media supplemented with 10% OADC (oleic acid/dextrose/catalase) growth supplement and incubated for up to 2 weeks at 37°C.ResultsThe method we have developed is fast (<10 mins) and highly sensitive (<1000 bacteria required); 96.7% of the Mtb complex strains (204/211) were correctly assigned as MTB complex and 91.7% (22/24) NTM species were correctly assigned based only on intact bacteria species-specific lipid profiling by MALDI-ToF MS.ConclusionsIntact bacter
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