Single nucleotide polymorphisms of Toll-like receptors and susceptibility to infectious diseases.

Toll-like receptors (TLRs) are the best-studied family of pattern-recognition receptors (PRRs), whose task is to rapidly recognize evolutionarily conserved structures on the invading microorganisms. Through binding to these patterns, TLRs trigger a number of proinflammatory and anti-microbial responses, playing a key role in the first line of defence against the pathogens also promoting adaptive immunity responses. Growing amounts of data suggest that single nucleotide polymorphisms (SNPs) on the various human TLR proteins are associated with altered susceptibility to infection. This review summarizes the role of TLRs in innate immunity, their ligands and signalling and focuses on the TLR SNPs which have been linked to infectious disease susceptibility.

Medico-legal implications of sleep apnoea syndrome: driving license regulations in Europe.

BACKGROUND: Sleep apnoea syndrome (SAS), one of the main medical causes of excessive daytime sleepiness, has been shown to be a risk factor for traffic accidents. Treating SAS results in a normalized rate of traffic accidents. As part of the COST Action B-26, we looked at driving license regulations, and especially at its medical aspects in the European region. METHODS: We obtained data from Transport Authorities in 25 countries (Austria, AT; Belgium, BE; Czech Republic, CZ; Denmark, DK; Estonia, EE; Finland, FI; France, FR; Germany, DE; Greece, GR; Hungary, HU; Ireland, IE; Italy, IT; Lithuania, LT; Luxembourg, LU; Malta, MT; Netherlands, NL; Norway, EC; Poland, PL; Portugal, PT; Slovakia, SK; Slovenia, SI; Spain, ES; Sweden, SE; Switzerland, CH; United Kingdom, UK). RESULTS: Driving license regulations date from 1997 onwards. Excessive daytime sleepiness is mentioned in nine, whereas sleep apnoea syndrome is mentioned in 10 countries. A patient with untreated sleep apnoea is always considered unfit to drive. To recover the driving capacity, seven countries rely on a physician's medical certificate based on symptom control and compliance with therapy, whereas in two countries it is up to the patient to decide (on his doctor's advice) to drive again. Only FR requires a normalized electroencephalography (EEG)-based Maintenance of Wakefulness Test for professional drivers. Rare conditions (e.g., narcolepsy) are considered a driving safety risk more frequently than sleep apnoea syndrome. CONCLUSION: Despite the available scientific evidence, most countries in Europe do not include sleep apnoea syndrome or excessive daytime sleepiness among the specific medical conditions to be considered when judging whether or not a person is fit to drive. A unified European Directive seems desirable.

Functional dissection of the ParB homologue (KorB) from IncP-1 plasmid RK2.

Active partitioning of low-copy number plasmids requires two proteins belonging to the ParA and ParB families and a cis-acting site which ParB acts upon. Active separation of clusters of plasmid molecules to the defined locations in the cell before cell division ensures stable inheritance of the plasmids. The central control operon of IncP-1 plasmids codes for regulatory proteins involved in the global transcriptional control of operons for vegetative replication, stable maintenance and conjugative transfer. Two of these proteins, IncC and KorB, also play a role in active partitioning, as the ParA and ParB homologues, respectively. Here we describe mapping the regions in KorB responsible for four of its different functions: dimerisation, DNA binding, repression of transcription and interaction with IncC. For DNA binding, amino acids E151 to T218 are essential, while repression depends not only on DNA binding but, additionally, on the adjacent region amino acids T218 to R255. The C-terminus of KorB is the main dimerisation domain but a secondary oligomerisation region is located centrally in the region from amino acid I174 to T218. Using three different methods (potentiation of transcriptional repression, potentiation of DNA binding and activation in the yeast two-hybrid system) we identify this region as also responsible for interactions with IncC. This IncC-KorB contact differs in location from the ParA-ParB/SopA-SopB interactions in P1/F but is similar to these systems in lying close to a masked oligomerisation determinant.

The hierarchy of KorB binding at its 12 binding sites on the broad-host-range plasmid RK2 and modulation of this binding by IncC1 protein.

IncC and KorB proteins of broad-host-range plasmid RK2 are members of the ParA-ParB families of proteins needed for stable partitioning of bacterial chromosomes and plasmids. KorB also functions as a global regulator of expression of RK2 genes. It recognises and binds to a palindromic operator, O(B), found 12 times on RK2 DNA (O(B)1-O(B)12). We performed detailed studies on the binding of KorB to the 12 operators and showed that they fall into three groups (A, B, C) based on the binding strength of KorB. The highest affinity site is O(B)10, which occurs in the promoter transcribing genes for replication, trfAp. Purified IncC1 potentiated KorB binding to all O(B) sites except O(B)3, a site involved in partitioning. Using O(B)10 as a test system, we showed that IncC1 increases the stability of the KorB-DNA complex. The 5 bp sequences flanking the 13mer O(B) site were found to affect KorB binding and IncC1 potentiation activity. Study of hybrid operators indicated that flanking sequences on one side only were sufficient to specify the difference between O(B)10 and O(B)3. Replacement of adenine by guanine at positions -8 and -10 from the O(B)10 centre of symmetry was needed to convert it from the highest-affinity group (A) to the medium-affinity group (B) on the basis of KorB binding. These changes also eliminated potentiation by IncC1. The -8 and -10 positions from the centre of O(B)3 symmetry are occupied by guanines and this may provide part of the specificity of IncC1 behaviour on KorB binding. Studies on a series of synthetic operators suggested that KorB contacts O(B) flanking sequences, and that IncC1 may alter the conformation of multimeric KorB so that it is better able to make these contacts, thus stabilising the complexes once formed.

Design, synthesis, and conformational study of biologically active photolabeled analogues of the main immunogenic region of the acetylcholine receptor.

Photoaffinity labeling is a powerful tool for the characterization of the molecular basis of ligand binding to acceptor molecules, which provides important insights for mapping the bimolecular interfaces. The autoimmune disease myasthenia gravis is caused by autoantibodies against the acetylcholine receptor (AChR). The majority of the anti-AChR antibodies bind to the "main immunogenic region" (MIR) of the AChR. To identify the contact points between the complementarity determining regions of the anti-MIR antibodies that recognize the MIR contact sites of the AChR, we present here three photoreactive dodecapeptide MIR analogues containing the photolabel p-benzoyl-L-phenylalanine (Bpa) moiety, either in position 1 or 11. The structure of the produced 12-mers was analyzed using two-dimensional (1)H-NMR spectroscopy, whereas their binding to anti-MIR monoclonal antibodies (mAbs) was determined by immunochemical assays. In all cases the modifications resulted in conservation of the beta-turn conformation of the N-terminus, which has been proved essential for antibody recognition and increased anti-MIR binding relative to the MIR decapeptide.

Conserved C-terminal region of global repressor KorA of broad-host-range plasmid RK2 is required for co-operativity between KorA and a second RK2 global regulator, KorB.

KorA and KorB proteins of IncP1 plasmid RK2 are encoded in the central control region (ccr) of the plasmid and act as global regulators of plasmid genes for replication, transfer and stable inheritance. KorA represses seven promoters on RK2, by binding to a defined operator site, OA, which always occurs in promoter regions. KorB recognises another operator, OB, which is found 12 times on the RK2 genome, but not always in promoter regions. At five of the KorA-regulated promoters, an OBsequence is also present. The presence of both KorA and KorB leads to severely decreased promoter activity. By measuring repression at different levels of KorA and KorB alone and in combination, we showed that there is at least 3. 4-fold co-operativity between them at korApin vivo. Testing the ability of previously isolated KorA mutants to act in a co-operative way in the presence of KorB in vivo or in vitro showed that the C-terminal part of KorA between amino acid positions 68 and 83 is required for this co-operativity. This region is part of a segment that is highly conserved between KorA and two other RK2 proteins, TrbA and KlcB. We propose that this conserved region may provide the basis for co-operativity with KorB either indirectly, by modulating DNA structure near the KorB binding site, or directly by serving as the "recognition" patch of each protein by KorB. It may thus serve as a key domain in allowing a sensitive response of the global circuits to changes in repressor concentration and thus modulation of replication, transfer and maintenance.

IncC of broad-host-range plasmid RK2 modulates KorB transcriptional repressor activity In vivo and operator binding in vitro.

The korAB operon of broad-host-range plasmid RK2 encodes five genes, two of which, incC and korB, belong to the parA and parB families, respectively, of genome partitioning functions. Both korB and a third gene, korA, are responsible for coordinate regulation of operons encoding replication, transfer, and stable inheritance functions. Overexpression of incC alone caused rapid displacement of RK2. Using two different reporter systems, we show that incC modulates the action of KorB. Using promoter fusions to the reporter gene xylE, we show that incC potentiates the repression of transcription by korB. This modulation of korB activity was only observed with incC1, which encodes the full-length IncC (364 amino acids [aa]), whereas no effect was observed with incC2, which encodes a polypeptide of 259 aa that lacks the N-terminal 105 aa. Using bacterial extracts with IncC1 and IncC2 or IncC1 purified through the use of a His6 tail and Ni-agarose chromatography, we showed that IncC1 potentiates the binding of KorB to DNA at representative KorB operators. The ability of IncC to stabilize KorB-DNA complexes suggests that these two proteins work together in the global regulation of many operons on the IncP-1 genomes, as well in plasmid partitioning.

Mutational analysis of the global regulator KorA of broad-host-range plasmid RK2.

KorA protein encoded in the central control region of IncP plasmid RK2 binds to seven operators on the plasmid genome and acts as a global repressor of genes for replication and stable inheritance functions. At trfAp, the promoter for plasmid replication genes, KorA also causes derepression of trbAp, the promoter for trbA, encoding another global regulator (TrbA), which controls genes required for conjugative transfer. Both KorB, a second global repressor encoded in the central control region, and TrbA also act in the trfAp-trbAp region to down-regulate trfAp, but neither of these extra repressors allows derepression of trbAp. To initiate a functional dissection of KorA, we used random mutagenesis and a positive selection system to identify korA mutants which no longer repressed trfAp. Nine single amino acid changes were obtained, which did not affect polypeptide length or apparent stability. These clustered either in the N-terminal region of the protein (region I) or in the putative HTH motif (region II). No changes were obtained in the C-terminal region (region III). Four truncated KorA proteins, with deletions either from the N-terminal or the C-terminal end, were also screened together with the single mutants. Both the band-shift assay with trfAp DNA and the in vivo promoter-probe assays with either trfAp or trbAp showed that none of the region II mutants could bind to DNA and repress the promoter. The region I mutants with a conservative amino acid substitution retained some DNA binding and repressor activity, as well as the ability to dimerise. However, an in vivo system to detect trans-dominance of the mutants indicated that one region I point mutant together with the two N-terminally truncated mutants had lost their dimerisation ability. Deletions into the basic C terminus of KorA did not abolish dimerisation. The results implicate region I in dimerisation, region II in DNA binding and region III in a yet unspecified role, possibly interaction with other proteins such as KorB.

Conservation of the genetic switch between replication and transfer genes of IncP plasmids but divergence of the replication functions which are major host-range determinants.

The trfA operon of broad-host-range IncP plasmids is essential to activate the origin of vegetative replication in diverse species. The trb operon encodes most of the apparatus for mating pair formation, the first step in conjugative transfer. Comparison of the nucleotide sequence of the IncP beta plasmid R751 presented here with the equivalent IncP alpha sequence identifies conserved features of the organization and regulation of the trfA operon and the region controlling expression of the trb operon. As in IncP alpha plasmids, these operons are transcribed from a bidirectional promoter region consisting of trfAp for the trfA operon and trbAp and trbBp for the trb operon. The KorA-dependent switch between the trfA and trbA promoters is conserved as is the trbA gene encoding the third IncP global regulator. The intergenic region between trbA and trbB shows very little sequence identity between the two plasmids but the spacing, the KorB operator, the trbB promoter, and the existence of a hairpin loop (albeit of different actual sequence) which sequesters the trbB ribosome binding site are all conserved. The trfA operon encodes two ORFs. The first ORF is highly conserved and encodes a putative single-stranded DNA binding protein (Ssb). The second, trfA, contains two translational starts as in the IncP alpha plasmids, generating related polypeptides of 406 (TrfA1) and 282 (TrfA2) amino acids. TrfA2 is very similar to the IncP alpha product, whereas the N-terminal region of TrfA1 shows very little similarity to the equivalent region of IncP alpha TrfA1. This region has been implicated in the ability of IncP alpha plasmids to replicate efficiently in Pseudomonas aeruginosa. A TcR derivative of R751 was constructed and shown not to establish itself efficiently in P. aeruginosa at 37 degrees C, although it did establish itself inefficiently at lower temperatures, underlining the importance of this region in the adaptation of the plasmid to the host.