Outer Membrane-associated Proteins Research Articles

Insecticidal pilin subunit from the insect pathogen Xenorhabdus nematophila.

Xenorhabdus nematophila is an insect pathogen and produces protein toxins which kill the larval host. Previously, we characterized an orally toxic, large, outer membrane-associated protein complex from the culture medium of X. nematophila. Here, we describe the cloning, expression, and characterization of a 17-kDa pilin subunit of X. nematophila isolated from that protein complex. The gene was amplified by PCR, cloned, and expressed in Escherichia coli. The recombinant protein was refolded in vitro in the absence of its cognate chaperone by using a urea gradient. The protein oligomerized during in vitro refolding, forming multimers. Point mutations in the conserved N-terminal residues of the pilin protein greatly destabilized its oligomeric organization, demonstrating the importance of the N terminus in refolding and oligomerization of the pilin subunit by donor strand complementation. The recombinant protein was cytotoxic to cultured Helicoverpa armigera larval hemocytes, causing agglutination and subsequent release of the cytoplasmic enzyme lactate dehydrogenase. The agglutination of larval cells by the 17-kDa protein was inhibited by several sugar derivatives. The biological activity of the purified recombinant protein indicated that it has a conformation similar to that of the native protein. The 17-kDa pilin subunit was found to be orally toxic to fourth- or fifth-instar larvae of an important crop pest, H. armigera, causing extensive damage to the midgut epithelial membrane. To our knowledge, this is first report describing an insecticidal pilin subunit of a bacterium.

Characterization of a lipoprotein, NilC, required by Xenorhabdus nematophila for mutualism with its nematode host.

Xenorhabdus nematophila is a gamma-proteobacterial mutualist of an insect-pathogenic nematode, Steinernema carpocapsae. X. nematophila requires nilC, a gene predicted to encode an outer membrane lipoprotein of unknown function, for colonization of its nematode host. Characterization of NilC, described here, demonstrated it is a 28 kDa lipoprotein directed to the periplasm by an N-terminal signal sequence. Lipidation and processing of NilC occurs by a mechanism that is conserved in proteobacteria. This work also showed NilC is membrane associated and oriented towards the periplasm of X. nematophila and is produced as an outer membrane-associated protein when expressed in Escherichia coli. Expression analyses revealed that nilC transcription is directly or indirectly repressed by Lrp, and this regulatory link may explain the nematode mutualism defect of a previously identified lrp::Tn5 mutant. An lrp::Tn5 mutant produces an additional nilC transcript, not observed in wild-type cells growing in vitro, and produces approximately 75-fold more nilC than wild-type cells in late stationary phase. These fundamental characterizations of nilC expression and nilC localization and processing events have provided firm bases for understanding the role of this colonization factor in the X. nematophila/S. carpocapsae microbe-host interaction.

Correlation between resistance of Pseudomonas aeruginosa to quaternary ammonium compounds and expression of outer membrane protein OprR.

The adaptation mechanism of Pseudomonas aeruginosa ATCC 10145 to quaternary ammonium compounds (QACs) was investigated. A P. aeruginosa strain with adapted resistance to QACs was developed by a standard broth dilution method. It was revealed that P. aeruginosa exhibited remarkable resistance to N-dodecylpyridinium iodide (P-12), whose structure is similar to that of a common disinfectant, cetylpyridinium chloride. Adapted resistance to benzalkonium chloride (BAC), which is commonly used as a disinfectant, was also observed in P. aeruginosa. Moreover, the P-12-resistant strain exhibited cross-resistance to BAC. Analysis of the outer membrane protein of the P-12-resistant strain by two-dimensional polyacrylamide gel electrophoresis showed a significant increase in the level of expression of a protein (named OprR) whose molecular mass was approximately 26 kDa. The actual function of OprR is not yet clear; however, OprR was expected to be an outer membrane-associated protein with homology to lipoproteins of other bacterial species, according to a search of the National Center for Biotechnology Information website with the BLAST program by use of the N-terminal sequence of OprR. A correlation between the level of expression of OprR and the level of resistance of P. aeruginosa to QACs was observed by using a PA2800 gene knockout mutant derived from the P-12-resistant strain. The knockout mutant recovered susceptibility not only to P-12 but also to BAC. These results suggested that OprR significantly participated in the adaptation of P. aeruginosa to QACs, such as P-12 and BAC.

Hemin binding, functional expression, and complementation analysis of Pap 31 from Bartonella henselae.

Growth of Bartonella henselae is strongly heme dependent, and B. henselae is unable to synthesize heme itself. At least five outer membrane-associated proteins from B. henselae bind hemin, including the 31-kDa protein designated Pap31. The gene of this protein was heterologously expressed in Escherichia coli M15(pREP4) and detected with monoclonal antibodies in the outer membrane fraction. Complementation of the hemA-deficient mutant E. coli K-12 EB53 (aroB tsx malT hemA) with pap31 demonstrated that this protein is involved in heme acquisition and may be an important virulence factor in the pathogenesis of B. henselae.

Bacterial–host-cell tethers

The dynamic interface between pathogenic bacteria and host cells is a focus of many current studies. Agrobacterium tumefaciens elaborates T pili on its surface that function in the directed type IV secretion of bacterial products into plant host cells. To elucidate the mechanism by which direct cell-to-cell transfer is accomplished, Christie's group have focused on outer membrane-associated proteins encoded by the virB gene cluster that might be responsible for contact and possibly channel formation.

Isolation and Characterization of a Putative Hemin-binding Protein from Prevotella intermedia

Iron is essential for the growth, reproduction, and survival of all prokaryotic and eukaryotic species1-3). Iron has also been shown to be essential for the initiation and progression of several infectious diseases1). In order to survive therefore, prokaryotes and eukaryotes have devised unique mechanisms to compete for, and sequester the small amounts of iron which are available to them. Eukaryotic cells compete for iron by synthesizing large number of high-affinity iron-binding proteins (i.e., transferrin, lactoferrin), hemoglobin-binding protein (haptoglobin), heme-binding proteins (hemopexin, albumin), and iron-storage proteins (ferritin, hemosiderin) which are capable of competing for, and storing iron1,4-8). Therefore, in order for host-associated bacteria to survive, they must be capable of competing with the host’s large number of highaffinity iron-binding proteins. Prokaryotic cells compete with eukaryotes for this iron by employing two mechanisms: synthesizing their own high-affinity binding and transport proteins, siderophores, and outer membrane associated iron-binding protein. The siderophores are low molecular weight proteins, which bind iron by chelating it from the host, and transferring it to the specific outer membrane receptors which then transport it directly into the cell9). Several Gram-negative bacteria contain, in addition to, or in place of siderophores, iron (hemin) binding proteins in their outer membrane1021). These outer membrane-associated iron-binding proteins are capable of binding iron directly, and transporting it across the outer membrane into the cell. For the most part, these outer membrane expressed iron-binding proteins are expressed during iron stress, and are formed in response to very low levels of iron in the environment. While there are several studies which have dealt with the hemin-binding and hemin-regulated proteins in the oral pathogen, Porphyromonas gingivalis20,22-25), there is no such studies which has studied hemin-binding in Prevotella intermedia. In the study presented here, we have identified, purified, and characterized a putative hemin-binding protein in Prevotella intermedia.

Expression of membrane-associated proteins by strains of enteroaggregative Escherichia coli

Certain strains of enteroaggregative Escherichia coli express an outer membrane-associated protein, involved with the adhesion of these bacteria to HEp-2 cells. Strains of enteroaggregative E. coli hybridising with DNA probes for aggregative adhesion, diffuse adhesion and aggregative adhesion fimbriae II expressed an outer membrane-associated protein of 18 kDa regulated by magnesium ions. Strains hybridising with the aggregative adhesion probe only expressed a 20-kDa outer membrane-associated protein regulated by calcium and magnesium. The present study describes two populations of enteroaggregative E. coli which appear to adhere to HEp-2 cells by expressing antigenically distinct, negatively charged membrane-associated proteins.

Expression of membrane-associated proteins by strains of enteroaggregative Escherichia coli.

Certain strains of enteroaggregative Escherichia coli express an outer membrane-associated protein, involved with the adhesion of these bacteria to HEp-2 cells. Strains of enteroaggregative E. coli hybridising with DNA probes for aggregative adhesion, diffuse adhesion and aggregative adhesion fimbriac II expressed an outer membrane-associated protein of 18 kDa regulated by magnesium ions. Strains hybridising with the aggregative adhesion probe only expressed a 20-kDa outer membrane-associated protein regulated by calcium and magnesium. The present study describes two populations of enteroaggregative E. coli which appear to adhere to HEp-2 cells by expressing antigenically distinct, negatively charged membrane-associated proteins.

Characterization of genetic exchanges between various highly divergent tbpBs, having occurred in Neisseria meningitidis.

Transferrin-binding protein B (TbpB) from Neisseria is an outer membrane-associated extracellular protein involved in iron capture during bacterial infection. The tbpB genes display extensive divergences throughout the open reading frame (ORF) that have presumably been selected under the pressure of the immune system. Early studies suggested that they could possibly constitute two distantly related groups of genes (sharing less than 57% identical nt). However, the analysis of one tbpB suggested the existence of a greater genetic diversity, and the occurrence of horizontal genetic exchanges leading to rearrangements of highly divergent ORFs. This study has confirmed this and revealed the occurrence of genetic exchanges having involved at least three types of very distantly related tbpBs. These rearrangements resulted from recombination events having occurred at very similar positions within an ORF region encoding a highly structured protein domain, probably due to constraints imposed by protein function and mode(s) of folding. These new data also provide valuable tools for epidemiological studies and evaluation of TbpBs as candidate vaccines.

Magnesium ions are required for HEp-2 cell adhesion by enteroaggregative strains of Escherichia coli O126:H27 and O44:H18

Enteroaggregative strains of Escherichia coli, belonging to serotypes O44:H18 and O126:H27, were used to show that magnesium ions were essential for the adhesion of these enteroaggregative strains to HEp-2 cells. The removal of Mg 2+ ions from culture media was correlated with the inability of strains to produce an outer membrane-associated protein of 18 kDa and a pellicle. It was concluded that magnesium ions were directly involved with the expression of an 18 kDa outer membrane-associated protein by strains of E. coli O126:H27 and O44:H18, and that the outer membrane-associated protein was involved in both HEp-2 adhesion and pellicle formation.

Cloning and functional analysis of the P97 swine cilium adhesin gene of Mycoplasma hyopneumoniae.

Colonization of the swine respiratory tract by Mycoplasma hyopneumoniae is accomplished by specific binding to the cilia of the mucosal epithelial cells. Previous studies have implicated a 97-kDa outer membrane-associated protein, P97, that appeared to mediate this interaction. In order to further define the role of P97 in adherence to porcine cilia, the structural gene was cloned and sequenced, and the recombinant products were analyzed. Monoclonal antibodies were used to identify recombinant clones in a genomic library expressed in an opal suppressor host because of alternate codon usage by mycoplasmas. The gene coding for P97 was then identified by Tn1000 mutagenesis of recombinant clones. DNA sequence analysis revealed an open reading frame coding for a 124.9-kDa protein with a hydrophobic transmembrane spanning domain. The N-terminal sequence of purified P97 mapped at amino acid position 195 of the translated sequence, indicating that a processing event had occurred in M. hyopneumoniae. Both recombinant P97 protein expressed in an Escherichia coli opal suppressor host and M. hyopneumoniae bound specifically to swine cilia, and the binding was inhibited by heparin and fucoidan, thus supporting the hypothesis that P97 was actively involved in binding to swine cilia in vivo.

Enteroaggregative strains ofEscherichia colibelonging to serotypes 0126:H27 and 044:H18 express antigenically similar 18 kDa outer membrane-associated proteins

Outer membrane-associated proteins of 18 kDa were expressed by enteroaggregative Escherichia coli (EAggEC) belonging to serotypes O126:H27 and O44:H18, which hybridized with a probe derived from a plasmid necessary for enteroaggregative adhesion. The 18 kDa proteins expressed by strains of E. coli, belonging to these serotypes, were surface exposed and antigenically similar but not structurally identical.

Enteroaggregative strains of Escherichia coli belonging to serotypes 0126:H27 and 044:H18 express antigenically similar 18 kDa outer membrane-associated proteins

Outer membrane-associated proteins of 18 kDa were expressed by enteroaggregative Escherichia coli (EAggEC) belonging to serotypes 0126:H27 and O44:H18, which hybridized with a probe derived from a plasmid necessary for enteroaggregative adhesion. The 18 kDa proteins expressed by strains of E. coli, belonging to these serotypes, were surface exposed and antigenically similar but not structurally identical.

Variable serum immunoglobulin responses against different Borrelia burgdorferi sensu lato species in a population at risk for and patients with Lyme disease

Borrelia burgdorferi sensu lato species display considerable antigenic polymorphism. In order to evaluate the importance of this antigenic heterogeneity in the serodiagnosis of Lyme disease, the serum immunoglobulin G response in 148 healthy individuals from an area in northern Sweden where Lyme disease is endemic and in 40 American patients with Lyme disease was assessed. In a seroprevalence study, the control group included 173 individuals from a region of northern Sweden where Lyme disease is not endemic. The two enzyme immunoassays used were based on outer membrane-associated proteins of either B. burgdorferi sensu stricto or Borrelia garinii. The Swedish populations were also screened for antiflagellum seroreactivity. The individuals from the area of endemicity were significantly more seropositive for the subcellular protein fraction of the local B. garinii isolate NBS16 than the control group (11.5 versus 2.9%; P = 0.005) but were not significantly more positive for the other antigens used. In contrast, American patients with Lyme disease were significantly more reactive against the North American B. burgdorferi sensu stricto strain B31 than against B. garinii NBS16 (57.5 versus 15.0%; P = 0.0001). Immunoblot analysis suggests that the borrelial outer surface protein C is involved in triggering the production of species-specific antibody during localized Lyme disease. We conclude that a species-specific immune response develops during infection with Lyme disease Borrelia spp. Thus, the reliability of a serological investigation of Lyme disease increases when one measures antibody titers against the outer membrane proteins of Lyme disease Borrelia spp. occurring in a particular geographic region.

Changes in the surface of Leptospira interrogans serovar grippotyphosa during in vitro cultivation

Surface components of virulent and attenuated Leptospira interrogans serovar grippotyphosa were compared by using Triton X-114 solubilization and phase partitioning, immunoprecipitation of intact organisms, and freeze-fracture electron microscopy. Removal of the leptospiral outer membrane by using 0.1% Triton X-114 was demonstrated by whole-mount electron microscopy and by essentially complete solubilization of a lipopolysaccharidelike substance (LLS) from the outer membrane. Triton X-114 (0.1%) did not solubilize subsurface proteins, such as endoflagellar filaments or penicillin-binding proteins, which are markers for the periplasmic space and inner membrane, respectively. Triton X-114 solubilized material from both the virulent and attenuated strains, which partitioned into the hydrophobic, detergent phase, contained LLS and major proteins of 41 and 44 kDa, which were also immunoprecipitable from intact organisms. The virulent strain contained greater amounts of an LLS component with an apparent molecular mass of 30 kDa (R(f) = 0.57), whereas the attenuated strain contained larger amounts of an LLS component with an apparent molecular mass of 20 kDa (R(f) = 0.74). Differences in protein components between virulent and attenuated organisms were also detected; whereas the 41- and 44-kDa proteins were immunoprecipitated in equal amounts from both the virulent and attenuated strains, a 33-kDa protein was immunoprecipitated in significantly greater amounts from the attenuated strain. Quantitation of outer membrane particle density by freeze-fracture electron microscopy showed that both strains had a low transmembrane outer membrane protein content compared with that of typical gram-negative bacteria. The virulent and attenuated strains had 443 and 990 particles (P less than 0.000001) per micron, respectively, in the concave outer membrane fracture face. These findings suggest that in vitro cultivation of L. interrogans is accompanied by quantitative and qualitative changes in both LLS and outer membrane-associated proteins.

Characterization of the hlyB gene and its role in the production of the EI Tor haemolysin of Vibrio choierae O1

El Tor strains of Vibrio cholerae are capable of producing a haemolysin which they actively secrete into the growth medium. This requires translation to produce the protein at the surface of the cytoplasmic membrane and translocation across this membrane, the periplasmic space and the outer membrane. The mechanism by which this occurs is poorly understood. In addition to the structural gene for the haemolysin (hlyA), we have cloned a second adjacent gene, hlyB. By site-directed mutagenesis, specific hlyB mutants have been constructed. These mutants are defective in the secretion of HlyA in the early to mid-exponential phase of growth and the haemolysin becomes trapped within the cell and is only released in stationary phase. Nucleotide sequence analysis and cell fractionations reveal HlyB to be a 60.3 kD putative outer membrane-associated protein.

Immunochemical and biological properties of the outer membrane-associated lipopolysaccharide and protein of Rochalimaea quintana.

The outer membrane complex of Rochalimaea quintana was isolated by use of ethylene-eiaminetetraacetate and was compared biochemically and biologically both with lipopolysaccharide (LPS) isolated by phenol-water extraction of whole organisms and with lipids isolated by chloroform-methanol extractions of the phenol-water insoluble residues. The outer membrane consisted of protein and LPS components, as distinguished by precipitin tests with sera from patients with trench fever or tests with hyperimmune animal sera. The outer membrane protein component, but not LPS, also reacted with sera from infections with Rickettsia tsutsugamushi. The LPS contained 2-keto-3-deoxy-octonate and heptose. The outer membrane and phenol-extracted LPS were reactive in the chick embryo lethality test, limulus assay, and complement activation. Outer-membrane activity was confined to the LPS component. The lipid extracts were reactive in chick embryo lethality and limulus assays, but did not activate complement.

Identification of an outer membrane-associated protein ofAquaspirillum serpensby immunoelectron microscopy

Identification of an outer membrane-associated protein of<i>Aquaspirillum serpens</i>by immunoelectron microscopy

Identification of an outer membrane-associated protein of Aquaspirillum serpens by immunoelectron microscopy

Identification of an outer membrane-associated protein of Aquaspirillum serpens by immunoelectron microscopy