19 results found
Bernal P, MurilloTorres M, Allsopp LP, 2020, Integrating signals to drive T6SS killing, Environmental Microbiology, Vol: 22, Pages: 4520-4523, ISSN: 1462-2912
Allsopp LP, Bernal P, Nolan LM, et al., 2020, Causalities of war: the connection between type VI secretion system and microbiota, Cellular Microbiology, Vol: 22, Pages: 1-9, ISSN: 1462-5814
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.
Pissaridou P, Allsopp LP, Wettstadt S, et al., 2018, The Pseudomonas aeruginosa T6SS-VgrG1b spike is topped by a PAAR protein eliciting DNA damage to bacterial competitors, Proceedings of the National Academy of Sciences of the United States of America, Vol: 115, Pages: 12519-12524, ISSN: 0027-8424
The type VI secretion system (T6SS) is a supramolecular complex involved in the delivery of potent toxins during bacterial competition. Pseudomonas aeruginosa possesses three T6SS gene clusters and several hcp and vgrG gene islands, the latter encoding the spike at the T6SS tip. The vgrG1b cluster encompasses seven genes whose organization and sequences are highly conserved in P. aeruginosa genomes, except for two genes that we called tse7 and tsi7. We show that Tse7 is a Tox-GHH2 domain nuclease which is distinct from other T6SS nucleases identified thus far. Expression of this toxin induces the SOS response, causes growth arrest and ultimately results in DNA degradation. The cytotoxic domain of Tse7 lies at its C terminus, while the N terminus is a predicted PAAR domain. We find that Tse7 sits on the tip of the VgrG1b spike and that specific residues at the PAAR–VgrG1b interface are essential for VgrG1b-dependent delivery of Tse7 into bacterial prey. We also show that the delivery of Tse7 is dependent on the H1-T6SS cluster, and injection of the nuclease into bacterial competitors is deployed for interbacterial competition. Tsi7, the cognate immunity protein, protects the producer from the deleterious effect of Tse7 through a direct protein–protein interaction so specific that toxin/immunity pairs are effective only if they originate from the same P. aeruginosa isolate. Overall, our study highlights the diversity of T6SS effectors, the exquisite fitting of toxins on the tip of the T6SS, and the specificity in Tsi7-dependent protection, suggesting a role in interstrain competition.
Karampatzakis 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: 2055-5008
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.
Davies SK, Fearn S, Allsopp LP, et al., 2017, Visualizing Antimicrobials in BacterialBiofilms: Three-Dimensional BiochemicalImaging Using TOF-SIMS, mSphere, Vol: 2, ISSN: 2379-5042
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.
Allsopp LP, Wood TE, Howard SA, et al., 2017, RsmA and AmrZ orchestrate the assembly of all three type VI secretion systems in Pseudomonas aeruginosa, Proceedings of the National Academy of Sciences of the United States of America, Vol: 114, Pages: 7707-7712, ISSN: 1091-6490
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.
Bernal P, Allsopp LP, Filloux AAM, et al., 2017, The Pseudomonas putida T6SS is a plant warden against phytopathogens, The ISME Journal, Vol: 11, Pages: 972-987, ISSN: 1751-7362
Bacterial type VI secretion systems (T6SSs) are molecular weapons designed to deliver toxic effectors into prey cells. These nanomachines play an important role in inter-bacterial competition and provide advantages to T6SS active strains in polymicrobial environments. Here we analyse the genome of the biocontrol agent Pseudomonas putida KT2440 and identify three T6SS gene clusters (K1-, K2- and K3-T6SS). Besides, ten 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.
Wurpel DJ, Totsika M, Allsopp LP, et al., 2015, 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, Journal of Proteomics, Vol: 131, Pages: 177-189, ISSN: 1874-3919
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.
Easton DM, Allsopp LP, Minh-Duy P, et al., 2014, The intimin-like protein FdeC is regulated by H-NS and temperature in enterohemorrhagic Escherichia coli, Applied and Environmental Microbiology, Vol: 80, Pages: 7337-7347, ISSN: 0099-2240
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.
Hachani A, Allsopp LP, Oduko Y, et al., 2014, The VgrG Proteins Are "à la Carte" Delivery Systems for Bacterial Type VI Effectors, Journal of Biological Chemistry, Vol: 289, Pages: 17872-17884, ISSN: 1083-351X
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.
Wurpel DJ, Totsika M, Allsopp LP, et al., 2014, F9 Fimbriae of uropathogenic Escherichia coli are expressed at low temperature and recognise Gal beta 1-3GlcNAc-containing glycans, PLoS ONE, Vol: 9, ISSN: 1932-6203
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. UPECstrains typically possess a range of adhesins, with type 1 fimbriae and P fimbriae of the chaperone-usher class the bestcharacterised. We previously identified and characterised F9 as a new chaperone-usher fimbrial type that mediates biofilmformation. However, the regulation and specific role of F9 fimbriae remained to be determined in the context of wild-typeclinical 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 incomparison to the well-defined E. coli reference (ECOR) collection. In the prototypic UPEC strain CFT073, the globalregulator protein H-NS was identified as a transcriptional repressor of f9 gene expression at 37uC through its ability to binddirectly to the f9 promoter region. F9 fimbriae expression was demonstrated at 20uC, representing the first evidence offunctional F9 fimbriae expression by wild-type E. coli. Finally, glycan array analysis demonstrated that F9 fimbriae recogniseand bind to terminal Galb1-3GlcNAc structures.
Jones C, Allsopp L, Horlick J, et al., 2013, Subinhibitory concentration of kanamycin induces the pseudomonas aeruginosa type VI secretion system, PLoS ONE, Vol: 8, ISSN: 1932-6203
Pseudomonas aeruginosa is a Gram-negative bacterium found in natural environments including plants, soils and warm moist surfaces. This organism is also in the top ten of nosocomial pathogens, and prevalent in cystic fibrosis (CF) lung infections. The ability of P. aeruginosa to colonize a wide variety of environments in a lasting manner is associated with the formation of a resistant biofilm and the capacity to efficiently outcompete other microorganisms. Here we demonstrate that sub-inhibitory concentration of kanamycin not only induces biofilm formation but also induces expression of the type VI secretion genes in the H1-T6SS cluster. The H1-T6SS is known for its role in toxin production and bacterial competition. We show that the antibiotic induction of the H1-T6SS only occurs when a functional Gac/Rsm pathway is present. These observations may contribute to understand how P. aeruginosa responds to antibiotic producing competitors. It also suggests that improper antibiotic therapy may enhance P. aeruginosa colonization, including in the airways of CF patients.
Minh-Duy P, Peters KM, Sarkar S, et al., 2013, The serum resistome of a globally disseminated multidrug resistant Uropathogenic Escherichia coli clone, PLoS Genetics, Vol: 9, ISSN: 1553-7390
Escherichia coli ST131 is a globally disseminated, multidrug resistant clone responsible for a high proportion of urinary tractand bloodstream infections. The rapid emergence and successful spread of E. coli ST131 is strongly associated withantibiotic resistance; however, this phenotype alone is unlikely to explain its dominance amongst multidrug resistanturopathogens circulating worldwide in hospitals and the community. Thus, a greater understanding of the molecularmechanisms that underpin the fitness of E. coli ST131 is required. In this study, we employed hyper-saturated transposonmutagenesis in combination with multiplexed transposon directed insertion-site sequencing to define the essential genesrequired for in vitro growth and the serum resistome (i.e. genes required for resistance to human serum) of E. coli EC958, arepresentative 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 encodemembrane proteins or factors involved in lipopolysaccharide (LPS) biosynthesis. Targeted mutagenesis confirmed a role inserum resistance for 46 (82%) of these genes. The murein lipoprotein Lpp, along with two lipid A-core biosynthesis enzymesWaaP and WaaG, were most strongly associated with serum resistance. While LPS was the main resistance mechanismdefined 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 mechanismsthat enable them to survive in the bloodstream and cause disease.
Allsopp LP, Beloin C, Moriel DG, et al., 2012, Functional Heterogeneity of the UpaH Autotransporter Protein from Uropathogenic Escherichia coli, JOURNAL OF BACTERIOLOGY, Vol: 194, Pages: 5769-5782, ISSN: 0021-9193
Totsika M, Wells TJ, Beloin C, et al., 2012, Molecular Characterization of the EhaG and UpaG Trimeric Autotransporter Proteins from Pathogenic Escherichia coli, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Vol: 78, Pages: 2179-2189, ISSN: 0099-2240
Allsopp LP, Beloin C, Ulett GC, et al., 2012, Molecular Characterization of UpaB and UpaC, Two New Autotransporter Proteins of Uropathogenic Escherichia coli CFT073, INFECTION AND IMMUNITY, Vol: 80, Pages: 321-332, ISSN: 0019-9567
Easton DM, Totsika M, Allsopp LP, et al., 2011, Characterization of EhaJ, a new autotransporter protein from enterohemorrhagic and enteropathogenic Escherichia coli, Frontiers in Microbiology, Vol: 2, ISSN: 1664-302X
Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are diarrheagenic pathotypes of E. coli that cause gastrointestinal disease with the potentialfor life-threatening sequelae. While certain EHEC and EPEC virulence mechanisms havebeen extensively studied, the factors that mediate host colonization remain to be properlydefined. 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 otherAT-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-encodinggenes being highly prevalent. One previously uncharacterized AT-encoding gene, which wehave 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 thatEhaJ is glycosylated by EgtA. Expression of EhaJ in a recombinant E. coli strain, revealedEhaJ is located at the cell surface and in the presence of the egtA glycosyltransferasegene mediates strong biofilm formation in microtiter plate and flow cell assays. EhaJ alsomediated adherence to a range of extracellular matrix proteins, however this occurredindependent of glycosylation. We also demonstrate that EhaJ is expressed in a wild-typeEPEC strain following in vitro growth. However, deletion of ehaJ did not significantly alterits adherence or biofilm properties. In summary, EhaJ is a new glycosylated AT proteinfrom EPEC and EHEC. Further studies are required to elucidate the function of EhaJ incolonization and virulence.
Allsopp LP, Totsika M, Tree JJ, et al., 2010, UpaH Is a Newly Identified Autotransporter Protein That Contributes to Biofilm Formation and Bladder Colonization by Uropathogenic Escherichia coli CFT073, INFECTION AND IMMUNITY, Vol: 78, Pages: 1659-1669, ISSN: 0019-9567
Wells TJ, Sherlock O, Rivas L, et al., 2008, EhaA is a novel autotransporter protein of enterohemorrhagic Escherichia coli O157 : H7 that contributes to adhesion and biofilm formation, ENVIRONMENTAL MICROBIOLOGY, Vol: 10, Pages: 589-604, ISSN: 1462-2912
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