Phase variation in meningococcal lipooligosaccharide biosynthesis genes

Neisseria meningitidis expresses a range of lipooligosaccharide (LOS) structures, comprising of at least 13 immunotypes (ITs). Meningococcal LOS is subject to phase variation of its terminal structures allowing switching between ITs, which is proposed to have functional significance in disease. The objectives of this study were to investigate the repertoire of structures that can be expressed in clinical isolates, and to examine the role of phase-variable expression of LOS genes during invasive disease. Southern blotting was used to detect the presence of LOS biosynthetic genes in two collections of meningococci, a global set of strains previously assigned to lineages of greater or lesser virulence, and a collection of local clinical isolates which included paired throat and blood isolates from individual patients. Where the phase-variable genes lgtA, lgtC or IgtG were identified, they were amplified by PCR and the homopolymeric tracts, responsible for their phase-variable expression, were sequenced. The results revealed great potential for variation between alternate LOS structures in the isolates studied, with most strains capable of expressing several alternative terminal structures. The structures predicted to be currently expressed by the genotype of the strains agreed well with conventional immunotyping. No correlation was observed between the structural repertoire and virulence of the isolate. Based on the potential for LOS phase variation in the clinical collection and observations with the paired patient isolates, our data suggest that phase variation of LOS structures is not required for translocation between distinct compartments in the host. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.

Genetic characterization of pilin glycosylation and phase variation in Neisseria meningitidis

Pili of Neisseria meningitidis are a key virulence factor, being the major adhesin of this capsulate organism and contributing to specificity for the human host. Pili are post-translationally modified by addition of either an O-linked trisaccharide, Gal (beta1-4) Gal (alpha1-3) 2,4-diacetamido-2,4,6-trideoxyhexose or an O-linked disaccharide Gal (alpha1,3) GlcNAc. The role of these structures in meningococcal pathogenesis has not been resolved. In previous studies we identified two separate genetic loci, pglA and pglBCD, involved in pilin glycosylation. Putative functions have been allocated to these genes; however, there are not enough genes to account for the complete biosynthesis of the described structures, suggesting additional genes remain to be identified. In addition, it is not known why some strains express the trisaccharide structure and some the disaccharide structure. In order to find additional genes involved in the biosynthesis. of these structures, we used the recently published group A strain Z2491 and group B strain MC58 Neisseria meningitidis genomes and the unfinished Neisseria meningitidis group C strain FAM18 and Neisseria gonorrhoeae strain FA1090 genomes to identify novel genes involved in pilin glycosylation, based on homology to known oligosaccharide biosynthetic genes. We identified a new gene involved in pilin glycosylation designated pglE and examined four additional genes pgIB/B2, pglF, pglG and pglH. A strain survey revealed that pglE and pglF were present in each strain examined. The pglG, pglH and pgIB2 polymorphisms were not found in strain C311#3 but were present in a large number of clinical isolates. Insertional mutations were constructed in pglE and pglF in N. meningitidis strain C311#3, a strain with well-defined lipopolysaccharide (LPS) and pilin-linked glycan structures. Increased gel migration of the pilin subunit molecules of pglE and pglF mutants was observed by Western analysis, indicating truncation of the trisaccharide structure. Antisera specific for the C311#3 trisaccharide failed to react with pilin from these pglE and pglF mutants. GC-MS analysis of the sugar composition of the pglE mutant showed a reduction in galactose compared with C311#3 wild type. Analysis of amino acid sequence homologies has suggested specific roles for pglE and pglF in the biosynthesis of the trisaccharide structure. Further, we present evidence that pglE, which contains heptanucleotide repeats, is responsible for the phase variation between trisaccharide and disaccharide structures in strain C311#3 and other strains. We also present evidence that pglG, pglH and pgIB2 are potentially phase variable.

Phase variation in Flavobacterium psychrophilum : influence on host-pathogen interactions

-

Reversible phase variation in the phnE gene, which is required for phosphonate metabolism in Escherichia coli K-12

It is known that Escherichia coli K-12 is cryptic (Phn(-)) for utilization of methyl phosphonate (MePn) and that Phn(+) variants can be selected for growth on MePn as the sole P source. Variants arise from deletion via a possible slip strand mechanism of one of three direct 8-bp repeat sequences in phnE, which restores function to a component of a putative ABC type transporter. Here we show that Phn(+) variants are present at the surprisingly high frequency of >10(-2) in K-12 strains. Amplified-fragment length polymorphism analysis was used to monitor instability in phnE in various strains growing under different conditions. This revealed that, once selection for growth on MePn is removed, Phn(+) revertants reappear and accumulate at high levels through reinsertion of the 8-bp repeat element sequence. It appears that, in K-12, phnE contains a high-frequency reversible gene switch, producing phase variation which either allows ("on" form) or blocks ("off" form) MePn utilization. The switch can also block usage of other metabolizable alkyl phosphonates, including the naturally occurring 2-aminoethylphosphonate. All K-12 strains, obtained from collections, appear in the "off" form even when bearing mutations in mutS, mutD, or dnaQ which are known to enhance slip strand events between repetitive sequences. The ability to inactivate the phnE gene appears to be unique to K-12 strains since the B strain is naturally Phn(+) and lacks the inactivating 8-bp insertion in phnE, as do important pathogenic strains for which genome sequences are known and also strains isolated recently from environmental sources.

A novel phase variation mechanism in the meningococcus driven by a ligand-responsive repressor and differential spacing of distal promoter elements

Phase variable expression, mediated by high frequency reversible changes in the length of simple sequence repeats, facilitates adaptation of bacterial populations to changing environments and is frequently important in bacterial virulence. Here we elucidate a novel phase variable mechanism for NadA expression, an adhesin and invasin of Neisseria meningitidis. The NadR repressor protein binds to operators flanking the phase variable tract of the nadA promoter gene and contributes to the differential expression levels of phase variant promoters with different numbers of repeats, likely due to different spacing between operators. It is shown that IHF binds between these operators, and may permit looping of the promoter, allowing interaction of NadR at operators located distally or overlapping the promoter. The 4-hydroxyphenylacetic acid, a metabolite of aromatic amino acid catabolism that is secreted in saliva, induces nadA expression by inhibiting the DNA binding activity of the NadR repressor. When induced, only minor differences are evident between NadR-independent transcription levels of promoter phase variants, which are likely due to differential RNA polymerase contacts leading to altered promoter activity. These results suggest that NadA expression is under both stochastic and tight environmental-sensing regulatory control, and both regulations are mediated by the NadR repressor that and may be induced during colonization of the oropharynx where it plays a major role in the successful adhesion and invasion of the mucosa. Hence, simple sequence repeats in promoter regions may be a strategy used by host-adapted bacterial pathogens to randomly switch between expression states that may nonetheless still be induced by appropriate niche-specific signals.

Phase variation of the lpf operon is a mechanism to evade cross-immunity between Salmonella serotypes

Conventional wisdom holds that phase variation is a mechanism for immune evasion. However, despite fimbrial phase variation, mice previously exposed to Salmonella typhimurium are protected against a subsequent challenge. We evaluated whether lpf phase variation instead may be a mechanism to evade cross-immunity between Salmonella serotypes. Mice were immunized orally with S. typhimurium aroA mutants either that expressed the lpf operon (phase-on variant) or in which the entire lpf operon had been removed by deletion. During a subsequent challenge with virulent Salmonella enteritidis a selection against lpf phase-on variants was observed in mice previously exposed to S. typhimurium long polar fimbriae. Vaccination with S. typhimurium did not confer protection against challenge with S. enteritidis, presumably because lpf phase-off variants were able to evade cross-immunity. We propose that lpf phase variation is a mechanism to evade cross-immunity between Salmonella serotypes, thereby allowing their coexistence in a host population.

Étude des mécanismes moléculaires influençant la variation de phase des adhésines P, F1651 et CS31A présentes chez des souches d'Escherichia coli pathogènes.

F1651, les pili Pap et l’antigène CS31A associé aux antigènes de surface K88 sont tout trois des membres de la famille de type P des facteurs d’adhérence jouant un rôle prépondérant lors de l’établissement d’une maladie causée par des souches Escherichia coli pathogènes, en particulier des souches d’E. coli pathogènes extra-intestinales (ExPEC, Extra-intestinal pathogenic E. coli). Leur expression est sous le contrôle d’un mécanisme de régulation transcriptionnel dépendant de l’état de méthylation de l’ADN, résultant dans l’existence de deux populations définies, l’une exprimant l’adhésine (population ON) et l’autre ne l’exprimant pas (population OFF). Malgré de fortes identités de séquences, ces trois systèmes diffèrent l’un de l’autre, principalement par le pourcentage de cellules ON rencontrées. Ainsi, quand CS31A est systématiquement orienté vers un état considéré comme OFF, F1651 présente une phase ON particulièrement élevée et Pap montre deux états OFF et ON bien distincts, selon le phénotype de départ. La protéine régulatrice sensible à la leucine (Lrp, Leucine-responsive regulatory protein) joue un rôle essentiel dans la réversibilité de ce phénomène épigénétique et il est supposé que les différences de séquences au niveau de la région régulatrice modifient la localisation à ces sites de fixation de Lrp; ce qui résulte, en final, aux différences de phase existant entre CS31A, F1651 et Pap.À l’aide de divers techniques parmi lesquelles l’utilisation de gènes rapporteurs, mutagénèses dirigées et d’analyse des interactions ADN-protéines in vitro, nous montrons dans ce présent projet que la phase OFF prédominante chez CS31A est principalement due à une faible interaction de Lrp avec la région distale de l’opéron clp, et que la présence d’un homologue du régulateur local PapI joue un rôle également clef dans la production de CS31A. Dans le cas de F1651, nous montrons dans cette étude que le taux élevé de cellules en phase ON est dû à une altération dans le maintien de Lrp sur les sites répresseurs 1-3. Ceci est dû à la présence de deux nucléotides spécifiques, situé de part et d’autre du site répresseur 1, qui défavorisent la fixation de Lrp sur ce site précis. Tout comme dans le cas de CS31A, la formation d’un complexe, activateur ou répresseur de la phase ON, dépend également de l’action de du régulatuer local FooI, qui favorise alors le déplacement de Lrp des sites répresseurs 1-3 vers les sites activateurs 4-6.

Étude de la variation de phase des fimbriae F1651, Pap et CS31A et de l'impact des régulateurs homologues de PapI

Les Escherichia coli pathogènes extra-intestinaux (ExPEC) sont responsables d’une grande variété de maladies. Plus particulièrement, certaines souches ExPEC, du sous-groupe d’E. coli uropathogènes, sont porteuses de fimbriae de type P. Cette famille d’adhésines est soumise à une régulation transcriptionnelle appelée variation de phase; un mécanisme du tout ou rien. Il s’agit d’une compétition entre deux protéines régulatrices : la Dam méthylase et la nucléoprotéine Lrp. Ce mécanisme est aussi soumis à l’influence des régulateurs locaux PapB et PapI, deux régulateurs essentiels. Afin d’étudier PapI et ses homologues ainsi que leur impact sur la variation de phase des fimbriae F1651, Pap et CS31A. Grâce à une fusion chromosomique entre la région régulatrice de clp et les gènes lacZYA, nous avons étudié l’effet, en trans, de PapI et FooI qui ont pu restaurer la variation de phase avec une forte tendance pour la phase OFF. Pour étudier l’action de ces protéines sur foo et pap, nous avons utilisé un système utilisant gfp comme gène rapporteur de l’activité des promoteurs des opérons pap et foo. Cela a permis d’observer la variation de phase au niveau cellulaire par cytométrie en flux et en temps réel par microscopie à fluorescence. Ces expériences ont confirmé que la population de cellules F1651 positives a un phénotype d’expression de F1651 partielle alors que les cellules Pap sont en majorité en phase OFF. PapI et FooI n’ont pas la même influence sur la variation de phase, puisque FooI favorise une plus grande fréquence de variation de phase.

Phase variable restriction-modification systems in Moraxella catarrhalis

A repetitive DNA motif was used as a marker to identify novel genes in the mucosal pathogen Moraxella catarrhalis. There is a high prevalence of such repetitive motifs in virulence genes that display phase variable expression. Two repeat containing loci were identified using a digoxigenin-labelled 5'-(CAAC)(6)-3' oligonucleotide probe. The repeats are located in the methylase components of two distinct type III restriction-modification (R-M) systems. We suggest that the phase variable nature of these R-M systems indicates that they have an important role in the biology of M. catarrhalis. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.

Identification and characterization of pptA: a gene involved in the phase-variable expression of phosphorylcholine on pili of Neisseria meningitidis

Pili of pathogenic Neisseria are major virulence factors associated with adhesion, cytotoxicity, twitching motility, autoaggregation, and DNA transformation. Pili are modified posttranslationally by the addition of phosphorylcholine. However, no genes involved in either the biosynthesis or the transfer of phosphorylcholine in Neisseria meningitidis have been identified. In this study, we identified five candidate open reading frames (ORFs) potentially involved in the biosynthesis or transfer of phosphorylcholine to pilin in N. meningitidis. Insertional mutants were constructed for each ORF in N. meningitidis strain C311#3 to determine their effect on phosphorylcholine expression. The effect of the mutant ORFs on the modification by phosphorylcholine was analyzed by Western analysis with phosphorylcholine-specific monoclonal antibody TEPC-15. Analysis of the mutants showed that ORF NMB0415, now defined as pptA (pilin phosphorylcholine transferase A), is involved in the addition of phosphorylcholine to pilin in N. meningitidis. Additionally, the phase variation (high frequency on-off switching of expression) of phosphorylcholine on pilin is due to changes in a homopolymeric guanosine tract in pptA.

Genome-scale analysis of the non-cultivable Treponema pallidum reveals extensive within-patient genetic variation

Insights into the genomic adaptive traits of Treponema pallidum, the causative bacterium of syphilis, have long been hampered due to the absence of in vitro culture models and the constraints associated with its propagation in rabbits. Here, we have bypassed the culture bottleneck by means of a targeted strategy never applied to uncultivable bacterial human pathogens to directly capture whole-genome T. pallidum data in the context of human infection. This strategy has unveiled a scenario of discreet T. pallidum interstrain single-nucleotide-polymorphism-based microevolution, contrasting with a rampant within-patient genetic heterogeneity mainly targeting multiple phase-variable loci and a major antigen-coding gene (tprK). TprK demonstrated remarkable variability and redundancy, intra- and interpatient, suggesting ongoing parallel adaptive diversification during human infection. Some bacterial functions (for example, flagella- and chemotaxis-associated) were systematically targeted by both inter- and intrastrain single nucleotide polymorphisms, as well as by ongoing within-patient phase variation events. Finally, patient-derived genomes possess mutations targeting a penicillin-binding protein coding gene (mrcA) that had never been reported, unveiling it as a candidate target to investigate the impact on the susceptibility to penicillin. Our findings decode the major genetic mechanisms by which T. pallidum promotes immune evasion and survival, and demonstrate the exceptional power of characterizing evolving pathogen subpopulations during human infection.

Receptor-mediated endocytosis of Neisseria gonorrhoeae into primary human urethral epithelial cells: the role of the asialoglycoprotein receptor

Urethral epithelial cells are invaded by Neisseria gonorrhoeae during gonococcal infection in men. To understand further the mechanisms of gonococcal entry into host cells, we used the primary human urethral epithelial cells (PHUECs) tissue culture system recently developed by our laboratory. These studies showed that human asialoglycoprotein receptor (ASGP-R) and the terminal lactosamine of lacto-N-neotetraose-expressing gonococcal lipooligosaccharide (LOS) play an important role in invasion of PHUECs. Microscopy studies showed that ASGP-R traffics to the cell surface after gonococcal challenge. Co-localization of ASGP-R with gonococci was observed. As ASGP-R-mediated endocytosis is clathrin dependent, clathrin localization in PHUECs was examined after infection. Infected PHUECs showed increased clathrin recruitment and co-localization of clathrin and gonococci. Preincubating PHUECs in 0.3 M sucrose or monodansylcadaverine (MDC), which both inhibit clathrin-coated pit formation, resulted in decreased invasion. N. gonorrhoeae strain 1291 produces a single LOS glycoform that terminates with Gal(beta1-4)Glc-Nac(beta1-3)Gal(beta1-4)Glc (lacto-N-neotetraose). Invasion assays showed that strain 1291 invades significantly more than four isogenic mutants expressing truncated LOS. Sialylation of strain 1291 LOS inhibited invasion significantly. Preincubation of PHUECs in asialofetuin (ASF), an ASGP-R ligand, significantly reduced invasion. A dose-response reduction in invasion was observed in PHUECs preincubated with increasing concentrations of NaOH-deacylated 1291 LOS. These studies indicated that an interaction between lacto-N-neotetraose-terminal LOS and ASGP-R allows gonococcal entry into PHUECs.

Purification of post-translationally modified proteins from bacteria: homologous expression and purification of histidine-tagged pilin from Neisseria meningitidis

Until recently, glycosylation of proteins in prokaryotes was regarded as uncommon and thought to be limited to special cases such as S-layer proteins and some archeal outer membrane proteins. Now, there are an increasing number of reports of bacterial proteins that are glycosylated. Pilin of pathogenic Neisseria is one of the best characterised post-translation ally modified bacterial proteins, with four different types of modifications reported, including a novel glycosylation. Pilin monomers assemble to form pilus fibres, which are long protein filaments that protrude from the surface of bacterial cells and are key virulence factors. To aid in the investigation of these modifications, pure pilin is required. A number of pilin purification methods have been published, but none are appropriate for the routine purification of pilin from many different isolates. This study describes a novel, rapid, and simple method of pilin purification from Neisseria meningitidis C311#3, which facilitates the production of consistent quantities of pure, native pilin. A 6 x histidine tag was fused to the C-terminus of the pilin subunit structural gene, pilE, via homologous recombination placing the 6 x histidine-tagged allele in the chromosome of N. meningitidis C311#3. Pilin was purified under non-denaturing conditions via a two-step process using immobilised metal affinity chromatography (IMAC), followed by dye affinity chromatography. Analysis of the purified pilin confirmed that it retained both of the post-translational modifications examined. This novel approach may prove to be a generally applicable method for purification and analysis of post-translationally modified proteins in bacteria. (C) 2003 Elsevier Science (USA). All rights reserved.

On Rossby waves modified by weak sinusoidal shear

The spatial structure of beta-plane Rossby waves in a sinusoidal basic zonal flow U 0cos(γ,y) is determined analytically in the (stable) asymptotic limit of weak shear, U 0γ2 0/β≈1. The propagating neutral normal modes are found to take their greatest amplitude in the region of maximum westerly flow, while their most rapid phase variation is achieved in the region of maximum easterly flow. These results are shown to be consistent with what is obtained by ray-tracing methods in the limit of small meridional disturbance wavelength.

Spin coherence generation in negatively charged self-assembled (In,Ga)As quantum dots by pumping excited trion states

Spin coherence generation in an ensemble of negatively charged (In,Ga)As/GaAs quantum dots was investigated by picosecond time-resolved pump-probe spectroscopy measuring ellipticity. Robust coherence of the ground-state electron spins is generated by pumping excited charged exciton (trion) states. The phase of the coherent state, as evidenced by the spin ensemble precession about an external magnetic field, varies relative to spin coherence generation resonant with the ground state. The phase variation depends on the pump photon energy. It is determined by (a) pumping dominantly either singlet or triplet excited states, leading to a phase inversion, and (b) the subsequent carrier relaxation into the ground states. From the dependence of the precession phase and the measured g factors, information about the quantum dot shell splitting and the exchange energy splitting between triplet and singlet states can be extracted in the ensemble.