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.

Neurobiology of migraine.

Migraine is a complex disorder of the brain whose mechanisms are only now being unraveled. It is common, disabling and economically costly. The pain suggests an important role of the nociceptive activation, or the perception of activation, of trigeminal cranial, particularly intracranial afferents. Moreover, the involvement of a multi-sensory disturbance that includes light, sound and smells, as well as nausea, suggests the problem may involve central modulation of afferent traffic more broadly. Brain imaging studies in migraine point to the importance of sub-cortical structures in the underlying pathophysiology of the disorder. Migraine may thus be considered an inherited dysfunction of sensory modulatory networks with the dominant disturbance affecting abnormal processing of essentially normal neural traffic.

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.

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.

Identification of a PEST-like motif in listeriolysin O required for phagosomal escape and for virulence in Listeria monocytogenes.

The hly-encoded listeriolysin O (LLO) is a major virulence factor secreted by the intracellular pathogen Listeria monocytogenes, which plays a crucial role in the escape of bacteria from the phagosomal compartment. Here, we identify a putative PEST sequence close to the N-terminus of LLO and focus on the role of this motif in the biological activities of LLO. Two LLO variants were constructed: a deletion mutant protein, lacking the 19 residues comprising this sequence (residues 32-50), and a recombinant protein of wild-type size, in which all the P, E, S or T residues within this motif have been substituted. The two mutant proteins were fully haemolytic and were secreted in culture supernatants of L. monocytogenes in quantities comparable with that of the wild-type protein. Strikingly, both mutants failed to restore virulence to a hly-negative strain in vivo. In vitro assays showed that L. monocytogenes expressing the LLO deletion mutant was strongly impaired in its ability to escape from the phagosomal vacuole and, subsequently, to divide in the cytosol of infected cells. This work reveals for the first time that the N-terminal portion of LLO plays an important role in the development of the infectious process of L. monocytogenes.

Identification of new genes involved in the virulence of Listeria monocytogenes by signature-tagged transposon mutagenesis.

Listeria monocytogenes is a gram-positive, facultative intracellular pathogen that can cause severe food-born infections in humans and animals. We have adapted signature-tagged transposon mutagenesis to L. monocytogenes to identify new genes involved in virulence in the murine model of infection. We used transposon Tn1545 carried on the integrative vector pAT113. Forty-eight tagged transposons were constructed and used to generate banks of L. monocytogenes mutants. Pools of 48 mutants were assembled, taking one mutant from each bank, injected into mice, and screened for those affected in their multiplication in the brains of infected animals. From 2,000 mutants tested, 18 were attenuated in vivo. The insertions harbored by these mutants led to the identification of 10 distinct loci, 7 of which corresponded to previously unknown genes. The properties of four loci involving putative cell wall components were further studied in vitro and in vivo. The data suggested that these components are involved in bacterial invasion and multiplication in the brain.

Listeriolysin O as a reporter to identify constitutive and in vivo-inducible promoters in the pathogen Listeria monocytogenes.

Listeria monocytogenes is a facultative intracellular gram-positive bacterium capable of growing in the cytoplasm of infected host cells. Bacterial escape from the phagosomal vacuole of infected cells is mainly mediated by the pore-forming hemolysin listeriolysin O (LLO) encoded by hly. LLO-negative mutants of L. monocytogenes are avirulent in the mouse model. We have developed a genetic system with hly as a reporter gene allowing the identification of both constitutive and in vivo-inducible promoters of this pathogen. Genomic libraries were created by randomly inserting L. monocytogenes chromosomal fragments upstream of the promoterless hly gene cloned into gram-positive and gram-negative shuttle vectors and expressed in an LLO-negative mutant strain. With this hly-based promoter trap system, combined with access to the L. monocytogenes genome database, we identified 20 in vitro-transcribed genes, including genes encoding (i) p60, a previously known virulence gene, (ii) a putative new hemolysin, and (iii) two proteins of the general protein secretion pathway. By using the hly-based system as an in vivo expression technology tool, nine in vivo-induced loci of L. monocytogenes were identified, including genes encoding (i) the previously known in vivo-inducible phosphatidylinositol phospholipase C and (ii) a putative N-acetylglucosamine epimerase, possibly involved in teichoic acid biosynthesis. The use of hly as a reporter is a simple and powerful alternative to classical methods for transcriptional analysis to monitor promoter activity in L. monocytogenes.

In vivo and in vitro studies of transmembrane beta-strand deletion, insertion or substitution mutants of the Escherichia coli K-12 maltoporin.

LamB of Escherichia coli K12, also called maltoporin, is an outer membrane protein, which specifically facilitates the diffusion of maltose and maltodextrin through the bacterial outer membrane. Each monomer is composed of an 18-stranded antiparallel beta-barrel. In the present work, on the basis of the known X-ray structure of LamB, the effects of modifications of the beta-barrel domain of maltoporin were studied in vivo and in vitro. We show that: (i) the substitution of the pair of strands beta13-beta14 of the E. coli maltoporin with the corresponding pair of strands from the functionally related maltoporin of Salmonella typhimurium yielded a protein active in vivo and in vitro; and (ii) the removal of one pair of beta-strands (deletion beta13-beta14) from the E. coli maltoporin, or its replacement by a pair of strands from the general porin OmpF of E. coli, leads to recombinant proteins that lost in vivo maltoporin activities but still kept channel formation and carbohydrate binding in vitro. We also inserted into deletion beta13-beta14 the portion of the E. coli LamB protein comprising strands beta13 to beta16. This resulted in a protein expected to have 20 beta-strands and which completely lost all LamB-specific activities in vivo and in vitro.

The C-terminal portion of the tail fiber protein of bacteriophage lambda is responsible for binding to LamB, its receptor at the surface of Escherichia coli K-12.

Bacteriophage lambda adsorbs to its Escherichia coli K-12 host by interacting with LamB, its cell-surface receptor. We fused C-terminal portions of J, the tail fiber protein of lambda, to maltose-binding protein. Solid-phase binding assays demonstrated that a purified fusion protein comprising only the last 249 residues of J could bind to LamB trimers and inhibited recognition by anti-LamB antibodies. Electron microscopy further demonstrated that the fusion protein could also bind to LamB at the surface of intact cells. This interaction prevented lambda adsorption but affected only partially maltose uptake.

In vivo and in vitro studies of major surface loop deletion mutants of the Escherichia coli K-12 maltoporin: contribution to maltose and maltooligosaccharide transport and binding.

The trimeric protein LamB of Escherichia coli K-12 (maltoporin) specifically facilitates the diffusion of maltose and maltooligosaccharides through the outer membrane. Each monomer consists of an 18-stranded antiparallel beta-barrel with nine surface loops (L1 to L9). The effects on transport and binding of the deletion of some of the surface loops or of combinations of several of them were studied in vivo and in vitro. In vivo, single-, DeltaL4, DeltaL5, DeltaL6, and double-loop deletions, DeltaL4 + DeltaL5 and DeltaL5 + DeltaL6, abolished maltoporin functions, but not the double deletion DeltaL4 + DeltaL6 and the triple deletion DeltaL4 + DeltaL5 + DeltaL6. While deletion of the central variable portion of loop L9 (DeltaL9v) affected maltoporin function only moderately, the combination of DeltaL9v with the double deletion of loops L4 and L6 (triple deletion DeltaL4 + DeltaL6 + DeltaL9v) strongly impaired maltoporin function and resulted in sensitivity to large hydrophilic antibiotics without change in channel size as measured in vitro. In vitro, the carbohydrate-binding properties of the different loop mutants were studied in titration experiments using the asymmetric and symmetric addition of the mutant porins and of the carbohydrates to one or both sides of the lipid bilayer membranes. The deletion of loop L9v alone (LamBDeltaL9v), of two loops L4 and L6 (LamBDeltaL4 + DeltaL6), of three loops L4, L5 and L6 (LamBDeltaL4 + DeltaL5 + DeltaL6) or of L4, L6 and L9v (LamBDeltaL4 + DeltaL6 + DeltaL9v) had relatively little influence on the carbohydrate-binding properties of the mutant channels, and they had approximately similar binding properties for carbohydrate addition to both sides compared with only one side. The deletion of one of the loops L4 (LamBDeltaL4) or L6 (LamBDeltaL6) resulted in an asymmetric carbohydrate binding. The in vivo and in vitro results, together with those of the purification across the starch column, suggest that maltooligosaccharides enter the LamB channel from the cell surface side with the non-reducing end in advance. The absence of some of the loops leads to obstruction of the channel from the outside, which results in a considerable difference in the on-rate of carbohydrate binding from the extracellular side compared with that from the periplasmic side.

Immunogenicity of viral B-cell epitopes inserted into two surface loops of the Escherichia coli K12 LamB protein and expressed in an attenuated aroA strain of Salmonella typhimurium.

We previously developed a general procedure which allows the genetic coupling of a chosen foreign linear epitope in different 'permissive' sites of a carrier protein. By using the outer membrane protein LamB of Escherichia coli K12 as a carrier, we were able to express a number of different foreign epitopes at the bacterial surface. In the present work, taking advantage of the recent determination of the crystal structure of LamB, we inserted two model B-cell epitopes i.e.--the C3 epitope from poliovirus (residues 93 to 103 of VP1) and the preS2 epitope from hepatitis B virus, (residues 132 to 145)--at the tip of the most distal and largest surface exposed region of LamB (after residues 386, into loop L9). We also used two previously constructed LamB hybrids, corresponding to the insertion of the C3B or preSB epitope into permissive site 153 (lying in the middle of the fourth surface loop of LamB), to construct two LamB proteins corresponding to the simultaneous insertion of the two different epitopes (with one epitope per site). The LamB hybrids were placed under the control of the anaerobically inducible pnirB promoter and expressed in a LamB-negative derivative of the aroA attenuated strain of S. typhimurium, SL3261. In vitro, the recombinant proteins were expressed at a high level (up to 10% of whole cell proteins) and in vivo the recombinant plasmids were stably maintained. For both epitopes, genetic coupling at site 386 appeared to be more favorable for the induction of anti-epitope antibodies than coupling at site 153. Moreover, the LamB hybrid corresponding to the simultaneous insertion of the preSB epitope at site 153 and of the C3B epitope at site 386 allowed the induction of both anti-poliovirus and anti-hepatitis B antibodies.

A cluster of charged and aromatic residues in the C-terminal portion of maltoporin participates in sugar binding and uptake.

The maltoporin LamB of Escherichia coli K12 is a trimeric protein which facilitates the diffusion of maltose and maltodextrins through the bacterial outer membrane, and also acts as a non-specific porin for small hydrophilic molecules as well as a receptor for phages. Loop L9 (residues 375 to 405) is the most distal and largest surface-exposed loop of LamB. It comprises a central portion, which varies in size and sequence in the maltoporins of known sequence, flanked by two conserved regions containing charged and aromatic residues. In order to identify the residues within the proximal region that are specifically involved in sugar utilization, we used site-directed mutagenesis to change, individually, each of the charged (five) and aromatic (three) residues in the region 371 to 379 into alanine. None of the eight single amino acid substitutions affected the phage receptor activity of LamB. In contrast, they all affected, to variable extents, maltoporin functions. For all the mutants, very good correlations were observed between the effects on sugar binding and on in vivo uptake. In no case were maltoporin functions completely abolished. Mutants E374 A and W376 A were the most impaired (with over 60% reduction in dextrin binding and in vivo uptake of maltose and maltopentaose). These two mutations also led to an increased bacterial sensitivity to bacitracin and vancomycin. The functional and structural implications are discussed.

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.

Horizontal transfer of chromosomal DNA between the marine bacterium Vibrio furnissii and Escherichia coli revealed by sequence analysis.

Previous in silico analysis of the 67.4-76.0 minutes region of the Escherichia coli genome led to the identification of a gene cluster (named aga) comprising five genes encoding homologs of the mannose transporter of E. coli, a member of the sugar-specific phosphoenolypyruvate/sugar phosphotransferase system (PTS). In the present work, we compared the aga gene cluster of E. coli, which has been considered to be involved in N-acetylgalactosamine or N-acetylmannosamine transport and metabolism, to the region comprising the recently identified mannose transporter of the marine bacterium Vibrio furnissii. Our analysis revealed that the proteins encoded by three genes (agaV, agaW, and agaA), located in the proximal portion of the aga gene cluster, shared striking similarities with the proteins encoded by the manX (IIBMan), manY (IICMan), and manD (a putative deacetylase) genes of V. furnissii, respectively (70%-82.3% identity among the three pairs of proteins). Moreover, we found that the two following aga genes (agaS and agaY) were homologous to the sequences flanking the mannose operon of V. furnissii. These observations strongly support the idea of a horizontal transfer of the chromosomally encoded man operon of V. furnissii into the E. coli genome.

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.

Expression and immune response to foreign epitopes in bacteria. Perspectives for live vaccine development.

We previously developed a general procedure which allows the genetic coupling of a chosen foreign linear epitope in different regions of a carrier protein. By using as carriers, two bacterial envelope proteins, the LamB and MalE proteins of E. coli K12, we were able to express the same epitope in different sites of the two proteins and in different compartments of the bacteria. This allowed us to analyze the influence of the localization in E. coli cells of a foreign B-cell epitope on the induction of specific antibody responses, and the role of the molecular environment on the immunological properties of foreign B- or T-cell epitopes, using either purified hybrid proteins or live recombinant bacteria. Several LamB and MalE hybrid proteins were expressed in the aroA attenuated strain of S. typhimurium, SL3261. Immunizations of mice with live recombinant bacteria by the intravenous route showed that it was possible to induce humoral responses against inserted foreign sequences. In order to improve the in vivo stability of the plasmids carrying the different contructions, and to increase the amounts of recombinant LamB and MalE hybrid proteins expressed in vivo, the LamB and malE genes were placed under the control of the anaerobically inducible pnirBpromoter control. The genetic factors susceptible of influencing the immune response to recombinant Salmonella in mice were also studied.

The levanase operon of Bacillus subtilis expressed in Escherichia coli can substitute for the mannose permease in mannose uptake and bacteriophage lambda infection.

Bacteriophage lambda adsorbs to its Escherichia coli K-12 host by interacting with LamB, a maltose- and maltodextrin-specific porin of the outer membrane. LamB also serves as a receptor for several other bacteriophages. Lambda DNA requires, in addition to LamB, the presence of two bacterial cytoplasmic integral membrane proteins for penetration, namely, the IIC(Man) and IID(Man) proteins of the E. coli mannose transporter, a member of the sugar-specific phosphoenolpyruvate:sugar phosphotransferase system (PTS). The PTS transporters for mannose of E. coli, for fructose of Bacillus subtilis, and for sorbose of Klebsiella pneumoniae were shown to be highly similar to each other but significantly different from other PTS transporters. These three enzyme II complexes are the only ones to possess distinct IIC and IID transmembrane proteins. In the present work, we show that the fructose-specific permease encoded by the levanase operon of B. subtilis is inducible by mannose and allows mannose uptake in B. subtilis as well as in E. coli. Moreover, we show that the B. subtilis permease can substitute for the E. coli mannose permease cytoplasmic membrane components for phage lambda infection. In contrast, a series of other bacteriophages, also using the LamB protein as a cell surface receptor, do not require the mannose transporter for infection.

Topology of the membrane protein LamB by epitope tagging and a comparison with the X-ray model.

We previously developed a genetic approach to study, with a single antibody, the topology of the outer membrane protein LamB, an Escherichia coli porin with specificity towards maltodextrins and a receptor for bacteriophage lambda. Our initial procedure consisted of inserting at random the same reporter epitope (the C3 neutralization epitope from poliovirus) into permissive sites of LamB (i.e., sites which tolerate insertions without deleterious effects on the protein activities or the cell). A specific monoclonal antibody was then used to examine the position of the inserted epitope with respect to the protein and the membrane. In the present work, we set up a site-directed procedure to insert the C3 epitope at new sites in order to distinguish between two-dimensional folding models. This allowed us to identify two new surface loops of LamB and to predict another periplasmic exposed region. The results obtained by random and directed epitope tagging are analyzed in light of the recently published X-ray structure of the LamB protein. Study of 23 hybrid LamB-C3 proteins led to the direct identification of five of the nine external loops (L4, L5, L6, L7, and L9) and led to the prediction of four periplasmic loops (I1, I4, I5, and I8) of LamB. Nine of the hybrid proteins did not lead to topological conclusions, and none led to the wrong predictions or conclusions. The comparison indicates that parts of models based on secondary structure predictions alone are not reliable and points to the importance of experimental data in the establishment of outer membrane protein topological models. The advantages and limitations of genetic foreign epitope insertion for the study of integral membrane proteins are discussed.