Biomedical subjects

Jan Tommassen

Publications and source records attributed to Jan Tommassen.

At least 37 records · Page 2Linked to original sources

PubMed2004

Identification of an outer membrane protein required for the transport of lipopolysaccharide to the bacterial cell surface.

Lipopolysaccharide (LPS), also known as endotoxin due to its severe pathophysiological effects in infected subjects, is an essential component of the outer membrane (OM) of most Gram-negative bacteria. LPS is synthesized in the bacterial inner membrane, a process that is now well understood. In contrast, the mechanism of its transport to the outer leaflet of the OM has remained enigmatic. We demonstrate here that the OM protein, known as increased membrane permeability (Imp) or organic solvent tolerance protein, is involved in this process. An Imp-deficient mutant of Neisseria meningitidis was viable and produced severely reduced amounts of LPS. The limited amount of LPS that was still produced was not accessible to LPS-modifying enzymes expressed in the OM or added to the extracellular medium. We conclude therefore that Imp mediates the transport of LPS to the cell surface. The role of Imp in LPS biogenesis and its high conservation among Gram-negative bacteria make it an excellent target for the development of novel antibacterial compounds.

Bacterial Outer Membrane Proteins↗

PubMed2004

Three homologues, including two membrane-bound proteins, of the disulfide oxidoreductase DsbA in Neisseria meningitidis: effects on bacterial growth and biogenesis of functional type IV pili.

Many proteins, especially membrane and exported proteins, are stabilized by intramolecular disulfide bridges between cysteine residues without which they fail to attain their native functional conformation. The formation of these bonds is catalyzed in Gram-negative bacteria by enzymes of the Dsb system. Thus, the activity of DsbA has been shown to be necessary for many phenotypes dependent on exported proteins, including adhesion, invasion, and intracellular survival of various pathogens. The Dsb system in Neisseria meningitidis, the causative agent of cerebrospinal meningitis, has not, however, been studied. In a previous work where genes specific to N. meningitidis and not present in the other pathogenic Neisseria were isolated, a meningococcus-specific dsbA gene was brought to light (Tinsley, C. R., and Nassif, X. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11109-11114). Inactivation of this gene, however, did not result in deficits in the phenotypes commonly associated with DsbA. A search of available genome data revealed that the meningococcus contains three dsbA genes encoding proteins with different predicted subcellular locations, i.e. a soluble periplasmic enzyme and two membrane-bound lipoproteins. Cell fractionation experiments confirmed the localization in the inner membrane of the latter two, which include the previously identified meningococcus-specific enzyme. Mutational analysis demonstrated that the deletion of any single enzyme was compensated by the action of the remaining two on bacterial growth, whereas the triple mutant was unable to grow at 37 degrees C. Remarkably, however, the combined absence of the two membrane-bound enzymes led to a phenotype of sensitivity to reducing agents and loss of functionality of the pili. Although in many species a single periplasmic DsbA is sufficient for the correct folding of various proteins, in the meningococcus a membrane-associated DsbA is required for a wild type DsbA+ phenotype even in the presence of a functional periplasmic DsbA.

Amino Acid Sequence↗

PubMed2004

Structure of the translocator domain of a bacterial autotransporter.

Autotransporters are virulence-related proteins of Gram-negative bacteria that are secreted via an outer-membrane-based C-terminal extension, the translocator domain. This domain supposedly is sufficient for the transport of the N-terminal passenger domain across the outer membrane. We present here the crystal structure of the in vitro-folded translocator domain of the autotransporter NalP from Neisseria meningitidis, which reveals a 12-stranded beta-barrel with a hydrophilic pore of 10 x 12.5 A that is filled by an N-terminal alpha-helix. The domain has pore activity in vivo and in vitro. Our data are consistent with the model of passenger-domain transport through the hydrophilic channel within the beta-barrel, and inconsistent with a model for transport through a central channel formed by an oligomer of translocator domains. However, the dimensions of the pore imply translocation of the secreted domain in an unfolded form. An alternative model, possibly covering the transport of folded domains, is that passenger-domain transport involves the Omp85 complex, the machinery required for membrane insertion of outer-membrane proteins, on which autotransporters are dependent.

Amino Acid Sequence↗

PubMed2004

Lipase-specific foldases.

Lipases represent the most important class of enzymes used in biotechnology. Many bacteria produce and secrete lipases but the enzymes originating from Pseudomonas and Burkholderia species seem to be particularly useful for a wide variety of different biocatalytic applications. These enzymes are usually encoded in an operon together with a second gene which codes for a lipase-specific foldase, Lif, which is necessary to obtain enzymatically active lipase. A detailed analysis based on amino acid homology has suggested the classification of Lif proteins into four different families and also revealed the presence of a conserved motif, Rx1x2FDY(F/C)L(S/T)A. Recent experimental evidence suggests that Lifs are so-called steric chaperones, which exert their physiological function by lowering energetic barriers during the folding of their cognate lipases, thereby providing essential steric information needed to fold lipases into their enzymatically active conformation.

Amino Acid Sequence↗

PubMed2004

Biogenesis of the Gram-negative bacterial outer membrane.

Gram-negative bacteria are bounded by two membranes. The outer membrane consists of phospholipids, lipopolysaccharides, lipoproteins and integral outer membrane proteins, all of which are synthesized in the cytoplasm. Recently, much progress has been made in the elucidation of the mechanisms of transport of these molecules over the inner membrane, through the periplasm and into the outer membrane, in part by exploiting the extraordinary capacity of Neisseria meningitidis to survive without lipopolysaccharide.

Bacterial Outer Membrane Proteins↗

PubMed2004

Omp85, an evolutionarily conserved bacterial protein involved in outer-membrane-protein assembly.

The insertion of proteins into membranes generally requires the assistance of membrane proteins. A protein, designated Omp85 in Neisseria meningitidis, was shown to be required for the assembly of bacterial outer-membrane proteins. The protein is essential for the viability of the bacteria and is ubiquitous among Gram-negative bacteria. Omp85 depletion results in the accumulation of aggregates of unfolded outer-membrane proteins, and we argue that Omp85 is directly involved in outer-membrane-protein assembly. Omp85 shows sequence similarity with Toc75 of the chloroplast protein-import machinery, suggesting a common evolutionary origin.

Amino Acid Sequence↗

PubMed2004

Structure and electrophysiological properties of the YscC secretin from the type III secretion system of Yersinia enterocolitica.

YscC is the integral outer membrane component of the type III protein secretion machinery of Yersinia enterocolitica and belongs to the family of secretins. This group of proteins forms stable ring-like oligomers in the outer membrane, which are thought to function as transport channels for macromolecules. The YscC oligomer was purified after solubilization from the membrane with a nonionic detergent. Sodium dodecyl sulfate did not dissociate the oligomer, but it caused a change in electrophoretic mobility and an increase in protease susceptibility, indicating partial denaturation of the subunits within the oligomer. The mass of the homo-oligomer, as determined by scanning transmission electron microscopy, was approximately 1 MDa. Analysis of the angular power spectrum from averaged top views of negatively stained YscC oligomers revealed a 13-fold angular order, suggesting that the oligomer consists of 13 subunits. Reconstituted in planar lipid bilayers, the YscC oligomer displayed a constant voltage-independent conductance of approximately 3 nS, thus forming a stable pore. However, in vivo, the expression of YscC did not lead to an increased permeability of the outer membrane. Electron microscopy revealed that the YscC oligomer is composed of three domains, two stacked rings attached to a conical domain. This structure is consistent with the notion that the secretin forms the upper part of the basal body of the needle structure of the type III secreton.

Bacterial Outer Membrane Proteins↗

PubMed2004

Role of the pilot protein YscW in the biogenesis of the YscC secretin in Yersinia enterocolitica.

The YscC secretin is a major component of the type III protein secretion system of Yersinia enterocolitica and forms an oligomeric structure in the outer membrane. In a mutant lacking the outer membrane lipoprotein YscW, secretion is strongly reduced, and it has been proposed that YscW plays a role in the biogenesis of the secretin. To study the interaction between the secretin and this putative pilot protein, YscC and YscW were produced in trans in a Y. enterocolitica strain lacking all other components of the secretion machinery. YscW expression increased the yield of oligomeric YscC and was required for its outer membrane localization, confirming the function of YscW as a pilot protein. Whereas the pilot-binding site of other members of the secretin family has been identified in the C terminus, a truncated YscC derivative lacking the C-terminal 96 amino acid residues was functional and stabilized by YscW. Pulse-chase experiments revealed that approximately 30 min were required before YscC oligomerization was completed. In the absence of YscW, oligomerization was delayed and the yield of YscC oligomers was strongly reduced. An unlipidated form of the YscW protein was not functional, although it still interacted with the secretin and caused mislocalization of YscC even in the presence of wild-type YscW. Hence, YscW interacts with the unassembled YscC protein and facilitates efficient oligomerization, likely at the outer membrane.

Bacterial Outer Membrane Proteins↗

PubMed2004

Teasing apart structural determinants of 'toxicity' and 'adjuvanticity': implications for meningococcal vaccine development.

The use of lipopolysaccharide (LPS) as an adjuvant is limited by its high endotoxic activity. In particular, the fatty-acyl pattern of the lipid A part of LPS has been demonstrated to determine its biological activity. By genetic modification of the lipid A biosynthesis pathway in Neisseria meningitidis, a panel of recombinant strains with specific alterations in the lipid A acylation pattern, as well as a strain completely lacking LPS were isolated. Whereas all variations in the fatty-acyl pattern resulted in reduced endotoxic activity, as measured by TNF-alpha induction in the human macrophage cell line MM6, the adjuvant activity of the modified LPS was, in most cases, barely affected. The in vivo adjuvant properties of N. meningitidis wild-type and mutant LPS was found to correlate with induction of co-stimulatory molecules, in particular CD80 and CD40, and with IL-12 production by LPS-stimulated bone marrow-derived BALB/c dendritic cells in vitro. Our results suggest that the ability of LPS to stimulate pro-inflammatory cytokine induction is not necessarily linked to its adjuvant activity. The availability of this novel set of lipid A variants with improved pharmacological properties will be of great importance for the improvement of future outer membrane vesicle vaccines against N. meningitidis.

Animals↗

PubMed2003

Crystal structure of Neisserial surface protein A (NspA), a conserved outer membrane protein with vaccine potential.

The neisserial surface protein A (NspA) from Neisseria meningitidis is a promising vaccine candidate because it is highly conserved among meningococcal strains and induces bactericidal antibodies. NspA is a homolog of the Opa proteins, which mediate adhesion to host cells. Here, we present the crystal structure of NspA, determined to 2.55-A resolution. NspA forms an eight-stranded antiparallel beta-barrel. The four loops at the extracellular side of the NspA molecule form a long cleft, which contains mainly hydrophobic residues and harbors a detergent molecule, suggesting that the protein might function in the binding of hydrophobic ligands, such as lipids. In addition, the structure provides a starting point for structure-based vaccine design.

Amino Acid Sequence↗

PubMed2003

Role of a highly conserved bacterial protein in outer membrane protein assembly.

After transport across the cytoplasmic membrane, bacterial outer membrane proteins are assembled into the outer membrane. Meningococcal Omp85 is a highly conserved protein in Gram-negative bacteria, and its homolog Toc75 is a component of the chloroplast protein-import machinery. Omp85 appeared to be essential for viability, and unassembled forms of various outer membrane proteins accumulated upon Omp85 depletion. Immunofluorescence microscopy revealed decreased surface exposure of outer membrane proteins, which was particularly apparent at the cell-division planes. Thus, Omp85 is likely to play a role in outer membrane protein assembly.

Amino Acid Sequence↗

PubMed2003

Identification of a novel Gsp-related pathway required for secretion of the manganese-oxidizing factor of Pseudomonas putida strain GB-1.

The manganese-oxidizing factor of Pseudomonas putida strain GB-1 is associated with the outer membrane. One of the systems of protein transport across the outer membrane is the general secretory pathway (Gsp). The gsp genes are called xcp in Pseudomonas species. In a previous study, it was shown that mutation of the prepilin peptidase XcpA and of a homologue of the pseudopilin XcpT inhibited transport of the factor. In the present study, we describe the genomic region flanking the xcpT homologue (designated xcmT1). We show that xcmT1 is part of a two-gene operon that includes an xcpS homologue (designated xcmS). No other xcp-like genes are present in the regions flanking the xcmT1/xcmS cluster. We also characterized the site of transposon insertion of another transport mutant of P. putida GB-1. This insertion appeared to be located in a gene (designated xcmX) possibly encoding another pseudopilin-related protein. This xcmX is clustered with two other xcpT-related genes (designated xcmT2 and xcmT3) on one side and homologues of three csg genes (designated csmE, csmF and csmG) on the other side. The csg genes are involved in production of aggregative fibres in Escherichia coli and Salmonella typhimurium. A search for XcmX homologues revealed that the recently published genome of Ralstonia solanacearum and the unannotated genome of P. putida KT2440 contain comparable gene clusters with xcmX and xcp homologues that are different from the well-described 'regular'xcp/gsp clusters. They do contain xcpR and xcpQ homologues but, for example, homologues of xcpP, Y and Z are lacking. The results suggest a novel Xcp-related system for the transport of manganese-oxidizing enzymes to the cell surface.

Amino Acid Sequence↗

PubMed2003

A Neisserial autotransporter NalP modulating the processing of other autotransporters.

Autotransporters constitute a relatively simple secretion system in Gram-negative bacteria, depending for their translocation across the outer membrane only on a C-terminal translocator domain. We have studied a novel autotransporter serine protease, designated NalP, from Neisseria meningitidis strain H44/76, featuring a lipoprotein motif at the signal sequence cleavage site. Indeed, lipidation of NalP could be demonstrated, but the secreted 70 kDa domain of NalP lacked the lipid-moiety as a result of additional N-terminal processing. A nalP mutant showed a drastically altered profile of secreted proteins. Mass-spectrometric analysis of tryptic fragments identified the autotransporters IgA protease and App, a homologue of the adhesin Hap of Haemophilus influenzae, as the major secreted proteins. Two forms of both of these proteins were found in the culture supernatant of the wild-type strain, whereas only the lower molecular-weight forms predominated in the culture supernatant of the nalP mutant. The serine-protease active site of NalP was required for the modulation of the processing of these autotransporters. We propose that, apart from the autoproteolytic processing, NalP can process App and IgA protease and hypothesize that this function of NalP could contribute to the virulence of the organism.

Bacterial Outer Membrane Proteins↗

PubMed2003

Role of Pseudomonas putida tol-oprL gene products in uptake of solutes through the cytoplasmic membrane.

Proteins of the Tol-Pal (Tol-OprL) system play a key role in the maintenance of outer membrane integrity and cell morphology in gram-negative bacteria. Here we describe an additional role for this system in the transport of various carbon sources across the cytoplasmic membrane. Growth of Pseudomonas putida tol-oprL mutant strains in minimal medium with glycerol, fructose, or arginine was impaired, and the growth rate with succinate, proline, or sucrose as the carbon source was lower than the growth rate of the parental strain. Assays with radiolabeled substrates revealed that the rates of uptake of these compounds by mutant cells were lower than the rates of uptake by the wild-type strain. The pattern and amount of outer membrane protein in the P. putida tol-oprL mutants were not changed, suggesting that the transport defect was not in the outer membrane. Consistently, the uptake of radiolabeled glucose and glycerol in spheroplasts was defective in the P. putida tol-oprL mutant strains, suggesting that there was a defect at the cytoplasmic membrane level. Generation of a proton motive force appeared to be unaffected in these mutants. To rule out the possibility that the uptake defect was due to a lack of specific transporter proteins, the PutP symporter was overproduced, but this overproduction did not enhance proline uptake in the tol-oprL mutants. These results suggest that the Tol-OprL system is necessary for appropriate functioning of certain uptake systems at the level of the cytoplasmic membrane.

Bacterial Outer Membrane Proteins↗

PubMed2003

Interactions between phage-shock proteins in Escherichia coli.

Expression of the pspABCDE operon of Escherichia coli is induced upon infection by filamentous phage and by many other stress conditions, including defects in protein export. Expression of the operon requires the alternative sigma factor sigma54 and the transcriptional activator PspF. In addition, PspA plays a negative regulatory role, and the integral-membrane proteins PspB and PspC play a positive one. In this study, we investigated whether the suggested protein-protein interactions implicated in this complex regulatory network can indeed be demonstrated. Antisera were raised against PspB, PspC, and PspD, which revealed, in Western blotting experiments, that PspC forms stable sodium dodecyl sulfate-resistant dimers and that the hypothetical pspD gene is indeed expressed in vivo. Fractionation experiments showed that PspD localizes as a peripherally bound inner membrane protein. Cross-linking studies with intact cells revealed specific interactions of PspA with PspB and PspC, but not with PspD. Furthermore, affinity-chromatography suggested that PspB could bind PspA only in the presence of PspC. These data indicate that regulation of the psp operon is mediated via protein-protein interactions.

Bacterial Proteins↗

PubMed2002

PspE (phage-shock protein E) of Escherichia coli is a rhodanese.

The psp (phage-shock protein) operon of Escherichia coli is induced when the bacteria are infected by filamentous phage and under several other stress conditions. The physiological role of the individual Psp proteins is still not known. We demonstrate here that the last gene of the operon, pspE, encodes a thiosulfate:cyanide sulfurtransferase (EC 2.8.1.1; rhodanese). Kinetic analysis revealed that catalysis occurs via a double displacement mechanism as described for other rhodaneses. The K(m)s for SSO3(2-) and CN- were 4.6 and 27 mM, respectively.

Amino Acid Sequence↗

PubMed2002

Conformational analysis of opacity proteins from Neisseria meningitidis.

Opacity-associated (Opa) proteins are outer membrane proteins which play a critical role in the adhesion of pathogenic Neisseria spp. to epithelial and endothelial cells and polymorphonuclear neutrophils. The adherence is mainly mediated by the CD66-epitope-containing members of the carcinoembryonic-antigen family of human cell-adhesion molecules (CEACAM). For the analysis of the specific interactions of individual Opa proteins with their receptors, pure protein is needed in its native conformation. In this study, we describe the isolation and structural analysis of opacity proteins OpaJ129 and OpaB128 derived from Neisseria meningitidis strain H44/76. When the Opa proteins were produced with the phoE signal sequence in Escherichia coli, they were localized at the cell surface and the recombinant bacteria were found to specifically interact with CEACAM1. For refolding and purification, the proteins were overproduced without their signal sequences in E. coli, resulting in its cytoplasmic accumulation in the form of inclusion bodies. After solubilization of the inclusion bodies in urea, the proteins could be folded efficiently in vitro, under alkaline conditions by dilution in ethanolamine and the detergent n-dodecyl-N,N-dimethyl-1-ammonio-3-propanesulfonate (SB12). The structure of the refolded and purified proteins, determined by circular dichroism, indicated a high content of beta-sheet conformation, which is consistent with previously proposed topology models for Opa proteins. A clear difference was found between the binding of refolded vs. denatured OpaJ protein to the N-A1 domain of CEACAM1. Almost no binding was found with the denatured Opa protein, showing that the Opa-receptor interaction is conformation-dependent.

Antigens, CD↗

PubMed2002

The presence of a helix breaker in the hydrophobic core of signal sequences of secretory proteins prevents recognition by the signal-recognition particle in Escherichia coli.

Signal sequences often contain alpha-helix-destabilizing amino acids within the hydrophobic core. In the precursor of the Escherichia coli outer-membrane protein PhoE, the glycine residue at position -10 (Gly-10) is thought to be responsible for the break in the alpha-helix. Previously, we showed that substitution of Gly-10 by alpha-helix-promoting residues (Ala, Cys or Leu) reduced the proton-motive force dependency of the translocation of the precursor, but the actual role of the helix breaker remained obscure. Here, we considered the possibility that extension of the alpha-helical structure in the signal sequence resulting from the Gly-10 substitutions affects the targeting pathway of the precursor. Indeed, the mutations resulted in reduced dependency on SecB for targeting in vivo. In vitro cross-linking experiments revealed that the G-10L and G-10C mutant PhoE precursors had a dramatically increased affinity for P48, one of the constituents of the signal-recognition particle (SRP). Furthermore, in vitro cross-linking experiments revealed that the G-10L mutant protein is routed to the SecYEG translocon via the SRP pathway, the targeting pathway that is exploited by integral inner-membrane proteins. Together, these data indicate that the helix breaker in cleavable signal sequences prevents recognition by SRP and is thereby, together with the hydrophobicity of the signal sequence, a determinant of the targeting pathway.

Amino Acid Sequence↗