Publications by authors named "Alain Filloux"

145 Publications

The Breadth and Molecular Basis of Hcp-Driven Type VI Secretion System Effector Delivery.

mBio 2021 06 1;12(3):e0026221. Epub 2021 Jun 1.

Imperial College London, Department of Life Sciences, MRC Centre for Molecular Bacteriology and Infection, London, United Kingdom.

The type VI secretion system (T6SS) is a bacterial nanoscale weapon that delivers toxins into prey ranging from bacteria and fungi to animal hosts. The cytosolic contractile sheath of the system wraps around stacked hexameric rings of Hcp proteins, which form an inner tube. At the tip of this tube is a puncturing device comprising a trimeric VgrG topped by a monomeric PAAR protein. The number of toxins a single system delivers per firing event remains unknown, since effectors can be loaded on diverse sites of the T6SS apparatus, notably the inner tube and the puncturing device. Each VgrG or PAAR can bind one effector, and additional effector cargoes can be carried in the Hcp ring lumen. While many VgrG- and PAAR-bound toxins have been characterized, to date, very few Hcp-bound effectors are known. Here, we used 3 known Pseudomonas aeruginosa Hcp proteins (Hcp1 to -3), each of which associates with one of the three T6SSs in this organism (H1-T6SS, H2-T6SS, and H3-T6SS), to perform pulldown assays. We confirmed the known interactions of Hcp1 with Tse1 to -4, further copurified a Hcp1-Tse4 complex, and identified potential novel Hcp1-bound effectors. Moreover, we demonstrated that Hcp2 and Hcp3 can shuttle T6SS cargoes toxic to Escherichia coli. Finally, we used a Tse1-Bla chimera to probe the loading strategy for Hcp passengers and found that while large effectors can be loaded onto Hcp, the formed complex jams the system, abrogating T6SS function. The type VI secretion system (T6SS) is an effective weapon used by bacteria to outgrow or kill competitors. It can be used by endogenous commensal microbiota to prevent invasion by pathogens or by pathogens to overcome resident flora and successfully colonize a host or a specific environmental niche. The T6SS is a key contributor to this continuous arms race between organisms as it delivers a multitude of toxins directed at essential processes, such as nucleic acid synthesis and replication, cell wall and membrane integrity, protein synthesis, or cofactor abundance. Many T6SS toxins with unknown function remain to be discovered, whose yet-uncharacterized targets could be exploited for antimicrobial drug design. The systematic search for these toxins is not facilitated by the presence of readily recognizable T6SS motifs, and unbiased screening approaches are thus required. Here, we successfully used a known shuttle for cargo T6SS effectors, Hcp, as bait to identify uncharacterized toxins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/mBio.00262-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262886PMC
June 2021

A novel stabilization mechanism for the type VI secretion system sheath.

Proc Natl Acad Sci U S A 2021 02;118(7)

MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, SW7 2AZ London, United Kingdom;

The type VI secretion system (T6SS) is a phage-derived contractile nanomachine primarily involved in interbacterial competition. Its pivotal component, TssA, is indispensable for the assembly of the T6SS sheath structure, the contraction of which propels a payload of effector proteins into neighboring cells. Despite their key function, TssA proteins exhibit unexpected diversity and exist in two major forms, a short form (TssA) and a long form (TssA). While TssA proteins interact with a partner, called TagA, to anchor the distal end of the extended sheath, the mechanism for the stabilization of TssA-containing T6SSs remains unknown. Here we discover a class of structural components that interact with short TssA proteins and contribute to T6SS assembly by stabilizing the polymerizing sheath from the baseplate. We demonstrate that the presence of these components is important for full sheath extension and optimal firing. Moreover, we show that the pairing of each form of TssA with a different class of sheath stabilization proteins results in T6SS apparatuses that either reside in the cell for some time or fire immediately after sheath extension. We propose that this diversity in firing dynamics could contribute to the specialization of the T6SS to suit bacterial lifestyles in diverse environmental niches.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.2008500118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7896307PMC
February 2021

Dual role of a (p)ppGpp- and (p)ppApp-degrading enzyme in biofilm formation and interbacterial antagonism.

Mol Microbiol 2021 Jun 25;115(6):1339-1356. Epub 2021 Jan 25.

Center for Synthetic Microbiology (SYNMIKRO) & Faculty of Chemistry, Philipps-University Marburg, Marburg, Germany.

The guanosine nucleotide-based second messengers ppGpp and pppGpp (collectively: (p)ppGpp) enable adaptation of microorganisms to environmental changes and stress conditions. In contrast, the closely related adenosine nucleotides (p)ppApp are involved in type VI secretion system (T6SS)-mediated killing during bacterial competition. Long RelA-SpoT Homolog (RSH) enzymes regulate synthesis and degradation of (p)ppGpp (and potentially also (p)ppApp) through their synthetase and hydrolase domains, respectively. Small alarmone hydrolases (SAH) that consist of only a hydrolase domain are found in a variety of bacterial species, including the opportunistic human pathogen Pseudomonas aeruginosa. Here, we present the structure and mechanism of P. aeruginosa SAH showing that the enzyme promiscuously hydrolyses (p)ppGpp and (p)ppApp in a strictly manganese-dependent manner. While being dispensable for P. aeruginosa growth or swimming, swarming, and twitching motilities, its enzymatic activity is required for biofilm formation. Moreover, (p)ppApp-degradation by SAH provides protection against the T6SS (p)ppApp synthetase effector Tas1, suggesting that SAH enzymes can also serve as defense proteins during interbacterial competition.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/mmi.14684DOI Listing
June 2021

Role of Recipient Susceptibility Factors During Contact-Dependent Interbacterial Competition.

Front Microbiol 2020 12;11:603652. Epub 2020 Nov 12.

Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan.

Bacteria evolved multiple strategies to survive and develop optimal fitness in their ecological niche. They deployed protein secretion systems for robust and efficient delivery of antibacterial toxins into their target cells, therefore inhibiting their growth or killing them. To maximize antagonism, recipient factors on target cells can be recognized or hijacked to enhance the entry or toxicity of these toxins. To date, knowledge regarding recipient susceptibility (RS) factors and their mode of action is mostly originating from studies on the type Vb secretion system that is also known as the contact-dependent inhibition (CDI) system. Yet, recent studies on the type VI secretion system (T6SS), and the CDI by glycine-zipper protein (Cdz) system, also reported the emerging roles of RS factors in interbacterial competition. Here, we review these RS factors and their mechanistic impact in increasing susceptibility of recipient cells in response to CDI, T6SS, and Cdz. Past and future strategies for identifying novel RS factors are also discussed, which will help in understanding the interplay between attacker and prey upon secretion system-dependent competition. Understanding these mechanisms would also provide insights for developing novel antibacterial strategies to antagonize aggressive bacteria-killing pathogens.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2020.603652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7690452PMC
November 2020

Solving the Puzzle: Connecting a Heterologous T6SS Effector to a Spike Complex.

Front Cell Infect Microbiol 2020 23;10:291. Epub 2020 Jun 23.

MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, London, United Kingdom.

The type VI secretion system (T6SS) is a contractile injection apparatus that translocates a spike loaded with various effectors directly into eukaryotic and prokaryotic target cells. Such T6SS spike consists of a needle-shaped trimer of VgrG proteins topped by a conical and sharp PAAR protein that facilitates puncturing of the target membrane. T6SS-delivered effector proteins can be either fused to one of the two spike proteins or interact with either in a highly specific manner. In the T6SS effector Tde1 is targeted to its cognate VgrG1 protein. Here, we attempted to use a VgrG shuttle to deliver a heterologous T6SS effector by directing Tde1 onto a T6SS spike in . For this, we designed chimeras between VgrG1 from and VgrG1a from and showed that modification of the spike protein hampered T6SS functionality in the presence of the Tde1 effector complex. We provide evidence suggesting that Tde1 specifically binds to the VgrG spike in the heterologous environment and propose that there are additional requirements to allow proper effector delivery and translocation. Our work sheds light on complex aspects of the molecular mechanisms of T6SS delivery and highlights some limitations on how effectors can be translocated using this nanomachine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fcimb.2020.00291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7324665PMC
June 2021

Detection of Colistin Resistance in Using MALDIxin Test on the Routine MALDI Biotyper Sirius Mass Spectrometer.

Front Microbiol 2020 3;11:1141. Epub 2020 Jun 3.

MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London, United Kingdom.

Resistance to polymyxins in most Gram-negative bacteria arises from chemical modifications to the lipid A portion of their lipopolysaccharide (LPS) mediated by chromosomally encoded mutations or the recently discovered plasmid-encoded genes that have further complicated the landscape of colistin resistance. Currently, minimal inhibitory concentration (MIC) determination by broth microdilution, the gold standard for the detection of polymyxin resistance, is time consuming (24 h) and challenging to perform in clinical and veterinary laboratories. Here we present the use of the MALDIxin to detect colistin resistant using the MALDxin test on the routine matrix-assisted laser desorption ionization (MALDI) Biotyper Sirius system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2020.01141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7283459PMC
June 2020

Manipulating the type VI secretion system spike to shuttle passenger proteins.

PLoS One 2020 26;15(2):e0228941. Epub 2020 Feb 26.

MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, London, United Kingdom.

The type VI secretion system (T6SS) is a contractile injection apparatus that translocates a spike loaded with various effectors directly into eukaryotic or prokaryotic target cells. Pseudomonas aeruginosa can load either one of its three T6SSs with a variety of toxic bullets using different but specific modes. The T6SS spike, which punctures the bacterial cell envelope allowing effector transport, consists of a torch-like VgrG trimer on which sits a PAAR protein sharpening the VgrG tip. VgrG itself sits on the Hcp tube and all elements, packed into a T6SS sheath, are propelled out of the cell and into target cells. On occasion, effectors are covalent extensions of VgrG, PAAR or Hcp proteins, which are then coined "evolved" components as opposed to canonical. Here, we show how various passenger domains could be fused to the C terminus of a canonical VgrG, VgrG1a from P. aeruginosa, and be sent into the bacterial culture supernatant. There is no restriction on the passenger type, although the efficacy may vary greatly, since we used either an unrelated T6SS protein, β-lactamase, a covalent extension of an "evolved" VgrG, VgrG2b, or a Hcp-dependent T6SS toxin, Tse2. Our data further highlights an exceptional modularity/flexibility for loading the T6SS nano-weapon. Refining the parameters to optimize delivery of passenger proteins of interest would have attractive medical and industrial applications. This may for example involve engineering the T6SS as a delivery system to shuttle toxins into either bacterial pathogens or tumour cells which would be an original approach in the fight against antimicrobial resistant bacteria or cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0228941PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7043769PMC
May 2020

Causalities of war: The connection between type VI secretion system and microbiota.

Cell Microbiol 2020 03 12;22(3):e13153. Epub 2020 Jan 12.

Department of Life Sciences, MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, UK.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/cmi.13153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7540082PMC
March 2020

Complete Genome Sequence of Pseudomonas aeruginosa Reference Strain PAK.

Microbiol Resour Announc 2019 Oct 10;8(41). Epub 2019 Oct 10.

Wellcome Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom.

We report the complete genome of strain PAK, a strain which has been instrumental in the study of a range of virulence and pathogenesis factors and has been used for over 50 years as a laboratory reference strain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/MRA.00865-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6787321PMC
October 2019

Detection of Colistin Resistance in Escherichia coli by Use of the MALDI Biotyper Sirius Mass Spectrometry System.

J Clin Microbiol 2019 12 22;57(12). Epub 2019 Nov 22.

MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London, United Kingdom

Polymyxin antibiotics are a last-line treatment for multidrug-resistant Gram-negative bacteria. However, the emergence of colistin resistance, including the spread of mobile genes, necessitates the development of improved diagnostics for the detection of colistin-resistant organisms in hospital settings. The recently developed MALDIxin test enables detection of colistin resistance by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in less than 15 min but is not optimized for the mass spectrometers commonly found in clinical microbiology laboratories. In this study, we adapted the MALDIxin test for the MALDI Biotyper Sirius MALDI-TOF MS system (Bruker Daltonics). We optimized the sample preparation protocol by using a set of 6 mobile colistin resistance (MCR) protein-expressing clones and validated the assay with a collection of 40 clinical isolates, including 19 confirmed MCR protein producers, 12 colistin-resistant isolates that tested negative for commonly encountered genes (i.e., likely chromosomally resistant isolates), and 9 polymyxin-susceptible isolates. We calculated polymyxin resistance ratio (PRR) values from the acquired spectra; PRR values of 0, indicating polymyxin susceptibility, were obtained for all colistin-susceptible isolates, whereas positive PRR values, indicating resistance to polymyxins, were obtained for all resistant strains, independent of the genetic basis of resistance. Thus, we report a preliminary feasibility study showing that an optimized version of the MALDIxin test adapted for the routine MALDI Biotyper Sirius system provides an unbiased, fast, reliable, cost-effective, and high-throughput way of detecting colistin resistance in clinical isolates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JCM.01427-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6879293PMC
December 2019

Optimization of the MALDIxin test for the rapid identification of colistin resistance in Klebsiella pneumoniae using MALDI-TOF MS.

J Antimicrob Chemother 2020 01;75(1):110-116

MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London, SW7 2AZ, UK.

Background: With the dissemination of carbapenemase producers, a revival of colistin was observed for the treatment of infections caused by MDR Gram-negatives. Unfortunately, the increasing usage of colistin led to the emergence of resistance. In Klebsiella pneumoniae, colistin resistance arises through addition of 4-amino-l-arabinose (l-Ara4N) or phosphoethanolamine (pEtN) to the native lipid A. The underlying mechanisms involve numerous chromosome-encoded genes or the plasmid-encoded pEtN transferase MCR. Currently, detection of colistin resistance is time-consuming since it still relies on MIC determination by broth microdilution. Recently, a rapid diagnostic test based on MALDI-TOF MS detection of modified lipid A was developed (the MALDIxin test) and tested on Escherichia coli and Acinetobacter baumannii.

Objectives: Optimize the MALDIxin test for the rapid detection of colistin resistance in K. pneumoniae.

Methods: This optimization consists of an additional mild-acid hydrolysis of 15 min in 1% acetic acid. The optimized method was tested on a collection of 81 clinical K. pneumoniae isolates, including 49 colistin-resistant isolates (45 with chromosome-encoded resistance, 3 with MCR-related resistance and 1 with both mechanisms).

Results: The optimized method allowed the rapid (<30 min) identification of l-Ara4N- and pEtN-modified lipid A of K. pneumoniae, which are known to be the real triggers of polymyxin resistance. At the same time, it discriminates between chromosome-encoded and MCR-related polymyxin resistance.

Conclusions: The MALDIxin test has the potential to become an accurate tool for the rapid determination of colistin resistance in clinically relevant Gram-negative bacteria.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jac/dkz405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6910190PMC
January 2020

The Pseudomonas aeruginosa T6SS Delivers a Periplasmic Toxin that Disrupts Bacterial Cell Morphology.

Cell Rep 2019 10;29(1):187-201.e7

MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, London SW7 2AZ, UK. Electronic address:

The type VI secretion system (T6SS) is crucial in interbacterial competition and is a virulence determinant of many Gram-negative bacteria. Several T6SS effectors are covalently fused to secreted T6SS structural components such as the VgrG spike for delivery into target cells. In Pseudomonas aeruginosa, the VgrG2b effector was previously proposed to mediate bacterial internalization into eukaryotic cells. In this work, we find that the VgrG2b C-terminal domain (VgrG2b) elicits toxicity in the bacterial periplasm, counteracted by a cognate immunity protein. We resolve the structure of VgrG2b and confirm it is a member of the zinc-metallopeptidase family of enzymes. We show that this effector causes membrane blebbing at midcell, which suggests a distinct type of T6SS-mediated growth inhibition through interference with cell division, mimicking the impact of β-lactam antibiotics. Our study introduces a further effector family to the T6SS arsenal and demonstrates that VgrG2b can target both prokaryotic and eukaryotic cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2019.08.094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6899460PMC
October 2019

Looking inside an injection system.

Elife 2019 09 17;8. Epub 2019 Sep 17.

MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, London, United Kingdom.

The proteins injected by bacteria into eukaryotic organisms can lead to fates as diverse as death and metamorphosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.50815DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748822PMC
September 2019

Multiple Roles of c-di-GMP Signaling in Bacterial Pathogenesis.

Annu Rev Microbiol 2019 09;73:387-406

MRC Centre for Molecular Microbiology and Infection, Department of Life Sciences, Imperial College London, SW7 2AZ London, United Kingdom; email:

The intracellular signaling molecule cyclic di-GMP (c-di-GMP) regulates the lifestyle of bacteria and controls many key functions and mechanisms. In the case of bacterial pathogens, a wide variety of virulence lifestyle factors have been shown to be regulated by c-di-GMP. Evidence of the importance of this molecule for bacterial pathogenesis has become so great that new antimicrobial agents are tested for their capacity of targeting c-di-GMP signaling. This review summarizes the current knowledge on this topic and reveals its application for the development of new antivirulence intervention strategies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1146/annurev-micro-020518-115555DOI Listing
September 2019

Delivery of the Phospholipase Effectors PldA and PldB in a VgrG- and H2-T6SS-Dependent Manner.

Front Microbiol 2019 31;10:1718. Epub 2019 Jul 31.

MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, London, United Kingdom.

The bacterial pathogen uses three type VI secretion systems (T6SSs) to drive a multitude of effector proteins into eukaryotic or prokaryotic target cells. The T6SS is a supramolecular nanomachine, involving a set of 13 core proteins, which resembles the contractile tail of bacteriophages and whose tip is considered as a puncturing device helping to cross membranes. Effectors can attach directly to the T6SS spike which is composed of a VgrG (valine-glycine-rich proteins) trimer, of which produces several. We have previously shown that the master regulator RsmA controls the expression of all three T6SS gene clusters (H1-, H2- and H3-T6SS) and a range of remote and effector genes. We also demonstrated that specific interactions between VgrGs and various T6SS effectors are prerequisite for effector delivery in a process we called "à la carte delivery." Here, we provide an in-depth description on how the two H2-T6SS-dependent effectors PldA and PldB are delivered their cognate VgrGs, VgrG4b and VgrG5, respectively. We show that specific recognition of the VgrG C terminus is required and effector specificity can be swapped by exchanging these C-terminal domains. Importantly, we established that effector recognition by a cognate VgrG is not always sufficient to achieve successful secretion, but it is crucial to provide effector stability. This study highlights the complexity of effector adaptation to the T6SS nanomachine and shows how the VgrG tip can possibly be manipulated to achieve effector delivery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2019.01718DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6684961PMC
July 2019

PAAR proteins act as the 'sorting hat' of the type VI secretion system.

Microbiology (Reading) 2019 11;165(11):1203-1218

MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College, London, London, SW7 2AZ, UK.

Bacteria exist in polymicrobial environments and compete to prevail in a niche. The type VI secretion system (T6SS) is a nanomachine employed by Gram-negative bacteria to deliver effector proteins into target cells. Consequently, T6SS-positive bacteria produce a wealth of antibacterial effector proteins to promote their survival among a prokaryotic community. These toxins are loaded onto the VgrG-PAAR spike and Hcp tube of the T6SS apparatus and recent work has started to document the specificity of effectors for certain spike components. encodes several PAAR proteins, whose roles have been poorly investigated. Here we describe a phospholipase family antibacterial effector immunity pair from and demonstrate that a specific PAAR protein is necessary for the delivery of the effector and its cognate VgrG. Furthermore, the PAAR protein appears to restrict the delivery of other phospholipase effectors that utilise distinct VgrG proteins. We provide further evidence for competition for PAAR protein recruitment to the T6SS apparatus, which determines the identities of the delivered effectors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/mic.0.000842DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7376260PMC
November 2019

Cyclic di-GMP inactivates T6SS and T4SS activity in Agrobacterium tumefaciens.

Mol Microbiol 2019 08 4;112(2):632-648. Epub 2019 Jun 4.

MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK.

The Type VI secretion system (T6SS) is a bacterial nanomachine that delivers effector proteins into prokaryotic and eukaryotic preys. This secretion system has emerged as a key player in regulating the microbial diversity in a population. In the plant pathogen Agrobacterium tumefaciens, the signalling cascades regulating the activity of this secretion system are poorly understood. Here, we outline how the universal eubacterial second messenger cyclic di-GMP impacts the production of T6SS toxins and T6SS structural components. We demonstrate that this has a significant impact on the ability of the phytopathogen to compete with other bacterial species in vitro and in planta. Our results suggest that, as opposed to other bacteria, c-di-GMP turns down the T6SS in A. tumefaciens thus impacting its ability to compete with other bacterial species within the rhizosphere. We also demonstrate that elevated levels of c-di-GMP within the cell decrease the activity of the Type IV secretion system (T4SS) and subsequently the capacity of A. tumefaciens to transform plant cells. We propose that such peculiar control reflects on c-di-GMP being a key second messenger that silences energy-costing systems during early colonization phase and biofilm formation, while low c-di-GMP levels unleash T6SS and T4SS to advance plant colonization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/mmi.14279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6771610PMC
August 2019

Chronic infection by controlling inflammation.

Nat Microbiol 2019 03;4(3):378-379

The Cystic Fibrosis and Chronic Infection Group, National Heart and Lung Institute, Imperial College, London, UK.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41564-019-0397-6DOI Listing
March 2019

Biofilms 2018: A diversity of microbes and mechanisms.

J Bacteriol 2019 Feb 19. Epub 2019 Feb 19.

Department of Microbiology and Immunology, Loyola University Chicago, Maywood, Illinois USA.

The 8 ASM Conference on Biofilms was held in Washington D.C. on October 7-11, 2018. This very highly subscribed meeting represented a wide breadth of current research in biofilms, and included over 500 attendees, 12 sessions with 64 oral presentations, and four poster sessions with about 400 posters.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JB.00118-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6707918PMC
February 2019

The T6SS-VgrG1b spike is topped by a PAAR protein eliciting DNA damage to bacterial competitors.

Proc Natl Acad Sci U S A 2018 12 19;115(49):12519-12524. Epub 2018 Nov 19.

Department of Life Sciences, Medical Research Council Centre for Molecular Microbiology and Infection, Imperial College London, SW7 2AZ London, United Kingdom

The type VI secretion system (T6SS) is a supramolecular complex involved in the delivery of potent toxins during bacterial competition. possesses three T6SS gene clusters and several and gene islands, the latter encoding the spike at the T6SS tip. The cluster encompasses seven genes whose organization and sequences are highly conserved in genomes, except for two genes that we called and 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 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1814181115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298103PMC
December 2018

Rapid detection of colistin resistance in Acinetobacter baumannii using MALDI-TOF-based lipidomics on intact bacteria.

Sci Rep 2018 11 15;8(1):16910. Epub 2018 Nov 15.

MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London, SW7 2AZ, UK.

With the dissemination of extremely drug resistant bacteria, colistin is now considered as the last-resort therapy for the treatment of infection caused by Gram-negative bacilli (including carbapenemase producers). Unfortunately, the increase use of colistin has resulted in the emergence of resistance as well. In A. baumannii, colistin resistance is mostly caused by the addition of phosphoethanolamine to the lipid A through the action of a phosphoethanolamine transferase chromosomally-encoded by the pmrC gene, which is regulated by the two-component system PmrA/PmrB. In A. baumannii clinical isolate the main resistance mechanism to colistin involves mutations in pmrA, pmrB or pmrC genes leading to the overexpression of pmrC. Although, rapid detection of resistance is one of the key issues to improve the treatment of infected patient, detection of colistin resistance in A. baumannii still relies on MIC determination through microdilution, which is time-consuming (16-24 h). Here, we evaluated the performance of a recently described MALDI-TOF-based assay, the MALDIxin test, which allows the rapid detection of colistin resistance-related modifications to lipid A (i.e phosphoethanolamine addition). This test accurately detected all colistin-resistant A. baumannii isolates in less than 15 minutes, directly on intact bacteria with a very limited sample preparation prior MALDI-TOF analysis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-35041-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237936PMC
November 2018

Higher Prevalence of PldA, a -Kingdom H2-Type VI Secretion System Effector, in Clinical Isolates Responsible for Acute Infections and in Multidrug Resistant Strains.

Front Microbiol 2018 29;9:2578. Epub 2018 Oct 29.

EA7361 Structure, Dynamic, Function and Expression of Broad Spectrum β-Lactamases, LabEx Lermit, Faculty of Medicine, Université Paris Sud - Université Paris Saclay, Le Kremlin-Bicêtre, France.

can manipulate eukaryotic host cells using secreted effectors delivered by the type III or the type VI Secretion Systems (T3SS and T6SS). The T3SS allows the injection of bacterial effectors (Exo toxins) into eukaryotic cell. , encodes three T6SSs, H1-, H2- and H3-T6SS. The H1-T6SS is mainly involved in delivering toxins to kill bacterial competitors. Recently, two T6SS-secreted phospholipases D, PldA (H2-T6SS) and PldB (H3-T6SS), were identified as -kingdom virulence effectors, triggering both killing of bacterial competitors and internalization into non-phagocytic cells. We deciphered the prevalence of T3SS and T6SS effectors encoding genes in 185 clinical isolates responsible for infections (septicaemia, pulmonary infections, urinary tract infections, and chronic infections in CF patients), 47 environmental strains, and on 33 carbapenemase-producers. We included 107 complete genomes of available in public databases. The prevalence of is increased in clinical isolates responsible for severe acute infection and particularly in multi-drug resistant strains. In contrast, the prevalence was high (96.8%) in all isolates. Regarding T3SS effectors, and are present in nearly all isolates while and were found to be exclusive with a higher prevalence of strains in severe acute infections. The hypervirulent isolates are more prone to be , suggesting a role of PldA in virulence. Finally, we observed that extremely drug resistant isolates producing an IMP-type carbapenemase were all . Our results suggest that PldA might have a role during pulmonary infections and have been co-selected in multidrug resistant strains particularly IMP-producers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2018.02578DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6215852PMC
October 2018

Importance of flagella in acute and chronic Pseudomonas aeruginosa infections.

Environ Microbiol 2019 03 3;21(3):883-897. Epub 2018 Dec 3.

Institute of Molecular Bacteriology, Helmholtz Centre for Infection Research, Braunschweig, Germany.

Pseudomonas aeruginosa is an environmental microorganism and a causative agent of diverse acute and chronic, biofilm-associated infections. Advancing research-based knowledge on its adaptation to conditions within the human host is bound to reveal novel strategies and targets for therapeutic intervention. Here, we investigated the traits that P. aeruginosa PA14 as well as a virulence attenuated ΔlasR mutant need to survive in selected murine infection models. Experimentally, the genetic programs that the bacteria use to adapt to biofilm-associated versus acute infections were dissected by passaging transposon mutant libraries through mouse lungs (acute) or mouse tumours (biofilm-infection). Adaptive metabolic changes of P. aeruginosa were generally required during both infection processes. Counter-selection against flagella expression was observed during acute lung infections. Obviously, avoidance of flagella-mediated activation of host immunity is advantageous for the wildtype bacteria. For the ΔlasR mutant, loss of flagella did not confer a selective advantage. Apparently, other pathogenesis mechanisms are active in this virulence attenuated strain. In contrast, the infective process of P. aeruginosa in the chronic biofilm model apparently required expression of flagellin. Together, our findings imply that the host immune reactions against the infectious agent are very decisive for acuteness and duration of the infectious disease. They direct disease outcome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/1462-2920.14468DOI Listing
March 2019

A global genomic approach uncovers novel components for twitching motility-mediated biofilm expansion in Pseudomonas aeruginosa.

Microb Genom 2018 11 1;4(11). Epub 2018 Nov 1.

5​Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK.

Pseudomonas aeruginosa is an extremely successful pathogen able to cause both acute and chronic infections in a range of hosts, utilizing a diverse arsenal of cell-associated and secreted virulence factors. A major cell-associated virulence factor, the Type IV pilus (T4P), is required for epithelial cell adherence and mediates a form of surface translocation termed twitching motility, which is necessary to establish a mature biofilm and actively expand these biofilms. P. aeruginosa twitching motility-mediated biofilm expansion is a coordinated, multicellular behaviour, allowing cells to rapidly colonize surfaces, including implanted medical devices. Although at least 44 proteins are known to be involved in the biogenesis, assembly and regulation of the T4P, with additional regulatory components and pathways implicated, it is unclear how these components and pathways interact to control these processes. In the current study, we used a global genomics-based random-mutagenesis technique, transposon directed insertion-site sequencing (TraDIS), coupled with a physical segregation approach, to identify all genes implicated in twitching motility-mediated biofilm expansion in P. aeruginosa. Our approach allowed identification of both known and novel genes, providing new insight into the complex molecular network that regulates this process in P. aeruginosa. Additionally, our data suggest that the flagellum-associated gene products have a differential effect on twitching motility, based on whether components are intra- or extracellular. Overall the success of our TraDIS approach supports the use of this global genomic technique for investigating virulence genes in bacterial pathogens.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/mgen.0.000229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6321873PMC
November 2018

Rapid detection and discrimination of chromosome- and MCR-plasmid-mediated resistance to polymyxins by MALDI-TOF MS in Escherichia coli: the MALDIxin test.

J Antimicrob Chemother 2018 12;73(12):3359-3367

MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London, UK.

Background: Polymyxins are currently considered a last-resort treatment for infections caused by MDR Gram-negative bacteria. Recently, the emergence of carbapenemase-producing Enterobacteriaceae has accelerated the use of polymyxins in the clinic, resulting in an increase in polymyxin-resistant bacteria. Polymyxin resistance arises through modification of lipid A, such as the addition of phosphoethanolamine (pETN). The underlying mechanisms involve numerous chromosome-encoded genes or, more worryingly, a plasmid-encoded pETN transferase named MCR. Currently, detection of polymyxin resistance is difficult and time consuming.

Objectives: To develop a rapid diagnostic test that can identify polymyxin resistance and at the same time differentiate between chromosome- and plasmid-encoded resistances.

Methods: We developed a MALDI-TOF MS-based method, named the MALDIxin test, which allows the detection of polymyxin resistance-related modifications to lipid A (i.e. pETN addition), on intact bacteria, in <15 min.

Results: Using a characterized collection of polymyxin-susceptible and -resistant Escherichia coli, we demonstrated that our method is able to identify polymyxin-resistant isolates in 15 min whilst simultaneously discriminating between chromosome- and plasmid-encoded resistance. We validated the MALDIxin test on different media, using fresh and aged colonies and show that it successfully detects all MCR-1 producers in a blindly analysed set of carbapenemase-producing E. coli strains.

Conclusions: The MALDIxin test is an accurate, rapid, cost-effective and scalable method that represents a major advance in the diagnosis of polymyxin resistance by directly assessing lipid A modifications in intact bacteria.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jac/dky330DOI Listing
December 2018

-Induced Emergency Granulopoiesis Protects Zebrafish Larvae from Secondary Infection.

mBio 2018 06 26;9(3). Epub 2018 Jun 26.

Section of Microbiology, MRC Centre of Molecular Bacteriology and Infection (CMBI), Imperial College London, London, United Kingdom

Emergency granulopoiesis is a hematopoietic program of stem cell-driven neutrophil production used to counteract immune cell exhaustion following infection. is a Gram-negative enteroinvasive pathogen controlled by neutrophils. In this study, we use a -zebrafish () infection model to investigate emergency granulopoiesis We show that stem cell-driven neutrophil production occurs in response to infection and requires macrophage-independent signaling by granulocyte colony-stimulating factor (Gcsf). To test whether emergency granulopoiesis can function beyond homoeostasis to enhance innate immunity, we developed a reinfection assay using zebrafish larvae that have not yet developed an adaptive immune system. Strikingly, larvae primed with a sublethal dose of are protected against a secondary lethal dose of in a type III secretion system (T3SS)-dependent manner. Collectively, these results highlight a new role for emergency granulopoiesis in boosting host defense and demonstrate that zebrafish larvae can be a valuable model to investigate innate immune memory. is an important human pathogen of the gut. Emergency granulopoiesis is the enhanced production of neutrophils by hematopoietic stem and progenitor cells (HSPCs) upon infection and is widely considered a homoeostatic mechanism for replacing exhausted leukocytes. In this study, we developed a -zebrafish infection model to investigate stem cell-driven emergency granulopoiesis. We discovered that zebrafish initiate granulopoiesis in response to infection, via macrophage-independent signaling of granulocyte colony-stimulating factor (Gcsf). Strikingly, larvae primed with a sublethal dose of are protected against a secondary lethal dose of in a type III secretion system (T3SS)-dependent manner. Taken together, we show that zebrafish infection can be used to capture -mediated stem cell-driven granulopoiesis and provide a new model system to study stem cell biology Our results also highlight the potential of manipulating stem cell-driven granulopoiesis to boost innate immunity and combat infectious disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/mBio.00933-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6020294PMC
June 2018

TagF-mediated repression of bacterial type VI secretion systems involves a direct interaction with the cytoplasmic protein Fha.

J Biol Chem 2018 06 29;293(23):8829-8842. Epub 2018 Mar 29.

From the Institute of Plant and Microbial Biology and

The bacterial type VI secretion system (T6SS) delivers effectors into eukaryotic host cells or toxins into bacterial competitor for survival and fitness. The T6SS is positively regulated by the threonine phosphorylation pathway (TPP) and negatively by the T6SS-accessory protein TagF. Here, we studied the mechanisms underlying TagF-mediated T6SS repression in two distinct bacterial pathogens, and We found that in , T6SS toxin secretion and T6SS-dependent antibacterial activity are suppressed by a two-domain chimeric protein consisting of TagF and PppA, a putative phosphatase. Remarkably, this TagF domain is sufficient to post-translationally repress the T6SS, and this inhibition is independent of TPP. This repression requires interaction with a cytoplasmic protein, Fha, critical for activating T6SS assembly. In , PppA and TagF are two distinct proteins that repress T6SS in TPP-dependent and -independent pathways, respectively. TagF interacts with Fha1, suggesting that formation of this complex represents a conserved TagF-mediated regulatory mechanism. Using TagF variants with substitutions of conserved amino acid residues at predicted protein-protein interaction interfaces, we uncovered evidence that the TagF-Fha interaction is critical for TagF-mediated T6SS repression in both bacteria. TagF inhibits T6SS without affecting T6SS protein abundance in , but TagF overexpression reduces the protein levels of all analyzed T6SS components in Our results indicate that TagF interacts with Fha, which in turn could impact different stages of T6SS assembly in different bacteria, possibly reflecting an evolutionary divergence in T6SS control.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA117.001618DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5995506PMC
June 2018

Pore-forming activity of the Pseudomonas aeruginosa type III secretion system translocon alters the host epigenome.

Nat Microbiol 2018 03 5;3(3):378-386. Epub 2018 Feb 5.

MRC Centre for Molecular Microbiology and Infection, Department of Life Sciences, Imperial College London, London, UK.

Recent studies highlight that bacterial pathogens can reprogram target cells by influencing epigenetic factors. The type III secretion system (T3SS) is a bacterial nanomachine that resembles a syringe on the bacterial surface. The T3SS 'needle' delivers translocon proteins into eukaryotic cell membranes, subsequently allowing injection of bacterial effectors into the cytosol. Here we show that Pseudomonas aeruginosa induces early T3SS-dependent dephosphorylation and deacetylation of histone H3 in eukaryotic cells. This is not triggered by any of the P. aeruginosa T3SS effectors, but results from the insertion of the PopB-PopD translocon into the membrane. This suggests that the P. aeruginosa translocon is a genuine T3SS effector acting as a pore-forming toxin. We visualized the translocon plugged into the host cell membrane after the bacterium has left the site of contact, and demonstrate that subsequent ion exchange through this pore is responsible for histone H3 modifications and host cell subversion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41564-018-0109-7DOI Listing
March 2018

Atomic Structure of Type VI Contractile Sheath from Pseudomonas aeruginosa.

Structure 2018 02 4;26(2):329-336.e3. Epub 2018 Jan 4.

Section of Structural Biology, Department of Medicine, Imperial College London, London SW7 2AZ, UK. Electronic address:

Pseudomonas aeruginosa has three type VI secretion systems (T6SSs), H1-, H2-, and H3-T6SS, each belonging to a distinct group. The two T6SS components, TssB/VipA and TssC/VipB, assemble to form tubules that conserve structural/functional homology with tail sheaths of contractile bacteriophages and pyocins. Here, we used cryoelectron microscopy to solve the structure of the H1-T6SS P. aeruginosa TssB1C1 sheath at 3.3 Å resolution. Our structure allowed us to resolve some features of the T6SS sheath that were not resolved in the Vibrio cholerae VipAB and Francisella tularensis IglAB structures. Comparison with sheath structures from other contractile machines, including T4 phage and R-type pyocins, provides a better understanding of how these systems have conserved similar functions/mechanisms despite evolution. We used the P. aeruginosa R2 pyocin as a structural template to build an atomic model of the TssB1C1 sheath in its extended conformation, allowing us to propose a coiled-spring-like mechanism for T6SS sheath contraction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.str.2017.12.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5807055PMC
February 2018

Multiple Structures Disclose the Secretins' Secrets.

J Bacteriol 2018 03 7;200(5). Epub 2018 Feb 7.

Aix Marseille University, CNRS, IMM, LISM, Marseille, France.

Bacterial secretins are outer membrane proteins that provide a path for secreted proteins to access the cell exterior/surface. They are one of the core components of secretion machines and are found in type II and type III secretion systems (T2SS and T3SS, respectively). The secretins comprise giant ring-shaped homo-oligomers whose precise atomic organization was only recently deciphered thanks to spectacular developments in cryo-electron microscopy (cryo-EM) imaging techniques.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JB.00702-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5809689PMC
March 2018
-->