Periaqueductal gray calcitonin gene-related peptide modulates trigeminovascular neurons.

BACKGROUND: Calcitonin gene-related peptide (CGRP) receptor antagonism is an approach to migraine therapy. The locus of action of antimigraine treatment is not resolved. The objective was to investigate CGRP receptors in the ventrolateral periaqueductal gray (vlPAG) involved in the modulation of trigeminovascular nociception by descending influences on neurotransmission. METHODS: The presence of calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1), which form functional CGRP receptors, was investigated. CGRP and its receptor antagonists, olcegepant and CGRP (8-37), were microinjected into the vlPAG while changes of neural responses in the trigeminocervical complex (TCC) were monitored. RESULTS: Immunoreactivity indicated the presence of functional CGRP receptor components in the vlPAG and adjacent mesencephalic trigeminal nucleus. Inhibition of TCC responses to stimulation of dural afferents and ophthalmic cutaneous receptive fields after microinjection of bicuculline into vlPAG indicated a connection between the vlPAG and TCC neurons. CGRP facilitated these TCC responses, whereas olcegepant and CGRP (8-37) decreased them. CONCLUSIONS: CGRP and its receptor antagonists act on neurons in the region of vlPAG to influence nociceptive transmission in the TCC. This suggests CGRP receptor antagonists may act at loci outside of the TCC and reinforces the concept of migraine as a disorder of the brain.

Comparative genomics of Listeria species.

Listeria monocytogenes is a food-borne pathogen with a high mortality rate that has also emerged as a paradigm for intracellular parasitism. We present and compare the genome sequences of L. monocytogenes (2,944,528 base pairs) and a nonpathogenic species, L. innocua (3,011,209 base pairs). We found a large number of predicted genes encoding surface and secreted proteins, transporters, and transcriptional regulators, consistent with the ability of both species to adapt to diverse environments. The presence of 270 L. monocytogenes and 149 L. innocua strain-specific genes (clustered in 100 and 63 islets, respectively) suggests that virulence in Listeria results from multiple gene acquisition and deletion events.

Effect of point mutations on the in-vitro pore properties of maltoporin, a protein of Escherichia coli outer membrane.

Maltoporin (LamB protein), a protein of Escherichia coli outer membrane forms ionic channels with a selectivity for maltose and maltodextrins (Dargent et al., 1987). The effect of different point mutations on maltoporin pore properties was investigated in vitro with planar bilayers. The mutations belong to three classes in terms of selective maltose transport in vivo: class A (substitution at positions 259 and 382) does not affect maltose transport, class B (position 163 and 245) decreases maltose transport down to 20 to 30%, and class C (position 18) almost completely abolishes selective maltose transport. This in-vitro study reveals that class A does not affect the pore properties in contrast to class B substitutions. The class B maltoporins are still able to form channels but display some specific features and altered specificity for maltose and maltodextrins. The substitution (Gly18----Val) alters trimer stability and impedes pore function (class C mutant). Thus, there is a good correlation between the specific transport properties of the mutated maltoporins in vivo and their behavior in vitro. These data, in combination with the asymmetric orientation of the protein within the bilayer and topological considerations, indicate that residues 245 and 163 do not belong to the selectivity filter. Mutations at these sites cause hindrance at the mouth of the pore on the outer domain of maltoporin.

Further sequence analysis of the phage lambda receptor site. Possible implications for the organization of the lamB protein in Escherichia coli K12.

We present the DNA sequence alterations due to seven lamB missense mutations yielding resistance to phages lambda and K10. They reveal five different amino acid positions in the LamB protein. Three positions (245, 247 and 249) define a new region required for phage adsorption. The two other positions (148 and 152) belong to a region where mutations to phage resistance has already been detected. These two regions are hydrophilic and could belong to turns of the protein located at the surface of the cell. All the missense mutational alterations to phage resistance sequenced in the LamB protein correspond to 10 sites located in four different segments of the polypeptide chain. We discuss their location in terms of the notion of phage receptor site and of a working model for the organization of this protein in the outer membrane of Escherichia coli.

malM, a new gene of the maltose regulon in Escherichia coli K12. I. malM is the last gene of the malK-lamB operon and encodes a periplasmic protein.

The structure and expression of the distal part of the malK-lamB operon in Escherichia coli was studied. DNA sequencing was performed as far as a HinfI restriction site located 1313 base-pairs downstream from gene lamB. The open reading frame, formerly called molA, which begins 245 base-pairs downstream from gene lamB, is longer than was initially thought, and was renamed malM. It could encode a protein of 306 amino acid residues. The complete malM open reading frame was cloned under control of the tac 12 promoter. In maxicells, the resulting plasmid permitted tac12-promoted synthesis of two polypeptides, encoded by gene malM, with apparent molecular weights of 37 X 10(3) and 34.5 X 10(3). We provide strong evidence that the 34.5 X 10(3) Mr protein is derived from the 37 X 10(3) Mr protein by processing at the amino-terminal end, and that this processed form is located in the periplasmic space. We show that the chromosomal malM gene is expressed as part of the malK-lamB operon, and that its product is periplasmic. Finally, we demonstrate with nuclease S1 mapping experiments that the mRNA terminates at a typical rho-independent terminator located about 45 base-pairs beyond the end of gene malM, which is thus the last gene of the malK-lamB operon.

Maltose transport and starch binding in phage-resistant point mutants of maltoporin. Functional and topological implications.

The relationships between the bacteriophage lambda binding site, the starch binding site and the pore formed by maltoporin (LamB protein, lambda receptor protein) were investigated. Bacteria with single amino acid substitutions in the maltoporin sequence, which were previously shown to be strongly reduced in phage lambda sensitivity, were assayed for maltose- (and maltodextrin) selective pore functions. Maltose transport assays was performed at low substrate concentrations, under conditions where LamB is limiting for transport. It revealed three classes of mutants. Class A is composed of mutants with no effect on transport (substitutions at amino acid residues 154, 155, 259, 382 and 401); class B corresponds to mutants with a significant but variable reduction in transport (sites 148, 151, 152, 163, 164, 245, 247 and 250); class C is represented by a single mutant for which transport is almost completely abolished (site 18). Starch binding was assayed by two different methods that gave compatible results. In class A mutants, binding was normal, while no binding was observed in the class C mutant. Binding was impaired to various extents in category B mutants. There was a correlation between the level of impairment of starch binding and impairment of maltose transport, consistent with the notion that the residues influencing starch binding are inside, or in close proximity to, the pore. These results, together with previous data on starch-binding mutants that were not affected in phage binding (substitutions at residues 8, 74, 82, 118 and 121), suggest that the binding sites for starch and phage lambda overlap but are distinct. Mutations affecting transport and starch binding are located in the first third of the protein and in the region of residues 245 to 250. Mutations affecting phage adsorption are located mainly in the last two-thirds of the protein. The topological constraints suggested by the results with the available mutants altered in the lamB gene were used to propose a revised model of maltoporin folding across the outer membrane as well as to define the outlines of footprints of macromolecular binding sites (phage, starch and monoclonal antibodies) on the surface of the protein.

Nanopores: maltoporin channel as a sensor for maltodextrin and lambda-phage.

BACKGROUND: To harvest nutrition from the outside bacteria e.g. E. coli developed in the outer cell wall a number of sophisticated channels called porins. One of them, maltoporin, is a passive specific channel for the maltodextrin uptake. This channel was also named LamB as the bacterial virus phage Lambda mis-uses this channel to recognise the bacteria. The first step is a reversible binding followed after a lag phase by DNA injection. To date little is known about the binding capacity and less on the DNA injection mechanism. To elucidate the mechanism and to show the sensitivity of our method we reconstituted maltoporin in planar lipid membranes. Application of an external transmembrane electric field causes an ion current across the channel. Maltoporin channel diameter is around a few Angstroem. At this size the ion current is extremely sensitive to any modification of the channels surface. Protein conformational changes, substrate binding etc will cause fluctuations reflecting the molecular interactions with the channel wall. The recent improvement in ion current fluctuation analysis allows now studying the interaction of solutes with the channel on a single molecular level. RESULTS: We could demonstrate the asymmetry of the bacterial phage Lambda binding to its natural receptor maltoporin. CONCLUSION: We suggest that this type of measurement can be used as a new type of biosensors.

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.

Immunogenicity and antigenicity of conserved peptides from the envelope of HIV-1 expressed at the surface of recombinant bacteria.

We expressed peptides from the HIV-1 envelope protein at the surface of Escherichia coli by genetic insertions into an exposed loop of the outer membrane protein LamB. Recombinant bacteria expressing eight peptides from gp110 (pep1-pep8), conserved between HIV-1 and HIV-2, were used as live immunogens in rabbits by the intravenous route. The eight constructions elicited anti-LamB antibodies, showing that the hybrid proteins were immunogenic. One of them, LamB-pep8, gave rise to antibodies able to react with gp160 and to neutralize HIV-1 in vitro. We also show that this type of recombinant E. coli can provide a convenient reagent to monitor and characterize specific antibodies. Recombinant clones were used to test sera of seropositive individuals, as well as to narrow down the monoclonal antibody 110-1 recognition site to a cluster of eight residues at the carboxy-terminal end of gp110.

Versatility of a vector for expressing foreign polypeptides at the surface of gram-negative bacteria.

A wide variety of peptides in terms of length and sequence can be expressed at the surface of the bacterium Escherichia coli by genetic insertion into a 'permissive' site of the outer membrane protein LamB, used as a carrier. The resulting hybrid proteins essentially keep their biological activities with inserts of up to about 60 amino acid residues, and of a large range of predicted structures or hydrophobicities. This reflects a remarkable flexibility in the organization of the protein, but also in the export machinery. The method used to select such a permissive site is quite general and its potential to generate applications, including a versatile type of live bacterial vaccine, are discussed.

Creation of targets for proteolytic cleavage in the LamB protein of E coli K12 by genetic insertion of foreign sequences: implications for topological studies.

LamB, an integral outer membrane protein of E coli K12, is highly resistant to protease digestion. We had previously genetically inserted a foreign sequence corresponding to an epitope from the poliovirus next to amino acids 146, 153, 189, and 374 of LamB. In 3 cases (sites 146, 153, 374), insertion of the foreign peptide did not extensively affect the functions of LamB (and therefore folding). In 2 cases (sites 146 and 374) the polio virus epitope was detectable on the bacterial surface with a specific monoclonal antibody. We show here that the 4 modified proteins are sensitive to trypsin, including on intact cells. The sizes of the major cleavage products is that expected for proteolysis at or near the sequences inserted. In 1 case (site 153), this was directly demonstrated by protein sequencing. The results confirm the cell surface exposure of the regions of residues 153 and 374 and provide information on the regions around residues 146 and 189. Perspectives and limitations of this approach for fine studies on the mode of insertion of membrane proteins are briefly discussed.

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.

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.

Cloning of the J gene of bacteriophage lambda, expression and solubilization of the J protein: first in vitro studies on the interactions between J and LamB, its cell surface receptor.

Bacteriophage lambda adsorbs to its Escherichia coli K12 host by interacting with a specific cell surface receptor, the outer membrane protein LamB. Previous genetic analyses led us to define a set of residues at the surface of LamB, which belong to the lambda receptor site. Further genetic studies indicated that the C-terminal portion of J, the tail fibre protein of lambda, was directly involved in the recognition of the receptor site. The present work describe first in vitro studies on the interactions between J and LamB. The J gene of lambda was cloned into a plasmid vector under ptac promoter control and expressed in E. coli. We showed that J could be expressed at high levels (up to 28% of whole cell proteins), in an insoluble form. Anti-J antibodies, induced in rabbits immunized with insoluble J extracts, appeared to specifically neutralize lambda infection. Under defined conditions of extraction, the J protein was obtained in a soluble form. We showed that solubilized J was able to interact with LamB trimers in vitro. Implications for future studies on the interactions between LamB and J are discussed.

Studies of the anaerobically induced promoter pnirB and the improved expression of bacterial antigens.

The promoter of the Escherichia coli gene nirB is induced by both the presence of nitrite in the environment and by low oxygen tensions. It has been used to direct the high-level expression of heterologous proteins by E. coli strains in fermentors, and attenuated Salmonella strains expressing foreign proteins under nirB promoter (pnir) control have efficiently induced an immune response against these proteins. The genes encoding two different E. coli envelope proteins, the outer membrane protein LamB and the periplasmic protein MalE, were placed under pnir control on pBR322 derivatives, and both proteins were expressed at high levels during anaerobic growth. Our results showed that the expression level of MalE was influenced by the distance between the pnir promoter and the Shine-Dalgarno sequence: the highest levels were obtained by the longest constructs made; pnir directed a 4-fold increase in the level of MalE expression relative to the level reached by the previously described ptac-MalE expression vector. The best pnir construct produced 25 mg of MalE protein per 5 x 10(11) bacteria, which represents over 20% of total cell protein. Overexpression of MalE was well tolerated by E. coli, even under strict anaerobic conditions; for LamB, optimal induction was achieved under partial anaerobiosis. A MalE-HIV1 hybrid protein (33 residues from the V3 loop of HIV1 gp160 inserted into site 133 of MalE) was also overexpressed at a similar yield under pnir control, without apparent degradation of the hybrid protein. Moreover, when expressed in attenuated aroA S. typhimurium strain SL3261, the plasmids carrying malE and malE-HIV genes were stable in vitro and in vivo.

Further genetic analysis of the C-terminal external loop region in Escherichia coli maltoporin.

LamB specifically facilitates the diffusion of maltose and maltodextrins through the bacterial outer membrane, and acts as a general (i.e. non-specific) porin for small hydrophilic molecules (< 600 daltons). We reported previously that deletion of the last predicted external domain near the C-terminus of the Eschirichia coli LamB protein (residues 376 to 405), affected in vivo the binding and transport of maltodextrins (specific pore functions), and also increased bacterial sensitivity to large antibiotics. The residues covered by this deletion correspond almost exactly to the major cell surface loop of LamB on the structural model based on X-ray crystallography (loop L9, residues 375 to 405). The L9 loop comprises a large central portion, which varies in size and sequence between the LamB proteins from different species. This variable region is flanked by two highly charged and conserved portions, which overlap with the adjacent beta strands. To identify subregions in L9 that influence the pore properties of LamB, we constructed and analysed nine mutants in loop L9 and its flanking sequences. Deletion of the 23-amino-acids central variable portion of the loop (residues 379 to 401), and deletion of the downstream conserved region (residues 402 to 409), only moderately affected specific maltoporin function. In contrast, deletion of the conserved region (residues 372 to 378) upstream of the variable portion strongly decreased specific maltoporin function and also increased sensitivity to large antibiotics, accounting for most, if not all, of the effects of the complete deletion of L9.

Immunogenicity of foreign peptide epitopes expressed in bacterial envelope proteins.

We have used two bacterial proteins from Escherichia coli to express heterologous peptides. Both proteins are situated in the E. coli cell envelope but have different properties: LamB is an integral outer membrane protein, and MalE a soluble periplasmic protein. The peptides were expressed as genetic inserts within "permissive sites" of these recipient proteins, i.e. sites which allow the insertion of foreign peptides without affecting the biological properties of the host protein. In this paper, we summarize preliminary rules governing the immunogenicity of resulting LamB and MalE hybrid proteins when expressed in E. coli. We focus on two model epitopes: either peptide 132-145 from the preS(2) region of hepatitis B virus or peptide 93-103 from poliovirus VP1 capsid protein. We also present first results obtained when the same hybrid proteins were expressed in attenuated Salmonella typhimurium. Plasmids encoding the hybrid proteins were transferred to aroA S.typhimurium by electroporation. In vitro, the hybrid proteins could be expressed at high levels by S. typhimurium. Mice were immunized by parenteral and oral routes. The effect of the carrier protein and the level of its expression on the in vivo behaviour of the immunizing bacteria and on the immune response induced will be discussed.

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).

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.