Elimination of channel-forming activity by insertional inactivation of the p13 gene in Borrelia burgdorferi

P13 is a chromosomally encoded 13-kDa integral outer membrane protein of the Lyme disease agent, Borrelia burgdorferi. The aim of this study was to investigate the function of the P13 protein. Here, we inactivated the p13 gene by targeted mutagenesis and investigated the porin activities of outer membrane proteins by using lipid bilayer experiments. Channel-forming activity was lost in the p13 mutant compared to wild-type B. burgdorferi, indicating that P13 may function as a porin. We purified native P13 to homogeneity by fast performance liquid chromatography and demonstrated that pure P13 has channel-forming activity with a single-channel conductance in 1 M KCl of 3.5 nS, the same as the porin activity that was lost in the p13 mutant. Further characterization of the channel formed by P13 suggested that it is cation selective and voltage independent. In addition, no major physiological effects of the inactivated p13 gene could be detected under normal growth conditions. The inactivation of p13 is the first reported inactivation of a gene encoding an integral outer membrane protein in B. burgdorferi. Here, we describe both genetic and biophysical experiments indicating that P13 in B. burgdorferi is an outer membrane protein with porin activity.     

Porin activity of the native and recombinant outer membrane protein Oms28 of Borrelia burgdorferi.

The outer membrane-spanning (Oms) proteins of Borrelia burgdorferi have been visualized by freeze-fracture analysis but, until recently, not further characterized. We developed a method for the isolation of B. burgdorferi outer membrane vesicles and described porin activities with single-channel conductances of 0.6 and 12.6 nS in 1 M KCI. By using both nondenaturing isoelectric focusing gel electrophoresis and fast-performance liquid chromatography separation after detergent solubilization, we found that the 0.6-nS porin activity resided in a 28-kDa protein, designated Oms28. The oms28 gene was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence of Oms28 predicted a 257-amino-acid precursor protein with a putative 24-amino-acid leader peptidase I signal sequence. Processed Oms28 yielded a mature protein with a predicted molecular mass of 25,363 Da. When overproduced in Escherichia coli, the Oms28 porin fractionated in part to the outer membrane. Sodium dodecyl sulfate-polyacrylamide gel-purified recombinant Oms28 from E. coli retained functional activity as demonstrated by an average single-channel conductance of 1.1 nS in the planar lipid bilayer assay. These findings confirmed that Oms28 is a B. burgdorferi porin, the first to be described. As such, it is potential relevance to the pathogenesis of Lyme borreliosis and to the physiology of the spirochete.     

Elimination of channel-forming activity by insertional inactivation of the p66 gene in Borrelia burgdorferi

P66 is a chromosomally encoded 66-kDa integral outer membrane protein of the Lyme disease agent Borrelia burgdorferi exhibiting channel-forming activity. Herein, we inactivated and subsequently complemented the p66 gene in the B31-A (WT) strain. The P66 protein was also inactivated in two other channel-forming protein mutant strains, P13-18 (Deltap13) and Deltabba01, and then compared with the channel-forming activities of wild-type and various p66 mutant strains. We further investigated the ion-selectivity of native, purified P66. In conclusion, we show that the porin activity of P66 is eliminated by insertional inactivation and that this activity can be rescued by gene complementation.     

DipA, a pore-forming protein in the outer membrane of Lyme disease spirochetes exhibits specificity for the permeation of dicarboxylates

Lyme disease Borreliae are highly dependent on the uptake of nutrients provided by their hosts. Our study describes the identification of a 36 kDa protein that functions as putative dicarboxylate-specific porin in the outer membrane of Lyme disease Borrelia. The protein was purified by hydroxyapatite chromatography from Borrelia burgdorferi B31 and designated as DipA, for dicarboxylate-specific porin A. DipA was partially sequenced, and corresponding genes were identified in the genomes of B. burgdorferi B31, Borrelia garinii PBi and Borrelia afzelii PKo. DipA exhibits high homology to the Oms38 porins of relapsing fever Borreliae. B. burgdorferi DipA was characterized using the black lipid bilayer assay. The protein has a single-channel conductance of 50 pS in 1 M KCl, is slightly selective for anions with a permeability ratio for cations over anions of 0.57 in KCl and is not voltage-dependent. The channel could be partly blocked by different di- and tricarboxylic anions. Particular high stability constants up to about 28,000 l/mol (in 0.1 M KCl) were obtained among the 11 tested anions for oxaloacetate, 2-oxoglutarate and citrate. The results imply that DipA forms a porin specific for dicarboxylates which may play an important role for the uptake of specific nutrients in different Borrelia species.     

The BBA01 protein, a member of paralog family 48 from Borrelia burgdorferi, is potentially interchangeable with the channel-forming protein P13

The Borrelia burgdorferi genome exhibits redundancy, with many plasmid-carried genes belonging to paralogous gene families. It has been suggested that certain paralogs may be necessary in various environments and that they are differentially expressed in response to different conditions. The chromosomally located p13 gene which codes for a channel-forming protein belongs to paralog family 48, which consists of eight additional genes. Of the paralogous genes from family 48, the BBA01 gene has the highest homology to p13. Herein, we have inactivated the BBA01 gene in B. burgdorferi strain B31-A. This mutant shows no apparent phenotypic difference compared to the wild type. However, analysis of BBA01 in a C-terminal protease A (CtpA)-deficient background revealed that like P13, BBA01 is posttranslationally processed at its C terminus. Elevated BBA01 expression was obtained in strains with the BBA01 gene introduced on the shuttle vector compared to the wild-type strain. We could further demonstrate that BBA01 is a channel-forming protein with properties surprisingly similar to those of P13. The single-channel conductance, of about 3.5 nS, formed by BBA01 is comparable to that of P13, which together with the high degree of sequence similarity suggests that the two proteins may have similar and interchangeable functions. This is further strengthened by the up-regulation of the BBA01 protein and its possible localization in the outer membrane in a p13 knockout strain, thus suggesting that P13 can be replaced by BBA01.     

Specificity and role of the Borrelia burgdorferi CtpA protease in outer membrane protein processing

To further characterize the function of the Borrelia burgdorferi C-terminal protease CtpA, we used site-directed mutagenesis to alter the putative CtpA cleavage site of one of its known substrates, the outer membrane (OM) porin P13. These mutations resulted in only partial blockage of P13 processing. Ectopic expression of a C-terminally truncated P13 in B. burgdorferi indicated that the C-terminal peptide functions as a safeguard against misfolding or mislocalization prior to its proteolytic removal by CtpA. In a parallel study of Borrelia burgdorferi lipoprotein sorting mechanisms, we observed a lower-molecular-weight variant of surface lipoprotein OspC that was particularly prominent with OspC mutants that mislocalized to the periplasm or contained C-terminal epitope tags. Further investigation revealed that the variant resulted from C-terminal proteolysis by CtpA. Together, these findings indicate that CtpA rather promiscuously targets polypeptides that lack structurally constrained C termini, as proteolysis appears to occur independently of a specific peptide recognition sequence. Low-level processing of surface lipoproteins such as OspC suggests the presence of a CtpA-dependent quality control mechanism that may sense proper translocation of integral outer membrane proteins and surface lipoproteins by detecting the release of C-terminal peptides.     

Kinetic Properties of Purified Pseudomonas aeruginosa Phosphorylcholine Phosphatase Indicated That This Enzyme May Be Utilized by the Bacteria to Colonize in Different Environments

A protein oligomer with an approximate molecular weight of its 37-kDa monomer form was purified from the cell envelope fraction of Vibrio damsela cells. This oligomer exhibited strong porin activity when reconstituted into proteoliposomes with phosphatidyl choline. The functional properties for the 37-kDa protein suggest that it is a nonspecific or general porin, with an apparent pore size of 1.6 nm. This porin allows penetration of a variety of hydrophilic solutes according to their molecular mass. After electroelution, the oligomer was partially dissociated into monomers, whereas treatment with EDTA did not affect its dissociation. The monomers of the 37-kDa protein were not active in the reconstitution assay. The effect of culture media on the composition of the outer membrane protein of V. damsela was examined. Only one outer membrane protein with an apparent molecular weight of 37 kDa (37-kDa protein) was formed in cells grown in 3% NaCl–BHI broth and in 3% NaCl–nutrient broth with the addition of 2% glucose. Three outer membrane proteins, with apparent molecular weights of 37 kDa, 40 kDa, and 46 kDa, were produced in cells grown in 3% NaCl–nutrient broth. An additional outer membrane protein with an apparent molecular weight of 44 kDa (44-kDa protein) was found in cells grown in 3% NaCl–nutrient broth with the addition of 2% maltose. This protein was found to exhibit specificity to maltose derivatives. The results obtained in this study confirm the porin-like character of discussed proteins and give a basis for advanced study of those proteins. Received: 16 June 1998 / Accepted: 18 July 1998     

Non-heritable change of a spirochaete's phenotype by decoration of the cell surface with exogenous lipoproteins

Genetic transformation of Borrelia spp. is limited in development and has found application in only one species. For a non-genetic approach for manipulating the phenotype of these spirochaetes, we determined whether exogenous recombinant lipoproteins would incorporate in the cell's outer membrane. Using unlabelled or 125I-labelled Osp proteins, Osp-specific monoclonal antibodies, proteinase K and formaldehyde as reagents, we found that decoration of spirochaetes had the following characteristics. (i) Purified recombinant OspA or OspD lipoproteins associated with Borrelia burgdorferi and B. hermsii cells that lacked abundant lipoproteins of their own. (ii) This decoration of the cells with exogenous OspA did not affect cell's viability. (iii) The decoration was concentration and temperature dependent and stable for at least 24 h. (iv) Like native OspA, the recombinant OspA decorating the cells was accessible to antibodies and proteases and could be cross-linked to the integral outer membrane protein, P66. (v) Decoration of viable B. burgdorferi and B. hermsii with OspA rendered the cells susceptible to killing by OspA-specific antiserum. Such non-genetic alteration of the surface of a bacterium may be used to study functions and properties of lipoproteins in situ.     

Use of a nonpenetrating substrate analogue to study the molecular mechanism of the outer membrane dicarboxylate transport system in Escherichia coli K12

We have recently demonstrated that a cell-surface dicarboxylate-binding protein (DBP) is involved in the outer membrane dicarboxylate transport system in Escherichia coli K12. The present report deals with our findings relating to the mode of action of this protein, and the identity and properties of the outer membrane integral protein which is involved in the translocation of dicarboxylic acids across the hydrophobic regions of the outer membrane. By the use of a nonpenetrating succinate analogue, aspartate-dextran, and through reconstitution studies with purified DBP, the cell-surface DBP is found to play an important role in succinate influx but not efflux. Transport studies with major outer membrane protein mutants indicate that the matrix protein (also referred to as protein I or porin) is the only outer membrane integral protein actively involved in the outer membrane dicarboxylate transport system. In the absence of a functional DBP, porin translocates succinate in a relatively less efficient and nonspecific manner. A tentative working model is proposed for this transport system. In this model, the cell-surface DBP is depicted as the substrate recognition component of the otherwise nonspecific porin channel. Together, this "DBP-porin channel complex" forms an efficient, specific transport channel for dicarboxylic acids.     

Borrelia burgdorferi and other bacterial products induce expression and release of the urokinase receptor (CD87)

The urokinase-type plasminogen activator receptor (uPAR, CD87) is a highly glycosylated 55- to 60-kDa protein anchored to the cell membrane through a glycosylphosphatidylinositol moiety that promotes the acquisition of plasmin on the surface of cells and subsequent cell movement and migration by binding urokinase-type plasminogen activator. uPAR also occurs in a soluble form in body fluids and tumor extracts, and both membrane and soluble uPAR are overexpressed in patients with tumors. uPAR may be a factor in inflammatory disorders as well. We investigated whether Borrelia burgdorferi could stimulate up-regulation of cell membrane uPAR in vitro. B. burgdorferi, purified native outer surface protein A, and a synthetic outer surface protein A hexalipopeptide stimulated human monocytes to up-regulate membrane uPAR as measured by immunofluorescence/FACS and Western blot. The presence of soluble uPAR in culture supernatants, measured by Ag capture ELISA, was also observed. LPS from Salmonella typhimurium and lipotechoic acid from Streptococcus pyogenes also induced the up-regulation of both membrane and soluble uPAR protein by monocytes. Up-regulation of uPAR was induced by conditioned medium from B. burgdorferi/monocyte cocultures. The up-regulation of uPAR by B. burgdorferi was concomitant with an increase in uPAR mRNA, indicating that synthesis was de novo. The expression and release of uPAR in response to B. burgdorferi and other bacterial components suggests a role in the pathogenesis of Lyme disease as well as in other bacterial infections.     

Animal and human antibodies reactive with the outer surface protein A and B of Borrelia burgdorferi are borreliacidal, in vitro, in the presence of complement

Abstract Polyspecific antibodies present in ascitic fluids of mice (pMIAFs) immunized with whole Borrelia burgdorferi cells exerted borreliacidal activity in vitro when tested with complement and homologous antigen but not with heterologous B. hermsii . Similarly, monospecific mouse antibodies obtained by immunizing mice with purified preparations of outer surface protein A and B of B. burgdorferi were borreliacidal. On the contrary, mouse monospecific antibodies raised against the 41-kDa flagellar protein of B. burgdorferi did not kill borreliae in the presence of complement. A complement-mediated, in vitro, borreliacidal activity was observed in human sera from patients with Lyme disease when antibodies against OspA and/or OspB were detectable in sera by the Western blotting technique. The in vitro borreliacidal activity of human sera was evident after 14 h incubation with live B. burgdorferi spirochaetes and complement, whereas antibodies present in mouse immune ascitic fluids killed borreliae after 1 h incubation.     

Outer membrane porin protein of Haemophilus influenzae type b: pore size and subunit structure

The 40-kDa porin protein of Haemophilus influenzae type b was reconstituted into proteoliposomes. The relative rates of diffusion of small uncharged sugars across the channels formed by this protein were determined by measuring the rates of osmotic swelling of the liposomes. From these rates, a pore diameter of 1.8 nm was estimated using the Renkin equation. A chemical cross-linking technique was used to investigate the oligomeric structure of the 40-kDa porin. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis revealed the presence of porin dimers and trimers after reaction of the protein with dithio-bis-(succinimidyl propionate). These results confirmed that the porin of H. influenzae forms large water-filled channels and indicated that it probably exists as trimers in the outer membrane.     

Molecular and Structural Characterization of the HMP-AB Gene Encoding a Pore-Forming Protein from a Clinical Isolate of <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis>

The major outer membrane protein of Acinetobacter baumannii is the heat-modifiable protein HMP-AB, a porin with a large pore size allowing the penetration of solutes having a molecular weight of up to approximately 800 Da. Cross-linking experiments with glutardialdehyde failed to show any cross-linking between the monomers, a fact that proves again that this porin protein functions as a monomeric porin. The specific activity of this porin was found to be similar to that of other monomeric porins. Tryptic digestion of the outer membrane yielded a 23-kDa fragment of the HMP-AB protein that was resistant to further trypsin treatment. This observation indicates that HMP-AB is assembled in the membrane in a manner similar to monomeric porins. Cloning of the HMP-AB gene revealed an open reading frame of 1038 bp encoding a protein of 346 amino acids and a calculated molecular mass of 35,636 Da. The amino acid sequence and composition were typical of Gram-negative bacterial porins: a highly negative hydropathy index, absence of hydrophobic residue stretches, a slightly negative total charge, low instability index, high glycine content, and an absence of cysteine residues. Sequence comparison of HMP-AB with other outer membrane proteins revealed a clear homology with the monomeric outer membrane proteins, outer membrane protein A (OmpA) of Enterobacteria, and outer membrane protein F (OprF) of Pseudomonas sp. Secondary structure analysis indicated that HMP-AB has a 172-amino acid N-terminal domain that spans the outer membrane by eight amphiphilic beta strands and a C-terminal domain that apparently serves as an anchoring protein to the peptidoglycan layer. The results also indicate that HMP-AB belongs to the eight transmembrane beta-strand family of outer membrane proteins.     

Molecular characterization of Lactobacillus casei strains

Abstract The monoclonal antibody LA7 was raised against the species-specific Borrelia burgdorferi lipoprotein P22 (= IPLA7), which induces antibody formation in patients with Lyme arthritis. It is composed of 194 amino acids with a calculated molecular mass of 21.8 kDa. Its gene on the linear chromosome is 582 nucleotides in length. The aim of this study was to localize the protein P22 by immune electron microscopy. Immunolabeling of Borrelia burgdorferi with LA7 and an anti-mouse immunogold conjugate proved that P22 is an outer membrane protein. This finding was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis of the outer envelope fraction, which contained 99% of the P22 proteins.     

Pleiotropic effects of inactivating a carboxyl-terminal protease, CtpA, in Borrelia burgdorferi

A gene encoding a putative carboxyl-terminal protease (CtpA), an unusual type of protease, is present in the Borrelia burgdorferi B31 genome. The B. burgdorferi CtpA amino acid sequence exhibits similarities to the sequences of the CtpA enzymes of the cyanobacterium Synechocystis sp. strain PCC 6803 and higher plants and also exhibits similarities to the sequences of putative CtpA proteins in other bacterial species. Here, we studied the effect of ctpA gene inactivation on the B. burgdorferi protein expression profile. Total B. burgdorferi proteins were separated by two-dimensional gel electrophoresis, and the results revealed that six proteins of the wild type were not detected in the ctpA mutant and that nine proteins observed in the ctpA mutant were undetectable in the wild type. Immunoblot analysis showed that the integral outer membrane protein P13 was larger and had a more acidic pI in the ctpA mutant, which is consistent with the theoretical change in pI for P13 not processed at the carboxyl terminus. Matrix-assisted laser desorption ionization-time of flight data indicated that in addition to P13, the BB0323 protein may serve as a substrate for carboxyl-terminal processing by CtpA. Complementation analysis of the ctpA mutant provided strong evidence that the observed effect on proteins depended on inactivation of the ctpA gene alone. We show that CtpA in B. burgdorferi is involved in the processing of proteins such as P13 and BB0323 and that inactivation of ctpA has a pleiotropic effect on borrelial protein synthesis. To our knowledge, this is the first analysis of both a CtpA protease and different substrate proteins in a pathogenic bacterium.     

Expression, characterization, and crystallization of the pyrophosphate-dependent phosphofructo-1-kinase of Borrelia burgdorferi.

The two genes for the putative pyrophosphate-dependent phosphofructokinases (PPi-PFKs) of Borrelia burgdorferi were cloned by PCR and expressed in Escherichia coli, and their protein products were purified to near homogeneity. The larger of the two gene products, a 62-kDa protein, is an active PPi-PFK and exists in solution as a dimer. It has apparent K(m) values for fructose 6-P and PPi of 109 and 15 microM, respectively, and a pH optimum of 6.4 to 7.2. The 62-kDa protein was crystallized and subjected to preliminary diffraction analysis. The smaller gene product, a 48-kDa protein, exists in solution as a higher polymer and shows no ATP- or PPi-dependent activity, despite having a secondary structure as estimated by circular dichroism that is not significantly different from that of other PFKs.     

Identification of a candidate glycosaminoglycan-binding adhesin of the Lyme disease spirochete Borrelia burgdorferi

Binding of glycosaminoglycans (GAGs) by Borrelia burgdorferi, the Lyme disease spirochete, has the potential to promote the colonization of diverse tissues. GAG binding by B. burgdorferi is associated with haemagglutination and we have identified a 26 kDa protein, which we have termed Bgp (Borrelia GAG-binding protein), on the basis of its ability to bind to heparin and erythrocytes. Bgp was found in outer membrane fractions of B. burgdorferi and on the surface of intact bacteria, as assayed by labelling with a membrane-impermeable biotinylating agent or anti-Bgp antibodies. Purified recombinant Bgp agglutinated erythrocytes, binds to the same spectrum of GAGs as the B. burgdorferi strain from which the cloned bgp sequence was obtained, and inhibited B. burgdorferi binding to purified GAGs and to cultured mammalian cells. Thus, Bgp is a strong candidate for a GAG-binding adhesin of B. burgdorferi.     

Influence of Culture Conditions on Expression of the 40-Kilodalton Porin Protein of Vibrio anguillarum Serotype O2

Vibrio anguillarum serotype O2 strains express a 40-kDa outer membrane porin protein. Immunoblot analysis revealed that antigenic determinants of the V. anguillarum O2 40-kDa porin were conserved within bacterial species of the genus Vibrio. The relative amounts of the V. anguillarum O2 40-kDa porin were enhanced by growth of V. anguillarum O2 in CM9 medium containing 5 to 10% sucrose or 0.1 to 0.5 M NaCl. In contrast, the levels of the porin were significantly reduced when cells were grown at 37°C, and a novel 60-kDa protein was also observed. However, the osmolarity or ionic concentration of the growth medium did not influence expression of the 60-kDa protein. Growth in medium containing greater than 0.6 mM EDTA reduced production of the V. anguillarum O2 40-kDa porin and enhanced levels of a novel 19-kDa protein. Thus, expression of the V. anguillarum O2 40-kDa porin was osmoregulated and possibly coregulated by temperature. The N-terminal amino acid sequence of the V. anguillarum O2 40-kDa protein and the effect of environmental factors on the cellular levels of the porin suggested that the V. anguillarum O2 40-kDa porin was functionally similar to the OmpC porin of Escherichia coli. However, pore conductance assays revealed that the V. anguillarum O2 40-kDa porin was a general diffusion porin with a pore size in the range of that of the OmpF porin of E. coli.     

Characterization of an immunoreactive 93-kDa core protein of Borrelia burgdorferi with a human IgG monoclonal antibody

Lyme borreliosis is an infectious disease caused by the tick-borne spirochete Borrelia burgdorferi, which carries the potential for chronic infection. Ag on the etiologic Borrelia are currently being defined structurally and their ability to elicit immune responses delineated. EBV can be used to immortalize human B. burgdorferi-specific B cells from infected donors and generate antibodies against antigenic epitopes encountered in natural infection. A human mAb secreting EBV-transformed B cell line, D7, has been developed that is specific for a 93-kDa B. burgdorferi protein and has been used to characterize this potentially important Ag. D7 produces an IgG3 antibody that detects the 93-kDa Ag as well as smaller fragments at 46 kDa and lower molecular mass. The antibody detects similar epitopes on all B. burgdorferi isolates tested and on a Borrelia hermsii protein with molecular mass greater than 100 kDa but binds poorly to Treponema species. In contrast, polyclonal sera from Lyme disease patients show little binding to the homologous Ag in B. hermsii. Structurally, the 93-kDa protein is associated with the flagellum and may be firmly anchored in the protoplasmic cylinder. It is not solubilized by nonionic detergent treatment of the whole Borrelia. Antibodies against a comparable m.w. protein are present in sera from patients with both early and late infection. Thus, antibodies against this Ag are a sensitive and specific marker of Borrelia infection. This Ag is likely of structural importance and may represent a target of host defenses.