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Journal articleWitcomb LA, Czupryna J, Francis KP, et al., 2017,
Non-invasive three-dimensional imaging of Escherichia coli K1 infection using diffuse light imaging tomography combined with micro-computed tomography
, Methods, Vol: 127, Pages: 62-68, ISSN: 1046-2023In contrast to two-dimensional bioluminescence imaging, three dimensional diffuse light imaging tomography with integrated micro-computed tomography (DLIT-μCT) has the potential to realise spatial variations in infection patterns when imaging experimental animals dosed with derivatives of virulent bacteria carrying bioluminescent reporter genes such as the lux operon from the bacterium Photorhabdus luminescens. The method provides an opportunity to precisely localise the bacterial infection sites within the animal and enables the generation of four-dimensional movies of the infection cycle. Here, we describe the use of the PerkinElmer IVIS SpectrumCT in vivo imaging system to investigate progression of lethal systemic infection in neonatal rats following colonisation of the gastrointestinal tract with the neonatal pathogen Escherichia coli K1. We confirm previous observations that these bacteria stably colonize the colon and small intestine following feeding of the infectious dose from a micropipette; invading bacteria migrate across the gut epithelium into the blood circulation and establish foci of infection in major organs, including the brain. DLIT-μCT revealed novel multiple sites of colonisation within the alimentary canal, including the tongue, oesophagus and stomach, with penetration of the non-keratinised oesophageal epithelial surface, providing strong evidence of a further major site for bacterial dissemination. We highlight technical issues associated with imaging of infections in new born rat pups and show that the whole-body and organ bioburden correlates with disease severity.
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Journal articleTabib-Salazar A, Liu B, Shadrin A, et al., 2017,
Full shut-off of Escherichia coli RNA-polymerase by T7 phage requires a small phage-encoded DNA-binding protein
, Nucleic Acids Research, Vol: 45, Pages: 7697-7707, ISSN: 1362-4962Infection of Escherichia coli by the T7 phage leads to rapid and selective inhibition of the bacterial RNA polymerase (RNAP) by the 7 kDa T7 protein Gp2. We describe the identification and functional and structural characterisation of a novel 7 kDa T7 protein, Gp5.7, which adopts a winged helix-turn-helix-like structure and specifically represses transcription initiation from host RNAP-dependent promoters on the phage genome via a mechanism that involves interaction with DNA and the bacterial RNAP. Whereas Gp2 is indispensable for T7 growth in E. coli, we show that Gp5.7 is required for optimal infection outcome. Our findings provide novel insights into how phages fine-tune the activity of the host transcription machinery to ensure both successful and efficient phage progeny development.
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Journal articleImbert PRC, Louche A, Luizet J-B, et al., 2017,
A Pseudomonas aeruginosa TIR effector mediates immune evasion by targeting UBAP1 and TLR adaptors
, EMBO JOURNAL, Vol: 36, Pages: 1869-1887, ISSN: 0261-4189 -
Journal articlePallett MA, Crepin VF, Serafini N, et al., 2017,
Bacterial virulence factor inhibits caspase-4/11 activation in intestinal epithelial cells
, Mucosal Immunology, Vol: 10, Pages: 602-612, ISSN: 1935-3456The human pathogen enteropathogenic Escherichia coli (EPEC), as well as the mouse pathogen Citrobacter rodentium, colonize the gut mucosa via attaching and effacing lesion formation and cause diarrheal diseases. EPEC and C. rodentium type III secretion system (T3SS) effectors repress innate immune responses and infiltration of immune cells. Inflammatory caspases such as caspase-1 and caspase-4/11 are crucial mediators of host defense and inflammation in the gut via their ability to process cytokines such as interleukin (IL)-1β and IL-18. Here we report that the effector NleF binds the catalytic domain of caspase-4 and inhibits its proteolytic activity. Following infection of intestinal epithelial cells (IECs) EPEC inhibited caspase-4 and IL-18 processing in an NleF-dependent manner. Depletion of caspase-4 in IECs prevented the secretion of mature IL-18 in response to infection with EPECΔnleF. NleF-dependent inhibition of caspase-11 in colons of mice prevented IL-18 secretion and neutrophil influx at early stages of C. rodentium infection. Neither wild-type C. rodentium nor C. rodentiumΔnleF triggered neutrophil infiltration or IL-18 secretion in Cas11 or Casp1/11-deficient mice. Thus, IECs have a key role in modulating early innate immune responses in the gut via a caspase-4/11—IL-18 axis, which is targeted by virulence factors encoded by enteric pathogens.
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Journal articleKhara JS, Obuobi S, Wang Y, et al., 2017,
Disruption of drug-resistant biofilms using de novo designed short α-helicalantimicrobial peptides with idealized facial amphiphilicity
, Acta Biomaterialia, Vol: 57, Pages: 103-114, ISSN: 1878-7568The escalating threat of antimicrobial resistance has increased pressure to develop novel therapeutic strategies to tackle drug-resistant infections. Antimicrobial peptides have emerged as a promising class of therapeutics for various systemic and topical clinical applications. In this study, the de novo design of α-helical peptides with idealized facial amphiphilicities, based on an understanding of the pertinent features of protein secondary structures, is presented. Synthetic amphiphiles composed of the backbone sequence (X1Y1Y2X2)n, where X1 and X2 are hydrophobic residues (Leu or Ile or Trp), Y1 and Y2 are cationic residues (Lys), and n is the number repeat units (2 or 2.5 or 3), demonstrated potent broad-spectrum antimicrobial activities against clinical isolates of drug-susceptible and multi-drug resistant bacteria. Live-cell imaging revealed that the most selective peptide, (LKKL)3, promoted rapid permeabilization of bacterial membranes. Importantly, (LKKL)3 not only suppressed biofilm growth, but effectively disrupted mature biofilms after only 2 h of treatment. The peptides (LKKL)3 and (WKKW)3 suppressed the production of LPS-induced pro-inflammatory mediators to levels of unstimulated controls at low micromolar concentrations. Thus, the rational design strategies proposed herein can be implemented to develop potent, selective and multifunctional α-helical peptides to eradicate drug-resistant biofilm-associated infections.
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Journal articleLiew N, Mazon Moya MJ, Wierzbicki CJ, et al., 2017,
Chytrid fungus infection in zebrafish demonstrates that the pathogen can parasitize non-amphibian vertebrate hosts
, Nature Communications, Vol: 8, ISSN: 2041-1723Aquatic chytrid fungi threaten amphibian biodiversity worldwide owing to their ability to rapidly expand their geographical distributions and to infect a wide range of hosts. Combating this risk requires an understanding of chytrid host range to identify potential reservoirs of infection and to safeguard uninfected regions through enhanced biosecurity. Here we extend our knowledge on the host range of the chytrid Batrachochytrium dendrobatidis by demonstrating infection of a non-amphibian vertebrate host, the zebrafish. We observe dose-dependent mortality and show that chytrid can infect and proliferate on zebrafish tissue. We also show that infection phenotypes (fin erosion, cell apoptosis and muscle degeneration) are direct symptoms of infection. Successful infection is dependent on disrupting the zebrafish microbiome, highlighting that, as is widely found in amphibians, commensal bacteria confer protection against this pathogen. Collectively, our findings greatly expand the limited tool kit available to study pathogenesis and host response to chytrid infection.
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Journal articleHall A, Gollan B, Helaine S, 2017,
Toxin-antitoxin systems: reversible toxicity
, Current Opinion in Microbiology, Vol: 36, Pages: 102-110, ISSN: 1879-0364Toxin–antitoxin (TA) systems encoded on the plasmids and chromosomes of bacteria are emerging as key players in stress adaptation. In particular, they have been implicated in the induction of persisters non-growing cells that can evade antibiotic exposure. TA toxins operate by a diverse range of mechanisms, either destructive or conservative, leading to the reversible growth arrest of bacterial cells. Whilst the molecular mechanisms of intoxication are now well understood, we still have very little information on how corrupted cells reawaken. Alongside the phenomenon of conditional cooperativity, new evidence suggests that the effects of some TA toxins can be reversed, allowing non-growing cells to be detoxified and growth to resume.
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Journal articleBirchenough GMH, Dalgakiran F, Witcomb LA, et al., 2017,
Postnatal development of the small intestinal mucosa drives age-dependent, regio-selective susceptibility to Escherichia coli K1 infection
, Scientific Reports, Vol: 7, ISSN: 2045-2322The strong age dependency of neonatal systemic infection with Escherichia coli K1 can be replicated in the neonatal rat. Gastrointestinal (GI) colonization of two-day-old (P2) rats leads to invasion of the blood within 48 h of initiation of colonization; pups become progressively less susceptible to infection over the P2-P9 period. We show that, in animals colonized at P2 but not at P9, E. coli K1 bacteria gain access to the enterocyte surface in the mid-region of the small intestine and translocate through the epithelial cell monolayer by an intracellular pathway to the submucosa. In this region of the GI tract, the protective mucus barrier is poorly developed but matures to full thickness over P2-P9, coincident with the development of resistance to invasion. At P9, E. coli K1 bacteria are physically separated from villi by the mucus layer and their numbers controlled by mucus-embedded antimicrobial peptides, preventing invasion of host tissues.
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Journal articleMcCarthy RR, Mazon-Moya MJ, Moscoso JA, et al., 2017,
Cyclic-di-GMP regulates lipopolysaccharide modification and contributes to Pseudomonas aeruginosa immune evasion
, Nature Microbiology, Vol: 2, Pages: 1-10, ISSN: 2058-5276Pseudomonas aeruginosa is a Gram-negative bacterial pathogen associated with acute and chronic infections. The universal cyclic-di-GMP second messenger is instrumental in the switch from a motile lifestyle to resilient biofilm as in the cystic fibrosis lung. The SadC diguanylate cyclase is associated with this patho-adaptive transition. Here, we identify an unrecognized SadC partner, WarA, which we show is a methyltransferase in complex with a putative kinase, WarB. We established that WarA binds to cyclic-di-GMP, which potentiates its methyltransferase activity. Together, WarA and WarB have structural similarities with the bifunctional Escherichia coli lipopolysaccharide (LPS) O antigen regulator WbdD. Strikingly, WarA influences P. aeruginosa O antigen modal distribution and interacts with the LPS biogenesis machinery. LPS is known to modulate the immune response in the host, and by using a zebrafish infection model, we implicate WarA in the ability of P. aeruginosa to evade detection by the host.
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Journal articlePean CB, Schiebler M, Tan S, et al., 2017,
Regulation of phagocyte triglyceride by a STAT-ATG2 pathway controls mycobacterial infection
, Nature Communications, Vol: 8, Pages: 1-11, ISSN: 2041-1723Mycobacterium tuberculosis remains a global threat to human health yet the molecular mechanisms regulating immunity remain poorly understood. Cytokines can promote or inhibit mycobacterial survivalinside macrophages, andthe underlying mechanisms represent potential targets for host-directed therapies. Here we show that cytokine-STAT signaling promotesmycobacterial survivalwithin macrophages by deregulating lipid droplets via ATG2 repression. In Drosophilainfected withMycobacterium marinum,mycobacterium-induced STAT activitytriggered by unpaired-familycytokinesreduces Atg2 expression, permittingderegulation of lipid droplets. Increased Atg2expression, or reduced macrophage triglyceride biosynthesis,normalizes lipid deposition in infected phagocytes and reduces numbersof viable intracellular mycobacteria. In human macrophages,addition ofIL-6promotes mycobacterial survival and BCG-induced lipid accumulation by a similar, but probably not identical, mechanism. Our results reveal Atg2regulation as amechanism by which cytokines can control lipid droplet homeostasis and consequently resistance to mycobacterial infectionin Drosophila.
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