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A Charbit

Publications and source records attributed to A Charbit.

At least 37 records · Page 2Linked to original sources
PubMed1994

A model of maltodextrin transport through the sugar-specific porin, LamB, based on deletion analysis.

LamB facilitates the uptake of maltose and maltodextrins across the bacterial outer membrane and acts as a general porin for small molecules. Using directed deletion mutagenesis we removed several regions of the LamB polypeptide and identified a polypeptide loop that both constricts the maltoporin channel and binds maltodextrins. In conjunction with a second sugar binding site that we identified at the rim of the channel, these data clarify, for the first time, the mechanism of transport through a substrate-specific porin. Furthermore, unlike the transverse loops of general porins, which originate from a central location in their primary structure, the loop that regulates LamB permeability originates from a C-terminal site. Thus LamB represents a second distinct class of porins in the bacterial outer membrane that is differently organized and separately evolved from OmpF-type, general porins.

Amino Acid Sequence↗
PubMed1994

Genetic control of antibody responses induced against an antigen delivered by recombinant attenuated Salmonella typhimurium.

Recombinant derivatives of nonpathogenic bacteria such as attenuated Salmonella typhi have the potential to be used for delivery of heterologous antigens to the immune system. Genetic factors may modulate the immune responses to these live attenuated organisms and could therefore modify the immunogenicity of future human vaccines. In the present study, we compared the antibody responses of Ity or H-2 congenic strains of mice to a foreign antigen expressed by the murine attenuated aroA S. typhimurium strain. Our results demonstrate that the Ity gene may modulate the antibody responses to the foreign antigen but that the major genetic influence is exerted by H-2 genes, which control the capacity of mice to respond to the antigen expressed by recombinant attenuated Salmonella cells. This genetic control is related to differences in responsiveness of different strains of mice to low doses of antigen. Increasing the amount of foreign antigen expressed by recombinant Salmonella cells overcame the genetic restriction of these responses. These findings are potentially of great importance for the design of live vaccines for humans and show that care must be taken to optimize the amount of foreign antigen delivered to the immune system.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1994

A role for residue 151 of LamB in bacteriophage lambda adsorption: possible steric effect of amino acid substitutions.

LamB is the cell surface receptor for bacteriophage lambda. LamB missense mutations yielding resistance to lambda have been previously grouped in two classes. Class I mutants block growth of lambda with wild-type host range (lambda h+) but support growth of one-step extended-host-range mutants (lambda h). Class II mutants block lambda h but support growth of two-step extended host range mutants (lambda hh*). While Class I mutations occur at 11 different amino acid sites, in five distinct portions of LamB, all the Class II mutations analyzed previously correspond to the same G-to-D change at amino acid 151. We generated by in vitro mutagenesis four different new substitutions at site 151 (to S, V, R, and C). Two of the mutants (G-151-->V [G151V] and G151R) were of Class II, while the two others (G151S and G151C) were of Class I, demonstrating that not only the site but also the nature of the substitutions at residue 151 was critical for the phage sensitivity phenotypes. The introduction of a negatively charged, a positively charged, or an aliphatic nonpolar residue at site 151 of LamB prevented both lambda h+ and lambda h adsorption, indicating that the block is not due to a charge effect. In contrast to G151D, which was sensitive to all the lambda hh* phages, G151V and G151R conferred sensitivity to only four of the five lambda hh* phages. Thus, G151V and G151R represent a new subclass of Class II LamB mutations that is more restrictive with respect to the growth of lambda hh*. Our results agree with the hypothesis that residue 151 belongs to an accessibility gate controlling the access to the phage tight-binding site and that substitutions at this residue affect the access of the phage to the binding site in relation to the size of the substitute side chain (surface area): the most restrictive changes are G151V and G151R, followed to a lesser extent by G151D and they by G151S and G151C.

Bacterial Outer Membrane Proteins↗
PubMed1994

Adsorption of bacteriophage lambda on the LamB protein of Escherichia coli K-12: point mutations in gene J of lambda responsible for extended host range.

LamB is the cell surface receptor for bacteriophage lambda. LamB missense mutations yielding resistance to lambda group in two classes. Class I mutants block the growth of lambda with the wild-type host range (lambda h+) but support the growth of one-step host range mutants (lambda h). Class II mutants block lambda h but support the growth of two-step host range mutant (lambda hh*) phages. To identify amino acid residues in the J protein (the tail fiber of phage lambda) responsible for the extended host range phenotype of mutants of phage (lambda h+), we selected a series of one-step (lambda h) and two-step (lambda hh*) host range mutants and analyzed their corresponding J genes. Three different class I LamB missense mutants (mutations at sites 247, 245, and 148) were used to select 11 independent, new, one-step host range mutants (lambda h phages). DNA sequence analysis revealed a single-amino-acid change in each case. The 11 alterations affected only three residues in the distal part of J, corresponding to a Val-->Ala change at site 1077 in five cases, a Thr-->Met change at site 1040 in three cases, and a Leu-->Pro change at site 1127 in three cases. Recombination experiments confirmed that in the cases tested, the mutations identified were indeed responsible for the extended host range phenotype. The class II LamB mutant (Gly-->Asp at site 151) was used to select two-step extended host range mutants (lambda hh* phages) from three new lambda h phages, corresponding to different amino acid modifications in the J protein (at sites 1040, 1077, and 1127). The new lambda hh* phages analyzed corresponded to either double or triple point mutations located at the distal end of the J protein. In all, seven residues involved in the extended host range properties of lambda mutants were identified in the distal part of the J protein, suggesting that the last C-terminal portion of the J protein participates directly in the adsorption of the phage onto LamB. In agreement with the fact that the lambda h mutants (and the lambda hh* mutants) could grow on all of the lamB class I mutations tested, we found tha the nature of the J mutations did not depend on the LamB class I mutant used to select them. This is interpreted as meaning that the mutated residues in the J protein and in the LamB mutants are not involved in allele-specific protein-protein interactions. Rather, the LamB mutations would block a step in phage adsorption, and this block would be overcome by the mutations in the J protein.

Adsorption↗
PubMed1994

Immunogenicity of viral B- and T-cell epitopes expressed in recombinant bacterial proteins.

Foreign polypeptides can be expressed as genetic inserts in several permissive sites of MalE and LamB, two Escherichia coli envelope proteins. Several viral B and T-cell epitopes have been inserted in these proteins and we analyzed the role of the molecular environment on the immunogenicity of the foreign epitopes. These studies demonstrated that the antigenicity and immunogenicity of B-cell epitopes depend on their site of insertion in the carrier protein. Using bacteria expressing B-cell epitopes either at the cell surface or in the periplasm, it was also shown that the cellular location of a foreign B-cell epitope expressed by recombinant bacteria determines its T-cell dependent or independent characteristics. Analysis of in vivo immunogenicity of purified LamB or MalE hybrid proteins expressing two different T-cell epitopes established that the immunogenicity of recombinant T-cell epitopes may be strongly affected by both the insertion site and inserted adjacent residues. The in vitro analysis of specific T-cell hybridoma response to hybrid MalE proteins also showed that the molecular context of a T-cell determinant alters the diversity of its T-cell recognition.

ATP-Binding Cassette Transporters↗
PubMed1994

Molecular and cellular parameters controlling the immunogenicity of foreign B- or T-cell epitopes expressed by recombinant vectors.

Extensive work is being performed to develop live recombinant bacterial vaccines. The use of non-pathogenic bacteria or attenuated strains derived from pathogens may allow protection against the pathogen and at the same time induce immunity against one or several foreign antigens expressed by the recombinant micro-organism. Several bacteria such as attenuated Salmonella or BCG have been used successfully in several experimental models to induce protective immune responses against several pathogens. However, the presentation to the immune system of a foreign antigen in a context different from the natural one may greatly modify the characteristics of elicited immune responses. It is therefore of the utmost importance to establish rules concerning the influence of the vector on the immunogenicity of recombinant antigens. Using a bacterial system that allows the expression of genetically engineered hybrid proteins, we have analysed: 1) the role of the molecular environment on the immunogenicity of foreign B- or T-cell epitopes; 2) the influence of the cellular location of a foreign B-cell epitope on the induction of specific immune responses; 3) the role of the bacterial vector (E. coli or Salmonella typhimurium) on the isotypic characteristic of antibody responses induced against the recombinant antigen. These studies revealed the complexity of the mechanisms which control the immunogenicity of foreign B- or T-cell epitopes expressed by recombinant vectors.

ATP-Binding Cassette Transporters↗
PubMed1993

Immunodominance of a recombinant T-cell epitope depends on its molecular environment.

In the present study, we have investigated the influence of the molecular environment of a T-cell epitope on its immunogenicity. We genetically inserted into different sites of two bacterial recipient proteins, LamB or MalE, an immunodominant T-cell epitope: the 120-132 T-cell epitope from the PreS2 region of HBV. The T-cell epitope was introduced, either alone (PreS:T) or with an adjacent B-cell epitope (PreS:TB). After purification, the hybrid proteins were injected into mice and we studied the immunogenicity of recombinant T-cell epitopes by analyzing the in vitro proliferative responses of LN cells from these mice to the inserted peptides. The immunization of mice with recombinant MalE protein containing the PreS:T or PreS:TB peptides at two different sites induced strong peptide-specific proliferative responses, indicating that the insertion sites did not affect the immunodominance of the inserted T-cell epitope. A strong T-cell proliferative response was also obtained after immunization of mice with hybrid LamB protein containing the PreS:TB epitope at position 153. In contrast, the recombinant proteins which contained only the PreS:T epitope at positions 153 or 374 failed to stimulate T-cell responses. Therefore, this study demonstrates that the immunogenicity of recombinant T-cell epitopes may be strongly affected both by the insertion site and by inserted adjacent residues.

Amino Acid Sequence↗
PubMed1993

Immune responses to hybrid maltose-binding proteins.

The Escherichia coli maltose-binding protein is a highly versatile carrier protein allowing the construction of genetically engineered hybrid proteins. It accepts large fusions to both C- and N-termini as well as the insertion of shorter peptides at 'permissive sites' within the continuity of the protein. We have genetically inserted immunogenic peptides corresponding to defined viral B- and T-cell epitopes into two permissive sites: one at amino acid site 133, the other at site 303. The hybrid proteins are easily purifiable and immunogenic, inducing peptide-specific B- and T-cell responses. When delivered by live bacteria (E. coli K12 and aroA Salmonella typhimurium) antibody responses can be induced against both the MalE carrier and the inserted B-cell epitope. We discuss the induction of T-cell responses by bacterial delivery systems.

ATP-Binding Cassette Transporters↗
PubMed1993

Expression and immunogenicity of the V3 loop from the envelope of human immunodeficiency virus type 1 in an attenuated aroA strain of Salmonella typhimurium upon genetic coupling to two Escherichia coli carrier proteins.

A peptide comprising residues glu293 to ser334 from the principal neutralization determinant (V3 loop) of the envelope of human immunodeficiency virus type 1 (HIV1 LAVBRU isolate) has been inserted within internal permissive sites of either LamB or MalE, two envelope proteins from Escherichia coli K12. The MalE hybrid protein (MalE133-V3 loop) was stably expressed in the periplasm of Escherichia coli K12, and the V3 loop peptide was detectable on the surface of the native protein by an anti-gp160 monoclonal antibody (mAb 110-A). The disulfide bridge between the two cysteines of the loop was formed. In contrast, genetic coupling to the outer membrane protein LamB did not allow the expression of a stable hybrid protein, and major proteolytic cleavage products of the LamB153-V3 loop were detected by mAb 110-A. The two plasmid-encoded hybrid genes were transferred to an aroA mutant of Salmonella typhimurium. Constitutive expression of the MalE133-V3 loop had no detectable effect on cell growth and on the survival in vivo of the recipient strain. The LamB153-V3 loop was not stably expressed in Salmonella, either in vitro or in vivo. Live recombinant salmonellas expressing MalE-V3 and LamB-V3 loop hybrids were used to immunize mice. The MalE-V3 loop hybrid induced anti-HIV1 envelope antibodies detectable by Western blot and ELISA, while the anti-HIV1 envelope antibodies induced by the LamB-V3 loop hybrid were only detectable by Western blot. In addition, purified MalE-V3 loop hybrid protein was able to stimulate in vitro and induce in vivo a V3 loop-specific T-cell proliferative response.

AIDS Vaccines↗
PubMed1993

Construction and first characterization of two reciprocal hybrids between LamB from Escherichia coli K12 and Klebsiella pneumoniae.

The LamB proteins from Klebsiella pneumoniae and Escherichia coli K12 were previously shown to be highly homologous. The most conserved parts correspond to the N-proximal third and to the transmembranous portions of the molecule, while the variability occurred essentially within regions exposed to the cell surface or to the periplasm. Since the two proteins displayed identical in vitro trimer stability and in vivo pore properties, we tested whether the N-terminal parts of the two proteins could be exchanged and still allow the formation of stable and functional maltoporins. For that purpose, we expressed the LamB protein from K. pneumoniae in E. coli K12, and constructed two reciprocal hybrids between LamB from E. coli K12 and LamB from K. pneumoniae. The first hybrid (LamBE.c.-K.p.) is composed of residues 1 to 183 from LamBE.c. followed by residues 184 to 404 from LamBK.p. The second one comprises residues 1 to 183 from LamBK.p., followed by residues 184 to 421 from LamBE.c. (LamBK.p.-E.c.). Both hybrid proteins were correctly incorporated in the outer membrane of E. coli K12. Like the two parental LamB proteins, the two hybrids could be purified by affinity chromatography on a starch-sepharose column. The LamBE.c.-K.p. hybrid formed highly stable trimers, but was strongly impaired in its in vivo maltose transport function (15% of the wild-type level). The trimers formed by LamBK.p.-E.c. hybrid were less stable, but could be detected on the surface of intact cells by four anti-LamBE.c. monoclonal antibodies. This hybrid was also affected in its in vivo maltose transport function (30% of the wild-type level). As expected from the location of the residues critical for phage adsorption, both proteins had lost the phage receptor activity of the E. coli K12 LamB protein. We also examined whether LamBE.c. could form heterotrimers with LamBK.p., LamBK.p.-E.c., and LamBE.c.-K.p. In no case were heterotrimers detected, indicating that both terminal parts of the LamB protein are involved in homotrimer formation. All these data suggest that trimer formation and activity involve rare variable residues in the conserved regions and/or variable regions.

Bacterial Proteins↗
PubMed1993

Immunological relatedness of the LamB proteins among members of Enterobacteriaceae.

We have studied the immunological relatedness of LamB proteins from a wide range of enterobacterial species, using antibodies directed against denatured Escherichia coli K12 and Klebsiella pneumoniae LamB proteins (LamBE.c. and LamBK.p., respectively), and anti-peptide antibodies directed against 10 distinct loops of LamB from E. coli K12 predicted to protrude either side of the outer membrane. We have shown that a protein immunologically related to LamBE.c. and LamBK.p. was present in all members of Enterobacteriaceae tested. A protein recognized by several anti-peptide antibodies was identified in E. coli, Shigella sonnei, Salmonella typhimurium and Kleb. pneumoniae, as well as in two Citrobacter species, two Enterobacter species and Kluyvera ascorbata. The recognition patterns obtained with the anti-peptide antibodies were in agreement with the LamB protein sequence data available. They indicated that the cell surface and also the periplasmic loops of LamB are subject to great antigenic variability.

Amino Acid Sequence↗
PubMed1992

DNA sequence analysis of the lamB gene from Klebsiella pneumoniae: implications for the topology and the pore functions in maltoporin.

We have determined the sequence of the lamB gene from Klebsiella pneumoniae. It encodes the precursor to the LamB protein, a 429 amino acid polypeptide with maltoporin function. Comparison with the Escherichia coli LamB protein reveals a high degree of homology, with 325 residues strictly identical. The N-terminal third of the protein is the most conserved part of the molecule (1 change in the signal sequence, and 13 changes up to residue 146 of the mature protein). Differences between the two mature proteins are clustered mainly in six regions comprising residues 145-167, 173-187, 197-226, 237-300, 311-329, and 367-387 (K. pneumoniae LamB sequence). The most important changes were found in regions predicted by the two-dimensional model of LamB folding to form loops on the cell surface. In vivo maltose and maltodextrin transport properties of E. coli K12 and K. pneumoniae strains were identical. However, none of the E. coli K12 LamB-specific phages was able to plaque onto K. pneumoniae. Native K. pneumoniae LamB protein forms highly stable trimers. The protein could be purified by affinity chromatography on starch-Sepharose as efficiently as the E. coli K12 LamB protein, indicating a conservation of the binding site for dextrins. However, none of the monoclonal antibodies directed against native E. coli K12 LamB protein recognized native purified K. pneumoniae LamB protein. These data indicate that most of the variability occurs within exposed regions of the protein and provide additional support for the proposed model of LamB folding.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

The cellular location of a foreign B cell epitope expressed by recombinant bacteria determines its T cell-independent or T cell-dependent characteristics.

We have targeted two foreign B cell antigenic determinants to different locations in the Escherichia coli cell to examine what effect this had on antibody responses elicited by the recombinant bacteria. The two epitopes were the 132-145 peptide from the PreS2 region of hepatitis B virus and the C3 neutralization epitope of poliovirus type 1. They were each expressed in two forms either on the surface, as part of the outer-membrane protein LamB, or soluble in the periplasm, as part of the periplasmic protein MalE. When live bacteria expressing the foreign epitope at the cell surface were used for immunization of mice, they induced T cell-independent antibody responses characterized by a rapid induction of IgM and IgG antibodies. In contrast, when the same foreign epitope was inserted into the MalE protein, the antibody response was only detectable after 3 wk, belonged only to the IgG class and was strictly T cell dependent. This study has therefore identified two major pathways by which epitopes expressed by bacterial cells can stimulate specific antibody responses. The first pathway is mediated by direct activation of B cells by bacterial cell-surface Ag and does not require T cell help. The second pathway is T cell dependent and concerns Ag that can be released from the bacteria in a soluble form. We have also studied the effect of the exact position of the B cell antigenic determinant within the LamB protein and with respect to the outer membrane by comparing the immunogenicity of the PreS epitope inserted at three different permissive sites of LamB. The data indicated that to obtain an antibody response with intact bacteria, the epitope must be protruding sufficiently from the outside of the outer membrane. In contrast, when semipurified hybrid proteins were used as immunogen, the exact position of the B cell antigenic determinant within solubilized LamB protein does not influence its immunogenicity.

ATP-Binding Cassette Transporters↗
PubMed1991

A genetic system to elicit and monitor antipeptide antibodies without peptide synthesis.

We present a simple and flexible procedure to elicit and assay anti-peptide antibodies without peptide synthesis. It consists of expressing the peptide of interest in the form of a genetic insert within two different "recipient" bacterial proteins. One hybrid protein is used as immunogen for the induction of antibodies against the inserted peptide and the other as antigen for monitoring the anti-peptide antibodies raised. The two "recipient" proteins used are the MalE and the LamB proteins from E. coli. The MalE hybrid proteins can be affinity purified on an amylose column using mild nondenaturing conditions and can be crystalized for structural studies; LamB hybrid proteins express the inserted peptide on the cell surface so that intact bacteria can be used as a reagent. We chose, as a model peptide, a B-cell epitope from the pre-S(2) region of Hepatitis B virus. With both MalE and LamB hybrid proteins, high titres of anti-preS antibodies, able to react with native HBsAg particles, were induced in mice. The anti-peptide antibody titres recorded by ELISA were comparable to those obtained when either a synthetic peptide, or the hybrid proteins, were used as immobilized antigen.

Animals↗
PubMed1991

Permissive sites and topology of an outer membrane protein with a reporter epitope.

We are developing a genetic approach to study with a single antibody the folding and topology of LamB, an integral outer membrane protein from Escherichia coli K-12. This approach consists of inserting the same reporter foreign antigenic determinant (the C3 epitope from poliovirus) at different sites of LamB so that the resulting hybrid proteins have essentially kept the in vivo biological properties of LamB and therefore its cellular location and structure; the corresponding sites are called permissive sites. A specific monoclonal antibody can then be used to examine the position of the reporter epitope with respect to the protein and the membrane. We present an improved and efficient procedure that led us to identify eight new permissive sites in LamB. These sites appear to be distributed on both sides of the membrane. At one of them (after residue 253), the C3 epitope was detected on intact bacteria, providing the first direct argument for exposure of the corresponding LamB region at the cell surface. At this site as well as at four others (after residues 183, 219, 236, and 352), the C3 epitope could be detected with the C3 monoclonal antibody at the surface of the extracted trimeric LamB-C3 hybrid proteins. We provide a number of convergent arguments showing that the hybrid proteins are not strongly distorted with respect to the wild-type protein so that the conclusions drawn are also valid for this protein. These conclusions are essentially in agreement with the proposed folding model for the LamB protein. They agree, in particular, with the idea that regions 183 and 352 are exposed to the periplasm. In addition, they suggest that region 236 is buried at the external face of the outer membrane and that region 219 is exposed to the periplasm. Including the 3 sites previously determined, 11 permissive sites are now available in LamB, including 3 at the cell surface and most probably at least 3 in the periplasm. We discuss the nature of such sites, the generalization of this approach to other proteins, and possible applications.

Amino Acid Sequence↗
PubMed1991

Bacterial vectors to target and/or purify polypeptides: their use in immunological studies.

The construction of recombinant proteins by genetic engineering has opened new avenues in basic research (studies on protein organization, protein folding, immunogenicity of proteins, ...) and many different applications. Recombinant proteins which keep properties of both parental proteins are especially interesting. For example, if one protein--the vector protein--is targeted to a given cellular compartment, the other protein--the passenger--may be identically targeted. Also, if the vector protein can be purified by a simple affinity chromatographic procedure, this property may be extended to the passenger. The authors have developed a genetic procedure to detect "permissive" sites within potential vector proteins so that genetic fusion to these sites keep most or all biological properties of the vector. When they used LamB, an outer membrane protein from E. coli, foreign sequences could be expressed at the bacterial cell surface. This may lead to several types of applications: live bacterial vaccines, simple diagnostic tests, selection procedures for peptides with biological activity. When they used the MalE protein, a periplasmic maltose binding protein from E. coli, the passengers could be exported and purified in one-step high affinity chromatography in mild non-denaturing conditions. This led us to a simple preparation and purification scheme for the soluble part of the CD4 receptor for the Human Immunodeficiency Virus (HIV).

Bacterial Outer Membrane Proteins↗