Publications
236 results found
McCarthy RR, Valentini M, Filloux A, 2017, Contribution of Cyclic di-GMP in the Control of Type III and Type VI Secretion in Pseudomonas aeruginosa., Methods Mol Biol, Vol: 1657, Pages: 213-224
Bacteria produce toxins to enhance their competitiveness in the colonization of an environment as well as during an infection. The delivery of toxins into target cells is mediated by several types of secretion systems, among them our focus is Type III and Type VI Secretion Systems (T3SS and T6SS, respectively). A thorough methodology is provided detailing how to identify if cyclic di-GMP signaling plays a role in the P. aeruginosa toxin delivery mediated by T3SS or T6SS. This includes in vitro preparation of the samples for Western blot analysis aiming at detecting possible c-di-GMP-dependent T3SS/T6SS switch, as well as in vivo analysis using the model organism Galleria mellonella to demonstrate the ecological and pathogenic consequence of the switch between these two secretion systems.
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.
Heeb S, Camara M, Filloux A, et al., 2017, Professor Dieter Haas (1945-2017), FEMS Microbiology Reviews, Vol: 41, Pages: 597-598, ISSN: 0168-6445
Jakobsen TH, Warming AN, Vejborg RM, et al., 2017, A broad range quorum sensing inhibitor working through sRNA inhibition, Scientific Reports, Vol: 7, ISSN: 2045-2322
For the last decade, chemical control of bacterial virulence has received considerable attention. Ajoene, a sulfur-rich molecule from garlic has been shown to reduce expression of key quorum sensing regulated virulence factors in the opportunistic pathogen Pseudomonas aeruginosa. Here we show that the repressing effect of ajoene on quorum sensing occurs by inhibition of small regulatory RNAs (sRNA) in P. aeruginosa as well as in Staphylococcus aureus, another important human pathogen that employs quorum sensing to control virulence gene expression. Using various reporter constructs, we found that ajoene lowered expression of the sRNAs RsmY and RsmZ in P. aeruginosa and the small dual-function regulatory RNA, RNAIII in S. aureus, that controls expression of key virulence factors. We confirmed the modulation of RNAIII by RNA sequencing and found that the expression of many QS regulated genes encoding virulence factors such as hemolysins and proteases were lowered in the presence of ajoene in S. aureus. Importantly, our findings show that sRNAs across bacterial species potentially may qualify as targets of anti-virulence therapy and that ajoene could be a lead structure in search of broad-spectrum compounds transcending the Gram negative-positive borderline.
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.
Smith WD, Bardin E, Cameron L, et al., 2017, Current and future therapies for Pseudomonas aeruginosa infection in patients with cystic fibrosis, FEMS Microbiology Letters, Vol: 364, ISSN: 0378-1097
Pseudomonas aeruginosa opportunistically infects the airways of patients with cystic fibrosis and causes significant morbidity and mortality. Initial infection can often be eradicated though requires prompt detection and adequate treatment. Intermittent and then chronic infection occurs in the majority of patients. Better detection of P. aeruginosa infection using biomarkers may enable more successful eradication before chronic infection is established. In chronic infection P. aeruginosa adapts to avoid immune clearance and resist antibiotics via efflux pumps, β-lactamase expression, reduced porins and switching to a biofilm lifestyle. The optimal treatment strategies for P. aeruginosa infection are still being established, and new antibiotic formulations such as liposomal amikacin, fosfomycin in combination with tobramycin and inhaled levofloxacin are being explored. Novel agents such as the alginate oligosaccharide OligoG, cysteamine, bacteriophage, nitric oxide, garlic oil and gallium may be useful as anti-pseudomonal strategies, and immunotherapy to prevent infection may have a role in the future. New treatments that target the primary defect in cystic fibrosis, recently licensed for use, have been associated with a fall in P. aeruginosa infection prevalence. Understanding the mechanisms for this could add further strategies for treating P. aeruginosa in future.
Imbert PRC, Louche A, Luizet J-B, et al., 2017, A Pseudomonas aeruginosa TIR effector mediates immune evasion by targeting UBAP1 and TLR adaptors, EMBO JOURNAL, Vol: 36, Pages: 1869-1887, ISSN: 0261-4189
McCarthy RR, Mazon-Moya MJ, Moscoso JA, et al., 2017, Cyclic-di-GMP regulates lipopolysaccharide modification and contributes to Pseudomonas aeruginosa immune evasion, Nature Microbiology, Vol: 2, Pages: 1-10, ISSN: 2058-5276
Pseudomonas aeruginosa is a Gram-negative bacterial pathogen associated with acute and chronic infections. The universal cyclic-di-GMP second messenger is instrumental in the switch from a motile lifestyle to resilient biofilm as in the cystic fibrosis lung. The SadC diguanylate cyclase is associated with this patho-adaptive transition. Here, we identify an unrecognized SadC partner, WarA, which we show is a methyltransferase in complex with a putative kinase, WarB. We established that WarA binds to cyclic-di-GMP, which potentiates its methyltransferase activity. Together, WarA and WarB have structural similarities with the bifunctional Escherichia coli lipopolysaccharide (LPS) O antigen regulator WbdD. Strikingly, WarA influences P. aeruginosa O antigen modal distribution and interacts with the LPS biogenesis machinery. LPS is known to modulate the immune response in the host, and by using a zebrafish infection model, we implicate WarA in the ability of P. aeruginosa to evade detection by the host.
Bosi E, Fondi M, Orlandini V, et al., 2017, The pangenome of (Antarctic) Pseudoalteromonas bacteria: evolutionary and functional insights, BMC Genomics, Vol: 18, ISSN: 1471-2164
Background:Pseudoalteromonas is a genus of ubiquitous marine bacteria used as model organisms to study the biological mechanisms involved in the adaptation to cold conditions. A remarkable feature shared by these bacteria is their ability to produce secondary metabolites with a strong antimicrobial and antitumor activity. Despite their biotechnological relevance, representatives of this genus are still lacking (with few exceptions) an extensive genomic characterization, including features involved in the evolution of secondary metabolites production. Indeed, biotechnological applications would greatly benefit from such analysis.Results:Here, we analyzed the genomes of 38 strains belonging to different Pseudoalteromonas species and isolated from diverse ecological niches, including extreme ones (i.e. Antarctica). These sequences were used to reconstruct the largest Pseudoalteromonas pangenome computed so far, including also the two main groups of Pseudoalteromonas strains (pigmented and not pigmented strains). The downstream analyses were conducted to describe the genomic diversity, both at genus and group levels. This allowed highlighting a remarkable genomic heterogeneity, even for closely related strains. We drafted all the main evolutionary steps that led to the current structure and gene content of Pseudoalteromonas representatives. These, most likely, included an extensive genome reduction and a strong contribution of Horizontal Gene Transfer (HGT), which affected biotechnologically relevant gene sets and occurred in a strain-specific fashion. Furthermore, this study also identified the genomic determinants related to some of the most interesting features of the Pseudoalteromonas representatives, such as the production of secondary metabolites, the adaptation to cold temperatures and the resistance to abiotic compounds.Conclusions:This study poses the bases for a comprehensive understanding of the evolutionary trajectories followed in time by this peculiar bact
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.
Valentini M, Laventie BJ, Moscoso J, et al., 2016, The Diguanylate Cyclase HsbD Intersects with the HptB Regulatory Cascade to Control Pseudomonas aeruginosa Biofilm and Motility., PLOS Genetics, Vol: 12, ISSN: 1553-7390
The molecular basis of second messenger signaling relies on an array of proteins that synthesize, degrade or bind the molecule to produce coherent functional outputs. Cyclic di-GMP (c-di-GMP) has emerged as a eubacterial nucleotide second messenger regulating a plethora of key behaviors, like the transition from planktonic cells to biofilm communities. The striking multiplicity of c-di-GMP control modules and regulated cellular functions raised the question of signaling specificity. Are c-di-GMP signaling routes exclusively dependent on a central hub or can they be locally administrated? In this study, we show an example of how c-di-GMP signaling gains output specificity in Pseudomonas aeruginosa. We observed the occurrence in P. aeruginosa of a c-di-GMP synthase gene, hsbD, in the proximity of the hptB and flagellar genes cluster. We show that the HptB pathway controls biofilm formation and motility by involving both HsbD and the anti-anti-sigma factor HsbA. The rewiring of c-di-GMP signaling into the HptB cascade relies on the original interaction between HsbD and HsbA and on the control of HsbD dynamic localization at the cell poles.
Valentini M, Laventie BJ, Moscoso JA, et al., 2016, Correction: The Diguanylate Cyclase HsbD Intersects with the HptB Regulatory Cascade to Control Pseudomonas aeruginosa Biofilm and Motility., PLOS Genetics, Vol: 12, ISSN: 1553-7390
Rasheed M, Garnett J, Perez-Dorado I, et al., 2016, Crystal structure of the CupB6 adhesive tip from the chaperone-usher family of pili from Pseudomonas aeruginosa, Biochimica et Biophysica Acta - Protein Structure, Vol: 1864, Pages: 1500-1505, ISSN: 0005-2795
Pseudomonas aeruginosa is a Gram-negative opportunistic bacterial pathogen that can cause chronicinfection of the lungs of cystic fibrosis patients. Chaperone-usher systems in P. aeruginosa are knownto translocate and assemble adhesive pili on the bacterial surface and contribute to biofilm formationwithin the host. Here, we report the crystal structure of the tip adhesion subunit CupB6 from thecupB1-6 gene cluster. The tip domain is connected to the pilus via the N-terminal donor strand fromthe main pilus subunit CupB1. Although the CupB6 adhesion domain bears structural features similarto other CU adhesins it displays an unusual polyproline helix adjacent to a prominent surface pocket,which are likely the site for receptor recognition.
Filloux A, Freemont P, 2016, Structural biology: baseplates in contractile machines, Nature Microbiology, Vol: 1, ISSN: 2058-5276
Planamente S, Salih O, Manoli E, et al., 2016, TssA forms a gp6-like ring attached to the type VI secretion sheath, EMBO Journal, Vol: 35, Pages: 1613-1627, ISSN: 0261-4189
The type VI secretion system (T6SS) is a supra-molecular bacterial complex that resembles phage tails. It is a killing machine which fires toxins into target cells upon contraction of its TssBC sheath. Here, we show that TssA1 is a T6SS component forming dodecameric ring structures whose dimensions match those of the TssBC sheath and which can accommodate the inner Hcp tube. The TssA1 ring complex binds the T6SS sheath and impacts its behaviour in vivo. In the phage, the first disc of the gp18 sheath sits on a baseplate wherein gp6 is a dodecameric ring. We found remarkable sequence and structural similarities between TssA1 and gp6 C-termini, and propose that TssA1 could be a baseplate component of the T6SS. Furthermore, we identified similarities between TssK1 and gp8, the former interacting with TssA1 while the latter is found in the outer radius of the gp6 ring. These observations, combined with similarities between TssF and gp6N-terminus or TssG and gp53, lead us to propose a comparative model between the phage baseplate and the T6SS.
Holmes AH, Gill SK, Hui K, et al., 2016, Increased airway glucose increases airway bacterial load in hyperglycaemia, Scientific Reports, Vol: 6, ISSN: 2045-2322
Diabetes is associated with increased frequency of hospitalization due to bacterial lung infection.We hypothesize that increased airway glucose caused by hyperglycaemia leads to increasedbacterial loads. In critical care patients, we observed that respiratory tract bacterial colonisationis significantly more likely when blood glucose is high. We engineered mutants in genesaffecting glucose uptake and metabolism (oprB, gltK, gtrS and glk) in Pseudomonas aeruginosa,strain PAO1. These mutants displayed attenuated growth in minimal medium supplemented withglucose as the sole carbon source. The effect of glucose on growth in vivo was tested usingstreptozocin-induced, hyperglycaemic mice, which have significantly greater airway glucose.Bacterial burden in hyperglycaemic animals was greater than control animals when infected withwild type but not mutant PAO1. Metformin pre-treatment of hyperglycaemic animals reducedboth airway glucose and bacterial load. These data support airway glucose as a criticaldeterminant of increased bacterial load during diabetes.
Lund-Palau H, Turnbull AR, Bush A, et al., 2016, Pseudomonas aeruginosa infection in cystic fibrosis: pathophysiological mechanisms and therapeutic approaches, Expert Review of Respiratory Medicine, Vol: 10, Pages: 685-697, ISSN: 1747-6348
Pseudomonas aeruginosa is a remarkably versatile environmental bacterium with an extraordinary capacity to infect the cystic fibrosis (CF) lung. Infection with P. aeruginosa occurs early, and although eradication can be achieved following early detection, chronic infection occurs in over 60% of adults with CF. Chronic infection is associated with accelerated disease progression and increased mortality. Extensive research has revealed complex mechanisms by which P. aeruginosa adapts to and persists within the CF airway. Yet knowledge gaps remain, and prevention and treatment strategies are limited by the lack of sensitive detection methods and by a narrow armoury of antibiotics. Further developments in this field are urgently needed in order to improve morbidity and mortality in people with CF. Here, we summarize current knowledge of pathophysiological mechanisms underlying P. aeruginosa infection in CF. Established treatments are discussed, and an overview is offered of novel detection methods and therapeutic strategies in development.
Chambonnier G, Roux L, Redelberger D, et al., 2016, The Hybrid Histidine Kinase LadS Forms a Multicomponent Signal Transduction System with the GacS/GacA Two-Component System in Pseudomonas aeruginosa., PLOS Genetics, Vol: 12, ISSN: 1553-7390
In response to environmental changes, Pseudomonas aeruginosa is able to switch from a planktonic (free swimming) to a sessile (biofilm) lifestyle. The two-component system (TCS) GacS/GacA activates the production of two small non-coding RNAs, RsmY and RsmZ, but four histidine kinases (HKs), RetS, GacS, LadS and PA1611, are instrumental in this process. RetS hybrid HK blocks GacS unorthodox HK autophosphorylation through the formation of a heterodimer. PA1611 hybrid HK, which is structurally related to GacS, interacts with RetS in P. aeruginosa in a very similar manner to GacS. LadS hybrid HK phenotypically antagonizes the function of RetS by a mechanism that has never been investigated. The four sensors are found in most Pseudomonas species but their characteristics and mode of signaling may differ from one species to another. Here, we demonstrated in P. aeruginosa that LadS controls both rsmY and rsmZ gene expression and that this regulation occurs through the GacS/GacA TCS. We additionally evidenced that in contrast to RetS, LadS signals through GacS/GacA without forming heterodimers, either with GacS or with RetS. Instead, we demonstrated that LadS is involved in a genuine phosphorelay, which requires both transmitter and receiver LadS domains. LadS signaling ultimately requires the alternative histidine-phosphotransfer domain of GacS, which is here used as an Hpt relay by the hybrid kinase. LadS HK thus forms, with the GacS/GacA TCS, a multicomponent signal transduction system with an original phosphorelay cascade, i.e. H1LadS→D1LadS→H2GacS→D2GacA. This highlights an original strategy in which a unique output, i.e. the modulation of sRNA levels, is controlled by a complex multi-sensing network to fine-tune an adapted biofilm and virulence response.
Valentini M, Filloux A, 2016, Biofilms and cyclic di-GMP (c-di-GMP) signaling: lessons from Pseudomonas aeruginosa and other bacteria, Journal of Biological Chemistry, Vol: 291, Pages: 12547-12555, ISSN: 1083-351X
The cyclic-di-GMP (c-di-GMP) second messenger represents a signaling system that regulates many bacterial behaviors and is of key importance for driving the lifestyle switch between motile loner cells and biofilm formers. This review provides an up-to-date compendium of c-di-GMP pathways connected to biofilm formation, biofilm-associated motilities and other functionalities in the ubiquitous and opportunistic human pathogen Pseudomonas aeruginosa. This bacterium is frequently adopted as model organism to study bacterial biofilm formation. Importantly, its versatility and adaptation capabilities are linked with a broad range of complex regulatory networks, including a large set of genes involved in c-di-GMP biosynthesis, degradation and transmission.
Filloux A, Whitfield C, 2016, Editorial: The many wonders of the bacterial cell surface, FEMS MICROBIOLOGY REVIEWS, Vol: 40, Pages: 161-163, ISSN: 0168-6445
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filloux A, wood TE, Hachani A, 2015, Type VI secretion and anti-host effectors., Current Opinion in Microbiology, ISSN: 1879-0364
Larrouy-Maumus GJ, Abigail Clements, Alain Filloux, et al., 2015, Direct detection of lipid A on intact Gram-negative bacteria byMALDI-TOF mass spectrometry, Journal of Microbiological Methods, Vol: 120, Pages: 68-71, ISSN: 1872-8359
The purification and characterization of Gram-negative bacterial lipid A is tedious and time-consuming. Herein we report a rapid and sensitive method to identify lipid A directly on intact bacteria without any chemical treatment or purification, using an atypical solvent system to solubilize the matrix combined with MALDI-TOF mass spectrometry.
Jennings LK, Storek KM, Ledvina HE, et al., 2015, Pel is a cationic exopolysaccharide that cross-links extracellular DNA in the Pseudomonas aeruginosa biofilm matrix, Proceedings of the National Academy of Sciences of the United States of America, Vol: 112, Pages: 11353-11358, ISSN: 1091-6490
Biofilm formation is a complex, ordered process. In the opportunistic pathogen Pseudomonas aeruginosa, Psl and Pel exopolysaccharides and extracellular DNA (eDNA) serve as structural components of the biofilm matrix. Despite intensive study, Pel’s chemical structure and spatial localization within mature biofilms remain unknown. Using specialized carbohydrate chemical analyses, we unexpectedly found that Pel is a positively charged exopolysaccharide composed of partially acetylated 1→4 glycosidic linkages of N-acetylgalactosamine and N-acetylglucosamine. Guided by the knowledge of Pel’s sugar composition, we developed a tool for the direct visualization of Pel in biofilms by combining Pel-specific Wisteria floribunda lectin staining with confocal microscopy. The results indicate that Pel cross-links eDNA in the biofilm stalk via ionic interactions. Our data demonstrate that the cationic charge of Pel is distinct from that of other known P. aeruginosa exopolysaccharides and is instrumental in its ability to interact with other key biofilm matrix components.
Bosi E, Fondi M, Maida I, et al., 2015, Genome-scale phylogenetic and DNA composition analyses of Antarctic Pseudoalteromonas bacteria reveal inconsistencies in current taxonomic affiliation, Hydrobiologia, Vol: 761, Pages: 85-95, ISSN: 0018-8158
Bacteria belonging to the Pseudoalteromonas genus have important ecological implications in marine environments, playing a role in the control of microbial community as producers of bioactive molecules endowed with antifouling activity and able to antagonize larvae, fungi and bacteria, including important human pathogens. For these reasons, representatives of this genus are very promising for biotechnological and biomedical applications. In this work, we used different genome-scale approaches to infer the taxonomy of 38 Pseudoalteromonas representatives (most of which isolated from Antarctica) and whose complete genome has been sequenced. We show that an accurate re-evaluation of the real taxonomic relationships of Pseudoalteromonas representatives is needed since many inconsistencies with the current taxonomic annotation were observed. Moreover, data obtained with different genome-scale methods are consistent, confirming the reliability of the genomic approaches. On the basis of these data, we propose a re-annotation for some Pseudoalteromonas species. This proposal should be validated in the future by comparing the phenotypes of these strains.
Bouffartigues E, Moscoso JA, Duchesne R, et al., 2015, The absence of the Pseudomonas aeruginosa OprF protein leads to increased biofilm formation through variation in c-di-GMP level, Frontiers in Microbiology, Vol: 6, ISSN: 1664-302X
OprF is the major outer membrane porin in bacteria belonging to the Pseudomonas genus. In previous studies, we have shown that OprF is required for full virulence expression of the opportunistic pathogen Pseudomonas aeruginosa. Here, we describe molecular insights on the nature of this relationship and report that the absence of OprF leads to increased biofilm formation and production of the Pel exopolysaccharide. Accordingly, the level of c-di-GMP, a key second messenger in biofilm control, is elevated in an oprF mutant. By decreasing c-di-GMP levels in this mutant, both biofilm formation and pel gene expression phenotypes were restored to wild-type levels. We further investigated the impact on two small RNAs, which are associated with the biofilm lifestyle, and found that expression of rsmZ but not of rsmY was increased in the oprF mutant and this occurs in a c-di-GMP-dependent manner. Finally, the extracytoplasmic function (ECF) sigma factors AlgU and SigX displayed higher activity levels in the oprF mutant. Two genes of the SigX regulon involved in c-di-GMP metabolism, PA1181 and adcA (PA4843), were up-regulated in the oprF mutant, partly explaining the increased c-di-GMP level. We hypothesized that the absence of OprF leads to a cell envelope stress that activates SigX and results in a c-di-GMP elevated level due to higher expression of adcA and PA1181. The c-di-GMP level can in turn stimulate Pel synthesis via increased rsmZ sRNA levels and pel mRNA, thus affecting Pel-dependent phenotypes such as cell aggregation and biofilm formation. This work highlights the connection between OprF and c-di-GMP regulatory networks, likely via SigX (ECF), on the regulation of biofilm phenotypes.
Bernal P, Civantos C, Filloux A, et al., 2015, Type VI secretion in the plant growth promoting rhizobacteria Pseudomonas putida, FEMS Microbiology Congress 2015
BackgroundBacterial type VI secretion systems (T6SSs) are recently discovered nanomachines used to inject effectors into prokaryotic or eukaryotic cells. Therefore, T6SSs are involved in both inter-bacterial competition and bacterial pathogenesis.ObjectivesThe aim is the study of the T6SS of Pseudomonas putida a soil bacterium with the capacity to colonise the root of crop plants. The colonisation by this bacterium provides growth advantages to the plant and, importantly, protection against plant pathogens. This makes P. putida a relevant biocontrol agent. Since T6SS is mainly used by environmental bacteria for interbacterial competition, one might speculate that T6SSs play a relevant role in the biocontrol properties of P. putida. Methods• in silico analysis of P. putida KT2440 genome • Competition assays to determine H1-T6SS activity and for the identification of H1-T6SS targets.• Regulatory studies: qRT-PCR, transcriptional fusionsConclusionsThe in silico analysis has revealed the existence of three putative T6SSs (H1, H2, and H3). The clusters contain the genes encoding the conserved core components and some accessories, including regulatory proteins and toxins-immunity pairs. Additional T6SS-related genes are found scattered on the chromosome.By competition assays we have determined that H1-T6SS is active and that mutants in H1-T6SS structural components lack the ability to kill model prey strains. Moreover, the system can be used to kill serious phytopathogens such as Pseudomonas syringae in in vitro assays. Interestingly, the H1-T6SS is induced in stationary phase and controlled by the global regulators RetS and GacS-GacA, and by two alternative sigma factors, RpoS and RpoN.
Sana TG, Baumann C, Merdes A, et al., 2015, Internalization of Pseudomonas aeruginosa Strain PAO1 into Epithelial Cells Is Promoted by Interaction of a T6SS Effector with the Microtubule Network., mBio, Vol: 6, ISSN: 2150-7511
Invasion of nonphagocytic cells through rearrangement of the actin cytoskeleton is a common immune evasion mechanism used by most intracellular bacteria. However, some pathogens modulate host microtubules as well by a still poorly understood mechanism. In this study, we aim at deciphering the mechanisms by which the opportunistic bacterial pathogen Pseudomonas aeruginosa invades nonphagocytic cells, although it is considered mainly an extracellular bacterium. Using confocal microscopy and immunofluorescence, we show that the evolved VgrG2b effector of P. aeruginosa strain PAO1 is delivered into epithelial cells by a type VI secretion system, called H2-T6SS, involving the VgrG2a component. An in vivo interactome of VgrG2b in host cells allows the identification of microtubule components, including the γ-tubulin ring complex (γTuRC), a multiprotein complex catalyzing microtubule nucleation, as the major host target of VgrG2b. This interaction promotes a microtubule-dependent internalization of the bacterium since colchicine and nocodazole, two microtubule-destabilizing drugs, prevent VgrG2b-mediated P. aeruginosa entry even if the invasion still requires actin. We further validate our findings by demonstrating that the type VI injection step can be bypassed by ectopic production of VgrG2b inside target cells prior to infection. Moreover, such uncoupling between VgrG2b injection and bacterial internalization also reveals that they constitute two independent steps. With VgrG2b, we provide the first example of a bacterial protein interacting with the γTuRC. Our study offers key insight into the mechanism of self-promoting invasion of P. aeruginosa into human cells via a directed and specific effector-host protein interaction. IMPORTANCE: Innate immunity and specifically professional phagocytic cells are key determinants in the ability of the host to control P. aeruginosa infection. However, among various virulence strategies, includi
Aragon IM, Pérez-Mendoza D, Moscoso JA, et al., 2015, Diguanylate cyclase DgcP is involved in plant and human Pseudomonas spp. infections, Environmental Microbiology, Vol: 17, Pages: 4332-4351, ISSN: 1462-2920
The second messenger cyclic di-GMP (c-di-GMP) controls the transition between different lifestyles in bacterial pathogens. Here, we report the identification of DgcP (diguanylate cyclase conserved in Pseudomonads), whose activity in the olive tree pathogen Pseudomonas savastanoi pv. savastanoi is dependent on the integrity of its GGDEF domain. Furthermore, deletion of the dgcP gene revealed that DgcP negatively regulates motility and positively controls biofilm formation in both the olive tree pathogen P. savastanoi pv. savastanoi and the human opportunistic pathogen Pseudomonas aeruginosa. Overexpression of the dgcP gene in P. aeruginosa PAK led to increased exopolysaccharide production and upregulation of the type VI secretion system; in turn, it repressed the type III secretion system, which is a hallmark of chronic infections and persistence for P. aeruginosa. Deletion of the dgcP gene in P. savastanoi pv. savastanoi NCPPB 3335 and P. aeruginosa PAK reduced their virulence in olive plants and in a mouse acute lung injury model respectively. Our results show that diguanylate cyclase DgcP is a conserved Pseudomonas protein with a role in virulence, and confirm the existence of common c-di-GMP signalling pathways that are capable of regulating plant and human Pseudomonas spp. infections.
Garnett JA, Muhl D, Douse CH, et al., 2015, Structure-function analysis reveals that the Pseudomonas aeruginosa Tps4 two-partner secretion system is involved in CupB5 translocation, Protein Science, Vol: 24, Pages: 670-687, ISSN: 1469-896X
Pseudomonas aeruginosa is a Gram-negative opportunistic bacterium, synonymous withcystic fibrosis patients, which can cause chronic infection of the lungs. This pathogen is a modelorganism to study biofilms: a bacterial population embedded in an extracellular matrix that provideprotection from environmental pressures and lead to persistence. A number of Chaperone-UsherPathways, namely CupA-CupE, play key roles in these processes by assembling adhesive pili onthe bacterial surface. One of these, encoded by the cupB operon, is unique as it contains anonchaperone-usher gene product, CupB5. Two-partner secretion (TPS) systems are comprised ofa C-terminal integral membrane b-barrel pore with tandem N-terminal POTRA (POlypeptide TRansportAssociated) domains located in the periplasm (TpsB) and a secreted substrate (TpsA). UsingNMR we show that TpsB4 (LepB) interacts with CupB5 and its predicted cognate partner TpsA4(LepA), an extracellular protease. Moreover, using cellular studies we confirm that TpsB4 cantranslocate CupB5 across the P. aeruginosa outer membrane, which contrasts a previous observationthat suggested the CupB3 P-usher secretes CupB5. In support of our findings we also demonstratethat tps4/cupB operons are coregulated by the RocS1 sensor suggesting P. aeruginosa hasdeveloped synergy between these systems. Furthermore, we have determined the solutionstructureof the TpsB4-POTRA1 domain and together with restraints from NMR chemical shift mappingand in vivo mutational analysis we have calculated models for the entire TpsB4 periplasmic region in complex with both TpsA4 and CupB5 secretion motifs. The data highlight specific residuesfor TpsA4/CupB5 recognition by TpsB4 in the periplasm and suggest distinct roles for eachPOTRA domain.
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