Imperial College London

ProfessorAlainFilloux

Faculty of Natural SciencesDepartment of Life Sciences

Visiting Professor
 
 
 
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Contact

 

+44 (0)20 7594 9651a.filloux Website CV

 
 
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Location

 

1.47Flowers buildingSouth Kensington Campus

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Summary

 

Publications

Publication Type
Year
to

211 results found

Bullen NP, Sychantha D, Thang SS, Culviner PH, Rudzite M, Ahmad S, Shah VS, Filloux A, Prehna G, Whitney JCet al., 2022, An ADP-ribosyltransferase toxin kills bacterial cells by modifying structured non-coding RNAs, Molecular Cell, Vol: 82, Pages: 3484-3498.e11, ISSN: 1097-2765

ADP-ribosyltransferases (ARTs) were among the first identified bacterial virulence factors. Canonical ART toxins are delivered into host cells where they modify essential proteins, thereby inactivating cellular processes and promoting pathogenesis. Our understanding of ARTs has since expanded beyond protein-targeting toxins to include antibiotic inactivation and DNA damage repair. Here, we report the discovery of RhsP2 as an ART toxin delivered between competing bacteria by a type VI secretion system of Pseudomonas aeruginosa. A structure of RhsP2 reveals that it resembles protein-targeting ARTs such as diphtheria toxin. Remarkably, however, RhsP2 ADP-ribosylates 2'-hydroxyl groups of double-stranded RNA, and thus, its activity is highly promiscuous with identified cellular targets including the tRNA pool and the RNA-processing ribozyme, ribonuclease P. Consequently, cell death arises from the inhibition of translation and disruption of tRNA processing. Overall, our data demonstrate a previously undescribed mechanism of bacterial antagonism and uncover an unprecedented activity catalyzed by ART enzymes.

Journal article

Tortuel D, Tahrioui A, David A, Cambronel M, Nilly F, Clamens T, Maillot O, Barreau M, Feuilloley MGJ, Lesouhaitier O, Filloux A, Bouffartigues E, Cornelis P, Chevalier Set al., 2022, Pf4 Phage Variant Infection Reduces Virulence-Associated Traits in Pseudomonas aeruginosa, MICROBIOLOGY SPECTRUM, ISSN: 2165-0497

Journal article

Eilers K, Kuok Hoong Yam J, Morton R, Mei Hui Yong A, Brizuela J, Hadjicharalambous C, Liu X, Givskov M, Rice SA, Filloux Aet al., 2022, Phenotypic and integrated analysis of a comprehensive Pseudomonas aeruginosa PAO1 library of mutants lacking cyclic-di-GMP-related genes, Frontiers in Microbiology, Vol: 13, ISSN: 1664-302X

Pseudomonas aeruginosa is a Gram-negative bacterium that is able to survive and adapt in a multitude of niches as well as thrive within many different hosts. This versatility lies within its large genome of ca. 6 Mbp and a tight control in the expression of thousands of genes. Among the regulatory mechanisms widely spread in bacteria, cyclic-di-GMP signaling is one which influences all levels of control. c-di-GMP is made by diguanylate cyclases and degraded by phosphodiesterases, while the intracellular level of this molecule drives phenotypic responses. Signaling involves the modification of enzymes’ or proteins’ function upon c-di-GMP binding, including modifying the activity of regulators which in turn will impact the transcriptome. In P. aeruginosa, there are ca. 40 genes encoding putative DGCs or PDEs. The combined activity of those enzymes should reflect the overall c-di-GMP concentration, while specific phenotypic outputs could be correlated to a given set of dgc/pde. This notion of specificity has been addressed in several studies and different strains of P. aeruginosa. Here, we engineered a mutant library for the 41 individual dgc/pde genes in P. aeruginosa PAO1. In most cases, we observed a significant to slight variation in the global c-di-GMP pool of cells grown planktonically, while several mutants display a phenotypic impact on biofilm including initial attachment and maturation. If this observation of minor changes in c-di-GMP level correlating with significant phenotypic impact appears to be true, it further supports the idea of a local vs global c-di-GMP pool. In contrast, there was little to no effect on motility, which differs from previous studies. Our RNA-seq analysis indicated that all PAO1 dgc/pde genes were expressed in both planktonic and biofilm growth conditions and our work suggests that c-di-GMP networks need to be reconstructed for each strain separately and cannot be extrapolated from one to another.

Journal article

Filloux A, 2022, Bacterial protein secretion systems: Game of types, MICROBIOLOGY-SGM, Vol: 168, ISSN: 1350-0872

Journal article

Demmer J, Phillips B, Uhrig L, Filloux A, Allsopp L, Bublitz M, Meier Tet al., 2022, Structure of ATP synthase from ESKAPE pathogen Acinetobacter baumannii, Science Advances, Vol: 8, ISSN: 2375-2548

The global spread of multi-drug resistant Acinetobacter baumannii infections urgently calls for the identification of novel drug targets. We solved the cryo- electron microscopy structure of the F1Fo-ATP synthase from A. baumannii in three distinct conformational states. The nucleotide-converting F1 sub-complex reveals a specific self-inhibition mechanism, which supports a uni-directional ratchet mechanism to avoid wasteful ATP consumption. In the membrane-embedded Fo complex, the structure shows unique structural adaptations along both the entry and exit pathways of the proton-conducting a-subunit. These features, absent in mitochondrial ATP synthases, represent attractive targets for the development of next generation therapeutics that can act directly at the culmination of bioenergetics in this clinically relevant pathogen.

Journal article

Allsopp LP, Collins ACZ, Hawkins E, Wood TE, Filloux Aet al., 2022, RpoN/Sfa2-dependent activation of the Pseudomonas aeruginosa H2-T6SS and its cognate arsenal of antibacterial toxins., Nucleic Acids Research, Vol: 50, Pages: 227-243, ISSN: 0305-1048

Pseudomonas aeruginosa uses three type six secretion systems (H1-, H2- and H3-T6SS) to manipulate its environment, subvert host cells and for microbial competition. These T6SS machines are loaded with a variety of effectors/toxins, many being associated with a specific VgrG. How P. aeruginosa transcriptionally coordinates the main T6SS clusters and the multiple vgrG islands spread through the genome is unknown. Here we show an unprecedented level of control with RsmA repressing most known T6SS-related genes. Moreover, each of the H2- and H3-T6SS clusters encodes a sigma factor activator (SFA) protein called, Sfa2 and Sfa3, respectively. SFA proteins are enhancer binding proteins necessary for the sigma factor RpoN. Using a combination of RNA-seq, ChIP-seq and molecular biology approaches, we demonstrate that RpoN coordinates the T6SSs of P. aeruginosa by activating the H2-T6SS but repressing the H1- and H3-T6SS. Furthermore, RpoN and Sfa2 control the expression of the H2-T6SS-linked VgrGs and their effector arsenal to enable very effective interbacterial killing. Sfa2 is specific as Sfa3 from the H3-T6SS cannot complement loss of Sfa2. Our study further delineates the regulatory mechanisms that modulate the deployment of an arsenal of T6SS effectors likely enabling P. aeruginosa to adapt to a range of environmental conditions.

Journal article

Larrouy-Maumus G, Katy J, katheryn H, laurent D, markus K, Filloux A, Plesiat Pet al., 2021, Detection of colistin resistance in Pseudomonas aeruginosa using the MALDIxin test on the routine MALDI Biotyper Sirius mass spectrometer, Frontiers in Microbiology, Vol: 12, ISSN: 1664-302X

Colistin is frequently a last resort treatment for Pseudomonas aeruginosa infections caused by multidrug-resistant (MDR) and extensively drug resistant (XDR) strains, and detection of colistin resistance is essential for the management of infected patients. Therefore, we evaluated the recently developed MALDIxin test for the detection of colistin resistance in Pseudomonas aeruginosa clinical strains using the routine matrix-assisted laser desorption ionization (MALDI) Biotyper Sirius system. The test is based on the detection by mass spectrometry of modified lipid A by the addition of 4-amino-L-arabinose (L-ara4N) molecules on one or two phosphate groups, in strains resistant to colistin. Overproduction of L-Ara4N molecules is mainly due to the constitutive activation of the histidine kinase (PmrB) or the response regulator (PmrA) following an amino-acid substitution in clinical strains. The performance of the test was determined on a panel of 14 colistin-susceptible and 14 colistin-resistant Pseudomonas aeruginosa clinical strains, the reference strain PAO1 and positive control mutants PmrB (V28G), PmrB (D172), PhoQ (D240-247) and ParR (M59I). In comparison with the broth microdilution (BMD) method, all the susceptible strains (n=14) and 8/14 colistin-resistant strains were detected in less than 1 hour, directly on whole bacteria. The remaining resistant strains (n=6) were all detected after a short pre-exposure (4h) to colistin before sample preparation. Validation of the method on a larger panel of strains will be the next step before its use in diagnostics laboratories. Our data showed that the MALDIxin test offers rapid and efficient detection of colistin resistant Pseudomonas aeruginosa and is thus a valuable diagnostics tool to control the spread of these emerging resistant strains.

Journal article

Nolan LM, Cain AK, Clamens T, Furniss RCD, Manoli E, Sainz-Polo MA, Dougan G, Albesa-Jove D, Parkhill J, Mavridou DAI, Filloux Aet al., 2021, Identification of tse8 as a type VI secretion system toxin from pseudomonas aeruginosa that targets the bacterial transamidosome to inhibit protein synthesis in prey cells, Nature Microbiology, Vol: 6, Pages: 1199-+, ISSN: 2058-5276

The Type VI secretion system (T6SS) is a bacterial nanomachine that delivers toxic effectors to kill competitors or subvert some of their key functions. Here, we use transposon directed insertion–site sequencing to identify T6SS toxins associated with the H1-T6SS, one of the three T6SS machines found in Pseudomonas aeruginosa. This approach identified several putative toxin–immunity pairs, including Tse8–Tsi8. Full characterization of this protein pair demonstrated that Tse8 is delivered by the VgrG1a spike complex into prey cells where it targets the transamidosome, a multiprotein complex involved in protein synthesis in bacteria that lack either one, or both, of the asparagine and glutamine transfer RNA synthases. Biochemical characterization of the interactions between Tse8 and the transamidosome components GatA, GatB and GatC suggests that the presence of Tse8 alters the fine-tuned stoichiometry of the transamidosome complex, and in vivo assays demonstrate that Tse8 limits the ability of prey cells to synthesize proteins. These data expand the range of cellular components targeted by the T6SS by identifying a T6SS toxin affecting protein synthesis and validate the use of a transposon directed insertion site sequencing–based global genomics approach to expand the repertoire of T6SS toxins in T6SS-encoding bacteria.

Journal article

Howard SA, Furniss RCD, Bonini D, Amin H, Paracuellos P, Zlotkin D, R D Costa T, Levy A, A I Mavridou D, Filloux Aet al., 2021, The breadth and molecular basis of Hcp-driven type six secretion system (T6SS) effector delivery, mBio, Vol: 12, Pages: 1-19, ISSN: 2150-7511

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 in vivo 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.

Journal article

Steinchen W, Ahmad S, Valentini M, Eilers K, Majkini M, Altegoer F, Lechner M, Filloux A, Whitney JC, Bange Get al., 2021, Dual role of a (p)ppGpp- and (p)ppApp-degrading enzyme in biofilm formation and interbacterial antagonism, Molecular Microbiology, Vol: 115, Pages: 1339-1356, ISSN: 0950-382X

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.

Journal article

Bernal P, Furniss CD, Fecht S, Leung RCY, Spiga L, Mavridou DAI, Filloux ALAINet al., 2021, A novel stabilization mechanism for the type VI secretion system sheath, Proceedings of the National Academy of Sciences of USA, Vol: 118, ISSN: 0027-8424

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 (TssAS) and a long form (TssAL). While TssAL proteins interact with a partner, called TagA, to anchor the distal end of the extended sheath, the mechanism for the stabilization of TssAS-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.

Journal article

Lossi NS, Manoli E, Foerster A, Dajani R, Pape T, Freemont P, Filloux Aet al., 2021, The HsiB1C1 (TssB-TssC) complex of the pseudomonas aeruginosa Type VI secretion system forms a bacteriophage tail sheathlike structure, Journal of Biological Chemistry, Vol: 288, Pages: 7536-7548, ISSN: 0021-9258

Protein secretion systems in Gram-negative bacteria evolved into a variety of molecular nanomachines. They are related to cell envelope complexes, which are involved in assembly of surface appendages or transport of solutes. They are classified as types, the most recent addition being the type VI secretion system (T6SS). The T6SS displays similarities to bacteriophage tail, which drives DNA injection into bacteria. The Hcp protein is related to the T4 bacteriophage tail tube protein gp19, whereas VgrG proteins structurally resemble the gp27/gp5 puncturing device of the phage. The tube and spike of the phage are pushed through the bacterial envelope upon contraction of a tail sheath composed of gp18. In Vibrio cholerae it was proposed that VipA and VipB assemble into a tail sheathlike structure. Here we confirm these previous data by showing that HsiB1 and HsiC1 of the Pseudomonas aeruginosa H1-T6SS assemble into tubules resulting from stacking of cogwheel-like structures showing predominantly 12-fold symmetry. The internal diameter of the cogwheels is ∼100 Å, which is large enough to accommodate an Hcp tube whose external diameter has been reported to be 85 Å. The N-terminal 212 residues of HsiC1 are sufficient to form a stable complex with HsiB1, but the C terminus of HsiC1 is essential for the formation of the tubelike structure. Bioinformatics analysis suggests that HsiC1 displays similarities to gp18-like proteins in its C-terminal region. In conclusion, we provide further structural and mechanistic insights into the T6SS and show that a phage sheathlike structure is likely to be a conserved element across all T6SSs.

Journal article

Brinkman FSL, Winsor GL, Done RE, Filloux A, Francis VI, Goldberg JB, Greenberg EP, Han K, Hancock REW, Haney CH, Haeussler S, Klockgether J, Lamont IL, Levesque RC, Lory S, Nikel PI, Porter SL, Scurlock MW, Schweizer HP, Tuemmler B, Wang M, Welch Met al., 2021, The Pseudomonas aeruginosa whole genome sequence: A 20th anniversary celebration, ADVANCES IN MICROBIAL PHYSIOLOGY, VOL 79, Vol: 79, Pages: 25-88, ISSN: 0065-2911

Journal article

Lin H-H, Filloux A, Lai E-M, 2020, Role of recipient susceptibility factors during contact-dependent interbacterial competition, Frontiers in Microbiology, Vol: 11, Pages: 1-15, ISSN: 1664-302X

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.

Journal article

Sarah Wettstadt S, Lai E-M, Filloux AAM, 2020, Solving the puzzle: connecting a heterologous Agrobacterium tumefaciens T6SS effector to a Pseudomonas aeruginosa spike complex, Frontiers in Cellular and Infection Microbiology, Vol: 10, ISSN: 2235-2988

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 Agrobacterium tumefaciens 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 Pseudomonas aeruginosa. For this, we designed chimeras between VgrG1 from A. tumefaciens and VgrG1a from P. aeruginosa 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.

Journal article

Larrouy-Maumus G, Dortet L, Filloux A, bonnin, le hello, bonnet, kostrzewaet al., 2020, Detection of colistin resistance in Salmonella enterica using MALDIxin test on the routine MALDI Biotyper Sirius mass spectrometer, Frontiers in Microbiology, Vol: 11, Pages: 1-6, ISSN: 1664-302X

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 mcr 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 hours) and challenging to perform in clinical and veterinatryveterinary laboratories. Here we present the use of the MALDIxin to detect colistin resistant Salmonella enterica using the MALDxin test on the routine matrix-assisted laser desorption ionization (MALDI) Biotyper Sirius system.

Journal article

Bernal P, Furniss C, Fecht S, Leung RCY, Spiga L, Mavridou DAI, Filloux Aet al., 2020, Novel structural components generate distinct type VI secretion system anchoring modes, Publisher: bioRxiv

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 (TssA S ) and a long (TssA L ) TssA. Whilst TssA L proteins interact with a partner, called TagA, to anchor the distal end of the extended sheath, the mechanism for the stabilization of TssA S -containing T6SSs remains unknown. Here we discover a novel 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 a while or fire immediately after sheath extension, thus giving rise to different aggression behaviors. We propose that this functional diversity could contribute to the specialization of the T6SS to suit bacterial lifestyles in diverse environmental niches.

Working paper

Allsopp LP, Bernal P, Nolan LM, Filloux Aet 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.

Journal article

Wettstadt S, Filloux A, 2020, Manipulating the type VI secretion system spike to shuttle passenger proteins, PLoS One, Vol: 15, Pages: 1-20, ISSN: 1932-6203

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.

Journal article

Dortet L, Broda A, bernabeu S, GLUPCZYNSKI Y, bogaerts P, bonnin R, naas T, Filloux A, Larrouy-Maumus Get al., 2020, Optimization of the MALDIxin test for the rapid identification of colistin resistance in Klebsiella pneumoniae using MALDI-TOF-MS, Journal of Antimicrobial Chemotherapy, Vol: 75, Pages: 110-116, ISSN: 0305-7453

Background. With the dissemination of carbapenemase producers, a revival of colistin was observed for the treatment of infections caused by multidrug-resistant Gram-negatives. Unfortunately, the increasing usage of colistin led to the emergence of resistance. In Klebsiella pneumoniae, colistin resistance arises through addition of L-arabinose-4N (L-Ara4N) or phosphoethanolamine (pEtN) on the native lipid A. The underlying mechanisms involve numerous chromosome-encoded genes or the plasmid-encoded phosphoethanolamine 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 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 Klebsiella pneumoniae.Methods. This optimization consists on 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 strains among which 45 correspond to chromosome-encoded resistance, 3 MCR-related resistance and one isolate harbouring 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. In 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 diagnostic of colistin resistance in clinically-relevant Gram negative bacteria.

Journal article

Furniss C, Dortet L, Bolland W, drews O, sparbier K, bonnin R, Filloux A, kostrzewa M, Mavridou D, Larrouy-Maumus Get al., 2019, Detection of colistin resistance in Escherichia coli using the MALDI Biotyper Sirius mass spectrometry system, Journal of Clinical Microbiology, Vol: 57, Pages: 1-7, ISSN: 0095-1137

Polymyxin antibiotics are a last-line treatment for multidrug-resistant Gram-negative bacteria. However, the emergence of colistin resistance, including the spread of mobile mcr 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 MALDI-TOF mass spectrometry in less than 15 minutes, 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 mass spectrometry system (Bruker Daltonics). We optimized the sample preparation protocol using a set of 6 MCR-expressing Escherichia coli clones and validated the assay with a collection of 40 E. coli clinical isolates, including 19 confirmed MCR producers, 12 colistin-resistant isolates which tested negative for commonly encountered mcr genes (i.e. likely chromosomally-resistant isolates) and 9 polymyxin-susceptible isolates. We calculated Polymyxin resistance ratio (PRR) values from the acquired spectra; a PRR value of zero, indicating polymyxin susceptibility, was obtained for all colistin-susceptible E. coli 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, provides an unbiased, fast, reliable, cost-effective and high-throughput way of detecting colistin resistance in clinical E. coli isolates.

Journal article

Smith WD, Cameron SJ, Fletcher OL, Bardin E, Takats Z, Hogg C, Filloux A, Bush A, Davies JCet al., 2019, PSEUDOMONAS AERUGINOSA METABOLOME DIFFERENCES BETWEEN CF AND NON-CF BRONCHIECTASIS DETECTED USING DIRECT-FROM-SAMPLE MASS SPECTROMETRY, Pediatric Pulmonology, Publisher: WILEY, Pages: S313-S313, ISSN: 8755-6863

Conference paper

Cain AK, Nolan LM, Sullivan GJ, Whitchurch CB, Filloux A, Parkhill Jet al., 2019, Complete genome sequence of pseudomonas aeruginosa reference strain PAK, Microbiology Resource Announcements, Vol: 8, ISSN: 2576-098X

We report the complete genome of Pseudomonas aeruginosa strain PAK, a strain which has been instrumental in the study of a range of P. aeruginosa virulence and pathogenesis factors and has been used for over 50 years as a laboratory reference strain.

Journal article

Wood TE, Howard SA, Forster A, Nolan LM, Manoli E, Bullen NP, Yau HCL, Hachani A, Hayward RD, Whitney JC, Vollmer W, Freemont PS, Filloux Aet al., 2019, The Pseudomonas aeruginosa T6SS delivers a periplasmic toxin that disrupts bacterial cell morphology, Cell Reports, Vol: 29, Pages: 187-201.e7, ISSN: 2211-1247

The type VI secretion system (T6SS) is crucialin interbacterial competition and is avirulence determinant ofmany Gram-negative bacteria. Several T6SS effectorsarecovalently fused to secreted T6SS structural components such asthe VgrG spike for delivery into target cells.In Pseudomonas aeruginosa, theVgrG2b effector waspreviously proposedto mediatebacterial internalisation into eukaryotic cells. In this work, wefind that the VgrG2b C-terminal domain(VgrG2bC-ter) elicits toxicity in the bacterial periplasm, counteracted by a cognate immunity protein.We resolve thestructure of VgrG2bC-ter and confirm it is a member ofthezinc-metallopeptidasefamily of enzymes. We show that this effector causesmembrane blebbing atmidcell, whichsuggests a distincttype of T6SS-mediated growthinhibition through interference with cell division, mimicking the impact of β-lactam antibiotics. Ourstudyintroduces a further effector family to the T6SS arsenaland demonstrates that VgrG2b can target both prokaryotic and eukaryotic cells.

Journal article

Howard SA, Filloux A, 2019, Bacterial Protein Secretion: Looking inside an injection system, eLife, Vol: 8, Pages: 1-3, ISSN: 2050-084X

The proteins injected by bacteria into eukaryotic organisms can lead to fates as diverse as death and metamorphosis

Journal article

Valentini M, Filloux A, 2019, Multiple Roles of c-di-GMP Signaling in Bacterial Pathogenesis, Annual Review of Microbiology, Vol: 73, Pages: 387-406, ISSN: 0066-4227

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.

Journal article

Fuqua C, Filloux A, Ghigo J-M, Visick KLet al., 2019, Biofilms 2018: a diversity of microbes and mechanisms, Journal of Bacteriology, Vol: 201, Pages: e00118-e00119, ISSN: 0021-9193

The 8th 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.

Journal article

Wood TE, Howard SA, Wettstadt S, Filloux Aet al., 2019, PAAR proteins act as the ‘sorting hat’ of the type VI secretion system, Microbiology, Vol: 165, Pages: 1203-1218, ISSN: 1350-0872

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. Pseudomonas aeruginosa encodes several PAAR proteins, whose roles have been poorly investigated. Here we describe a phospholipase family antibacterial effector immunity pair from Pseudomonas aeruginosa 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.

Journal article

McCarthy RR, Yu M, Eilers K, Wang Y-C, Lai E-M, Filloux Aet al., 2019, Cyclic di-GMP inactivates T6SS and T4SS activity in Agrobacterium tumefaciens, Molecular Microbiology, Vol: 112, Pages: 632-648, ISSN: 0950-382X

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.

Journal article

Wettstadt S, Wood TE, Fecht S, Filloux Aet al., 2019, Delivery of the Pseudomonas aeruginosa phospholipase effectors PldA and PldB in a VgrG- and H2-T6SS-dependent manner, Frontiers in Microbiology, Vol: 10, Pages: 1-18, ISSN: 1664-302X

The bacterial pathogen Pseudomonas aeruginosa 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 P. aeruginosa 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 vgrG 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 via 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.

Journal article

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