29 results on '"Allsopp LP"'
Search Results
2. Integrating signals to drive T6SS killing
- Author
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Bernal, P, Murillo‐Torres, M, Allsopp, LP, and ESCMID (European Society of Clinical Microbiology and Infectious Diseases)
- Subjects
0603 Evolutionary Biology ,Microbiology ,0605 Microbiology - Published
- 2020
3. The VgrG Proteins Are 'à la Carte' Delivery Systems for Bacterial Type VI Effectors
- Author
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Hachani, A, Allsopp, LP, Oduko, Y, Filloux, A, Biotechnology and Biological Sciences Research Council (BBSRC), and Medical Research Council (MRC)
- Subjects
Biochemistry & Molecular Biology ,Bacterial Toxins ,VIBRIO-CHOLERAE ,Bacterial Toxin ,GENE FAMILY ,TOXIN ,Microbiology ,OPPORTUNISTIC PATHOGEN ,Bacterial Proteins ,VgrG ,Bacterial Genetics ,CRYSTAL-STRUCTURE ,Pseudomonas aeruginosa (P. aeruginosa) ,GRAM-NEGATIVE BACTERIA ,Bacterial Secretion Systems ,Bacterial Cell Envelope ,TAIL ,Type VI Secretion System ,Science & Technology ,Protein Translocation ,PSEUDOMONAS-AERUGINOSA ,SECRETION-SYSTEM ,11 Medical And Health Sciences ,06 Biological Sciences ,Pseudomonas aeruginosa ,TARGET-CELLS ,Carrier Proteins ,03 Chemical Sciences ,Life Sciences & Biomedicine - Abstract
The bacterial type VI secretion system (T6SS) is a supra-molecular complex akin to bacteriophage tails, with VgrG proteins acting as a puncturing device. The Pseudomonas aeruginosa H1-T6SS has been extensively characterized. It is involved in bacterial killing and in the delivery of three toxins, Tse1–3. Here, we demonstrate the independent contribution of the three H1-T6SS co-regulated vgrG genes, vgrG1abc, to bacterial killing. A putative toxin is encoded in the vicinity of each vgrG gene, supporting the concept of specific VgrG/toxin couples. In this respect, VgrG1c is involved in the delivery of an Rhs protein, RhsP1. The RhsP1 C terminus carries a toxic activity, from which the producing bacterium is protected by a cognate immunity. Similarly, VgrG1a-dependent toxicity is associated with the PA0093 gene encoding a two-domain protein with a putative toxin domain (Toxin_61) at the C terminus. Finally, VgrG1b-dependent killing is detectable upon complementation of a triple vgrG1abc mutant. The VgrG1b-dependent killing is mediated by PA0099, which presents the characteristics of the superfamily nuclease 2 toxin members. Overall, these data develop the concept that VgrGs are indispensable components for the specific delivery of effectors. Several additional vgrG genes are encoded on the P. aeruginosa genome and are not linked genetically to other T6SS genes. A closer inspection of these clusters reveals that they also encode putative toxins. Overall, these associations further support the notion of an original form of secretion system, in which VgrG acts as the carrier.
- Published
- 2014
4. Respiratory Epithelial Cell Surface Decoration Provides Defence to Bacterial Damage During Infection.
- Author
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Ellis HR and Allsopp LP
- Published
- 2024
- Full Text
- View/download PDF
5. Combined functional genomic and metabolomic approaches identify new genes required for growth in human urine by multidrug-resistant Escherichia coli ST131.
- Author
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Phan M-D, Schirra HJ, Nhu NTK, Peters KM, Sarkar S, Allsopp LP, Achard MES, Kappler U, and Schembri MA
- Subjects
- Humans, Escherichia coli genetics, Fluorides metabolism, Lipopolysaccharides metabolism, Genomics, Nucleotides metabolism, Lactates metabolism, Urinary Tract Infections microbiology, Escherichia coli Infections microbiology, Uropathogenic Escherichia coli genetics
- Abstract
Urinary tract infections (UTIs) are one of the most common bacterial infections in humans, with ~400 million cases across the globe each year. Uropathogenic Escherichia coli (UPEC) is the major cause of UTI and increasingly associated with antibiotic resistance. This scenario has been worsened by the emergence and spread of pandemic UPEC sequence type 131 (ST131), a multidrug-resistant clone associated with extraordinarily high rates of infection. Here, we employed transposon-directed insertion site sequencing in combination with metabolomic profiling to identify genes and biochemical pathways required for growth and survival of the UPEC ST131 reference strain EC958 in human urine (HU). We identified 24 genes required for growth in HU, which mapped to diverse pathways involving small peptide, amino acid and nucleotide metabolism, the stringent response pathway, and lipopolysaccharide biosynthesis. We also discovered a role for UPEC resistance to fluoride during growth in HU, most likely associated with fluoridation of drinking water. Complementary nuclear magnetic resonance (NMR)-based metabolomics identified changes in a range of HU metabolites following UPEC growth, the most pronounced being L-lactate, which was utilized as a carbon source via the L-lactate dehydrogenase LldD. Using a mouse UTI model with mixed competitive infection experiments, we demonstrated a role for nucleotide metabolism and the stringent response in UPEC colonization of the mouse bladder. Together, our application of two omics technologies combined with different infection-relevant settings has uncovered new factors required for UPEC growth in HU, thus enhancing our understanding of this pivotal step in the UPEC infection pathway., Importance: Uropathogenic Escherichia coli (UPEC) cause ~80% of all urinary tract infections (UTIs), with increasing rates of antibiotic resistance presenting an urgent threat to effective treatment. To cause infection, UPEC must grow efficiently in human urine (HU), necessitating a need to understand mechanisms that promote its adaptation and survival in this nutrient-limited environment. Here, we used a combination of functional genomic and metabolomic techniques and identified roles for the metabolism of small peptides, amino acids, nucleotides, and L-lactate, as well as the stringent response pathway, lipopolysaccharide biosynthesis, and fluoride resistance, for UPEC growth in HU. We further demonstrated that pathways involving nucleotide metabolism and the stringent response are required for UPEC colonization of the mouse bladder. The UPEC genes and metabolic pathways identified in this study represent targets for the development of innovative therapeutics to prevent UPEC growth during human UTI, an urgent need given the rapidly rising rates of global antibiotic resistance., Competing Interests: The authors declare no conflict of interest.
- Published
- 2024
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6. Killing in the name of: T6SS structure and effector diversity.
- Author
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Allsopp LP and Bernal P
- Subjects
- Bacteria genetics, Gram-Negative Bacteria genetics, Eukaryota, Bacterial Proteins genetics, Type VI Secretion Systems genetics
- Abstract
The life of bacteria is challenging, to endure bacteria employ a range of mechanisms to optimize their environment, including deploying the type VI secretion system (T6SS). Acting as a bacterial crossbow, this system delivers effectors responsible for subverting host cells, killing competitors and facilitating general secretion to access common goods. Due to its importance, this lethal machine has been evolutionarily maintained, disseminated and specialized to fulfil these vital functions. In fact, T6SS structural clusters are present in over 25 % of Gram-negative bacteria, varying in number from one to six different genetic clusters per organism. Since its discovery in 2006, research on the T6SS has rapidly progressed, yielding remarkable breakthroughs. The identification and characterization of novel components of the T6SS, combined with biochemical and structural studies, have revealed fascinating mechanisms governing its assembly, loading, firing and disassembly processes. Recent findings have also demonstrated the efficacy of this system against fungal and Gram-positive cells, expanding its scope. Ongoing research continues to uncover an extensive and expanding repertoire of T6SS effectors, the genuine mediators of T6SS function. These studies are shedding light on new aspects of the biology of prokaryotic and eukaryotic organisms. This review provides a comprehensive overview of the T6SS, highlighting recent discoveries of its structure and the diversity of its effectors. Additionally, it injects a personal perspective on avenues for future research, aiming to deepen our understanding of this combative system.
- Published
- 2023
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7. Diversity and prevalence of type VI secretion system effectors in clinical Pseudomonas aeruginosa isolates.
- Author
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Robinson LA, Collins ACZ, Murphy RA, Davies JC, and Allsopp LP
- Abstract
Pseudomonas aeruginosa is an opportunistic pathogen and a major driver of morbidity and mortality in people with Cystic Fibrosis (CF). The Type VI secretion system (T6SS) is a molecular nanomachine that translocates effectors across the bacterial membrane into target cells or the extracellular environment enabling intermicrobial interaction. P. aeruginosa encodes three T6SS clusters, the H1-, H2- and H3-T6SS, and numerous orphan islands. Genetic diversity of T6SS-associated effectors in P. aeruginosa has been noted in reference strains but has yet to be explored in clinical isolates. Here, we perform a comprehensive bioinformatic analysis of the pangenome and T6SS effector genes in 52 high-quality clinical P. aeruginosa genomes isolated from CF patients and housed in the Personalised Approach to P. aeruginosa strain repository. We confirm that the clinical CF isolate pangenome is open and principally made up of accessory and unique genes that may provide strain-specific advantages. We observed genetic variability in some effector/immunity encoding genes and show that several well-characterised vgrG and PAAR islands are absent from numerous isolates. Our analysis shows clear evidence of disruption to T6SS genomic loci through transposon, prophage, and mobile genetic element insertions. We identified an orphan vgrG island in P. aeruginosa strain PAK and five clinical isolates using in silico analysis which we denote vgrG7 , predicting a gene within this cluster to encode a Tle2 lipase family effector. Close comparison of T6SS loci in clinical isolates compared to reference P. aeruginosa strain PAO1 revealed the presence of genes encoding eight new T6SS effectors with the following putative functions: cytidine deaminase, lipase, metallopeptidase, NADase, and pyocin. Finally, the prevalence of characterised and putative T6SS effectors were assessed in 532 publicly available P. aeruginosa genomes, which suggests the existence of accessory effectors. Our in silico study of the P. aeruginosa T6SS exposes a level of genetic diversity at T6SS genomic loci not seen to date within P. aeruginosa, particularly in CF isolates. As understanding the effector repertoire is key to identifying the targets of T6SSs and its efficacy, this comprehensive analysis provides a path for future experimental characterisation of these mediators of intermicrobial competition and host manipulation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Robinson, Collins, Murphy, Davies and Allsopp.)
- Published
- 2023
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8. Differential Afa/Dr Fimbriae Expression in the Multidrug-Resistant Escherichia coli ST131 Clone.
- Author
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Alvarez-Fraga L, Phan MD, Goh KGK, Nhu NTK, Hancock SJ, Allsopp LP, Peters KM, Forde BM, Roberts LW, Sullivan MJ, Totsika M, Beatson SA, Ulett GC, and Schembri MA
- Subjects
- Humans, Adhesins, Bacterial metabolism, Anti-Bacterial Agents metabolism, Clone Cells, DNA Transposable Elements, Virulence genetics, Drug Resistance, Multiple, Bacterial genetics, Escherichia coli Infections genetics, Urinary Tract Infections genetics, Uropathogenic Escherichia coli genetics, Uropathogenic Escherichia coli pathogenicity
- Abstract
Many antibiotic resistant uropathogenic Escherichia coli (UPEC) strains belong to clones defined by their multilocus sequence type (ST), with ST131 being the most dominant. Although we have a good understanding of resistance development to fluoroquinolones and third-generation cephalosporins by ST131, our understanding of the virulence repertoire that has contributed to its global dissemination is limited. Here we show that the genes encoding Afa/Dr fimbriae, a group of adhesins strongly associated with UPEC that cause gestational pyelonephritis and recurrent cystitis, are found in approximately one third of all ST131 strains. Sequence comparison of the AfaE adhesin protein revealed a unique allelic variant carried by 82.9% of afa -positive ST131 strains. We identify the afa regulatory region as a hotspot for the integration of insertion sequence (IS) elements, all but one of which alter afa transcription. Close investigation demonstrated that the integration of an IS 1 element in the afa regulatory region leads to increased expression of Afa/Dr fimbriae, promoting enhanced adhesion to kidney epithelial cells and suggesting a mechanism for altered virulence. Finally, we provide evidence for a more widespread impact of IS 1 on ST131 genome evolution, suggesting that IS dynamics contribute to strain level microevolution that impacts ST131 fitness. IMPORTANCE E. coli ST131 is the most common antibiotic resistant UPEC clone associated with human urinary tract and bloodstream infections. Understanding the features of ST131 that have driven its global dissemination remains a critical priority if we are to counter its increasing antibiotic resistance. Here, we utilized a large collection of ST131 isolates to investigate the prevalence, regulation, and function of Afa/Dr fimbriae, a well-characterized UPEC colonization and virulence factor. We show that the afa genes are found frequently in ST131 and demonstrate how the integration of IS elements in the afa regulatory region modulates Afa expression, presenting an example of altered virulence capacity. We also exploit a curated set of ST131 genomes to map the integration of the antibiotic resistance-associated IS 1 element in the ST131 pangenome, providing evidence for its widespread impact on ST131 genome evolution.
- Published
- 2022
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9. Structure of ATP synthase from ESKAPE pathogen Acinetobacter baumannii .
- Author
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Demmer JK, Phillips BP, Uhrig OL, Filloux A, Allsopp LP, Bublitz M, and Meier T
- Subjects
- Adenosine Triphosphate metabolism, Cryoelectron Microscopy, Acinetobacter baumannii
- Abstract
The global spread of multidrug-resistant Acinetobacter baumannii infections urgently calls for the identification of novel drug targets. We solved the electron cryo-microscopy structure of the F
1 Fo -adenosine 5'-triphosphate (ATP) synthase from A. baumannii in three distinct conformational states. The nucleotide-converting F1 subcomplex reveals a specific self-inhibition mechanism, which supports a unidirectional ratchet mechanism to avoid wasteful ATP consumption. In the membrane-embedded Fo complex, the structure shows unique structural adaptations along both the entry and exit pathways of the proton-conducting a-subunit. These features, absent in mitochondrial ATP synthases, represent attractive targets for the development of next-generation therapeutics that can act directly at the culmination of bioenergetics in this clinically relevant pathogen.- Published
- 2022
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10. RpoN/Sfa2-dependent activation of the Pseudomonas aeruginosa H2-T6SS and its cognate arsenal of antibacterial toxins.
- Author
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Allsopp LP, Collins ACZ, Hawkins E, Wood TE, and Filloux A
- Subjects
- Bacterial Secretion Systems metabolism, Bacterial Toxins genetics, Bacterial Toxins metabolism, Gene Expression Regulation, Bacterial, Pseudomonas aeruginosa genetics, RNA Polymerase Sigma 54 genetics, Bacterial Secretion Systems genetics, Pseudomonas aeruginosa metabolism, RNA Polymerase Sigma 54 metabolism
- Abstract
Pseudomonas aeruginosa uses three type six secretion systems (H1-, H2- and H3-T6SS) to manipulate its environment, subvert host cells and for microbial competition. These T6SS machines are loaded with a variety of effectors/toxins, many being associated with a specific VgrG. How P. aeruginosa transcriptionally coordinates the main T6SS clusters and the multiple vgrG islands spread through the genome is unknown. Here we show an unprecedented level of control with RsmA repressing most known T6SS-related genes. Moreover, each of the H2- and H3-T6SS clusters encodes a sigma factor activator (SFA) protein called, Sfa2 and Sfa3, respectively. SFA proteins are enhancer binding proteins necessary for the sigma factor RpoN. Using a combination of RNA-seq, ChIP-seq and molecular biology approaches, we demonstrate that RpoN coordinates the T6SSs of P. aeruginosa by activating the H2-T6SS but repressing the H1- and H3-T6SS. Furthermore, RpoN and Sfa2 control the expression of the H2-T6SS-linked VgrGs and their effector arsenal to enable very effective interbacterial killing. Sfa2 is specific as Sfa3 from the H3-T6SS cannot complement loss of Sfa2. Our study further delineates the regulatory mechanisms that modulate the deployment of an arsenal of T6SS effectors likely enabling P. aeruginosa to adapt to a range of environmental conditions., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2022
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11. Antimicrobial Weapons of Pseudomonas aeruginosa.
- Author
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Nolan LM and Allsopp LP
- Subjects
- Humans, Bacterial Proteins genetics, Anti-Bacterial Agents pharmacology, Anti-Bacterial Agents therapeutic use, Pseudomonas aeruginosa genetics, Anti-Infective Agents pharmacology, Anti-Infective Agents therapeutic use
- Abstract
Pseudomonas aeruginosa is a robust and versatile organism capable of surviving and prospering in a diverse array of environments and is an opportunistic pathogen of humans. One reason for the success of this pathogen is the large arsenal of antimicrobial weapons that it possesses. Here we focus our attention on these antimicrobial weapons and how they give P. aeruginosa a survival edge in polymicrobial environments. We define antimicrobial weapons as components produced by P. aeruginosa that are used to kill, inhibit growth and/or subvert key cellular functions in other microbes. P. aeruginosa has a large and complex genome and encodes an armament of antimicrobial weapons that fall into two subclasses; those that are delivered directly to competing microbes using a contact-dependent method, and those that are secreted in a contact-independent manner into the environment to then be available to target neighbouring cells. This chapter provides an overview of the major antimicrobial weapons possessed by P. aeruginosa, captures recent advances in the field and discusses how these could be targeted as a therapeutic intervention, or potentially harnessed to combat infection., (© 2022. The Author(s), under exclusive license to Springer Nature Switzerland AG.)
- Published
- 2022
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12. Integrating signals to drive type VI secretion system killing.
- Author
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Bernal P, Murillo-Torres M, and Allsopp LP
- Published
- 2020
- Full Text
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13. Causalities of war: The connection between type VI secretion system and microbiota.
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Allsopp LP, Bernal P, Nolan LM, and Filloux A
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- Animals, Antibiosis, Bacterial Proteins physiology, Homeostasis, Host Microbial Interactions, Humans, Microbiota, Type VI Secretion Systems physiology
- Abstract
Microbiota niches have space and/or nutrient restrictions, which has led to the coevolution of cooperation, specialisation, and competition within the population. Different animal and environmental niches contain defined resident microbiota that tend to be stable over time and offer protection against undesired intruders. Yet fluxes can occur, which alter the composition of a bacterial population. In humans, the microbiota are now considered a key contributor to maintenance of health and homeostasis, and its alteration leads to dysbiosis. The bacterial type VI secretion system (T6SS) transports proteins into the environment, directly into host cells or can function as an antibacterial weapon by killing surrounding competitors. Upon contact with neighbouring cells, the T6SS fires, delivering a payload of effector proteins. In the absence of an immunity protein, this results in growth inhibition or death of prey leading to a competitive advantage for the attacker. It is becoming apparent that the T6SS has a role in modulating and shaping the microbiota at multiple levels, which is the focus of this review. Discussed here is the T6SS, its role in competition, key examples of its effect upon the microbiota, and future avenues of research., (© 2019 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd.)
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- 2020
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14. The Pseudomonas aeruginosa T6SS-VgrG1b spike is topped by a PAAR protein eliciting DNA damage to bacterial competitors.
- Author
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Pissaridou P, Allsopp LP, Wettstadt S, Howard SA, Mavridou DAI, and Filloux A
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- Bacterial Proteins genetics, Models, Molecular, Protein Conformation, Protein Interaction Domains and Motifs, Pseudomonas aeruginosa genetics, Bacterial Proteins metabolism, DNA Damage physiology, Gene Expression Regulation, Bacterial physiology, Pseudomonas aeruginosa metabolism, Type VI Secretion Systems physiology
- Abstract
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., Competing Interests: The authors declare no conflict of interest.
- Published
- 2018
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15. Probing the internal micromechanical properties of Pseudomonas aeruginosa biofilms by Brillouin imaging.
- Author
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Karampatzakis A, Song CZ, Allsopp LP, Filloux A, Rice SA, Cohen Y, Wohland T, and Török P
- Abstract
Biofilms are organised aggregates of bacteria that adhere to each other or surfaces. The matrix of extracellular polymeric substances that holds the cells together provides the mechanical stability of the biofilm. In this study, we have applied Brillouin microscopy, a technique that is capable of measuring mechanical properties of specimens on a micrometre scale based on the shift in frequency of light incident upon a sample due to thermal fluctuations, to investigate the micromechanical properties of an active, live Pseudomonas aeruginosa biofilm. Using this non-contact and label-free technique, we have extracted information about the internal stiffness of biofilms under continuous flow. No correlation with colony size was found when comparing the averages of Brillouin shifts of two-dimensional cross-sections of randomly selected colonies. However, when focusing on single colonies, we observed two distinct spatial patterns: in smaller colonies, stiffness increased towards their interior, indicating a more compact structure of the centre of the colony, whereas, larger (over 45 μm) colonies were found to have less stiff interiors.
- Published
- 2017
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16. Visualizing Antimicrobials in Bacterial Biofilms: Three-Dimensional Biochemical Imaging Using TOF-SIMS.
- Author
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Davies SK, Fearn S, Allsopp LP, Harrison F, Ware E, Diggle SP, Filloux A, McPhail DS, and Bundy JG
- Abstract
Bacterial biofilms are groups of bacteria that exist within a self-produced extracellular matrix, adhering to each other and usually to a surface. They grow on medical equipment and inserts such as catheters and are responsible for many persistent infections throughout the body, as they can have high resistance to many antimicrobials. Pseudomonas aeruginosa is an opportunistic pathogen that can cause both acute and chronic infections and is used as a model for research into biofilms. Direct biochemical methods of imaging of molecules in bacterial biofilms are of high value in gaining a better understanding of the fundamental biology of biofilms and biochemical gradients within them. Time of flight-secondary-ion mass spectrometry (TOF-SIMS) is one approach, which combines relatively high spatial resolution and sensitivity and can perform depth profiling analysis. It has been used to analyze bacterial biofilms but has not yet been used to study the distribution of antimicrobials (including antibiotics and the antimicrobial metal gallium) within biofilms. Here we compared two methods of imaging of the interior structure of P. aeruginosa in biological samples using TOF-SIMS, looking at both antimicrobials and endogenous biochemicals: cryosectioning of tissue samples and depth profiling to give pseudo-three-dimensional (pseudo-3D) images. The sample types included both simple biofilms grown on glass slides and bacteria growing in tissues in an ex vivo pig lung model. The two techniques for the 3D imaging of biofilms are potentially valuable complementary tools for analyzing bacterial infection. IMPORTANCE Modern analytical techniques are becoming increasingly important in the life sciences; imaging mass spectrometry offers the opportunity to gain unprecedented amounts of information on the distribution of chemicals in samples-both xenobiotics and endogenous compounds. In particular, simultaneous imaging of antibiotics (and other antimicrobial compounds) and bacterium-derived metabolites in complex biological samples could be very important in the future for helping to understand how sample matrices impact the survival of bacteria under antibiotic challenge. We have shown that an imaging mass spectrometric technique, TOF-SIMS, will be potentially extremely valuable for this kind of research in the future.
- Published
- 2017
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17. RsmA and AmrZ orchestrate the assembly of all three type VI secretion systems in Pseudomonas aeruginosa .
- Author
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Allsopp LP, Wood TE, Howard SA, Maggiorelli F, Nolan LM, Wettstadt S, and Filloux A
- Subjects
- Bacterial Proteins genetics, Bacterial Toxins chemistry, Cluster Analysis, Gene Deletion, Gene Regulatory Networks, Immune System, Lac Operon, Mutagenesis, Plasmids metabolism, RNA, Bacterial genetics, Transcription, Genetic, Type VI Secretion Systems genetics, Virulence genetics, Bacterial Proteins metabolism, Pseudomonas aeruginosa metabolism, Type VI Secretion Systems metabolism
- Abstract
The type VI secretion system (T6SS) is a weapon of bacterial warfare and host cell subversion. The Gram-negative pathogen Pseudomonas aeruginosa has three T6SSs involved in colonization, competition, and full virulence. H1-T6SS is a molecular gun firing seven toxins, Tse1-Tse7, challenging survival of other bacteria and helping P. aeruginosa to prevail in specific niches. The H1-T6SS characterization was facilitated through studying a P. aeruginosa strain lacking the RetS sensor, which has a fully active H1-T6SS, in contrast to the parent. However, study of H2-T6SS and H3-T6SS has been neglected because of a poor understanding of the associated regulatory network. Here we performed a screen to identify H2-T6SS and H3-T6SS regulatory elements and found that the posttranscriptional regulator RsmA imposes a concerted repression on all three T6SS clusters. A higher level of complexity could be observed as we identified a transcriptional regulator, AmrZ, which acts as a negative regulator of H2-T6SS. Overall, although the level of T6SS transcripts is fine-tuned by AmrZ, all T6SS mRNAs are silenced by RsmA. We expanded this concept of global control by RsmA to VgrG spike and T6SS toxin transcripts whose genes are scattered on the chromosome. These observations triggered the characterization of a suite of H2-T6SS toxins and their implication in direct bacterial competition. Our study thus unveils a central mechanism that modulates the deployment of all T6SS weapons that may be simultaneously produced within a single cell., Competing Interests: The authors declare no conflict of interest.
- Published
- 2017
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18. The Pseudomonas putida T6SS is a plant warden against phytopathogens.
- Author
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Bernal P, Allsopp LP, Filloux A, and Llamas MA
- Subjects
- Bacterial Proteins genetics, Biological Control Agents, Gene Expression Regulation, Bacterial, Plant Diseases microbiology, Pseudomonas putida genetics, Type VI Secretion Systems genetics, Type VI Secretion Systems physiology, Plant Diseases prevention & control, Pseudomonas putida physiology, Nicotiana microbiology, Xanthomonas campestris physiology
- Abstract
Bacterial type VI secretion systems (T6SSs) are molecular weapons designed to deliver toxic effectors into prey cells. These nanomachines have an important role in inter-bacterial competition and provide advantages to T6SS active strains in polymicrobial environments. Here we analyze the genome of the biocontrol agent Pseudomonas putida KT2440 and identify three T6SS gene clusters (K1-, K2- and K3-T6SS). Besides, 10 T6SS effector-immunity pairs were found, including putative nucleases and pore-forming colicins. We show that the K1-T6SS is a potent antibacterial device, which secretes a toxic Rhs-type effector Tke2. Remarkably, P. putida eradicates a broad range of bacteria in a K1-T6SS-dependent manner, including resilient phytopathogens, which demonstrates that the T6SS is instrumental to empower P. putida to fight against competitors. Furthermore, we observed a drastically reduced necrosis on the leaves of Nicotiana benthamiana during co-infection with P. putida and Xanthomonas campestris. Such protection is dependent on the activity of the P. putida T6SS. Many routes have been explored to develop biocontrol agents capable of manipulating the microbial composition of the rhizosphere and phyllosphere. Here we unveil a novel mechanism for plant biocontrol, which needs to be considered for the selection of plant wardens whose mission is to prevent phytopathogen infections.
- Published
- 2017
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19. Comparative proteomics of uropathogenic Escherichia coli during growth in human urine identify UCA-like (UCL) fimbriae as an adherence factor involved in biofilm formation and binding to uroepithelial cells.
- Author
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Wurpel DJ, Totsika M, Allsopp LP, Webb RI, Moriel DG, and Schembri MA
- Subjects
- Bacterial Adhesion physiology, Cell Adhesion Molecules metabolism, Epithelial Cells microbiology, Escherichia coli Proteins metabolism, Humans, Proteome metabolism, Biofilms growth & development, Fimbriae Proteins metabolism, Fimbriae, Bacterial metabolism, Urine microbiology, Uropathogenic Escherichia coli metabolism, Urothelium microbiology
- Abstract
Uropathogenic Escherichia coli (UPEC) are the primary cause of urinary tract infection (UTI) in humans. For the successful colonisation of the human urinary tract, UPEC employ a diverse collection of secreted or surface-exposed virulence factors including toxins, iron acquisition systems and adhesins. In this study, a comparative proteomic approach was utilised to define the UPEC pan and core surface proteome following growth in pooled human urine. Identified proteins were investigated for subcellular origin, prevalence and homology to characterised virulence factors. Fourteen core surface proteins were identified, as well as eleven iron uptake receptor proteins and four distinct fimbrial types, including type 1, P, F1C/S and a previously uncharacterised fimbrial type, designated UCA-like (UCL) fimbriae in this study. These pathogenicity island (PAI)-associated fimbriae are related to UCA fimbriae of Proteus mirabilis, associated with UPEC and exclusively found in members of the E. coli B2 and D phylogroup. We further demonstrated that UCL fimbriae promote significant biofilm formation on abiotic surfaces and mediate specific attachment to exfoliated human uroepithelial cells. Combined, this study has defined the surface proteomic profiles and core surface proteome of UPEC during growth in human urine and identified a new type of fimbriae that may contribute to UTI., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2016
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20. The Intimin-Like Protein FdeC Is Regulated by H-NS and Temperature in Enterohemorrhagic Escherichia coli.
- Author
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Easton DM, Allsopp LP, Phan MD, Moriel DG, Goh GK, Beatson SA, Mahony TJ, Cobbold RN, and Schembri MA
- Subjects
- Enterohemorrhagic Escherichia coli genetics, Enterohemorrhagic Escherichia coli physiology, Gene Expression Profiling, Temperature, Adhesins, Escherichia coli biosynthesis, Bacterial Proteins metabolism, Biofilms growth & development, DNA-Binding Proteins metabolism, Enterohemorrhagic Escherichia coli metabolism, Enterohemorrhagic Escherichia coli radiation effects, Gene Expression Regulation, Bacterial radiation effects
- Abstract
Enterohemorrhagic Escherichia coli (EHEC) is a Shiga-toxigenic pathogen capable of inducing severe forms of enteritis (e.g., hemorrhagic colitis) and extraintestinal sequelae (e.g., hemolytic-uremic syndrome). The molecular basis of colonization of human and animal hosts by EHEC is not yet completely understood, and an improved understanding of EHEC mucosal adherence may lead to the development of interventions that could disrupt host colonization. FdeC, also referred to by its IHE3034 locus tag ECOK1_0290, is an intimin-like protein that was recently shown to contribute to kidney colonization in a mouse urinary tract infection model. The expression of FdeC is tightly regulated in vitro, and FdeC shows promise as a vaccine candidate against extraintestinal E. coli strains. In this study, we characterized the prevalence, regulation, and function of fdeC in EHEC. We showed that the fdeC gene is conserved in both O157 and non-O157 EHEC and encodes a protein that is expressed at the cell surface and promotes biofilm formation under continuous-flow conditions in a recombinant E. coli strain background. We also identified culture conditions under which FdeC is expressed and showed that minor alterations of these conditions, such as changes in temperature, can significantly alter the level of FdeC expression. Additionally, we demonstrated that the transcription of the fdeC gene is repressed by the global regulator H-NS. Taken together, our data suggest a role for FdeC in EHEC when it grows at temperatures above 37°C, a condition relevant to its specialized niche at the rectoanal junctions of cattle., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)
- Published
- 2014
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21. The VgrG proteins are "à la carte" delivery systems for bacterial type VI effectors.
- Author
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Hachani A, Allsopp LP, Oduko Y, and Filloux A
- Subjects
- Bacterial Proteins genetics, Bacterial Toxins genetics, Carrier Proteins genetics, Pseudomonas aeruginosa genetics, Bacterial Proteins metabolism, Bacterial Secretion Systems physiology, Bacterial Toxins metabolism, Carrier Proteins metabolism, Pseudomonas aeruginosa metabolism
- Abstract
The bacterial type VI secretion system (T6SS) is a supra-molecular complex akin to bacteriophage tails, with VgrG proteins acting as a puncturing device. The Pseudomonas aeruginosa H1-T6SS has been extensively characterized. It is involved in bacterial killing and in the delivery of three toxins, Tse1-3. Here, we demonstrate the independent contribution of the three H1-T6SS co-regulated vgrG genes, vgrG1abc, to bacterial killing. A putative toxin is encoded in the vicinity of each vgrG gene, supporting the concept of specific VgrG/toxin couples. In this respect, VgrG1c is involved in the delivery of an Rhs protein, RhsP1. The RhsP1 C terminus carries a toxic activity, from which the producing bacterium is protected by a cognate immunity. Similarly, VgrG1a-dependent toxicity is associated with the PA0093 gene encoding a two-domain protein with a putative toxin domain (Toxin_61) at the C terminus. Finally, VgrG1b-dependent killing is detectable upon complementation of a triple vgrG1abc mutant. The VgrG1b-dependent killing is mediated by PA0099, which presents the characteristics of the superfamily nuclease 2 toxin members. Overall, these data develop the concept that VgrGs are indispensable components for the specific delivery of effectors. Several additional vgrG genes are encoded on the P. aeruginosa genome and are not linked genetically to other T6SS genes. A closer inspection of these clusters reveals that they also encode putative toxins. Overall, these associations further support the notion of an original form of secretion system, in which VgrG acts as the carrier., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2014
- Full Text
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22. F9 fimbriae of uropathogenic Escherichia coli are expressed at low temperature and recognise Galβ1-3GlcNAc-containing glycans.
- Author
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Wurpel DJ, Totsika M, Allsopp LP, Hartley-Tassell LE, Day CJ, Peters KM, Sarkar S, Ulett GC, Yang J, Tiralongo J, Strugnell RA, Jennings MP, and Schembri MA
- Subjects
- Acetylglucosamine metabolism, Adhesins, Bacterial genetics, Adhesins, Bacterial metabolism, Amino Acid Sequence, Bacterial Adhesion, Base Sequence, Biofilms, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism, Evolution, Molecular, Fimbriae, Bacterial metabolism, Galactose metabolism, Gene Expression Regulation, Bacterial, Genes, Bacterial, Molecular Sequence Data, Operon, Phylogeny, Transcription, Genetic, Uropathogenic Escherichia coli metabolism, Fimbriae, Bacterial genetics, Polysaccharides metabolism, Uropathogenic Escherichia coli genetics
- Abstract
Uropathogenic Escherichia coli (UPEC) is the leading causative agent of urinary tract infections (UTI) in the developed world. Among the major virulence factors of UPEC, surface expressed adhesins mediate attachment and tissue tropism. UPEC strains typically possess a range of adhesins, with type 1 fimbriae and P fimbriae of the chaperone-usher class the best characterised. We previously identified and characterised F9 as a new chaperone-usher fimbrial type that mediates biofilm formation. However, the regulation and specific role of F9 fimbriae remained to be determined in the context of wild-type clinical UPEC strains. In this study we have assessed the distribution and genetic context of the f9 operon among diverse E. coli lineages and pathotypes and demonstrated that f9 genes are significantly more conserved in a UPEC strain collection in comparison to the well-defined E. coli reference (ECOR) collection. In the prototypic UPEC strain CFT073, the global regulator protein H-NS was identified as a transcriptional repressor of f9 gene expression at 37°C through its ability to bind directly to the f9 promoter region. F9 fimbriae expression was demonstrated at 20°C, representing the first evidence of functional F9 fimbriae expression by wild-type E. coli. Finally, glycan array analysis demonstrated that F9 fimbriae recognise and bind to terminal Galβ1-3GlcNAc structures.
- Published
- 2014
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23. The serum resistome of a globally disseminated multidrug resistant uropathogenic Escherichia coli clone.
- Author
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Phan MD, Peters KM, Sarkar S, Lukowski SW, Allsopp LP, Gomes Moriel D, Achard ME, Totsika M, Marshall VM, Upton M, Beatson SA, and Schembri MA
- Subjects
- Gene Expression Regulation, Bacterial, Humans, Molecular Epidemiology, Mutagenesis, Uropathogenic Escherichia coli pathogenicity, Virulence drug effects, Virulence genetics, beta-Lactamases genetics, Blood microbiology, Drug Resistance, Multiple, Bacterial genetics, Urinary Tract Infections microbiology, Uropathogenic Escherichia coli genetics
- Abstract
Escherichia coli ST131 is a globally disseminated, multidrug resistant clone responsible for a high proportion of urinary tract and bloodstream infections. The rapid emergence and successful spread of E. coli ST131 is strongly associated with antibiotic resistance; however, this phenotype alone is unlikely to explain its dominance amongst multidrug resistant uropathogens circulating worldwide in hospitals and the community. Thus, a greater understanding of the molecular mechanisms that underpin the fitness of E. coli ST131 is required. In this study, we employed hyper-saturated transposon mutagenesis in combination with multiplexed transposon directed insertion-site sequencing to define the essential genes required for in vitro growth and the serum resistome (i.e. genes required for resistance to human serum) of E. coli EC958, a representative of the predominant E. coli ST131 clonal lineage. We identified 315 essential genes in E. coli EC958, 231 (73%) of which were also essential in E. coli K-12. The serum resistome comprised 56 genes, the majority of which encode membrane proteins or factors involved in lipopolysaccharide (LPS) biosynthesis. Targeted mutagenesis confirmed a role in serum resistance for 46 (82%) of these genes. The murein lipoprotein Lpp, along with two lipid A-core biosynthesis enzymes WaaP and WaaG, were most strongly associated with serum resistance. While LPS was the main resistance mechanism defined for E. coli EC958 in serum, the enterobacterial common antigen and colanic acid also impacted on this phenotype. Our analysis also identified a novel function for two genes, hyxA and hyxR, as minor regulators of O-antigen chain length. This study offers novel insight into the genetic make-up of E. coli ST131, and provides a framework for future research on E. coli and other Gram-negative pathogens to define their essential gene repertoire and to dissect the molecular mechanisms that enable them to survive in the bloodstream and cause disease., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2013
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24. Functional heterogeneity of the UpaH autotransporter protein from uropathogenic Escherichia coli.
- Author
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Allsopp LP, Beloin C, Moriel DG, Totsika M, Ghigo JM, and Schembri MA
- Subjects
- Bacterial Adhesion, Biofilms growth & development, Computational Biology, DNA, Bacterial chemistry, DNA, Bacterial genetics, Extracellular Matrix Proteins metabolism, Molecular Sequence Data, Mutant Proteins genetics, Mutant Proteins metabolism, Protein Binding, Protein Structure, Tertiary, Sequence Analysis, DNA, Sequence Deletion, Uropathogenic Escherichia coli genetics, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism, Genetic Variation, Uropathogenic Escherichia coli enzymology, Uropathogenic Escherichia coli physiology, Virulence Factors genetics, Virulence Factors metabolism
- Abstract
Uropathogenic Escherichia coli (UPEC) is responsible for the majority of urinary tract infections (UTI). To cause a UTI, UPEC must adhere to the epithelial cells of the urinary tract and overcome the shear flow forces of urine. This function is mediated primarily by fimbrial adhesins, which mediate specific attachment to host cell receptors. Another group of adhesins that contributes to UPEC-mediated UTI is autotransporter (AT) proteins. AT proteins possess a range of virulence properties, such as adherence, aggregation, invasion, and biofilm formation. One recently characterized AT protein of UPEC is UpaH, a large adhesin-involved-in-diffuse-adherence (AIDA-I)-type AT protein that contributes to biofilm formation and bladder colonization. In this study we characterized a series of naturally occurring variants of UpaH. We demonstrate that extensive sequence variation exists within the passenger-encoding domain of UpaH variants from different UPEC strains. This sequence variation is associated with functional heterogeneity with respect to the ability of UpaH to mediate biofilm formation. In contrast, all of the UpaH variants examined retained a conserved ability to mediate binding to extracellular matrix (ECM) proteins. Bioinformatic analysis of the UpaH passenger domain identified a conserved region (UpaH(CR)) and a hydrophobic region (UpaH(HR)). Deletion of these domains reduced biofilm formation but not the binding to ECM proteins. Despite variation in the upaH sequence, the transcription of upaH was repressed by a conserved mechanism involving the global regulator H-NS, and mutation of the hns gene relieved this repression. Overall, our findings shed new light on the regulation and functions of the UpaH AT protein.
- Published
- 2012
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25. Molecular characterization of the EhaG and UpaG trimeric autotransporter proteins from pathogenic Escherichia coli.
- Author
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Totsika M, Wells TJ, Beloin C, Valle J, Allsopp LP, King NP, Ghigo JM, and Schembri MA
- Subjects
- Adhesins, Escherichia coli metabolism, Amino Acid Sequence, Bacterial Adhesion, Caco-2 Cells, Cell Line, Tumor, Colon cytology, Colon microbiology, Epithelial Cells metabolism, Escherichia coli Infections microbiology, Escherichia coli O157 genetics, Escherichia coli O157 metabolism, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism, Extracellular Matrix metabolism, Fimbriae Proteins genetics, Fimbriae Proteins metabolism, Gene Expression Regulation, Bacterial, Humans, Molecular Sequence Data, Rectum cytology, Rectum microbiology, Sequence Analysis, DNA, Urinary Bladder cytology, Urinary Bladder microbiology, Uropathogenic Escherichia coli genetics, Uropathogenic Escherichia coli metabolism, Virulence Factors metabolism, Adhesins, Escherichia coli genetics, Biofilms growth & development, Epithelial Cells microbiology, Escherichia coli O157 pathogenicity, Uropathogenic Escherichia coli pathogenicity, Virulence Factors genetics
- Abstract
Trimeric autotransporter proteins (TAAs) are important virulence factors of many Gram-negative bacterial pathogens. A common feature of most TAAs is the ability to mediate adherence to eukaryotic cells or extracellular matrix (ECM) proteins via a cell surface-exposed passenger domain. Here we describe the characterization of EhaG, a TAA identified from enterohemorrhagic Escherichia coli (EHEC) O157:H7. EhaG is a positional orthologue of the recently characterized UpaG TAA from uropathogenic E. coli (UPEC). Similarly to UpaG, EhaG localized at the bacterial cell surface and promoted cell aggregation, biofilm formation, and adherence to a range of ECM proteins. However, the two orthologues display differential cellular binding: EhaG mediates specific adhesion to colorectal epithelial cells while UpaG promotes specific binding to bladder epithelial cells. The EhaG and UpaG TAAs contain extensive sequence divergence in their respective passenger domains that could account for these differences. Indeed, sequence analyses of UpaG and EhaG homologues from several E. coli genomes revealed grouping of the proteins in clades almost exclusively represented by distinct E. coli pathotypes. The expression of EhaG (in EHEC) and UpaG (in UPEC) was also investigated and shown to be significantly enhanced in an hns isogenic mutant, suggesting that H-NS acts as a negative regulator of both TAAs. Thus, while the EhaG and UpaG TAAs contain some conserved binding and regulatory features, they also possess important differences that correlate with the distinct pathogenic lifestyles of EHEC and UPEC.
- Published
- 2012
- Full Text
- View/download PDF
26. Molecular characterization of UpaB and UpaC, two new autotransporter proteins of uropathogenic Escherichia coli CFT073.
- Author
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Allsopp LP, Beloin C, Ulett GC, Valle J, Totsika M, Sherlock O, Ghigo JM, and Schembri MA
- Subjects
- Adhesins, Bacterial genetics, Animals, Biofilms growth & development, Cloning, Molecular, DNA, Bacterial genetics, Escherichia coli Infections microbiology, Escherichia coli Infections pathology, Escherichia coli K12 genetics, Escherichia coli K12 pathogenicity, Escherichia coli Proteins genetics, Extracellular Matrix Proteins metabolism, Female, Fimbriae Proteins metabolism, Gene Expression Profiling, Membrane Transport Proteins genetics, Mice, Mice, Inbred C57BL, Polymerase Chain Reaction, Urinary Tract Infections microbiology, Urinary Tract Infections pathology, Virulence Factors genetics, Adhesins, Bacterial metabolism, Escherichia coli Proteins metabolism, Membrane Transport Proteins metabolism, Uropathogenic Escherichia coli genetics, Uropathogenic Escherichia coli pathogenicity, Virulence Factors metabolism
- Abstract
Uropathogenic Escherichia coli (UPEC) is the primary cause of urinary tract infection (UTI) in the developed world. The major factors associated with virulence of UPEC are fimbrial adhesins, which mediate specific attachment to host receptors and trigger innate host responses. Another group of adhesins is represented by the autotransporter (AT) subgroup of proteins. The genome-sequenced prototype UPEC strain CFT073 contains 11 putative AT-encoding genes. In this study, we have performed a detailed molecular characterization of two closely related AT adhesins from CFT073: UpaB (c0426) and UpaC (c0478). PCR screening revealed that the upaB and upaC AT-encoding genes are common in E. coli. The upaB and upaC genes were cloned and characterized in a recombinant E. coli K-12 strain background. This revealed that they encode proteins located at the cell surface but possess different functional properties: UpaB mediates adherence to several ECM proteins, while UpaC expression is associated with increased biofilm formation. In CFT073, upaB is expressed while upaC is transcriptionally repressed by the global regulator H-NS. In competitive colonization experiments employing the mouse UTI model, CFT073 significantly outcompeted its upaB (but not upaC) isogenic mutant strain in the bladder. This attenuated phenotype was also observed in single-challenge experiments, where deletion of the upaB gene in CFT073 significantly reduced early colonization of the bladder.
- Published
- 2012
- Full Text
- View/download PDF
27. Characterization of EhaJ, a New Autotransporter Protein from Enterohemorrhagic and Enteropathogenic Escherichia coli.
- Author
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Easton DM, Totsika M, Allsopp LP, Phan MD, Idris A, Wurpel DJ, Sherlock O, Zhang B, Venturini C, Beatson SA, Mahony TJ, Cobbold RN, and Schembri MA
- Abstract
Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are diarrheagenic pathotypes of E. coli that cause gastrointestinal disease with the potential for life-threatening sequelae. While certain EHEC and EPEC virulence mechanisms have been extensively studied, the factors that mediate host colonization remain to be properly defined. Previously, we identified four genes (ehaA, ehaB, ehaC, and ehaD) from the prototypic EHEC strain EDL933 that encode for proteins that belong to the autotransporter (AT) family. Here we have examined the prevalence of these genes, as well as several other AT-encoding genes, in a collection of EHEC and EPEC strains. We show that the complement of AT-encoding genes in EHEC and EPEC strains is variable, with some AT-encoding genes being highly prevalent. One previously uncharacterized AT-encoding gene, which we have termed ehaJ, was identified in 12/44 (27%) of EHEC and 2/20 (10%) of EPEC strains. The ehaJ gene lies immediately adjacent to a gene encoding a putative glycosyltransferase (referred to as egtA). Western blot analysis using an EhaJ-specific antibody indicated that EhaJ is glycosylated by EgtA. Expression of EhaJ in a recombinant E. coli strain, revealed EhaJ is located at the cell surface and in the presence of the egtA glycosyltransferase gene mediates strong biofilm formation in microtiter plate and flow cell assays. EhaJ also mediated adherence to a range of extracellular matrix proteins, however this occurred independent of glycosylation. We also demonstrate that EhaJ is expressed in a wild-type EPEC strain following in vitro growth. However, deletion of ehaJ did not significantly alter its adherence or biofilm properties. In summary, EhaJ is a new glycosylated AT protein from EPEC and EHEC. Further studies are required to elucidate the function of EhaJ in colonization and virulence.
- Published
- 2011
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- View/download PDF
28. UpaH is a newly identified autotransporter protein that contributes to biofilm formation and bladder colonization by uropathogenic Escherichia coli CFT073.
- Author
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Allsopp LP, Totsika M, Tree JJ, Ulett GC, Mabbett AN, Wells TJ, Kobe B, Beatson SA, and Schembri MA
- Subjects
- Animals, Cloning, Molecular, DNA, Bacterial chemistry, DNA, Bacterial genetics, Disease Models, Animal, Escherichia coli Proteins genetics, Female, Gene Deletion, Humans, Mice, Mice, Inbred C57BL, Molecular Sequence Data, Protein Structure, Secondary, Protein Structure, Tertiary, Sequence Analysis, DNA, Urinary Tract Infections microbiology, Virulence Factors genetics, Biofilms growth & development, Escherichia coli Proteins physiology, Urinary Bladder microbiology, Uropathogenic Escherichia coli pathogenicity, Virulence Factors physiology
- Abstract
Escherichia coli is the primary cause of urinary tract infection (UTI) in the developed world. The major factors associated with virulence of uropathogenic E. coli (UPEC) are fimbrial adhesins, which mediate specific attachment to host receptors and trigger innate host responses. Another group of adhesins is represented by the autotransporter (AT) subgroup of proteins. In this study, we identified a new AT-encoding gene, termed upaH, present in a 6.5-kb unannotated intergenic region in the genome of the prototypic UPEC strain CFT073. Cloning and sequencing of the upaH gene from CFT073 revealed an intact 8.535-kb coding region, contrary to the published genome sequence. The upaH gene was widely distributed among a large collection of UPEC isolates as well as the E. coli Reference (ECOR) strain collection. Bioinformatic analyses suggest beta-helix as the predominant structure in the large N-terminal passenger (alpha) domain and a 12-strand beta-barrel for the C-terminal beta-domain of UpaH. We demonstrated that UpaH is expressed at the cell surface of CFT073 and promotes biofilm formation. In the mouse UTI model, deletion of the upaH gene in CFT073 and in two other UPEC strains did not significantly affect colonization of the bladder in single-challenge experiments. However, in competitive colonization experiments, CFT073 significantly outcompeted its upaH isogenic mutant strain in urine and the bladder.
- Published
- 2010
- Full Text
- View/download PDF
29. EhaA is a novel autotransporter protein of enterohemorrhagic Escherichia coli O157:H7 that contributes to adhesion and biofilm formation.
- Author
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Wells TJ, Sherlock O, Rivas L, Mahajan A, Beatson SA, Torpdahl M, Webb RI, Allsopp LP, Gobius KS, Gally DL, and Schembri MA
- Subjects
- Adhesins, Escherichia coli chemistry, Escherichia coli O157 genetics, Escherichia coli Proteins metabolism, Adhesins, Escherichia coli metabolism, Bacterial Adhesion genetics, Biofilms growth & development, Escherichia coli O157 physiology, Protein Transport
- Abstract
Autotransporter (AT) proteins have been identified in many Gram-negative pathogens and are unique in that their primary sequence is sufficient to direct their transport across the bacterial membrane system. Where characterized they are uniformly associated with virulence. Using conserved AT motifs as a search tool, four putative AT proteins were identified in the Enterohemorrhagic Escherichia coli O157:H7 EDL933 genome. The genes encoding these proteins (z0402/ehaA, z0469/ehaB, z3487/ehaC and z3948/ehaD) were PCR amplified, cloned and expressed in an E. coli K-12 MG1655flu background. Preliminary characterization revealed that ehaA, ehaB and ehaD encode proteins associated with increased biofilm formation. One of these genes (ehaA) resides on a genomic island in E. coli O157:H7 strains EDL933 and Sakai. Over-expression of EhaA in E. coli K-12 demonstrated it is located at the cell surface and resulted in the formation of large cell aggregates, promoted significant biofilm formation and mediated adhesion to primary epithelial cells of the bovine terminal rectum. The expression of ehaA was demonstrated in E. coli EDL933 by RT-PCR. An EhaA-specific antibody revealed the EhaA protein was expressed in 24/50 generic Shiga toxin-producing E. coli (STEC) strains of various serotypes including O157:H7. However, the deletion of ehaA from E. coli EDL933 and a STEC strain from serotype O111:H(-) did not affect biofilm growth. Our results suggest that EhaA may contribute to adhesion, colonization and biofilm formation by E. coli O157:H7 and possibly other STEC serotypes.
- Published
- 2008
- Full Text
- View/download PDF
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