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Journal articleWen KY, Cameron L, Chappell J, et al., 2017,
A Cell-Free Biosensor for Detecting Quorum Sensing Molecules in P. aeruginosa-Infected Respiratory Samples.
, ACS Synthetic Biology, Vol: 6, Pages: 2293-2301, ISSN: 2161-5063Synthetic biology designed cell-free biosensors are a promising new tool for the detection of clinically relevant biomarkers in infectious diseases. Here, we report that a modular DNA-encoded biosensor in cell-free protein expression systems can be used to measure a bacterial biomarker of Pseudomonas aeruginosa infection from human sputum samples. By optimizing the cell-free system and sample extraction, we demonstrate that the quorum sensing molecule 3-oxo-C12-HSL in sputum samples from cystic fibrosis lungs can be quantitatively measured at nanomolar levels using our cell-free biosensor system, and is comparable to LC-MS measurements of the same samples. This study further illustrates the potential of modular cell-free biosensors as rapid, low-cost detection assays that can inform clinical practice.
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Journal articleBerger C, Crepin V, Roumeliotis TI, et 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: 1550-4131The 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.
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Journal articleMcCarthy RR, Valentini M, Filloux A, 2017,
Contribution of Cyclic di-GMP in the Control of Type III and Type VI Secretion in Pseudomonas aeruginosa.
, Methods Mol Biol, Vol: 1657, Pages: 213-224Bacteria produce toxins to enhance their competitiveness in the colonization of an environment as well as during an infection. The delivery of toxins into target cells is mediated by several types of secretion systems, among them our focus is Type III and Type VI Secretion Systems (T3SS and T6SS, respectively). A thorough methodology is provided detailing how to identify if cyclic di-GMP signaling plays a role in the P. aeruginosa toxin delivery mediated by T3SS or T6SS. This includes in vitro preparation of the samples for Western blot analysis aiming at detecting possible c-di-GMP-dependent T3SS/T6SS switch, as well as in vivo analysis using the model organism Galleria mellonella to demonstrate the ecological and pathogenic consequence of the switch between these two secretion systems.
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Journal articleKarampatzakis A, Song CZ, Allsopp LP, et al., 2017,
Probing the internal micromechanical properties of Pseudomonas aeruginosa biofilms by Brillouin imaging.
, NPJ Biofilms Microbiomes, Vol: 3, ISSN: 2055-5008Biofilms 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.
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Journal articleHeeb S, Camara M, Filloux A, et al., 2017,
Professor Dieter Haas (1945-2017)
, FEMS Microbiology Reviews, Vol: 41, Pages: 597-598, ISSN: 0168-6445 -
Journal articleJakobsen TH, Warming AN, Vejborg RM, et al., 2017,
A broad range quorum sensing inhibitor working through sRNA inhibition
, Scientific Reports, Vol: 7, ISSN: 2045-2322For the last decade, chemical control of bacterial virulence has received considerable attention. Ajoene, a sulfur-rich molecule from garlic has been shown to reduce expression of key quorum sensing regulated virulence factors in the opportunistic pathogen Pseudomonas aeruginosa. Here we show that the repressing effect of ajoene on quorum sensing occurs by inhibition of small regulatory RNAs (sRNA) in P. aeruginosa as well as in Staphylococcus aureus, another important human pathogen that employs quorum sensing to control virulence gene expression. Using various reporter constructs, we found that ajoene lowered expression of the sRNAs RsmY and RsmZ in P. aeruginosa and the small dual-function regulatory RNA, RNAIII in S. aureus, that controls expression of key virulence factors. We confirmed the modulation of RNAIII by RNA sequencing and found that the expression of many QS regulated genes encoding virulence factors such as hemolysins and proteases were lowered in the presence of ajoene in S. aureus. Importantly, our findings show that sRNAs across bacterial species potentially may qualify as targets of anti-virulence therapy and that ajoene could be a lead structure in search of broad-spectrum compounds transcending the Gram negative-positive borderline.
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Journal articleSarkar P, Switzer A, Peters C, et al., 2017,
Phenotypic consequences of RNA polymerase dysregulation in Escherichiacoli
, Nucleic Acids Research, Vol: 45, Pages: 11131-11143, ISSN: 1362-4962Many bacterial adaptive responses to changes in growth conditions due to biotic and abiotic factors involve reprogramming of gene expression at the transcription level. The bacterial RNA polymerase (RNAP), which catalyzes transcription, can thus be considered as the major mediator of cellular adaptive strategies. But how do bacteria respond if a stress factor directly compromises the activity of the RNAP? We used a phage-derived small protein to specifically perturb bacterial RNAP activity in exponentially growing Escherichia coli. Using cytological profiling, tracking RNAP behavior at single-molecule level and transcriptome analysis, we reveal that adaptation to conditions that directly perturb bacterial RNAP performance can result in a biphasic growth behavior and thereby confer the ‘adapted’ bacterial cells an enhanced ability to tolerate diverse antibacterial stresses. The results imply that while synthetic transcriptional rewiring may confer bacteria with the intended desirable properties, such approaches may also collaterally allow them to acquire undesirable traits.
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Journal articlerouse S, hawthorne, berry, et al., 2017,
A new class of hybrid secretion system is employed in Pseudomonas amyloid biogenesis
, Nature Communications, Vol: 8, ISSN: 2041-1723Gram-negative bacteria possess specialised biogenesis machineries that facilitate the export of amyloid subunits for construction of a biofilm matrix. The secretion of bacterial functional amyloid requires a bespoke outer-membrane protein channel through which unfolded amyloid substrates are translocated. Here, we combine X-ray crystallography, native mass spectrometry, single-channel electrical recording, molecular simulations and circular dichroism measurements to provide high-resolution structural insight into the functional amyloid transporter from Pseudomonas, FapF. FapF forms a trimer of gated β-barrel channels in which opening is regulated by a helical plug connected to an extended coil-coiled platform spanning the bacterial periplasm. Although FapF represents a unique type of secretion system, it shares mechanistic features with a diverse range of peptide translocation systems. Our findings highlight alternative strategies for handling and export of amyloid protein sequences.
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Journal articleJennings E, Thurston TLM, Holden DW, 2017,
Salmonella SPI-2 Type III Secretion System Effectors: Molecular Mechanisms And Physiological Consequences
, CELL HOST & MICROBE, Vol: 22, Pages: 217-231, ISSN: 1931-3128- Author Web Link
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Journal articleFurniss RCD, Clements A, 2017,
Regulation of the Locus of Enterocyte Effacement in Attaching and Effacing Pathogens.
, Journal of Bacteriology, Vol: 200, ISSN: 0021-9193Attaching 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.
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