Biomedical subjects

Laurent Claret

Publications and source records attributed to Laurent Claret.

8 recordsLinked to original sources

PubMed2005

Strong decrease in invasive ability and outer membrane vesicle release in Crohn's disease-associated adherent-invasive Escherichia coli strain LF82 with the yfgL gene deleted.

Adherent-invasive Escherichia coli strain LF82 recovered from a chronic lesion of a patient with Crohn's disease is able to invade cultured intestinal epithelial cells. Three mutants with impaired ability to invade epithelial cells had the Tn5phoA transposon inserted in the yfgL gene encoding the YfgL lipoprotein. A yfgL- negative isogenic mutant showed a marked decrease both in its ability to invade Intestine-407 cells and in the amount of the outer membrane proteins OmpA and OmpC in the culture supernatant, as shown by analysis of the culture supernatant protein contents by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Transcomplementation of the LF82-DeltayfgL isogenic mutant with the cloned yfgL gene restored invasion ability and outer membrane protein release in the culture supernatant. The outer membrane proteins in the culture supernatant of strain LF82 resulted from the formation of vesicles. This was shown by Western blot analysis of periplasmic and outer membrane fraction markers typically found in outer membrane vesicles and by transmission electron microscopic analysis of ultracentrifuged cell-free LF82 supernatant pellets, indicating the presence of vesicles with a bilayered structure surrounding a central electron-dense core. Thus, deletion of the yfgL gene in strain LF82 resulted in a decreased ability to invade intestinal epithelial cells and a decreased release of outer membrane vesicles.

Bacterial Adhesion↗

PubMed2004

Involvement of lipoprotein NlpI in the virulence of adherent invasive Escherichia coli strain LF82 isolated from a patient with Crohn's disease.

Escherichia coli strain LF82 recovered from a chronic lesion of a patient with Crohn's disease (CD) is able to adhere to and invade cultured intestinal epithelial cells and to replicate within macrophages. One mutant selected for its impaired ability to invade epithelial cells had an insertion of a Tn phoA transposon within the nlpI gene encoding the lipoprotein NlpI. A NlpI-negative isogenic mutant showed a 35-fold decrease in its ability to adhere to and a 45-fold decrease in its ability to invade Intestine-407 cells, but its ability to survive and to replicate within macrophages was similar to that of wild-type strain LF82. In addition, this mutant did not express flagella and synthesized very small amounts of type 1 pili. Downregulation of type 1 pili in the NlpI-negative mutant resulted from a preferential switch toward the OFF position of the invertible DNA element located upstream of the fim operon. The FimB and FimE recombinases act in concert to control the switch, and a large decrease in fimB and fimE mRNA levels was observed. The absence of flagellar structures correlated with a drastic 19-fold decrease in the fliC mRNA level, regardless of the FlhD(2)C(2) transcriptional regulator and of the sigma(28) transcription factor. The key role of NlpI in virulence is independent of type 1 pili and motility, since induced type 1 pilus expression and/or forced contact between bacteria and intestinal epithelial cells did not restore the ability of the NlpI mutant to adhere to and to invade intestinal epithelial cells.

Bacterial Adhesion↗

PubMed2003

The FliS chaperone selectively binds the disordered flagellin C-terminal D0 domain central to polymerisation.

Assembly of each Salmonella typhimurium flagellum filament requires export and polymerisation of ca. 30000 flagellin (FliC) subunits. This is facilitated by the cytosolic chaperone FliS, which binds to the 494 residue FliC and inhibits its polymerisation. Yeast two-hybrid assays, co-purification and affinity blotting showed that FliS binds specifically to the C-terminal 40 amino acid component of the disordered D0 domain central to polymerisation. Without FliS binding, the C-terminus is degraded. Our data provide further support for the view that FliS is a domain-specific bodyguard preventing premature monomer interaction.

Bacterial Proteins↗

PubMed2003

Regulatory and functional co-operation of flagella and type 1 pili in adhesive and invasive abilities of AIEC strain LF82 isolated from a patient with Crohn's disease.

Genetic determinants that co-operate with type 1 pili to mediate invasion were sought for in adherent-invasive Escherichia coli strain LF82 isolated from a patient with Crohn's disease. Two mutants selected for their impaired ability to invade epithelial cells carried insertions of a TnphoA transposon within genes of the flagellar regulon. An isogenic mutant LF82-DeltafliC deleted for the flagellin-encoding gene did not adhere, did not invade and, surprisingly, expressed only a few type 1 pili. Type 1 pili downregulation resulted from a preferential switch towards the off-position of the invertible DNA element located upstream of the fim operon. This was also correlated with a decrease in the flagellar regulator flhDC mRNA levels, suggesting that the transcriptional regulator FlhD2C2 could control type 1 pili expression directly or indirectly. Transformation with a cloned fim operon allowed bypass of the type 1 pili downexpression in the LF82-DeltafliC mutant. Thus, we showed that flagella play a direct role in the adhesion process via active motility. In addition to downregulating type 1 pili expression, flagella also play an undefined role in strain LF82 invasion, which is not restricted to motility or flagellar structure, but could be related to co-ordinate expression of invasive determinants.

Bacterial Adhesion↗

PubMed2003

Oligomerization and activation of the FliI ATPase central to bacterial flagellum assembly.

FliI is the peripheral membrane ATPase pivotal to the type III protein export mechanism underlying the assembly of the bacterial flagellum. Gel filtration and multiangle light scattering showed that purified soluble native FliI protein was in a monomeric state but, in the presence of ATP, FliI showed a propensity to oligomerize. Electron microscopy revealed that FliI assembles to a ring structure, the yield of which was increased by the presence of a non-hydrolysable ATP analogue. Single particle analysis of the resulting electron micrograph images, to which no symmetry was applied, showed that the FliI ring structure has sixfold symmetry and an external diameter of approximately 10 nm. The oligomeric ring has a central cavity of 2.5-3.0 nm, which is comparable to the known diameter of the flagellar export channel into which export substrates feed. Enzymatic activity of the FliI ATPase showed positive co-operativity, establishing that oligomerization and enzyme activity are coupled. Escherichia coli phospholipids increased enzyme co-operativity, and in vitro cross-linking demonstrated that they promoted FliI multimerization. The data reveal central facets of the structure and action of the flagellar assembly ATPase and, by extension, the homologous ATPases of virulence-related type III export systems.

Adenosine Triphosphatases↗

PubMed2002

Interaction of the atypical prokaryotic transcription activator FlhD2C2 with early promoters of the flagellar gene hierarchy.

The transcriptional activator FlhD2C2 is the master regulator of bacterial flagellum biogenesis and swarming migration, activating the "early" class II promoters of the large flagellar gene hierarchy. Using primer extensions, band-shift assays, and enzymatic and chemical footprinting, we describe the binding of the FlhD2C2 heterotetramer to the promoter regions of four class II flagella operons, fliAZ, flhBA and the divergent flgAMN and flgBCD(EFGHIJ). Each of the promoter regions was bound by a single heterotetramer, i.e. the flgAMN and flgBCD operons are characterised by a single FlhD2C2 binding site. Binding affinity differed, and correlated with previously reported promoter strength and order of activation. Methylation protection and interference, and depurination and depyrimidation interference provided a detailed map of critical bases within a common 46-59bp DNaseI footprint overlapping the promoter -35 sequences. These data and compilation of the 12 known class II promoter sequences of Escherichia coli, Proteus mirabilis and Salmonella typhimurium allowed determination of a FlhD2C2 binding site with pseudo symmetry, comprising two 17-18bp inverted repeats, each a consensus FlhD2C2 box, separated by a 10-11bp spacer. DNaseI hypersensitivity indicated that binding may cause a conformational change in the promoter regions. Only the FlhC subunit can bind DNA independently, but the specificity and stability of the interaction is strengthened by FlhD. Here, photo-crosslinking established that both FlhC and the stabilising FlhD contact the DNA within the FlhD2C2 tetramer. Our data suggest that specificity of recognition and stability of the FlhD2C2/DNA complex require protein-protein interaction and interaction of both FlhC and FlhD subunits with DNA. These characteristics of the FlhD and FlhC subunits in the FlhD2C2/DNA complex are strikingly atypical of prokaryotic regulators.

Bacterial Proteins↗

PubMed2002

Intrinsic membrane targeting of the flagellar export ATPase FliI: interaction with acidic phospholipids and FliH.

The specialised ATPase FliI is central to export of flagellar axial protein subunits during flagellum assembly. We establish the normal cellular location of FliI and its regulatory accessory protein FliH in motile Salmonella typhimurium, and ascertain the regions involved in FliH(2)/FliI heterotrimerisation. Both FliI and FliH localised to the cytoplasmic membrane in the presence and in the absence of proteins making up the flagellar export machinery and basal body. Membrane association was tight, and FliI and FliH interacted with Escherichia coli phospholipids in vitro, both separately and as the preformed FliH(2)/FliI complex, in the presence or in the absence of ATP. Yeast two-hybrid analysis and pull-down assays revealed that the C-terminal half of FliH (H105-235) directs FliH homodimerisation, and interacts with the N-terminal region of FliI (I1-155), which in turn has an intra-molecular interaction with the remainder of the protein (I156-456) containing the ATPase domain. The FliH105-235 interaction with FliI was sufficient to exert the FliH-mediated down-regulation of ATPase activity. The basal ATPase activity of isolated FliI was stimulated tenfold by bacterial (acidic) phospholipids, such that activity was 100-fold higher than when bound by FliH in the absence of phospholipids. The results indicate similarities between FliI and the well-characterised SecA ATPase that energises general protein secretion. They suggest that FliI and FliH are intrinsically targeted to the inner membrane before contacting the flagellar secretion machinery, with both FliH105-235 and membrane phospholipids interacting with FliI to couple ATP hydrolysis to flagellum assembly.

Adenosine Triphosphatases↗

PubMed2002

Swarming-coupled expression of the Proteus mirabilis hpmBA haemolysin operon.

The HpmA haemolysin toxin of Proteus mirabilis is encoded by the hpmBA locus and its production is upregulated co-ordinately with the synthesis and assembly of flagella during differentiation into hyperflagellated swarm cells. Primer extension identified a sigma(70) promoter upstream of hpmB that was upregulated during swarming. Northern blotting indicated that this promoter region was also required for concomitant transcription of the immediately distal hpmA gene, and that the unstable hpmBA transcript generated a stable hpmA mRNA and an unstable hpmB mRNA. Transcriptional luxAB fusions to the DNA regions 5' of the hpmB and hpmA genes confirmed that hpmB sigma(70) promoter activity increased in swarm cells, and that there was no independent hpmA promoter. Increased transcription of the hpmBA operon in swarm cells was dependent upon a 125 bp sequence 5' of the sigma(70) promoter -35 hexamer. This sequence spans multiple putative binding sites for the leucine-responsive regulatory protein (Lrp), and band-shift assays with purified Lrp confirmed the presence of at least two such sites. The influence on hpmBA expression of the key swarming positive regulators FlhD(2)C(2) (encoded by the flagellar master operon), Lrp, and the membrane-located upregulator of the master operon, UmoB, was examined. Overexpression of each of these regulators moderately increased hpmBA transcription in wild-type P. mirabilis, and the hpmBA operon was not expressed in any of the flhDC, lrp or umoB mutants. Expression in the mutants was not recovered by cross-complementation, i.e. by overexpression of FlhD(2)C(2), Lrp or UmoB. Expression of the zapA protease virulence gene, which like hpmBA is also upregulated in swarm cells, did not require Lrp, but like flhDC it was upregulated by UmoB. The results indicate intersecting pathways of control linking virulence gene expression and swarm cell differentiation.

Amino Acid Sequence↗