Imperial College London

Dr Abigail Clements

Faculty of Natural SciencesDepartment of Life Sciences

Senior Lecturer
 
 
 
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Contact

 

+44 (0)20 7594 7681a.clements

 
 
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Location

 

1.42Flowers buildingSouth Kensington Campus

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Summary

 

Publications

Publication Type
Year
to

42 results found

Leung PB, Matanza XM, Roche B, Ha KP, Cheung HC, Appleyard S, Collins T, Flanagan O, Marteyn BS, Clements Aet al., 2024, Shigella sonnei utilises colicins during inter-bacterial competition., Microbiology (Reading), Vol: 170

The mammalian colon is one of the most densely populated habitats currently recognised, with 1011-1013 commensal bacteria per gram of colonic contents. Enteric pathogens must compete with the resident intestinal microbiota to cause infection. Among these enteric pathogens are Shigella species which cause approximately 125 million infections annually, of which over 90 % are caused by Shigella flexneri and Shigella sonnei. Shigella sonnei was previously reported to use a Type VI Secretion System (T6SS) to outcompete E. coli and S. flexneri in in vitro and in vivo experiments. S. sonnei strains have also been reported to harbour colicinogenic plasmids, which are an alternative anti-bacterial mechanism that could provide a competitive advantage against the intestinal microbiota. We sought to determine the contribution of both T6SS and colicins to the anti-bacterial killing activity of S. sonnei. We reveal that whilst the T6SS operon is present in S. sonnei, there is evidence of functional degradation of the system through SNPs, indels and IS within key components of the system. We created strains with synthetically inducible T6SS operons but were still unable to demonstrate anti-bacterial activity of the T6SS. We demonstrate that the anti-bacterial activity observed in our in vitro assays was due to colicin activity. We show that S. sonnei no longer displayed anti-bacterial activity against bacteria that were resistant to colicins, and removal of the colicin plasmid from S. sonnei abrogated anti-bacterial activity of S. sonnei. We propose that the anti-bacterial activity demonstrated by colicins may be sufficient for niche competition by S. sonnei within the gastrointestinal environment.

Journal article

Matanza XM, Clements A, 2023, Pathogenicity and virulence of Shigella sonnei: A highly drug-resistant pathogen of increasing prevalence., Virulence, Vol: 14

Shigella spp. are the causative agent of shigellosis (or bacillary dysentery), a diarrhoeal disease characterized for the bacterial invasion of gut epithelial cells. Among the 4 species included in the genus, Shigella flexneri is principally responsible for the disease in the developing world while Shigella sonnei is the main causative agent in high-income countries. Remarkably, as more countries improve their socioeconomic conditions, we observe an increase in the relative prevalence of S. sonnei. To date, the reasons behind this change in aetiology depending on economic growth are not understood. S. flexneri has been widely used as a model to study the pathogenesis of the genus, but as more research data are collected, important discrepancies with S. sonnei have come to light. In comparison to S. flexneri, S. sonnei can be differentiated in numerous aspects; it presents a characteristic O-antigen identical to that of one serogroup of the environmental bacterium Plesiomonas shigelloides, a group 4 capsule, antibacterial mechanisms to outcompete and displace gut commensal bacteria, and a poorer adaptation to an intracellular lifestyle. In addition, the World Health Organization (WHO) have recognized the significant threat posed by antibiotic-resistant strains of S. sonnei, demanding new approaches. This review gathers knowledge on what is known about S. sonnei within the context of other Shigella spp. and aims to open the door for future research on understanding the increasing spread of this pathogen.

Journal article

Wong JLC, Romano M, Kerry LE, Kwong H-S, Low W-W, Brett SJ, Clements A, Beis K, Frankel Get al., 2023, Author Correction: OmpK36-mediated Carbapenem resistance attenuates ST258 Klebsiella pneumoniae in vivo., Nat Commun, Vol: 14

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

Singh S, Wilksch JJ, Dunstan RA, Mularski A, Wang N, Hocking D, Jebeli L, Cao H, Clements A, Jenney AWJ, Lithgow T, Strugnell RAet al., 2022, LPS O Antigen Plays a Key Role in Klebsiella pneumoniae Capsule Retention, MICROBIOLOGY SPECTRUM, Vol: 10, ISSN: 2165-0497

Journal article

David S, Wong JLC, Sanchez-Garrido J, Kwong H-S, Low WW, Morecchiato F, Giani T, Rossolini GM, Brett SJ, Clements A, Beis K, Aanensen DM, Frankel Get al., 2022, Widespread emergence of OmpK36 loop 3 insertions among multidrug-resistant clones of Klebsiella pneumoniae., PLoS Pathogens, Vol: 18, Pages: 1-23, ISSN: 1553-7366

Mutations in outer membrane porins act in synergy with carbapenemase enzymes to increase carbapenem resistance in the important nosocomial pathogen, Klebsiella pneumoniae (KP). A key example is a di-amino acid insertion, Glycine-Aspartate (GD), in the extracellular loop 3 (L3) region of OmpK36 which constricts the pore and restricts entry of carbapenems into the bacterial cell. Here we combined genomic and experimental approaches to characterise the diversity, spread and impact of different L3 insertion types in OmpK36. We identified L3 insertions in 3588 (24.1%) of 14,888 KP genomes with an intact ompK36 gene from a global collection. GD insertions were most common, with a high concentration in the ST258/512 clone that has spread widely in Europe and the Americas. Aspartate (D) and Threonine-Aspartate (TD) insertions were prevalent in genomes from Asia, due in part to acquisitions by KP sequence types ST16 and ST231 and subsequent clonal expansions. By solving the crystal structures of novel OmpK36 variants, we found that the TD insertion causes a pore constriction of 41%, significantly greater than that achieved by GD (10%) or D (8%), resulting in the highest levels of resistance to selected antibiotics. We show that in the absence of antibiotics KP mutants harbouring these L3 insertions exhibit both an in vitro and in vivo competitive disadvantage relative to the isogenic parental strain expressing wild type OmpK36. We propose that this explains the reversion of GD and TD insertions observed at low frequency among KP genomes. Finally, we demonstrate that strains expressing L3 insertions remain susceptible to drugs targeting carbapenemase-producing KP, including novel beta lactam-beta lactamase inhibitor combinations. This study provides a contemporary global view of OmpK36-mediated resistance mechanisms in KP, integrating surveillance and experimental data to guide treatment and drug development strategies.

Journal article

David S, Wong JLC, Sanchez-Garrido J, Kwong H-S, Low WW, Morecchiato F, Giani T, Rossolini GM, Brett SJ, Clements A, Beis K, Aanensen D, Frankel Get al., 2022, Widespread emergence of OmpK36 loop 3 insertions among multidrug-resistant clones of <i>Klebsiella pneumoniae</i>

<jats:title>Abstract</jats:title><jats:p>Mutations in outer membrane porins act in synergy with carbapenemase enzymes to increase carbapenem resistance in the important nosocomial pathogen, <jats:italic>Klebsiella pneumoniae</jats:italic> (KP). A key example is a di-amino acid insertion, Glycine-Aspartate (GD), in the extracellular loop 3 (L3) region of OmpK36 which constricts the pore and restricts entry of carbapenems into the bacterial cell. Here we combined genomic and experimental approaches to characterise the diversity, spread and impact of different L3 insertion types in OmpK36. We identified L3 insertions in 3588 (24.1%) of 14,888 KP genomes with an intact <jats:italic>ompK36</jats:italic> gene from a global collection. GD insertions were most common, with a high concentration in the ST258/512 clone that has spread widely in Europe and the Americas. Aspartate (D) and Threonine-Aspartate (TD) insertions were prevalent in genomes from Asia, due in part to acquisitions by ST16 and ST231 and subsequent clonal expansions. By solving the crystal structures of novel OmpK36 variants, we found that the TD insertion causes a pore constriction of 41%, significantly greater than that achieved by GD (10%) or D (8%), resulting in the highest levels of resistance to selected antibiotics. In a murine pneumonia model, KP mutants harbouring L3 insertions have a competitive disadvantage relative to a strain expressing wild-type OmpK36 in the absence of antibiotics. This explains the reversion of GD and TD insertions observed at low frequency among KP genomes. Finally, we demonstrate that strains expressing L3 insertions remain susceptible to drugs targeting carbapenemase-producing KP, including novel beta lactam-beta lactamase inhibitor combinations. This study provides a contemporary global view of OmpK36-mediated resistance mechanisms in KP, integrating surveillance and experimental data to guide treatment and drug development strat

Working paper

Wong JLC, David S, Sanchez-Garrido J, Woo JZ, Low WW, Morecchiato F, Giani T, Rossolini GM, Brett SJ, Clements A, Aanensen DM, Rouskin S, Frankel Get al., 2022, Recurrent emergence of carbapenem resistance in <i>Klebsiella pneumoniae</i> mediated by an inhibitory <i>ompK36</i> mRNA secondary structure

<jats:title>Abstract</jats:title><jats:p>Outer membrane porins in Gram-negative bacteria facilitate antibiotic influx. In <jats:italic>Klebsiella pneumoniae</jats:italic> (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&gt;t) in <jats:italic>ompK36.</jats:italic> We show that the 25c&gt;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&gt;t transition tips the balance towards treatment failure, thus accounting for its recurrent emergence. Mechanistically, the 25c&gt;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 <jats:italic>ompK36</jats:italic> mutation mediating translational suppression in response to antibiotic pressure.</jats:p>

Working paper

Chatterjee S, Lekmeechai S, Constantinou N, Grzybowska EA, Kozik Z, Choudhary JS, Berger CN, Frankel G, Clements Aet al., 2021, The type III secretion system effector EspO of enterohaemorrhagic <i>Escherichia coli</i> inhibits apoptosis through an interaction with HAX-1, CELLULAR MICROBIOLOGY, Vol: 23, ISSN: 1462-5814

Journal article

Jégouzo SAF, Nelson C, Hardwick T, Wong STA, Lau NKK, Neoh GKE, Castellanos-Rueda R, Huang Z, Mignot B, Hirdaramani A, Howitt A, Frewin K, Shen Z, Fox RJ, Wong R, Ando M, Emony L, Zhu H, Holder A, Werling D, Krishnan N, Robertson BD, Clements A, Taylor ME, Drickamer Ket al., 2020, Mammalian lectin arrays for screening host–microbe interactions, Journal of Biological Chemistry, Vol: 295, Pages: 4541-4555, ISSN: 0021-9258

Many members of the C-type lectin family of glycan-binding receptors have been ascribed roles in the recognition of microorganisms and serve as key receptors in the innate immune response to pathogens. Other mammalian receptors have become targets through which pathogens enter target cells. These receptor roles have often been documented with binding studies involving individual pairs of receptors and microorganisms. To provide a systematic overview of interactions between microbes and the large complement of C-type lectins, here we developed a lectin array and suitable protocols for labeling of microbes that could be used to probe this array. The array contains C-type lectins from cow, chosen as a model organism of agricultural interest for which the relevant pathogen–receptor interactions have not been previously investigated in detail. Screening with yeast cells and various strains of both Gram-positive and -negative bacteria revealed distinct binding patterns, which in some cases could be explained by binding to lipopolysaccharides or capsular polysaccharides, but in other cases they suggested the presence of novel glycan targets on many of the microorganisms. These results are consistent with interactions previously ascribed to the receptors, but they also highlight binding to additional sugar targets that have not previously been recognized. Our findings indicate that mammalian lectin arrays represent unique discovery tools for identifying both novel ligands and new receptor functions.

Journal article

Sanchez Garrido J, Slater SL, Clements A, Shenoy A, Frankel Get al., 2020, Vying for the control of inflammasomes: the cytosolic frontier of enteric bacterial pathogen - host interactions, Cellular Microbiology, Vol: 22, Pages: 1-19, ISSN: 1462-5814

Enteric pathogen-host interactions occur at multiple interfaces,includingthe intestinal epitheliumand deeper organsof the immune system. Microbial ligands and activities are detected by host sensorsthat elicit a range of immune responses. Membrane-bound Toll-Like Receptors (TLRs) and cytosolic inflammasomepathways are key signal transducers that trigger production of pro-inflammatory molecules such as cytokines and chemokinesand regulate cell deathin response to infection. In recent years, the inflammasomes have emerged as a key frontier in the tusslebetween bacterial pathogens and the host. Inflammasomes are complexes that activate caspase-1and are regulated by related caspases, such as caspase-11, -4, -5 and -8.Importantly, enteric bacterial pathogens can actively engage or evade inflammasome signalling systems. Extracellular, vacuolar and cytosolic bacteria have developed divergent strategies to subvert inflammasomes. While some pathogens take advantage of inflammasomeactivation(e.g. Listeria monocytogenes, Helicobacter pylori), others(e.g. E. coli, Salmonella, Shigella, Yersinia sp.) deploy a range of virulence factors, mainly type 3 secretion system (T3SS) effectors, that subvert or inhibit inflammasomes. In this review we focus on inflammasomepathwaysand their immune functions and discuss how enteric bacterial pathogens interact with them.These studies have not only shed light on the inflammasome-mediated immunity, but also the exciting area of mammalian cytosolic immune

Journal article

Watson J, Sanchez-garrido J, Goddard P, Torraca V, Mostowy S, Shenoy A, Clements Aet al., 2019, Shigella sonnei O-Antigen Inhibits Internalization, Vacuole Escape, and Inflammasome Activation, mBio, Vol: 10, Pages: 1-14, ISSN: 2150-7511

Two Shigella species, flexneri and sonnei, cause approximately 90% of bacterial dysentery worldwide. While S. flexneri is the dominant species in low-income countries, S. sonnei causes the majority of infections in middle and high-income countries. S. flexneri is a prototypiccytosolic bacterium; once intracellular it rapidly escapes the phagocytic vacuole and causes pyroptosis of macrophages, which is important for pathogenesis and bacterial spread. By contrast little is known about the invasion, vacuole escape and induction of pyroptosis during S. sonnei infection of macrophages. We demonstrate that S. sonnei causes substantially less pyroptosis in human primary monocyte-derived macrophages and THP1 cells. This is due to reduced bacterial uptake and lower relative vacuole escape, which results in fewer cytosolic S. sonnei and hence reduced activation of caspase-1 inflammasomes. Mechanistically, the O-antigen, which in S. sonnei is contained in both the lipopolysaccharide and the capsule, was responsible for reduced uptake and the T3SS was required for vacuole escape. Our findings suggest that S. sonnei has adapted to an extracellular lifestyle by incorporating multiple layers of O-antigen onto its surface compared to other Shigella species.

Journal article

Torraca V, Kaforou M, Watson J, Duggan GM, Guerrero-Gutierrez H, Krokowski S, Hollinshead M, Clarke TB, Mostowy RJ, Tomlinson GS, Sancho-Shimizu V, Clements A, Mostowy Set al., 2019, Shigella sonnei infection of zebrafish reveals that O-antigen mediates neutrophil tolerance and dysentery incidence, PLoS Pathogens, Vol: 15, Pages: 1-26, ISSN: 1553-7366

Shigella flexneri is historically regarded as the primary agent of bacillary dysentery, yet the closely-related Shigella sonnei is replacing S. flexneri, especially in developing countries. The underlying reasons for this dramatic shift are mostly unknown. Using a zebrafish (Danio rerio) model of Shigella infection, we discover that S. sonnei is more virulent than S. flexneri in vivo. Whole animal dual-RNAseq and testing of bacterial mutants suggest that S. sonnei virulence depends on its O-antigen oligosaccharide (which is unique among Shigella species). We show in vivo using zebrafish and ex vivo using human neutrophils that S. sonnei O-antigen can mediate neutrophil tolerance. Consistent with this, we demonstrate that O-antigen enables S. sonnei to resist phagolysosome acidification and promotes neutrophil cell death. Chemical inhibition or promotion of phagolysosome maturation respectively decreases and increases neutrophil control of S. sonnei and zebrafish survival. Strikingly, larvae primed with a sublethal dose of S. sonnei are protected against a secondary lethal dose of S. sonnei in an O-antigen-dependent manner, indicating that exposure to O-antigen can train the innate immune system against S. sonnei. Collectively, these findings reveal O-antigen as an important therapeutic target against bacillary dysentery, and may explain the rapidly increasing S. sonnei burden in developing countries.

Journal article

Watson JL, Sanchez-Garrido J, Goddard PJ, Torraca V, Mostowy S, Shenoy AR, Clements Aet al., 2019, <i>Shigella sonnei</i>O-antigen inhibits internalisation, vacuole escape and inflammasome activation

<jats:title>Abstract</jats:title><jats:p>Two<jats:italic>Shigella</jats:italic>species,<jats:italic>flexneri</jats:italic>and<jats:italic>sonnei</jats:italic>, cause approximately 90% of bacterial dysentery worldwide. While<jats:italic>S. flexneri</jats:italic>is the dominant species in low-income countries,<jats:italic>S. sonnei</jats:italic>causes the majority of infections in middle and high-income countries.<jats:italic>S. flexneri</jats:italic>is a prototypic cytosolic bacterium; once intracellular it rapidly escapes the phagocytic vacuole and causes pyroptosis of macrophages, which is important for pathogenesis and bacterial spread. By contrast little is known about the invasion, vacuole escape and induction of pyroptosis during<jats:italic>S. sonnei</jats:italic>infection of macrophages. We demonstrate that<jats:italic>S. sonnei</jats:italic>causes substantially less pyroptosis in human primary monocyte-derived macrophages and THP1 cells. This is due to reduced bacterial uptake and lower relative vacuole escape, which results in fewer cytosolic<jats:italic>S. sonnei</jats:italic>and hence reduced activation of caspase-1 inflammasomes. Mechanistically, the O-antigen, which in<jats:italic>S. sonnei</jats:italic>is contained in both the lipopolysaccharide and the capsule, was responsible for reduced uptake and the T3SS was required for vacuole escape. Our findings suggest that<jats:italic>S. sonnei</jats:italic>has adapted to an extracellular lifestyle by incorporating additional O-antigen into its surface structures compared to other<jats:italic>Shigella</jats:italic>species.</jats:p>

Working paper

Wong JLC, Romano M, Kerry LE, Kwong H-S, Low W-W, Brett SJ, Clements A, Beis K, Frankel Get al., 2019, OmpK36-mediated Carbapenem resistance attenuates ST258 <i>Klebsiella pneumoniae</i> in vivo, NATURE COMMUNICATIONS, Vol: 10, ISSN: 2041-1723

Journal article

Torraca V, Kaforou M, Watson J, Duggan GM, Guerrero-Gutierrez H, Krokowski S, Hollinshead M, Clarke TB, Mostowy RJ, Tomlinson GS, Sancho-Shimizu V, Clements A, Mostowy Set al., 2019, <i>Shigella sonnei</i>infection of zebrafish reveals that O-antigen mediates neutrophil tolerance and dysentery incidence

<jats:title>Abstract</jats:title><jats:p><jats:italic>Shigella flexneri</jats:italic>is historically regarded as the primary agent of bacillary dysentery, yet the closely-related<jats:italic>Shigella sonnei</jats:italic>is replacing<jats:italic>S. flexneri</jats:italic>, especially in developing countries. The underlying reasons for this dramatic shift are mostly unknown. Using a zebrafish (<jats:italic>Danio rerio</jats:italic>) model of<jats:italic>Shigella</jats:italic>infection, we discover that<jats:italic>S. sonnei</jats:italic>is more virulent than<jats:italic>S. flexneri in vivo</jats:italic>. Whole animal dual-RNAseq and testing of bacterial mutants suggest that<jats:italic>S. sonnei</jats:italic>virulence depends on its O-antigen oligosaccharide (which is unique among<jats:italic>Shigella</jats:italic>species). We show<jats:italic>in vivo</jats:italic>using zebrafish and<jats:italic>ex vivo</jats:italic>using human neutrophils that<jats:italic>S. sonnei</jats:italic>O-antigen can mediate neutrophil tolerance. Consistent with this, we demonstrate that O-antigen enables<jats:italic>S. sonnei</jats:italic>to resist phagolysosome acidification and promotes neutrophil cell death. Chemical inhibition or promotion of phagolysosome maturation respectively decreases and increases neutrophil control of<jats:italic>S. sonnei</jats:italic>and zebrafish survival. Strikingly, larvae primed with a sublethal dose of<jats:italic>S. sonnei</jats:italic>are protected against a secondary lethal dose of<jats:italic>S. sonnei</jats:italic>in an O-antigen-dependent manner, indicating that exposure to O-antigen can train the innate immune system against<jats:italic>S. sonnei</jats:italic>. Collectively, these findings reveal O-antigen as an important therapeutic

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

Torres VVL, Heinz E, Stubenrauch CJ, Wilksch JJ, Cao H, Yang J, Clements A, Dunstan RA, Alcock F, Webb CT, Dougan G, Strugnell RA, Hay ID, Lithgow Tet al., 2018, An investigation into the Omp85 protein BamK in hypervirulent <i>Klebsiella pneumoniae</i>, and its role in outer membrane biogenesis, MOLECULAR MICROBIOLOGY, Vol: 109, Pages: 584-599, ISSN: 0950-382X

Journal article

Shenoy AR, Furniss RCD, Goddard PJ, Clements Aet al., 2018, Modulation of host cell processes by T3SS effectors, Escherichia coli, a Versatile Pathogen, Editors: Frankel, Ron, Publisher: Springer Verlag

Two of the enteric Escherichia coli pathotypes-enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC)-have a conserved type 3 secretion system which is essential for virulence. The T3SS is used to translocate between 25 and 50 bacterial proteins directly into the host cytosol where they manipulate a variety of host cell processes to establish a successful infection. In this chapter, we discuss effectors from EPEC/EHEC in the context of the host proteins and processes that they target-the actin cytoskeleton, small guanosine triphosphatases and innate immune signalling pathways that regulate inflammation and cell death. Many of these translocated proteins have been extensively characterised, which has helped obtain insights into the mechanisms of pathogenesis of these bacteria and also understand the host pathways they target in more detail. With increasing knowledge of the positive and negative regulation of host signalling pathways by different effectors, a future challenge is to investigate how the specific effector repertoire of each strain cooperates over the course of an infection.

Book chapter

Watson J, Jenkins C, Clements A, 2018, Shigella sonnei does not utilize amoebae as a protective host, Applied and Environmental Microbiology, Vol: 84, ISSN: 0099-2240

Shigella flexneri and Shigella sonnei bacteria cause the majority of all shigellosis cases worldwide. However, their distributions differ, with S. sonnei predominating in middle- and high-income countries and S. flexneri predominating in low-income countries. One proposed explanation for the continued range expansion of S. sonnei is that it can survive in amoebae, which could provide a protective environment for the bacteria. In this study, we demonstrate that while both S. sonnei and S. flexneri can survive coculture with the free-living amoebae Acanthamoebae castellanii, bacterial growth is predominantly extracellular. All isolates of Shigella were degraded following phagocytosis by A. castellanii, unlike those of Legionella pneumophila, which can replicate intracellularly. Our data suggest that S. sonnei is not able to use amoebae as a protective host to enhance environmental survival. Therefore, alternative explanations for S. sonnei emergence need to be considered.IMPORTANCE The distribution of Shigella species closely mirrors a country's socioeconomic conditions. With the transition of many populous nations from low- to middle-income countries, S. sonnei infections have emerged as a major public health issue. Understanding why S. sonnei infections are resistant to improvements in living conditions is key to developing methods to reduce exposure to this pathogen. We show that free-living amoebae are not likely to be environmental hosts of S. sonnei, as all Shigella strains tested were phagocytosed and degraded by amoebae. Therefore, alternative scenarios are required to explain the emergence and persistence of S. sonnei infections.

Journal article

Berger C, Crepin V, Roumeliotis TI, Wright JC, Carson D, Pevsner-Fischer M, Furniss RCD, Dougan G, Bachash M, Yu L, Clements A, Collins JW, Elinav E, larrouy-maumus G, Choudhary JS, Frankel GMet al., 2017, Citrobacter rodentium subverts ATP flux 1 and cholesterol homeostasis in 2 intestinal epithelial cell in vivo, Cell Metabolism, Vol: 26, Pages: 738-752.e6, ISSN: 1550-4131

The intestinal epithelial cells (IECs) that line the gut form a robust line of defense against ingested pathogens. We investigated the impact of infection with the enteric pathogen Citrobacter rodentium on mouse IEC metabolism using global proteomic and targeted metabolomics and lipidomics. The major signatures of the infection were upregulation of the sugar transporter Sglt4, aerobic glycolysis, and production of phosphocreatine, which mobilizes cytosolic energy. In contrast, biogenesis of mitochondrial cardiolipins, essential for ATP production, was inhibited, which coincided with increased levels of mucosal O2 and a reduction in colon-associated anaerobic commensals. In addition, IECs responded to infection by activating Srebp2 and the cholesterol biosynthetic pathway. Unexpectedly, infected IECs also upregulated the cholesterol efflux proteins AbcA1, AbcG8, and ApoA1, resulting in higher levels of fecal cholesterol and a bloom of Proteobacteria. These results suggest that C. rodentium manipulates host metabolism to evade innate immune responses and establish a favorable gut ecosystem.

Journal article

Furniss RCD, Clements A, 2017, Regulation of the Locus of Enterocyte Effacement in Attaching and Effacing Pathogens., Journal of Bacteriology, Vol: 200, ISSN: 0021-9193

Attaching and Effacing (AE) pathogens colonise the gut mucosa using a Type Three Secretion System (T3SS) and a suite of effector proteins. The Locus of Enterocyte Effacement (LEE) is the defining genetic feature of the AE pathogens, encoding the T3SS and the core effector proteins necessary for pathogenesis. Extensive research has revealed a complex regulatory network that senses and responds to a myriad of host and microbiota-derived signals in the infected gut to control transcription of the LEE. These signals include microbiota-liberated sugars and metabolites in the gut lumen, molecular oxygen at the gut epithelium and host hormones. Recent research has revealed that AE pathogens also perceive physical signals, such as attachment to the epithelium, and that the act of effector translocation remodels gene expression in infecting bacteria. In this review we summarise our knowledge to date and present an integrated view of how chemical, geographical and physical cues regulate the virulence program of AE pathogens during infection.

Journal article

Mavridou DAI, Gonzalez D, Clements A, Foster KRet al., 2016, The pUltra plasmid series: a robust and flexible tool for fluorescent labeling of Enterobacteria, Plasmid, Vol: 87-88, Pages: 65-71, ISSN: 1095-9890

Fluorescent labeling has been an invaluable tool for the study of living organisms andbacterial species are no exception to this. Here we present and characterize the pUltraplasmids which express constitutively a fluorescent protein gene (GFP, RFP, YFP or CFP)from a strong synthetic promoter and are suitable for the fluorescent labeling of a broad rangeof Enterobacteria. The amount of expressed fluorophore from these genetic constructs issuch, that the contours of the cells can be delineated on the basis of the fluorescent signalonly. In addition, labeling through the pUltra plasmids can be used successfully forfluorescence and confocal microscopy while unambiguous distinction of cells labeled withdifferent colors can be carried out efficiently by microscopy or flow cytometry. We comparethe labeling provided by the pUltra plasmids with that of another plasmid series encodingfluorescent proteins and we show that the pUltra constructs are vastly superior in signalintensity and discrimination power without having any detectable growth rate effects for thebacterial population. We also use the pUltra plasmids to produce mixtures of differentiallylabeled pathogenic Escherichia, Shigella and Salmonella species which we test duringinfection of mammalian cells. We find that even inside the host cell, different strains can bedistinguished effortlessly based on their fluorescence. We, therefore, conclude that the pUltraplasmids are a powerful labeling tool especially useful for complex biological experimentssuch as the visualization of ecosystems of different bacterial species or of enteric pathogensin contact with their hosts.

Journal article

Furniss RCD, Slater S, Frankel G, Clements Aet al., 2016, Enterohaemorrhagic E. coli modulates an ARF6:Rab35 signalling axis to prevent recycling endosome maturation during infection, Journal of Molecular Biology, Vol: 428, Pages: 3399-3407, ISSN: 1089-8638

Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC/EHEC) manipulate a plethora of host cell processes to establish infection of the gut mucosa. This manipulation is achieved via the injection of bacterial effector proteins into host cells using a Type III secretion system. We have previously reported that the conserved EHEC and EPEC effector EspG disrupts recycling endosome function, reducing cell surface levels of host receptors through accumulation of recycling cargo within the host cell. Here we report that EspG interacts specifically with the small GTPases ARF6 and Rab35 during infection. These interactions target EspG to endosomes and prevent Rab35-mediated recycling of cargo to the host cell surface. Furthermore, we show that EspG has no effect on Rab35-mediated uncoating of newly formed endosomes, and instead leads to the formation of enlarged EspG/TfR/Rab11 positive, EEA1/Clathrin negative stalled recycling structures. Thus, this paper provides a molecular framework to explain how EspG disrupts recycling whilst also reporting the first known simultaneous targeting of ARF6 and Rab35 by a bacterial pathogen.

Journal article

Larrouy-Maumus GJ, Abigail Clements, Alain Filloux, Ronan R McCarthy, Serge Mostowyet al., 2015, Direct detection of lipid A on intact Gram-negative bacteria byMALDI-TOF mass spectrometry, Journal of Microbiological Methods, Vol: 120, Pages: 68-71, ISSN: 1872-8359

The purification and characterization of Gram-negative bacterial lipid A is tedious and time-consuming. Herein we report a rapid and sensitive method to identify lipid A directly on intact bacteria without any chemical treatment or purification, using an atypical solvent system to solubilize the matrix combined with MALDI-TOF mass spectrometry.

Journal article

Young JC, Clements A, Lang AE, Garnett JA, Munera D, Arbeloa A, Pearson J, Hartland EL, Matthews SJ, Mousnier A, Barry DJ, Way M, Schlosser A, Aktories K, Frankel Get al., 2014, The Escherichia coli effector EspJ blocks Src kinase activity via amidation and ADP ribosylation, Nature Communications, Vol: 5, ISSN: 2041-1723

The hallmark of enteropathogenic Escherichia coli (EPEC) infection is the formation of actin-rich pedestal-like structures, which are generated following phosphorylation of the bacterial effector Tir by cellular Src and Abl family tyrosine kinases. This leads to recruitment of the Nck–WIP–N-WASP complex that triggers Arp2/3-dependent actin polymerization in the host cell. The same phosphorylation-mediated signalling network is also assembled downstream of the Vaccinia virus protein A36 and the phagocytic Fc-gamma receptor FcγRIIa. Here we report that the EPEC type-III secretion system effector EspJ inhibits autophosphorylation of Src and phosphorylation of the Src substrates Tir and FcγRIIa. Consistent with this, EspJ inhibits actin polymerization downstream of EPEC, Vaccinia virus and opsonized red blood cells. We identify EspJ as a unique adenosine diphosphate (ADP) ribosyltransferase that directly inhibits Src kinase by simultaneous amidation and ADP ribosylation of the conserved kinase-domain residue, Src E310, resulting in glutamine-ADP ribose.

Journal article

Clements A, Stoneham CA, Furniss RCD, Frankel Get al., 2014, Enterohaemorrhagic <i>Escherichia coli</i> inhibits recycling endosome function and trafficking of surface receptors, CELLULAR MICROBIOLOGY, Vol: 16, Pages: 1693-1705, ISSN: 1462-5814

Journal article

Raymond B, Young JC, Pallett M, Endres RG, Clements A, Frankel Get al., 2013, Subversion of trafficking, apoptosis, and innate immunity by type III secretion system effectors, TRENDS IN MICROBIOLOGY, Vol: 21, Pages: 430-439, ISSN: 0966-842X

Journal article

Velkov T, Soon RL, Chong PL, Huang JX, Cooper MA, Azad MAK, Baker MA, Thompson PE, Roberts K, Nation RL, Clements A, Strugnell RA, Li Jet al., 2013, Molecular basis for the increased polymyxin susceptibility of <i>Klebsiella pneumoniae</i> strains with under-acylated lipid A, INNATE IMMUNITY, Vol: 19, Pages: 265-277, ISSN: 1753-4259

Journal article

Clements A, Berger CN, Lomma M, Frankel Get al., 2013, Type 3 secretion effectors, Escherichia coliPathotypes and Principles of Pathogenesis, Editors: Donnenberg, ISBN: 978-0-12-397048-0

Book chapter

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