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  • Journal article
    Miguel-Romero L, Alqasmi M, Bacarizo J, Tan JA, Cogdell RJ, Chen J, Byron O, Christie GE, Marina A, Penades Jet al., 2022,

    Non-canonical Staphylococcus aureus pathogenicity island repression

    , Nucleic Acids Research, Vol: 50, Pages: 11109-11127, ISSN: 0305-1048

    Mobile genetic elements control their life cycles by the expression of a master repressor, whose function must be disabled to allow the spread of these elements in nature. Here we describe an unprecedented repression-derepression mechanism involved in the transfer of Staphylococcus aureus pathogenicity islands. Contrary to the classical phage and SaPI repressors, which are dimers, the SaPI1 repressor StlSaPI1 presents a unique tetramericconformation never seen before. Importantly, not just one but two tetramers are required for SaPI1 repression, which increases the novelty of the system. To derepress SaPI1, the phage-encoded protein Sri binds to and induces a conformational change in the DNA binding domains of StlSaPI1, preventing the binding of the repressor to its cognate StlSaPI1 sites. Finally, our findings demonstrate that this system is not exclusive to SaPI1 but widespread in nature. Overall, our results characterise a novel repression-induction system involved in the transfer of MGE-encoded virulence factors in nature.

  • Journal article
    Tickle ARH, Ledger EVK, Edwards AM, 2022,

    Human serum induces daptomycin tolerance in<i>Enterococcus faecalis</i>and viridans group streptococci

    <jats:title>Abstract</jats:title><jats:p>Daptomycin is a membrane-targeting lipopeptide antibiotic used in the treatment of infective endocarditis caused by multidrug-resistant Gram-positive bacteria such as<jats:italic>Staphylococcus aureus</jats:italic>, enterococci and viridans group streptococci. Despite demonstrating excellent<jats:italic>in vitro</jats:italic>activity and a low prevalence of resistant isolates, treatment failure is a significant concern, particularly for enterococcal infection. We have shown recently that human serum triggers daptomycin tolerance in<jats:italic>S. aureus</jats:italic>, but it was not clear if a similar phenotype occurred in other major infective endocarditis pathogens. We found that<jats:italic>Enterococcus faecalis, Streptococcus gordonii</jats:italic>or<jats:italic>Streptococcus mutans</jats:italic>grown under standard laboratory conditions were efficiently killed by daptomycin, whereas bacteria pre-incubated in human serum survived exposure to the antibiotic, with &gt;99% cells remaining viable. Incubation of enterococci or streptococci in serum led to peptidoglycan accumulation, as shown by increased incorporation of the fluorescent D-amino analogue HADA. Inhibition of peptidoglycan accumulation using the antibiotic fosfomycin resulted in a &gt;10-fold reduction in serum-induced daptomycin tolerance, demonstrating the important contribution of the cell wall to the phenotype. We also identified a small contribution to daptomycin tolerance in<jats:italic>E. faecalis</jats:italic>from cardiolipin synthases, although this may reflect the inherent susceptibility of cardiolipin-deficient mutants. In summary, serum-induced daptomycin tolerance is a consistent phenomenon between Gram-positive infective endocarditis pathogens, but it may be mitigated using currently available antibiotic combination therapy.</jats:p>

  • Conference paper
    Mullish BH, Paizs P, Alexander J, Verigos E, McDonald JAK, Ford L, Maneta-Stavrakaki S, Sani M, Roberts LA, Chrysostomou D, Kinross J, Monaghan T, Marchesi JR, Kao D, Takats Zet al., 2022,

    Intestinal microbiota transplant for recurrent Clostridioides difficile infection restores microbial arylsulfatases and sulfatide degradation: a novel mechanism of efficacy?

    , UEG Week 2022, Pages: 823-823
  • Journal article
    Thabet MA, Penadés JR, Haag AF, 2022,

    The ClpX protease is essential for removing the CI master repressor and completing prophage induction in <i>Staphylococcus aureus</i>

    <jats:title>Abstract</jats:title><jats:p>Bacteriophages (phages) are the predominant biological entities on the planet and play an important role in the spread of bacterial virulence, pathogenicity, and antimicrobial resistance. After infection, temperate phages can integrate in the bacterial chromosome thanks to the expression of the prophage-encoded CI master repressor. Upon SOS induction, and promoted by RecA*, CI auto-cleaves generating two fragments, one containing the N-terminal domain (NTD), which retains strong DNA-binding capacity, and other corresponding to the C-terminal part of the protein. However, it is unknown how the CI NTD is removed, a process that is essential to allow prophage induction. Here we identify for the first time that the specific interaction of the ClpX protease with the CI NTD repressor fragment is essential and sufficient for prophage activation after SOS-mediated CI autocleavage, defining the final stage in the prophage induction cascade. Our results provide unexpected roles for the bacterial protease ClpX in phage biology.</jats:p>

  • Journal article
    Farne H, Glanville N, Johnson N, Kebadze T, Aniscenko J, Regis E, Zhu J, Trujillo-Torralbo M-B, Kon OM, Mallia P, Prevost A, Edwards M, Johnston S, Singanayagam A, Jackson Det al., 2022,

    Effect of CRTH2 antagonism on the response to experimental rhinovirus infection in asthma: a pilot randomized controlled trial

    , Thorax, Vol: 77, Pages: 950-959, ISSN: 0040-6376

    Background and aimsThe CRTH2 antagonist timapiprant improved lung function and asthma control in a phase 2 study, with evidence suggesting reduced exacerbations. We aimed to assess whether timapiprant attenuated or prevented asthma exacerbations induced by experimental rhinovirus (RV) infection. We furthermore hypothesized that timapiprant would dampen RV-induced type 2 inflammation and consequently improve antiviral immune responses.MethodsAtopic patients with partially controlled asthma on maintenance inhaled corticosteroids were randomized to timapiprant (n=22) or placebo (n=22) and challenged with RV-A16 three weeks later. The primary endpoint was the cumulative lower respiratory symptom score over the 14 days post-infection. Upper respiratory symptoms, spirometry, airway hyperresponsiveness, exhaled nitric oxide, RV-A16 virus load and soluble mediators in upper and lower airways samples, and CRTH2 staining in bronchial biopsies were additionally assessed before and during RV-A16 infection.ResultsSix subjects discontinued the study and eight were not infected; outcomes were assessed in 16 timapiprant- and 14 placebo-treated, successfully infected subjects. There were no differences between treatment groups in clinical exacerbation severity including cumulative lower respiratory symptom score day 0-14 (difference 3.0 (95% CI -29.0 to 17.0), P=0.78), virus load, antiviral immune responses, or RV-A16-induced airway inflammation other than in the bronchial biopsies, where CRTH2 staining was increased during RV-A16 infection in the placebo- but not the timapiprant-treated group. Timapiprant had a favourable safety profile, with no deaths, serious adverse events, or drug-related withdrawals.ConclusionTimapiprant treatment had little impact on the clinicopathological changes induced by RV-A16 infection in partially controlled asthma.

  • Journal article
    Mishra V, Crespo-Puig A, McCarthy C, Masonou T, Glegola-Madejska I, Dejoux A, Dow G, Eldridge MJG, Marinelli LH, Meng M, Wang S, Bennison DJ, Shenoy ARet al., 2022,

    IL-1β turnover by TRIP12 and AREL1 ubiquitin ligases and UBE2L3 limits inflammation

    <jats:title>ABSTRACT</jats:title><jats:p>The cytokine interleukin-1β (IL-1β) has pivotal roles in antimicrobial immunity, but also incites inflammatory pathology. Bioactive IL-1β is released following proteolytic maturation of the pro-IL-1β precursor by caspase-1 inflammasomes. UBE2L3/UBCH7, a conserved ubiquitin conjugating enzyme, promotes pro-IL-1β ubiquitylation and proteasomal disposal. However, UBE2L3 actions<jats:italic>in vivo</jats:italic>and ubiquitin ligases involved in this process are unknown. Here we report that deletion of<jats:italic>Ube2l3</jats:italic>in mice markedly reduces pro-IL-1β turnover in macrophages, leading to excessive mature IL-1β production, neutrophilic inflammation and disease symptoms following inflammasome activation. A family-wide siRNA screen identified two ubiquitin ligases, TRIP12 and AREL1, which we show add K27-, K29- and K33- poly-ubiquitin chains on lysine residues in the ‘pro’ domain and destabilise pro-IL-1β. Mutation of ubiquitylation sites increased pro-IL-1β stability, but did not affect proteolysis by caspase-1. The extent of mature IL-1β production is therefore determined by precursor abundance, and UBE2L3, TRIP12 and AREL1 limit inflammation by shrinking the cellular pool of pro-IL-1β. Our study has uncovered fundamental processes governing IL-1β homeostasis and provided molecular insights that could be exploited to mitigate its adverse actions in disease.</jats:p>

  • Journal article
    Wong J, David S, Sanchez Garrido J, Woo J, Low WW, Morecchiato F, Giani T, Rossolini GM, Beis K, Brett S, Clements A, Aaenensen D, Rouskin S, Frankel Get al., 2022,

    Recurrent emergence of Klebsiella pneumoniae carbapenem resistance mediated by an inhibitory ompK36 mRNA secondary structure

    , Proceedings of the National Academy of Sciences of USA, Vol: 119, Pages: 1-12, ISSN: 0027-8424

    Outer membrane porins in Gram-negative bacteria facilitate antibiotic influx. In Klebsiella pneumoniae (KP), modifications in the porin OmpK36 are implicated in increasing resistance to carbapenems. Analysis of large KP genome collections, encompassing major healthcare-associated clones, revealed the recurrent emergence of a synonymous cytosine to thymine transition at position 25 (25c>t) in ompK36. We show that the 25c>t transition increases carbapenem resistance through depletion of OmpK36 from the outer membrane. The mutation attenuates KP in a murine pneumonia model, which accounts for its limited clonal expansion observed by phylogenetic analysis. However, in the context of carbapenem treatment, the 25c>t transition tips the balance towards treatment failure, thus accounting for its recurrent emergence. Mechanistically, the 25c>t transition mediates an intramolecular mRNA interaction between a uracil encoded by 25t and the first adenine within the Shine-Dalgarno sequence. This specific interaction leads to the formation of an RNA stem structure, which obscures the ribosomal binding site thus disrupting translation. While mutations reducing OmpK36 expression via transcriptional silencing are known, we uniquely demonstrate the repeated selection of a synonymous ompK36 mutation mediating translational suppression in response to antibiotic pressure.

  • Journal article
    Asai M, Li Y, Spiropoulos J, Cooley W, Everest D, Kendall S, Martin C, Robertson B, Langford P, Newton Set al., 2022,

    Galleria mellonella as an infection model for the virulent Mycobacterium tuberculosis H37Rv

    , Virulence, Vol: 13, Pages: 1543-1557, ISSN: 2150-5594

    Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is a leading cause of infectious disease mortality. Animal infection models have contributed substantially to our understanding of TB, yet their biological and non-biological limitations are a research bottleneck. There is a need for more ethically acceptable, economical, and reproducible TB infection models capable of mimicking key aspects of disease. Here we demonstrate and present a basic description of how Galleria mellonella (the greater wax moth, Gm) larvae can be used as a low cost, rapid and ethically more acceptable model for TB research. This is the first study to infect Gm with the fully virulent MTB H37Rv, the most widely used strain in research. Infection of Gm with MTB resulted in a symptomatic lethal infection, the virulence of which differed from both attenuated Mycobacterium bovis BCG and auxotrophic MTB strains. The Gm-MTB model can also be used for anti-TB drug screening, although CFU enumeration from Gm is necessary for confirmation of mycobacterial load reducing activity of the tested compound. Furthermore, comparative virulence of MTB isogenic mutants can be determined in Gm. However, comparison of mutant phenotypes in Gm against conventional models must consider the limitations of innate immunity. Our findings indicate that Gm will be a practical, valuable and advantageous additional model to be used alongside existing models to advance tuberculosis research.

  • Journal article
    Weng Y, Shepherd D, Liu Y, Krishnan N, Robertson BD, Platt N, Larrouy-Maumus G, Platt FMet al., 2022,

    Inhibition of the Niemann-Pick C1 protein is a conserved feature of multiple strains of pathogenic mycobacteria

    , Nature Communications, Vol: 13, Pages: 1-16, ISSN: 2041-1723

    Mycobacterium tuberculosis (Mtb) survives and replicates within host macrophages (MΦ) and subverts multiple antimicrobial defense mechanisms. Previously, we reported that lipids shed by pathogenic mycobacteria inhibit NPC1, the lysosomal membrane protein deficient in the lysosomal storage disorder Niemann-Pick disease type C (NPC). Inhibition of NPC1 leads to a drop in lysosomal calcium levels, blocking phagosome-lysosome fusion leading to mycobacterial survival. We speculated that the production of specific cell wall lipid(s) that inhibit NPC1 could have been a critical step in the evolution of pathogenicity. We therefore investigated whether lipid extracts from clinical Mtb strains from multiple Mtb lineages, Mtb complex (MTBC) members and non-tubercular mycobacteria (NTM) inhibit the NPC pathway. We report that inhibition of the NPC pathway was present in all clinical isolates from Mtb lineages 1, 2, 3 and 4, Mycobacterium bovis and the NTM, Mycobacterium abscessus and Mycobacterium avium. However, lipid extract from Mycobacterium canettii, which is considered to resemble the common ancestor of the MTBC did not inhibit the NPC1 pathway. We conclude that the evolution of NPC1 inhibitory mycobacterial cell wall lipids evolved early and post divergence from Mycobacterium canettii-related mycobacteria and that this activity contributes significantly to the promotion of disease.

  • Journal article
    Alqurainy N, Miguel-Romero L, Moura de Sousa J, Chen J, Rocha EPC, Fillol-Salom A, Penadés JRet al., 2022,

    A widespread family of phage-inducible chromosomal islands only steals bacteriophage tails to spread in nature

    <jats:title>Abstract</jats:title><jats:p>Phage satellites interfere with helper phage packaging through the production of small-capsids, where only satellites can be packaged. So far, in all the analysed systems, the satellite-sized capsids are composed of phage proteins. Here we report the first demonstration that a family of phage-inducible chromosomal island (PICIs), a type of satellites, encodes all the proteins required for both the production of the small-sized capsids and the exclusive packaging of the PICIs into these capsids. Therefore, this new family, that we have named cf-PICIs (<jats:underline>c</jats:underline>apsid forming PICIs), only requires phage tails to generate infective PICI particles. Remarkably, the representative cf-PICI reproduces without cost for their helper phages, suggesting that the relationship between these elements is not parasitic but commensalistic. Finally, our phylogenomic studies indicate that cf-PICIs are present both in Gram-positive and Gram-negative bacteria and have evolved at least three times independently to spread widely into the satellite universe.</jats:p>

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