Imperial College London

DrDespoinaMavridou

Faculty of Natural SciencesDepartment of Life Sciences

Visiting Researcher
 
 
 
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Contact

 

d.mavridou

 
 
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1.45Flowers buildingSouth Kensington Campus

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Summary

 

Publications

Publication Type
Year
to

50 results found

Gadar K, de Dios R, Kadeřábková N, Prescott TAK, Mavridou DAI, McCarthy RRet al., 2023, Disrupting iron homeostasis can potentiate colistin activity and overcome colistin resistance mechanisms in Gram-Negative Bacteria., Commun Biol, Vol: 6

Acinetobacter baumannii is a Gram-negative priority pathogen that can readily overcome antibiotic treatment through a range of intrinsic and acquired resistance mechanisms. Treatment of carbapenem-resistant A. baumannii largely relies on the use of colistin in cases where other treatment options have been exhausted. However, the emergence of resistance against this last-line drug has significantly increased amongst clinical strains. In this study, we identify the phytochemical kaempferol as a potentiator of colistin activity. When administered singularly, kaempferol has no effect on growth but does impact biofilm formation. Nonetheless, co-administration of kaempferol with sub-inhibitory concentrations of colistin exposes bacteria to a metabolic Achilles heel, whereby kaempferol-induced dysregulation of iron homeostasis leads to bacterial killing. We demonstrate that this effect is due to the disruption of Fenton's reaction, and therefore to a lethal build-up of toxic reactive oxygen species in the cell. Furthermore, we show that this vulnerability can be exploited to overcome both intrinsic and acquired colistin resistance in clinical strains of A. baumannii and E. coli in vitro and in the Galleria mellonella model of infection. Overall, our findings provide a proof-of-principle demonstration that targeting iron homeostasis is a promising strategy for enhancing the efficacy of colistin and overcoming colistin-resistant infections.

Journal article

Randall JR, DuPai CD, Cole TJ, Davidson G, Groover KE, Slater SL, Mavridou DAI, Wilke CO, Davies BWet al., 2023, Designing and identifying β-hairpin peptide macrocycles with antibiotic potential, SCIENCE ADVANCES, Vol: 9, ISSN: 2375-2548

Journal article

Parker JK, Gu R, Estrera GA, Kirkpatrick B, Rose DT, Mavridou DA, Mondy KE, Davies BWet al., 2023, Carbapenem-Resistant and ESBL-Producing Enterobacterales Emerging in Central Texas, INFECTION AND DRUG RESISTANCE, Vol: 16, Pages: 1249-1261, ISSN: 1178-6973

Journal article

Stelzl LS, Kritsiligkou P, Mehdipour AR, Baldwin AJ, Ferguson SJ, Mavridou DAI, Sansom MSP, Redfield Cet al., 2022, Molecular determinants of dynamic protein-protein interactions in the functional cycle of the membrane protein DsbD

<jats:title>Abstract</jats:title><jats:p>Molecular recognition is of central importance in biology. The molecular determinants shaping recognition of one protein domain by another are incompletely understood, especially in the context of the complex function of molecular machines. Here, we combine NMR experiments and molecular dynamics simulations to elucidate the determinants of recognition of the C-terminal (cDsbD) domain of the transmembrane reductant conductor DsbD by its cognate partner, the N-terminal domain of the protein (nDsbD). As part of the natural cycle of this oxidoreductase, which effectively transfers electrons from the cytoplasm to the periplasm of Gram-negative bacteria, cDsbD and nDsbD toggle between oxidised and reduced states, something that modulates the affinity of the domains for each other and prevents otherwise unproductive reactions. We find that the redox state of cDsbD determines the dissociation rate of cDsbD-nDsbD complexes. Molecular dynamics simulations demonstrate how the redox-state of the active site determines the stability of inter-domain hydrogen bonds and thus the dissociation rate. AlphaFold modelling and atomistic molecular dynamics simulations of full-length DsbD in a realistic bacterial membrane again highlights the close proximity of the periplasmic domains and the importance of tuning the strength of the interactions of the periplasmic domains to enable electron transfer to cognate periplasmic partners such as CcmG. Our AlphaFold models are consistent with <jats:italic>in vivo</jats:italic> functional assays of DsbD mutants, which together help to reveal for the first-time a putative binding site for thioredoxin on the cytoplasmic side of DsbD.</jats:p>

Journal article

Kaderabkova N, Bharathwaj M, Furniss RCD, Gonzalez D, Palmer T, Mavridou DAIet al., 2022, The biogenesis of ?-lactamase enzymes, MICROBIOLOGY-SGM, Vol: 168, ISSN: 1350-0872

Journal article

Mavridou D, Furniss RCD, Kaderabkova N, 2022, Blocking protein folding to fight antibiotic resistance, TheScienceBreaker, Vol: 8

<jats:p>Antibiotics are the cornerstone of modern medicine, but their effectiveness is threatened by the growing problem of antibiotic resistance. Recent research has identified a new strategy with the potential to restore the effectiveness of a range of currently used antibiotics, including drugs of last resort.</jats:p>

Journal article

Furniss RCD, Kaderabkova N, Barker D, Bernal P, Maslova E, Antwi AAA, McNeil HE, Pugh HL, Dortet L, Blair JMA, Larrouy-Maumus G, McCarthy RR, Gonzalez D, Mavridou DAet al., 2022, Breaking antimicrobial resistance by disrupting extracytoplasmic protein folding, ELIFE, Vol: 11, ISSN: 2050-084X

Journal article

Humphrey M, Larrouy-Maumus GJ, Furniss RCD, Mavridou DA, Sabnis A, Edwards AMet al., 2021, Colistin resistance in Escherichia coli confers protection of the cytoplasmic but not outer membrane from the polymyxin antibiotic, MICROBIOLOGY-SGM, Vol: 167, Pages: 1-9, ISSN: 1350-0872

Colistin is a polymyxin antibiotic of last resort for the treatment of infections caused by multi-drug-resistant Gram-negative bacteria. By targeting lipopolysaccharide (LPS), the antibiotic disrupts both the outer and cytoplasmic membranes, leading to bacterial death and lysis. Colistin resistance in Escherichia coli occurs via mutations in the chromosome or the acquisition of mobilized colistin-resistance (mcr) genes. Both these colistin-resistance mechanisms result in chemical modifications to the LPS, with positively charged moieties added at the cytoplasmic membrane before the LPS is transported to the outer membrane. We have previously shown that MCR-1-mediated LPS modification protects the cytoplasmic but not the outer membrane from damage caused by colistin, enabling bacterial survival. However, it remains unclear whether this observation extends to colistin resistance conferred by other mcr genes, or resistance due to chromosomal mutations. Using a panel of clinical E. coli that had acquired mcr −1, –1.5, −2, –3, −3.2 or −5, or had acquired polymyxin resistance independently of mcr genes, we found that almost all isolates were susceptible to colistin-mediated permeabilization of the outer, but not cytoplasmic, membrane. Furthermore, we showed that permeabilization of the outer membrane of colistin-resistant isolates by the polymyxin is in turn sufficient to sensitize bacteria to the antibiotic rifampicin, which normally cannot cross the LPS monolayer. These findings demonstrate that colistin resistance in these E. coli isolates is due to protection of the cytoplasmic but not outer membrane from colistin-mediated damage, regardless of the mechanism of resistance.

Journal article

Nolan LM, Cain AK, Clamens T, Furniss RCD, Manoli E, Sainz-Polo MA, Dougan G, Albesa-Jove D, Parkhill J, Mavridou DAI, Filloux Aet al., 2021, Identification of tse8 as a type VI secretion system toxin from pseudomonas aeruginosa that targets the bacterial transamidosome to inhibit protein synthesis in prey cells, Nature Microbiology, Vol: 6, Pages: 1199-+, ISSN: 2058-5276

The Type VI secretion system (T6SS) is a bacterial nanomachine that delivers toxic effectors to kill competitors or subvert some of their key functions. Here, we use transposon directed insertion–site sequencing to identify T6SS toxins associated with the H1-T6SS, one of the three T6SS machines found in Pseudomonas aeruginosa. This approach identified several putative toxin–immunity pairs, including Tse8–Tsi8. Full characterization of this protein pair demonstrated that Tse8 is delivered by the VgrG1a spike complex into prey cells where it targets the transamidosome, a multiprotein complex involved in protein synthesis in bacteria that lack either one, or both, of the asparagine and glutamine transfer RNA synthases. Biochemical characterization of the interactions between Tse8 and the transamidosome components GatA, GatB and GatC suggests that the presence of Tse8 alters the fine-tuned stoichiometry of the transamidosome complex, and in vivo assays demonstrate that Tse8 limits the ability of prey cells to synthesize proteins. These data expand the range of cellular components targeted by the T6SS by identifying a T6SS toxin affecting protein synthesis and validate the use of a transposon directed insertion site sequencing–based global genomics approach to expand the repertoire of T6SS toxins in T6SS-encoding bacteria.

Journal article

Humphrey M, Larrouy-Maumus GJ, Furniss RCD, Mavridou DAI, Sabnis A, Edwards AMet al., 2021, Colistin resistance in <i>Escherichia coli</i> confers protection of the cytoplasmic but not outer membrane from the polymyxin antibiotic

<jats:title>Abstract</jats:title><jats:p>Colistin is a polymyxin antibiotic of last resort for the treatment of infections caused by multi-drug resistant Gram-negative bacteria. By targeting lipopolysaccharide (LPS), the antibiotic disrupts both the outer and cytoplasmic membranes, leading to lysis and bacterial death. Colistin resistance in <jats:italic>Escherichia coli</jats:italic> occurs via mutations in the chromosome or the acquisition of mobilised colistin resistance (<jats:italic>mcr</jats:italic>) genes. Both these colistin resistance mechanisms result in chemical modifications to the LPS, with positively charged moieties added at the cytoplasmic membrane before the LPS is transported to the outer membrane. We have previously shown that MCR-1-mediated LPS modification protects the cytoplasmic but not the outer membrane from damage caused by colistin, enabling bacterial survival. However, it remains unclear whether this observation extends to colistin resistance conferred by other <jats:italic>mcr</jats:italic> genes, or resistance due to chromosomal mutations. Using a panel of clinical <jats:italic>E. coli</jats:italic> that had acquired <jats:italic>mcr</jats:italic> -1, -1.5, -2, -3, -3.2 or -5, or had acquired polymyxin resistance independently of <jats:italic>mcr</jats:italic> genes, we found that almost all isolates were susceptible to colistin-mediated permeabilisation of the outer, but not cytoplasmic, membrane. Furthermore, we showed that permeabilisation of the outer membrane of colistin resistant isolates by the polymyxin is in turn sufficient to sensitise bacteria to the antibiotic rifampicin, which normally cannot cross the LPS monolayer. These findings demonstrate that colistin resistance in <jats:italic>E. coli</jats:italic> is typically due to protection of the cytoplasmic but not outer membrane from colistin-mediated damage, regardless of th

Working paper

Slater SL, Mavridou DAI, 2021, Harnessing the potential of bacterial oxidative folding to aid protein production, MOLECULAR MICROBIOLOGY, Vol: 116, Pages: 16-28, ISSN: 0950-382X

Journal article

Howard SA, Furniss RCD, Bonini D, Amin H, Paracuellos P, Zlotkin D, R D Costa T, Levy A, A I Mavridou D, Filloux Aet al., 2021, The breadth and molecular basis of Hcp-driven type six secretion system (T6SS) effector delivery, mBio, Vol: 12, Pages: 1-19, ISSN: 2150-7511

The 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.

Journal article

Sabnis A, Haggard K, Kloeckner A, Becce M, Evans L, Furniss R, Mavridou D, Stevens M, Murphy R, Davies J, Clarke T, Edwards Aet al., 2021, Colistin kills bacteria by targeting lipopolysaccharide in the cytoplasmic membrane, eLife, Vol: 10, Pages: 1-26, ISSN: 2050-084X

Colistin is an antibiotic of last resort, but has poor efficacy and resistance is a growing problem. Whilst it is well established that colistin disrupts the bacterial outer membrane (OM) by selectively targeting lipopolysaccharide (LPS), it was unclear how this led to bacterial killing. We discovered that MCR-1 mediated colistin resistance in Escherichia coli is due to modified LPS at the cytoplasmic rather than OM. In doing so, we also demonstrated that colistin exerts bactericidal activity by targeting LPS in the cytoplasmic membrane (CM). We then exploited this information to devise a new therapeutic approach. Using the LPS transport inhibitor murepavadin, we were able to cause LPS accumulation in the CM of Pseudomonas aeruginosa, which resulted in increased susceptibility to colistin in vitro and improved treatment efficacy in vivo. These findings reveal new insight into the mechanism by which colistin kills bacteria, providing the foundations for novel approaches to enhance therapeutic outcomes.

Journal article

Bernal P, Furniss CD, Fecht S, Leung RCY, Spiga L, Mavridou DAI, Filloux ALAINet al., 2021, A novel stabilization mechanism for the type VI secretion system sheath, Proceedings of the National Academy of Sciences of USA, Vol: 118, ISSN: 0027-8424

The type VI secretion system (T6SS) is a phage-derived contractile nanomachine primarily involved in interbacterial competition. Its pivotal component, TssA, is indispensable for the assembly of the T6SS sheath structure, the contraction of which propels a payload of effector proteins into neighboring cells. Despite their key function, TssA proteins exhibit unexpected diversity and exist in two major forms, a short form (TssAS) and a long form (TssAL). While TssAL proteins interact with a partner, called TagA, to anchor the distal end of the extended sheath, the mechanism for the stabilization of TssAS-containing T6SSs remains unknown. Here we discover a class of structural components that interact with short TssA proteins and contribute to T6SS assembly by stabilizing the polymerizing sheath from the baseplate. We demonstrate that the presence of these components is important for full sheath extension and optimal firing. Moreover, we show that the pairing of each form of TssA with a different class of sheath stabilization proteins results in T6SS apparatuses that either reside in the cell for some time or fire immediately after sheath extension. We propose that this diversity in firing dynamics could contribute to the specialization of the T6SS to suit bacterial lifestyles in diverse environmental niches.

Journal article

Kumar RK, Meiller-Legrand TA, Alcinesio A, Gonzalez D, Mavridou DA, Meacock OJ, Smith WPJ, Zhou L, Kim W, Pulcu GS, Bayley H, Foster KRet al., 2021, Droplet printing reveals the importance of micron-scale structure for bacterial ecology, NATURE COMMUNICATIONS, Vol: 12

Journal article

Ha KP, Clarke RS, Kim G-L, Brittan JL, Rowley JE, Mavridou DAI, Parker D, Clarke TB, Nobbs AH, Edwards AMet al., 2020, Staphylococcal DNA repair Is required for infection, mBio, Vol: 11, ISSN: 2150-7511

To cause infection, Staphylococcus aureus must withstand damage caused by host immune defenses. However, the mechanisms by which staphylococcal DNA is damaged and repaired during infection are poorly understood. Using a panel of transposon mutants, we identified the rexBA operon as being important for the survival of Staphylococcus aureus in whole human blood. Mutants lacking rexB were also attenuated for virulence in murine models of both systemic and skin infections. We then demonstrated that RexAB is a member of the AddAB family of helicase/nuclease complexes responsible for initiating the repair of DNA double-strand breaks. Using a fluorescent reporter system, we were able to show that neutrophils cause staphylococcal DNA double-strand breaks through reactive oxygen species (ROS) generated by the respiratory burst, which are repaired by RexAB, leading to the induction of the mutagenic SOS response. We found that RexAB homologues in Enterococcus faecalis and Streptococcus gordonii also promoted the survival of these pathogens in human blood, suggesting that DNA double-strand break repair is required for Gram-positive bacteria to survive in host tissues. Together, these data demonstrate that DNA is a target of host immune cells, leading to double-strand breaks, and that the repair of this damage by an AddAB-family enzyme enables the survival of Gram-positive pathogens during infection.IMPORTANCE To cause infection, bacteria must survive attack by the host immune system. For many bacteria, including the major human pathogen Staphylococcus aureus, the greatest threat is posed by neutrophils. These immune cells ingest the invading organisms and try to kill them with a cocktail of chemicals that includes reactive oxygen species (ROS). The ability of S. aureus to survive this attack is crucial for the progression of infection. However, it was not clear how the ROS damaged S. aureus and how the bacterium repaired this damage. In this work, we show that ROS cause breaks

Journal article

Kumar RK, Meiller-Legrand TA, Alcinesio A, Gonzalez D, Mavridou DAI, Meacock OJ, Smith WPJ, Zhou L, Kim W, Su Pulcu G, Bayley H, Foster KRet al., 2020, Droplet printing reveals the importance of micron-scale structure for bacterial ecology

<jats:title>Abstract</jats:title><jats:p>Bacteria often live in diverse communities where the spatial arrangement of strains and species is considered critical for their ecology, including whether strains can coexist, which are ecologically dominant, and how productive they are as a community<jats:sup>1,2</jats:sup>. However, a test of the importance of spatial structure requires manipulation at the fine scales at which this structure naturally occurs<jats:sup>3–8</jats:sup>. Here we develop a droplet-based printing method to arrange different bacterial genotypes across a sub-millimetre array. We use this to test the importance of fine-scale spatial structure by printing strains of the gut bacterium <jats:italic>Escherichia coli</jats:italic> that naturally compete with one another using protein toxins<jats:sup>9,10</jats:sup>. This reveals that the spatial arrangement of bacterial genotypes is important for ecological outcomes. Toxin-producing strains largely eliminate susceptible non-producers when genotypes are well-mixed. However, printing strains side-by-side creates an ecological refuge such that susceptible strains can coexist with toxin producers, even to the extent that a susceptible strain outnumbers the toxin producer. Head-to-head competitions between toxin producers also reveals strong effects, where spatial structure can make the difference between one strain winning and mutual destruction. Finally, we print different potential barriers between two competing strains to understand why space is so important. This reveals the importance of processes that limit the free diffusion of molecules. Specifically, we show that cells closest to a toxin producer bind to and capture toxin molecules, which creates a refuge for their clonemates. Our work provides a new method to generate customised bacterial communities with defined spatial distributions, and reveals that micron-scale changes in t

Journal article

Ha KP, Clarke R, Kim G-L, Brittan J, Rowley J, Mavridou D, Parker D, Clarke T, Nobbs A, Edwards Aet al., 2020, Staphylococcal DNA repair is required for infection, Publisher: bioRxiv

To cause infection, Staphylococcus aureus must withstand damage caused by host immune defences. However, the mechanisms by which staphylococcal DNA is damaged and repaired during infection are poorly understood. Using a panel of transposon mutants, we identified the rexBA operon as important for the survival of Staphylococcus aureus in whole human blood. Mutants lacking rexB were also attenuated for virulence in murine models of both systemic and skin infections. We then demonstrated that RexAB is a member of the AddAB family of helicase/nuclease complexes responsible for initiating the repair of DNA double strand breaks. Using a fluorescent reporter system, we were able to show that neutrophils cause staphylococcal DNA double strand breaks through ROS generated by the respiratory burst, which are repaired by RexAB, leading to induction of the mutagenic SOS response. We found that RexAB homologues in Enterococcus faecalis and Streptococcus gordonii also promoted survival of these pathogens in human blood, suggesting that DNA double strand break repair is required for Gram-positive bacteria to survive in host tissues. Together, these data demonstrate that DNA is a target of host immune cells, leading to double-strand breaks, and that repair of this damage by an AddAB-family enzyme enables the survival of Gram-positive pathogens during infection.

Working paper

Stelzl LS, Mavridou DA, Saridakis E, Gonzalez D, Baldwin AJ, Ferguson SJ, Sansom MS, Redfield Cet al., 2020, Local frustration determines loop opening during the catalytic cycle of an oxidoreductase., eLife, Vol: 9, Pages: 1-27, ISSN: 2050-084X

Local structural frustration, the existence of mutually exclusive competing interactions, may explain why some proteins are dynamic while others are rigid. Frustration is thought to underpin biomolecular recognition and the flexibility of protein-binding sites. Here, we show how a small chemical modification, the oxidation of two cysteine thiols to a disulfide bond, during the catalytic cycle of the N-terminal domain of the key bacterial oxidoreductase DsbD (nDsbD), introduces frustration ultimately influencing protein function. In oxidized nDsbD, local frustration disrupts the packing of the protective cap-loop region against the active site allowing loop opening. By contrast, in reduced nDsbD the cap loop is rigid, always protecting the active-site thiols from the oxidizing environment of the periplasm. Our results point toward an intricate coupling between the dynamics of the active-site cysteines and of the cap loop which modulates the association reactions of nDsbD with its partners resulting in optimized protein function.

Journal article

Bernal P, Furniss C, Fecht S, Leung RCY, Spiga L, Mavridou DAI, Filloux Aet al., 2020, Novel structural components generate distinct type VI secretion system anchoring modes, Publisher: bioRxiv

The type VI secretion system (T6SS) is a phage-derived contractile nanomachine primarily involved in interbacterial competition. Its pivotal component, TssA, is indispensable for the assembly of the T6SS sheath structure, the contraction of which propels a payload of effector proteins into neighboring cells. Despite their key function, TssA proteins exhibit unexpected diversity and exist in two major forms, a short (TssA S ) and a long (TssA L ) TssA. Whilst TssA L proteins interact with a partner, called TagA, to anchor the distal end of the extended sheath, the mechanism for the stabilization of TssA S -containing T6SSs remains unknown. Here we discover a novel class of structural components that interact with short TssA proteins and contribute to T6SS assembly by stabilizing the polymerizing sheath from the baseplate. We demonstrate that the presence of these components is important for full sheath extension and optimal firing. Moreover, we show that the pairing of each form of TssA with a different class of sheath stabilization proteins results in T6SS apparatuses that either reside in the cell for a while or fire immediately after sheath extension, thus giving rise to different aggression behaviors. We propose that this functional diversity could contribute to the specialization of the T6SS to suit bacterial lifestyles in diverse environmental niches.

Working paper

Furniss RCD, Kostrzewa M, Mavridou DAI, Larrouy-Maumus Get al., 2020, The clue is in the lipid A: Rapid detection of colistin resistance, PLoS Pathogens, Vol: 16, ISSN: 1553-7366

Journal article

Gonzalez D, Mavridou D, 2019, Making the best of aggression, the many dimensions of bacterial toxin regulation, Trends in Microbiology, Vol: 27, Pages: 897-905, ISSN: 0966-842X

Most bacteria use toxins to exclude competitors.As the synthesis and delivery of these moleculesentail considerablecostsfor the producers, theirexpressionis tightly regulated, often by molecular systems detecting physiological stressesor environment-specific cues.However, the ecological connection between such systemsand competitive behaviorsis not always clear. Here, we review the regulation of antibacterial toxins and propose a conceptual framework organizing the decision-making processes controlling toxin production. As bacteria are unable to precisely identify their competitors,we argue that toxin regulation primarily responds to cues directly or indirectly associated with the presence of competing strains. The density and fitnessof the producing populational so play a role in the decision-making process.Overall, we contendthat optimal toxin production strategies involvemonitoring of both self and foe.

Journal article

Furniss C, Dortet L, Bolland W, drews O, sparbier K, bonnin R, Filloux A, kostrzewa M, Mavridou D, Larrouy-Maumus Get al., 2019, Detection of colistin resistance in Escherichia coli using the MALDI Biotyper Sirius mass spectrometry system, Journal of Clinical Microbiology, Vol: 57, Pages: 1-7, ISSN: 0095-1137

Polymyxin antibiotics are a last-line treatment for multidrug-resistant Gram-negative bacteria. However, the emergence of colistin resistance, including the spread of mobile mcr genes, necessitates the development of improved diagnostics for the detection of colistin-resistant organisms in hospital settings. The recently developed MALDIxin test enables detection of colistin resistance by MALDI-TOF mass spectrometry in less than 15 minutes, but is not optimized for the mass spectrometers commonly found in clinical microbiology laboratories. In this study, we adapted the MALDIxin test for the MALDI Biotyper Sirius MALDI-TOF mass spectrometry system (Bruker Daltonics). We optimized the sample preparation protocol using a set of 6 MCR-expressing Escherichia coli clones and validated the assay with a collection of 40 E. coli clinical isolates, including 19 confirmed MCR producers, 12 colistin-resistant isolates which tested negative for commonly encountered mcr genes (i.e. likely chromosomally-resistant isolates) and 9 polymyxin-susceptible isolates. We calculated Polymyxin resistance ratio (PRR) values from the acquired spectra; a PRR value of zero, indicating polymyxin susceptibility, was obtained for all colistin-susceptible E. coli isolates, whereas positive PRR values, indicating resistance to polymyxins, were obtained for all resistant strains independent of the genetic basis of resistance. Thus, we report a preliminary feasibility study showing that an optimized version of the MALDIxin test, adapted for the routine MALDI Biotyper Sirius, provides an unbiased, fast, reliable, cost-effective and high-throughput way of detecting colistin resistance in clinical E. coli isolates.

Journal article

Pissaridou P, Allsopp LP, Wettstadt S, Howard SA, Mavridou DAI, Filloux Aet al., 2018, The Pseudomonas aeruginosa T6SS-VgrG1b spike is topped by a PAAR protein eliciting DNA damage to bacterial competitors, Proceedings of the National Academy of Sciences of the United States of America, Vol: 115, Pages: 12519-12524, ISSN: 0027-8424

The type VI secretion system (T6SS) is a supramolecular complex involved in the delivery of potent toxins during bacterial competition. Pseudomonas aeruginosa possesses three T6SS gene clusters and several hcp and vgrG gene islands, the latter encoding the spike at the T6SS tip. The vgrG1b cluster encompasses seven genes whose organization and sequences are highly conserved in P. aeruginosa genomes, except for two genes that we called tse7 and tsi7. We show that Tse7 is a Tox-GHH2 domain nuclease which is distinct from other T6SS nucleases identified thus far. Expression of this toxin induces the SOS response, causes growth arrest and ultimately results in DNA degradation. The cytotoxic domain of Tse7 lies at its C terminus, while the N terminus is a predicted PAAR domain. We find that Tse7 sits on the tip of the VgrG1b spike and that specific residues at the PAAR–VgrG1b interface are essential for VgrG1b-dependent delivery of Tse7 into bacterial prey. We also show that the delivery of Tse7 is dependent on the H1-T6SS cluster, and injection of the nuclease into bacterial competitors is deployed for interbacterial competition. Tsi7, the cognate immunity protein, protects the producer from the deleterious effect of Tse7 through a direct protein–protein interaction so specific that toxin/immunity pairs are effective only if they originate from the same P. aeruginosa isolate. Overall, our study highlights the diversity of T6SS effectors, the exquisite fitting of toxins on the tip of the T6SS, and the specificity in Tsi7-dependent protection, suggesting a role in interstrain competition.

Journal article

Dortet L, Bonnin RA, Pennisi I, Gauthier L, Jousset AB, Dabos L, Furniss RCD, Mavridou DAI, Bogaerts P, Glupczynski Y, Potron A, Plesiat P, Beyrouthy R, Robin F, Bonnet R, Naas T, Filloux A, Larrouy-Maumus Get al., 2018, Rapid detection and discrimination of chromosome-and MCR-plasmid-mediated resistance to polymyxins by MALDI-TOF MS in Escherichia coli: the MALDIxin test, Journal of Antimicrobial Chemotherapy, Vol: 73, Pages: 3359-3367, ISSN: 0305-7453

BackgroundPolymyxins are currently considered a last-resort treatment for infections caused by MDR Gram-negative bacteria. Recently, the emergence of carbapenemase-producing Enterobacteriaceae has accelerated the use of polymyxins in the clinic, resulting in an increase in polymyxin-resistant bacteria. Polymyxin resistance arises through modification of lipid A, such as the addition of phosphoethanolamine (pETN). The underlying mechanisms involve numerous chromosome-encoded genes or, more worryingly, a plasmid-encoded pETN transferase named MCR. Currently, detection of polymyxin resistance is difficult and time consuming.ObjectivesTo develop a rapid diagnostic test that can identify polymyxin resistance and at the same time differentiate between chromosome- and plasmid-encoded resistances.MethodsWe developed a MALDI-TOF MS-based method, named the MALDIxin test, which allows the detection of polymyxin resistance-related modifications to lipid A (i.e. pETN addition), on intact bacteria, in <15 min.ResultsUsing a characterized collection of polymyxin-susceptible and -resistant Escherichia coli, we demonstrated that our method is able to identify polymyxin-resistant isolates in 15 min whilst simultaneously discriminating between chromosome- and plasmid-encoded resistance. We validated the MALDIxin test on different media, using fresh and aged colonies and show that it successfully detects all MCR-1 producers in a blindly analysed set of carbapenemase-producing E. coli strains.ConclusionsThe MALDIxin test is an accurate, rapid, cost-effective and scalable method that represents a major advance in the diagnosis of polymyxin resistance by directly assessing lipid A modifications in intact bacteria.

Journal article

Sabnis A, Hagart KLH, Klöckner A, Becce M, Evans LE, Furniss RCD, Mavridou DAI, Murphy R, Stevens MM, Davies JC, Larrouy-Maumus GJ, Clarke TB, Edwards AMet al., 2018, Colistin kills bacteria by targeting lipopolysaccharide in the cytoplasmic membrane

<jats:title>Summary</jats:title><jats:p>Colistin is an antibiotic of last resort, but has poor efficacy and resistance is a growing problem. Whilst it is well established that colistin disrupts the bacterial outer membrane by selectively targeting lipopolysaccharide (LPS), it was unclear how this led to bacterial killing. We discovered that MCR-1 mediated colistin resistance is due to modified LPS at the cytoplasmic rather than outer membrane. In doing so, we also demonstrated that colistin exerts bactericidal activity by targeting LPS in the cytoplasmic membrane. We then exploited this information to devise a new therapeutic approach. Using the LPS transport inhibitor murepavadin, we were able to cause LPS accumulation in the cytoplasmic membrane, which resulted in increased susceptibility to colistin <jats:italic>in vitro</jats:italic> and improved treatment efficacy <jats:italic>in vivo</jats:italic>. These findings reveal new insight into the mechanism by which colistin kills bacteria, providing the foundations for novel approaches to enhance therapeutic outcomes.</jats:p>

Working paper

Furniss RCD, Low WW, Mavridou DAI, Dagley LF, Webb AI, Tate E, Clements Aet al., 2018, Plasma membrane profiling during enterohemorrhagic E. coli infection reveals that the metalloprotease StcE cleaves CD55 from host epithelial surfaces, Journal of Biological Chemistry, Vol: 293, Pages: 17188-17199, ISSN: 0021-9258

Enterohemorrhagic Escherichia coli (EHEC) is one of several E. coli pathotypes that infect the intestinal tract and cause disease. Formation of the characteristic attaching and effacing (A/E) lesion on the surface of infected cells causes significant remodelling of the host cell surface, however limited information is available about changes at the protein level. Here we employed "plasma membrane profiling", a quantitative cell-surface proteomics technique, to identify host proteins whose cell-surface levels are altered during infection. Using this method, we quantified more than 1100 proteins, 280 of which showed altered cell-surface levels after exposure to EHEC. 22 host proteins were significantly reduced on the surface of infected epithelial cells. These included both known and unknown targets of EHEC infection. The complement decay-accelerating factor CD55 exhibited the greatest reduction in cell surface levels during infection. We showed by flow cytometry and Western blot analysis that CD55 is cleaved from the cell surface by the EHEC-specific protease StcE, and found that StcE-mediated CD55 cleavage results in increased neutrophil adhesion to the apical surface of intestinal epithelial cells. This suggests that StcE alters host epithelial surfaces to depress neutrophil transepithelial migration during infection. This work is the first report of the global manipulation of the epithelial cell surface by a bacterial pathogen and illustrates the power of quantitative cell-surface proteomics in uncovering critical aspects of bacterial infection biology.

Journal article

Shevket SH, Gonzalez D, Cartwright JL, Kleanthous C, Ferguson SJ, Redfield C, Mavridou Det al., 2018, The CcmC-CcmE interaction during cytochrome c maturation by System I is driven by protein-protein and not protein-heme contacts, Journal of Biological Chemistry, Vol: 293, Pages: 16778-16790, ISSN: 0021-9258

Cytochromes c are ubiquitous proteins, essential for life in most organisms. Their distinctive characteristic is the covalent attachment of heme to their polypeptide chain. This post-translational modification is performed by a dedicated protein system, which in many Gram-negative bacteria and plant mitochondria is a nine-protein apparatus (CcmA–I) called System I. Despite decades of study, mechanistic understanding of the protein–protein interactions in this highly complex maturation machinery is still lacking. Here, we focused on the interaction of CcmC, the protein that sources the heme cofactor, with CcmE, the pivotal component of System I responsible for the transfer of the heme to the apocytochrome. Using in silico analyses, we identified a putative interaction site between these two proteins (residues Asp47, Gln50, and Arg55 on CcmC; Arg73, Asp101, and Glu105 on CcmE), and we validated our findings by in vivo experiments in Escherichia coli. Moreover, employing NMR spectroscopy, we examined whether a heme-binding site on CcmE contributes to this interaction and found that CcmC and CcmE associate via protein–protein rather than protein–heme contacts. The combination of in vivo site-directed mutagenesis studies and high-resolution structural techniques enabled us to determine at the residue level the mechanism for the formation of one of the key protein complexes for cytochrome c maturation by System I.

Journal article

Gonzalez D, Sabnis A, Foster K, Mavridou Det al., 2018, Costs and benefits of provocation in bacterial warfare, Proceedings of the National Academy of Sciences, Vol: 115, Pages: 7593-7598, ISSN: 0027-8424

Bacteria live in dense environments where competition for space and resources is fierce. For this reason, they often use diffusible toxins to eliminate closely related strains. Some toxins trigger systematic retaliation, raising the question of the role of provocation in bacterial warfare. We combine mathematical modeling and experiments to study the costs and benefits of provocation. In one-to-one encounters, provocation is costly as it leads to strong counterattacks. However, with three or more strains present, provocation can provide benefits via a “divide-and-conquer” effect, whereby a strain forces its opponents to wipe each other out. This effect could be harnessed as a targeted antibacterial approach; adding low levels of certain antibiotics to communities can promote warfare and cross-elimination between strains.

Journal article

Murphey AC, Mavridou DAI, Hodgkin J, Ferguson SJet al., 2018, The heme auxotroph Caenorhabditis elegans can cleave the thioether bonds of c-type cytochromes, FEBS Letters, Vol: 592, Pages: 928-938, ISSN: 0014-5793

Heme is essential and synthesised via highly-regulated processes. For this reason, most organisms strive to recycle it or acquire it from their environment. When heme is bound to proteins non-covalently, degradation of the polypeptide is sufficient to release it. However, in some hemoproteins, such as c-type cytochromes, heme is covalently bound to the protein backbone. We use the heme auxotroph Caenorhabditis elegans to investigate if cytochromes c can be a heme source, and we show that this organism must encode a novel system, which specifically cleaves the thioether bonds of c-type cytochromes. We also find that at limiting heme concentrations, while somatic tissues develop normally the germline fails to proliferate, suggesting the presence of a heme-sensing checkpoint in C. elegans.

Journal article

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