Publications
236 results found
Mikkelsen H, McMullan R, Filloux A, 2011, The pseudomonas aeruginosa reference strain PA14 displays increased virulence due to a mutation in ladS, PLoS ONE, Vol: 6, ISSN: 1932-6203
Pseudomonas aeruginosa is a pathogen that causes acute and chronic infections in a variety of hosts. The pathogenic potential of P. aeruginosa is strain-dependent. PA14 is a highly virulent strain that causes disease in a wide range of organisms, whereas PAO1 is moderately virulent. Although PA14 carries pathogenicity islands that are absent in PAO1, the presence or absence of specific gene clusters is not predictive of virulence. Here, we show that the virulent strain PA14 has an acquired mutation in the ladS gene. This mutation has a deleterious impact on biofilm, while it results in elevated type III secretion system (T3SS) activity and increased cytotoxicity towards mammalian cells. These phenotypes can be reverted by repairing the ladS mutation on the PA14 genome. The RetS/LadS/GacS signaling cascade is associated with virulence and the switch between acute and chronic infections. RetS is a sensor that down-regulates biofilm formation and up-regulates the T3SS. Mutations in retS are acquired in strains isolated from chronically infected cystic fibrosis patients and lead to hyperbiofilm formation and reduced cytotoxicity. Conversely, the LadS sensor promotes biofilm formation and represses the T3SS. We conclude that the ladS mutation is partly responsible for the high cytotoxicity of PA14, and our findings corroborate the central role of RetS and LadS in the switch between acute and chronic infections. Given the extensive use of the reference strain PA14 in infection and virulence models, the bias caused by the ladS mutation on the observed phenotypes will be crucial to consider in future research.
Moscoso JA, Mikkelsen H, Heeb S, et al., 2011, The <i>Pseudomonas aeruginosa</i> sensor RetS switches Type III and Type VI secretion via c-di-GMP signalling, ENVIRONMENTAL MICROBIOLOGY, Vol: 13, Pages: 3128-3138, ISSN: 1462-2912
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- Citations: 193
Lossi NS, Dajani R, Freemont P, et al., 2011, Structure-function analysis of HsiF, a gp25-like component of the type VI secretion system, in <i>Pseudomonas aeruginosa</i>, MICROBIOLOGY-SGM, Vol: 157, Pages: 3292-3305, ISSN: 1350-0872
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- Citations: 40
De Luca G, Barakat M, Ortet P, et al., 2011, The cyst-dividing bacterium ramlibacter tataouinensis TTB310 genome reveals a well-stocked toolbox for adaptation to a desert environment, PLoS ONE, Vol: 6, ISSN: 1932-6203
Ramlibacter tataouinensis TTB310T (strain TTB310), a betaproteobacterium isolated from a semi-arid region of South Tunisia (Tataouine), is characterized by the presence of both spherical and rod-shaped cells in pure culture. Cell division of strain TTB310 occurs by the binary fission of spherical “cyst-like” cells (“cyst-cyst” division). The rod-shaped cells formed at the periphery of a colony (consisting mainly of cysts) are highly motile and colonize a new environment, where they form a new colony by reversion to cyst-like cells. This unique cell cycle of strain TTB310, with desiccation tolerant cyst-like cells capable of division and desiccation sensitive motile rods capable of dissemination, appears to be a novel adaptation for life in a hot and dry desert environment. In order to gain insights into strain TTB310's underlying genetic repertoire and possible mechanisms responsible for its unusual lifestyle, the genome of strain TTB310 was completely sequenced and subsequently annotated. The complete genome consists of a single circular chromosome of 4,070,194 bp with an average G+C content of 70.0%, the highest among the Betaproteobacteria sequenced to date, with total of 3,899 predicted coding sequences covering 92% of the genome. We found that strain TTB310 has developed a highly complex network of two-component systems, which may utilize responses to light and perhaps a rudimentary circadian hourglass to anticipate water availability at the dew time in the middle/end of the desert winter nights and thus direct the growth window to cyclic water availability times. Other interesting features of the strain TTB310 genome that appear to be important for desiccation tolerance, including intermediary metabolism compounds such as trehalose or polyhydroxyalkanoate, and signal transduction pathways, are presented and discussed.
Filloux A, 2011, Protein secretion systems in Pseudomonas aeruginosa: an essay on diversity, evolution, and function, Frontiers in Microbiology, Vol: 2, ISSN: 1664-302X
Protein secretion systems are molecular nanomachines used by Gram-negative bacteria to thrive within their environment. They are used to release enzymes that hydrolyze complex carbon sources into usable compounds, or to release proteins that capture essential ions such as iron. They are also used to colonize and survive within eukaryotic hosts, causing acute or chronic infections, subverting the host cell response and escaping the immune system. In this article, the opportunistic human pathogen Pseudomonas aeruginosa is used as a model to review the diversity of secretion systems that bacteria have evolved to achieve these goals. This diversity may result from a progressive transformation of cell envelope complexes that initially may not have been dedicated to secretion. The striking similarities between secretion systems and type IV pili, flagella, bacteriophage tail, or efflux pumps is a nice illustration of this evolution. Differences are also needed since various secretion configurations call for diversity. For example, some proteins are released in the extracellular medium while others are directly injected into the cytosol of eukaryotic cells. Some proteins are folded before being released and transit into the periplasm. Other proteins cross the whole cell envelope at once in an unfolded state. However, the secretion system requires conserved basic elements or features. For example, there is a need for an energy source or for an outer membrane channel. The structure of this review is thus quite unconventional. Instead of listing secretion types one after each other, it presents a melting pot of concepts indicating that secretion types are in constant evolution and use basic principles. In other words, emergence of new secretion systems could be predicted the way Mendeleïev had anticipated characteristics of yet unknown elements.
Durand E, Alphonse S, Brochier-Armanet C, et al., 2011, The Assembly Mode of the Pseudopilus <i>A HALLMARK TO DISTINGUISH A NOVEL SECRETION SYSTEM SUBTYPE</i>, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 286, Pages: 24407-24416
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- Citations: 17
Mikkelsen H, Sivaneson M, Filloux A, 2011, Key two-component regulatory systems that control biofilm formation in <i>Pseudomonas aeruginosa</i>, ENVIRONMENTAL MICROBIOLOGY, Vol: 13, Pages: 1666-1681, ISSN: 1462-2912
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- Citations: 158
Filoux A, 2011, The bacterial type VI secretion system: On the bacteriophage trail, Microbiology Today, Vol: 38, Pages: 96-101, ISSN: 1464-0570
Sivaneson M, Mikkelsen H, Ventre I, et al., 2011, Two-component regulatory systems in Pseudomonas aeruginosa: an intricate network mediating fimbrial and efflux pump gene expression, Molecular Microbiology, Vol: 79, Pages: 1353-1366, ISSN: 0950-382X
Pseudomonas aeruginosa is responsible for chronic and acute infections in humans. Chronic infections are associated with production of fimbriae and the formation of a biofilm. The two‐component system Roc1 is named after its role in the regulation of cup genes, which encode components of a machinery allowing assembly of fimbriae. A non‐characterized gene cluster, roc2, encodes components homologous to the Roc1 system. We show that cross‐regulation occurs between the Roc1 and Roc2 signalling pathways. We demonstrate that the sensors RocS2 and RocS1 converge on the response regulator RocA1 to control cupC gene expression. This control is independent of the response regulator RocA2. Instead, we show that these sensors act via the RocA2 response regulator to repress the mexAB‐oprM genes. These genes encode a multidrug efflux pump and are upregulated in the rocA2 mutant, which is less susceptible to antibiotics. It has been reported that in cystic fibrosis lungs, in which P. aeruginosa adopts the biofilm lifestyle, most isolates have an inactive MexAB‐OprM pump. The concomitant RocS2‐dependent upregulation of cupC genes (biofilm formation) and downregulation of mexAB‐oprM genes (antibiotic resistance) is in agreement with this observation. It suggests that the Roc systems may sense the environment in the cystic fibrosis lung.
Giraud C, Bernard CS, Calderon V, et al., 2011, The PprA-PprB two-component system activates CupE, the first non-archetypal <i>Pseudomonas aeruginosa</i> chaperone-usher pathway system assembling fimbriae, ENVIRONMENTAL MICROBIOLOGY, Vol: 13, Pages: 666-683, ISSN: 1462-2912
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- Citations: 55
Hachani A, Lossi NS, Hamilton A, et al., 2011, Type VI Secretion System in Pseudomonas aeruginosa: Secretion and Multimerization of VgrG Proteins, Journal of Biological Chemistry, Vol: 286, ISSN: 1083-351X
Pseudomonas aeruginosa is a Gram-negative bacterium causing chronic infections in cystic fibrosis patients. Such infections are associated with an active type VI secretion system (T6SS), which consists of about 15 conserved components, including the AAA+ ATPase, ClpV. The T6SS secretes two categories of proteins, VgrG and Hcp. Hcp is structurally similar to a phage tail tube component, whereas VgrG proteins show similarity to the puncturing device at the tip of the phage tube. In P. aeruginosa, three T6SSs are known. The expression of H1-T6SS genes is controlled by the RetS sensor. Here, 10 vgrG genes were identified in the PAO1 genome, among which three are co-regulated with H1-T6SS, namely vgrG1a/b/c. Whereas VgrG1a and VgrG1c were secreted in a ClpV1-dependent manner, secretion of VgrG1b was ClpV1-independent. We show that VgrG1a and VgrG1c form multimers, which confirmed the VgrG model predicting trimers similar to the tail spike. We demonstrate that Hcp1 secretion requires either VgrG1a or VgrG1c, which may act independently to puncture the bacterial envelope and give Hcp1 access to the surface. VgrG1b is not required for Hcp1 secretion. Thus, VgrG1b does not require H1-T6SS for secretion nor does H1-T6SS require VgrG1b for its function. Finally, we show that VgrG proteins are required for secretion of a genuine H1-T6SS substrate, Tse3. Our results demonstrate that VgrG proteins are not only secreted components but are essential for secretion of other T6SS substrates. Overall, we emphasize variability in behavior of three P. aeruginosa VgrGs, suggesting that, although very similar, distinct VgrGs achieve specific functions.
Cai X, Wang R, Filloux A, et al., 2011, Structural and functional characterization of pseudomonas aeruginosa CupB chaperones, PLoS ONE, Vol: 6, ISSN: 1932-6203
Pseudomonas aeruginosa, an important human pathogen, is estimated to be responsible for ∼10% of nosocomial infections worldwide. The pathogenesis of P. aeruginosa starts from its colonization in the damaged tissue or medical devices (e.g. catheters, prothesis and implanted heart valve etc.) facilitated by several extracellular adhesive factors including fimbrial pili. Several clusters containing fimbrial genes have been previously identified on the P. aeruginosa chromosome and named cup [1]. The assembly of the CupB pili is thought to be coordinated by two chaperones, CupB2 and CupB4. However, due to the lack of structural and biochemical data, their chaperone activities remain speculative. In this study, we report the 2.5 Å crystal structure of P. aeruginosa CupB2. Based on the structure, we further tested the binding specificity of CupB2 and CupB4 towards CupB1 (the presumed major pilus subunit) and CupB6 (the putative adhesin) using limited trypsin digestion and strep-tactin pull-down assay. The structural and biochemical data suggest that CupB2 and CupB4 might play different, but not redundant, roles in CupB secretion. CupB2 is likely to be the chaperone of CupB1, and CupB4 could be the chaperone of CupB4:CupB5:CupB6, in which the interaction of CupB4 and CupB6 might be mediated via CupB5.
Coulon C, Vinogradov E, Filloux A, et al., 2010, Chemical analysis of cellular and extracellular carbohydrates of a biofilm-forming strain pseudomonas aeruginosa PA14, PLoS ONE, Vol: 5, ISSN: 1932-6203
BackgroundPseudomonas aeruginosa is a Gram-negative bacterium and an opportunistic pathogen, which causes persisting life-threatening infections in cystic fibrosis (CF) patients. Biofilm mode of growth facilitates its survival in a variety of environments. Most P. aeruginosa isolates, including the non-mucoid laboratory strain PA14, are able to form a thick pellicle, which results in a surface-associated biofilm at the air-liquid (A–L) interface in standing liquid cultures. Exopolysaccharides (EPS) are considered as key components in the formation of this biofilm pellicle. In the non-mucoid P. aeruginosa strain PA14, the “scaffolding” polysaccharides of the biofilm matrix, and the molecules responsible for the structural integrity of rigid A–L biofilm have not been identified. Moreover, the role of LPS in this process is unclear, and the chemical structure of the LPS O-antigen of PA14 has not yet been elucidated.Principal FindingsIn the present work we carried out a systematic analysis of cellular and extracellular (EC) carbohydrates of P. aeruginosa PA14. We also elucidated the chemical structure of the LPS O-antigen by chemical methods and 2-D NMR spectroscopy. Our results showed that it is composed of linear trisaccharide repeating units, identical to those described for P. aeruginosa Lanýi type O:2a,c (Lanýi-Bergman O-serogroup 10a, 10c; IATS serotype 19) and having the following structure: -4)-α-L-GalNAcA-(1–3)-α-D-QuiNAc-(1–3)- α-L-Rha-(1-. Furthermore, an EC O-antigen polysaccharide (EC O-PS) and the glycerol-phosphorylated cyclic β-(1,3)-glucans were identified in the culture supernatant of PA14, grown statically in minimal medium. Finally, the extracellular matrix of the thick biofilm formed at the A-L interface contained, in addition to eDNA, important quantities (at least ∼20% of dry weight) of LPS-like material.ConclusionsWe characterized the chemical structure of the LPS O-anti
Bleves S, Viarre V, Salacha R, et al., 2010, Protein secretion systems in <i>Pseudomonas aeruginosa</i>: A wealth of pathogenic weapons, INTERNATIONAL JOURNAL OF MEDICAL MICROBIOLOGY, Vol: 300, Pages: 534-543, ISSN: 1438-4221
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- Citations: 215
Filloux A, 2010, Secretion Signal and Protein Targeting in Bacteria: a Biological Puzzle, JOURNAL OF BACTERIOLOGY, Vol: 192, Pages: 3847-3849, ISSN: 0021-9193
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- Citations: 28
Filloux A, 2010, A Variety of Bacterial Pili Involved in Horizontal Gene Transfer, JOURNAL OF BACTERIOLOGY, Vol: 192, Pages: 3243-3245, ISSN: 0021-9193
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- Citations: 24
Sadovskaya I, Vinogradov E, Li J, et al., 2010, High-level antibiotic resistance in <i>Pseudomonas aeruginosa</i> biofilm: the <i>ndvB</i> gene is involved in the production of highly glycerol-phosphorylated β-(1→3)-glucans, which bind aminoglycosides, GLYCOBIOLOGY, Vol: 20, Pages: 895-904, ISSN: 0959-6658
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- Citations: 78
Salacha R, Kovacic F, Brochier-Armanet C, et al., 2010, The <i>Pseudomonas aeruginosa</i> patatin-like protein PlpD is the archetype of a novel Type V secretion system, ENVIRONMENTAL MICROBIOLOGY, Vol: 12, Pages: 1498-1512, ISSN: 1462-2912
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- Citations: 73
Vincent F, Round A, Reynaud A, et al., 2010, Distinct oligomeric forms of the <i>Pseudomonas aeruginosa</i> RetS sensor domain modulate accessibility to the ligand binding site, ENVIRONMENTAL MICROBIOLOGY, Vol: 12, Pages: 1775-1786, ISSN: 1462-2912
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- Citations: 33
Bordi C, Lamy M-C, Ventre I, et al., 2010, Regulatory RNAs and the HptB/RetS signalling pathways fine-tune Pseudomonas aeruginosa pathogenesis, Molecular Microbiology, Vol: 76, Pages: 1427-1443, ISSN: 1365-2958
Bacterial pathogenesis often depends on regulatory networks, two-component systems and small RNAs (sRNAs). In Pseudomonas aeruginosa, the RetS sensor pathway downregulates expression of two sRNAs, rsmY and rsmZ. Consequently, biofilm and the Type Six Secretion System (T6SS) are repressed, whereas the Type III Secretion System (T3SS) is activated. We show that the HptB signalling pathway controls biofilm and T3SS, and fine-tunes P. aeruginosa pathogenesis. We demonstrate that RetS and HptB intersect at the GacA response regulator, which directly controls sRNAs production. Importantly, RetS controls both sRNAs, whereas HptB exclusively regulates rsmY expression. We reveal that HptB signalling is a complex regulatory cascade. This cascade involves a response regulator, with an output domain belonging to the phosphatase 2C family, and likely an anti-anti-σ factor. This reveals that the initial input in the Gac system comes from several signalling pathways, and the final output is adjusted by a differential control on rsmY and rsmZ. This is exemplified by the RetS-dependent but HptB-independent control on T6SS. We also demonstrate a redundant action of the two sRNAs on T3SS gene expression, while the impact on pel gene expression is additive. These features underpin a novel mechanism in the fine-tuned regulation of gene expression.
Alphonse S, Durand E, Douzi B, et al., 2010, Structure of the <i>Pseudomonas aeruginosa</i> XcpT pseudopilin, a major component of the type II secretion system, JOURNAL OF STRUCTURAL BIOLOGY, Vol: 169, Pages: 75-80, ISSN: 1047-8477
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- Citations: 27
Kowalska K, Soscia C, Combe H, et al., 2010, The C-terminal amphipathic α-helix of <i>Pseudomonas aeruginosa</i> PelC outer membrane protein is required for its function, BIOCHIMIE, Vol: 92, Pages: 33-40, ISSN: 0300-9084
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- Citations: 15
Haas D, Camara M, Bitter W, et al., 2010, Pseudomonas Volume 6molecular Microbiology and Biodiversity, Publisher: Springer
Douzi B, Durand E, Bernard C, et al., 2009, The XcpV/GspI Pseudopilin Has a Central Role in the Assembly of a Quaternary Complex within the T2SS Pseudopilus, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 284, Pages: 34580-34589
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- Citations: 51
Viarre V, Cascales E, Ball G, et al., 2009, HxcQ Liposecretin Is Self-piloted to the Outer Membrane by Its N-terminal Lipid Anchor, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 284, Pages: 33815-33823, ISSN: 0021-9258
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- Citations: 56
Filloux A, Sansonetti P, 2009, Emmanuelle Caron Obituary, CELLULAR MICROBIOLOGY, Vol: 11, Pages: 1423-1424, ISSN: 1462-5814
Garvis S, Munder A, Ball G, et al., 2009, Caenorhabditis elegans semi-automated liquid screen reveals a specialized role for the chemotaxis gene cheB2 in pseudomonas aeruginosa virulence, PLoS Pathogens, Vol: 5, ISSN: 1553-7366
Pseudomonas aeruginosa is an opportunistic human pathogen that causes infections in a variety of animal and plant hosts. Caenorhabditis elegans is a simple model with which one can identify bacterial virulence genes. Previous studies with C. elegans have shown that depending on the growth medium, P. aeruginosa provokes different pathologies: slow or fast killing, lethal paralysis and red death. In this study, we developed a high-throughput semi-automated liquid-based assay such that an entire genome can readily be scanned for virulence genes in a short time period. We screened a 2,200-member STM mutant library generated in a cystic fibrosis airway P. aeruginosa isolate, TBCF10839. Twelve mutants were isolated each showing at least 70% attenuation in C. elegans killing. The selected mutants had insertions in regulatory genes, such as a histidine kinase sensor of two-component systems and a member of the AraC family, or in genes involved in adherence or chemotaxis. One mutant had an insertion in a cheB gene homologue, encoding a methylesterase involved in chemotaxis (CheB2). The cheB2 mutant was tested in a murine lung infection model and found to have a highly attenuated virulence. The cheB2 gene is part of the chemotactic gene cluster II, which was shown to be required for an optimal mobility in vitro. In P. aeruginosa, the main player in chemotaxis and mobility is the chemotactic gene cluster I, including cheB1. We show that, in contrast to the cheB2 mutant, a cheB1 mutant is not attenuated for virulence in C. elegans whereas in vitro motility and chemotaxis are severely impaired. We conclude that the virulence defect of the cheB2 mutant is not linked with a global motility defect but that instead the cheB2 gene is involved in a specific chemotactic response, which takes place during infection and is required for P. aeruginosa pathogenicity.
Mikkelsen H, Ball G, Giraud C, et al., 2009, Expression of pseudomonas aeruginosa CupD fimbrial genes Is antagonistically controlled by RcsB and the EAL-containing PvrR response regulators, PLoS ONE, Vol: 4, ISSN: 1932-6203
Pseudomonas aeruginosa is a gram-negative pathogenic bacterium with a high adaptive potential that allows proliferation in a broad range of hosts or niches. It is also the causative agent of both acute and chronic biofilm-related infections in humans. Three cup gene clusters (cupA-C), involved in the assembly of cell surface fimbriae, have been shown to be involved in biofilm formation by the P. aeruginosa strains PAO1 or PAK. In PA14 isolates, a fourth cluster, named cupD, was identified within a pathogenicity island, PAPI-I, and may contribute to the higher virulence of this strain. Expression of the cupA genes is controlled by the HNS-like protein MvaT, whereas the cupB and cupC genes are under the control of the RocS1A1R two-component system. In this study, we show that cupD gene expression is positively controlled by the response regulator RcsB. As a consequence, CupD fimbriae are assembled on the cell surface, which results in a number of phenotypes such as a small colony morphotype, increased biofilm formation and decreased motility. These behaviors are compatible with the sessile bacterial lifestyle. The balance between planktonic and sessile lifestyles is known to be linked to the intracellular levels of c-di-GMP with high levels favoring biofilm formation. We showed that the EAL domain-containing PvrR response regulator counteracts the activity of RcsB on cupD gene expression. The action of PvrR is likely to involve c-di-GMP degradation through phosphodiesterase activity, confirming the key role of this second messenger in the balance between bacterial lifestyles. The regulatory network between RcsB and PvrR remains to be elucidated, but it stands as a potential model system to study how the equilibrium between the two lifestyles could be influenced by therapeutic agents that favor the planktonic lifestyle. This would render the pathogen accessible for the immune system or conventional antibiotic treatment.
Bernard CS, Bordi C, Termine E, et al., 2009, Organization and PprB-Dependent Control of the <i>Pseudomonas aeruginosa</i> tad Locus, Involved in Flp Pilus Biology, JOURNAL OF BACTERIOLOGY, Vol: 191, Pages: 1961-1973, ISSN: 0021-9193
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- Citations: 56
Filloux A, 2009, The type VI secretion system: a tubular story, EMBO Journal, Vol: 28, Pages: 309-310, ISSN: 0261-4189
The molecular mechanism by which proteins are secreted in gram‐negative bacteria is supported by nanomachines called type I to type VI secretion systems (T1SS to T6SS). Previous data suggested that the ClpV ATPase energizes the transport of Hcp and VgrG proteins through the T6SS secretion channel. The identification of a yet unknown tubular structure, which interacts with ClpV, adds another level of complexity and challenges our vision of the T6SS working model.
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