Neisseria meningitidis Lacking the Major Porins PorA and PorB Is Viable and Modulates Apoptosis and the Oxidative Burst of Neutrophils.

The bacterial pathogen Neisseria meningitidis expresses two major outer-membrane porins. PorA expression is subject to phase-variation (high frequency, random, on-off switching), and both PorA and PorB are antigenically variable between strains. PorA expression is variable and not correlated with meningococcal colonisation or invasive disease, whereas all naturally-occurring strains express PorB suggesting strong selection for expression. We have generated N. meningitidis strains lacking expression of both major porins, demonstrating that they are dispensable for bacterial growth in vitro. The porAB mutant strain has an exponential growth rate similar to the parental strain, as do the single porA or porB mutants, but the porAB mutant strain does not reach the same cell density in stationary phase. Proteomic analysis suggests that the double mutant strain exhibits compensatory expression changes in proteins associated with cellular redox state, energy/nutrient metabolism, and membrane stability. On solid media, there is obvious growth impairment that is rescued by addition of blood or serum from mammalian species, particularly heme. These porin mutants are not impaired in their capacity to inhibit both staurosporine-induced apoptosis and a phorbol 12-myristate 13-acetate-induced oxidative burst in human neutrophils suggesting that the porins are not the only bacterial factors that can modulate these processes in host cells.

EXACT2: the semantics of biomedical protocols.

BACKGROUND: The reliability and reproducibility of experimental procedures is a cornerstone of scientific practice. There is a pressing technological need for the better representation of biomedical protocols to enable other agents (human or machine) to better reproduce results. A framework that ensures that all information required for the replication of experimental protocols is essential to achieve reproducibility. To construct EXACT2 we manually inspected hundreds of published and commercial biomedical protocols from several areas of biomedicine. After establishing a clear pattern for extracting the required information we utilized text-mining tools to translate the protocols into a machine amenable format. We have verified the utility of EXACT2 through the successful processing of previously 'unseen' (not used for the construction of EXACT2)protocols. METHODS: We have developed the ontology EXACT2 (EXperimental ACTions) that is designed to capture the full semantics of biomedical protocols required for their reproducibility. RESULTS: The paper reports on a fundamentally new version EXACT2 that supports the semantically-defined representation of biomedical protocols. The ability of EXACT2 to capture the semantics of biomedical procedures was verified through a text mining use case. In this EXACT2 is used as a reference model for text mining tools to identify terms pertinent to experimental actions, and their properties, in biomedical protocols expressed in natural language. An EXACT2-based framework for the translation of biomedical protocols to a machine amenable format is proposed. CONCLUSIONS: The EXACT2 ontology is sufficient to record, in a machine processable form, the essential information about biomedical protocols. EXACT2 defines explicit semantics of experimental actions, and can be used by various computer applications. It can serve as a reference model for for the translation of biomedical protocols in natural language into a semantically-defined format.

Transcriptomic analysis reveals calcium regulation of specific promoter motifs in Arabidopsis.

Increases in intracellular calcium concentration ([Ca(2+)](c)) mediate plant responses to stress by regulating the expression of genes encoding proteins that confer tolerance. Several plant stress genes have previously been shown to be calcium-regulated, and in one case, a specific promoter motif Abscisic Acid Responsive-Element (ABRE) has been found to be regulated by calcium. A comprehensive survey of the Arabidopsis thaliana transcriptome for calcium-regulated promoter motifs was performed by measuring the expression of genes in Arabidopsis seedlings responding to three calcium elevations of different characteristics, using full genome microarray analysis. This work revealed a total of 269 genes upregulated by [Ca(2+)](c) in Arabidopsis. Bioinformatic analysis strongly indicated that at least four promoter motifs were [Ca(2+)](c)-regulated in planta. We confirmed this finding by expressing in plants chimeric gene constructs controlled exclusively by these cis-elements and by testing the necessity and sufficiency of calcium for their expression. Our data reveal that the C-Repeat/Drought-Responsive Element, Site II, and CAM box (along with the previously identified ABRE) promoter motifs are calcium-regulated. The identification of these promoter elements targeted by the second messenger intracellular calcium has implications for plant signaling in response to a variety of stimuli, including cold, drought, and biotic stress.

Economic and health impact modelling of a whole genome sequencing-led intervention strategy for bacterial healthcare-associated infections for England and for the USA.

Bacterial healthcare-associated infections (HAIs) are a substantial source of global morbidity and mortality. The estimated cost associated with HAIs ranges from $35 to $45 billion in the USA alone. The costs and accessibility of whole genome sequencing (WGS) of bacteria and the lack of sufficiently accurate, high-resolution, scalable and accessible analysis for strain identification are being addressed. Thus, it is timely to determine the economic viability and impact of routine diagnostic bacterial genomics. The aim of this study was to model the economic impact of a WGS surveillance system that proactively detects and directs interventions for nosocomial infections and outbreaks compared to the current standard of care, without WGS. Using a synthesis of published models, inputs from national statistics, and peer-reviewed articles, the economic impacts of conducting a WGS-led surveillance system addressing the 11 most common nosocomial pathogen groups in England and the USA were modelled. This was followed by a series of sensitivity analyses. England was used to establish the baseline model because of the greater availability of underpinning data, and this was then modified using USA-specific parameters where available. The model for the NHS in England shows bacterial HAIs currently cost the NHS around £3 billion. WGS-based surveillance delivery is predicted to cost £61.1 million associated with the prevention of 74 408 HAIs and 1257 deaths. The net cost saving was £478.3 million, of which £65.8 million were from directly incurred savings (antibiotics, consumables, etc.) and £412.5 million from opportunity cost savings due to re-allocation of hospital beds and healthcare professionals. The USA model indicates that the bacterial HAI care baseline costs are around $18.3 billion. WGS surveillance costs $169.2 million, and resulted in a net saving of ca.$3.2 billion, while preventing 169 260 HAIs and 4862 deaths. From a 'return on investment' perspective, the model predicts a return to the hospitals of £7.83 per £1 invested in diagnostic WGS in the UK, and US$18.74 per $1 in the USA. Sensitivity analyses show that substantial savings are retained when inputs to the model are varied within a wide range of upper and lower limits. Modelling a proactive WGS system addressing HAI pathogens shows significant improvement in morbidity and mortality while simultaneously achieving substantial savings to healthcare facilities that more than offset the cost of implementing diagnostic genomics surveillance.

A role for BELLRINGER in cell wall development is supported by loss-of-function phenotypes.

BACKGROUND: Homeodomain transcription factors play critical roles in metazoan development. BELLRINGER (BLR), one such transcription factor, is involved in diverse developmental processes in Arabidopsis, acting in vascular differentiation, phyllotaxy, flower and fruit development. BLR also has a redundant role in meristem maintenance. Cell wall remodelling underpins many of these processes, and BLR has recently been shown to regulate expression of PECTIN METHYL-ESTERASE 5 (PME5), a cell wall modifying enzyme in control of phyllotaxy. We have further explored the role of BLR in plant development by analysing phenotypes and gene expression in a series of plants over-expressing BLR, and generating combinatorial mutants with blr, brevipedicellus (bp), a member of the KNOX1 family of transcription factors that has previously been shown to interact with blr, and the homeodomain transcription factor revoluta (rev), required for radial patterning of the stem. RESULTS: Plants over-expressing BLR exhibited a wide range of phenotypes. Some were defective in cell size and demonstrated misregulation of genes predominantly affecting cell wall development. Other lines with more extreme phenotypes failed to generate lateral organs, consistent with BLR repressing transcription in the shoot apex. Cell wall dynamics are also affected in blr mutant plants, and BLR has previously been shown to regulate vascular development in conjunction with BP. We found that when bp and blr were combined with rev, a set of defects was observed that were distinct from those of bp blr lines. In these triple mutants xylem development was most strikingly affected, resulting in an almost complete lack of vessels and xylem parenchyma with secondary thickening. CONCLUSIONS: Our data support a role for BLR in ordering the shoot apex and, in conjunction with BP and REV, playing a part in determining the composition and organisation of the vascular system. Microarray analysis strongly indicates that the striking vascular phenotypes of blr bp rev triple mutants and plants over-expressing BLR result from the misregulation of a suite of genes, targets of BLR in wild type plants, that determine cell size and structure in the developing vasculature.

MicroRNA expression in Sezary syndrome: identification, function, and diagnostic potential.

MicroRNAs are commonly aberrantly expressed in many cancers. Very little is known of their role in T-cell lymphoma, however. We therefore elucidated the complete miRNome of purified T cells from 21 patients diagnosed with Sézary Syndrome (SzS), a rare aggressive primary cutaneous T-cell (CD4(+)) lymphoma. Unsupervised cluster analysis of microarray data revealed that the microRNA expression profile was distinct from CD4(+) T-cell controls and B-cell lymphomas. The majority (104 of 114) of SzS-associated microRNAs (P < .05) were down-regulated and their expression pattern was largely consistent with previously reported genomic copy number abnormalities and were found to be highly enriched (P < .001) for aberrantly expressed target genes. Levels of miR-223 distinguished SzS samples (n = 32) from healthy controls (n = 19) and patients with mycosis fungoides (n = 11) in more than 90% of samples. Furthermore, we demonstrate that the down-regulation of intronically encoded miR-342 plays a role in the pathogenesis of SzS by inhibiting apoptosis, and describe a novel mechanism of regulation for this microRNA via binding of miR-199a* to its host gene. We also provide the first in vivo evidence for down-regulation of the miR-17-92 cluster in malignancy and demonstrate that ectopic miR-17-5p expression increases apoptosis and decreases cell proliferation in SzS cells.

Primary cutaneous anaplastic large cell lymphoma shows a distinct miRNA expression profile and reveals differences from tumor-stage mycosis fungoides.

The miRNA expression profiles of skin biopsies from 14 primary cutaneous anaplastic large cell lymphoma (C-ALCL) patients were analysed with miRNA microarrays using the same control group of 12 benign inflammatory dermatoses (BID) as previously used to study the miRNA expression profile of tumor-stage mycosis fungoides (MF). We identified 13 differentially expressed miRNAs between C-ALCL and BID. The up-regulation of miR-155, miR-27b, miR-30c and miR-29b in C-ALCL was validated by miRNA-Q-PCR on independent study groups. Additionally, the miRNA expression profiles of C-ALCL were compared with those of tumor-stage MF. Although miRNA microarray analysis did not identify statistically significant differentially expressed miRNAs, miRNA-Q-PCR demonstrated statistically significantly differential expression of miR-155, miR-27b, miR-93, miR-29b and miR-92a between tumor-stage MF and C-ALCL. This study, the first describing the miRNA expression profile of C-ALCL, reveals differences with tumor-stage MF, suggesting a different contribution to the pathogenesis of these lymphomas.

Defects in lamin B1 expression or processing affect interphase chromosome position and gene expression.

Radial organization of nuclei with peripheral gene-poor chromosomes and central gene-rich chromosomes is common and could depend on the nuclear boundary as a scaffold or position marker. To test this, we studied the role of the ubiquitous nuclear envelope (NE) component lamin B1 in NE stability, chromosome territory position, and gene expression. The stability of the lamin B1 lamina is dependent on lamin endoproteolysis (by Rce1) but not carboxymethylation (by Icmt), whereas lamin C lamina stability is not affected by the loss of full-length lamin B1 or its processing. Comparison of wild-type murine fibroblasts with fibroblasts lacking full-length lamin B1, or defective in CAAX processing, identified genes that depend on a stable processed lamin B1 lamina for normal expression. We also demonstrate that the position of mouse chromosome 18 but not 19 is dependent on such a stable nuclear lamina. The results implicate processed lamin B1 in the control of gene expression as well as chromosome position.

Structure of the regulatory domain of the LysR family regulator NMB2055 (MetR-like protein) from Neisseria meningitidis.

The crystal structure of the regulatory domain of NMB2055, a putative MetR regulator from Neisseria meningitidis, is reported at 2.5 Šresolution. The structure revealed that there is a disulfide bond inside the predicted effector-binding pocket of the regulatory domain. Mutation of the cysteines (Cys103 and Cys106) that form the disulfide bond to serines resulted in significant changes to the structure of the effector pocket. Taken together with the high degree of conservation of these cysteine residues within MetR-related transcription factors, it is suggested that the Cys103 and Cys106 residues play an important role in the function of MetR regulators.

Genome sequence of Rhodobacter sphaeroides Strain WS8N.

Rhodobacter sphaeroides is a metabolically diverse photosynthetic alphaproteobacterium found ubiquitously in soil and freshwater habitats. Here we present the annotated genome sequence of R. sphaeroides WS8N.

Tolerogenicity is not an absolute property of a dendritic cell.

Pharmacological modulation is known to temper the immune capacity of DC, enhancing the notion that modulated Ag-bearing DC might be used therapeutically to induce tolerance. We have investigated phenotypic features shared by such DC, and queried their potential to tolerize in different settings. Immature, IL-10, TGF-beta and 1alpha,25-dihydroxyvitamin D(3)-modulated BMDC all induced tolerance to male skin in female TCR transgenic A1.RAG mice, and the modulated DC also tolerized after exposure to the TLR4-ligand LPS. Transcript profiling revealed that this was achieved despite retaining much of the normal LPS-maturation response. No shared tolerance-associated transcripts could be identified. Equivalent BMDC could not tolerize in Marilyn TCR-transgenic mice. Simultaneous presentation of both A1.RAG and Marilyn peptide-Ag (Dby-H2E(k) and Dby-H2A(b)) on immature (C57BL/6JxCBA/Ca) F1 BMDC also only achieved tolerance in A1.RAG mice. Both strains registered Ag, but Foxp3(+) Treg were only induced in A1.RAG mice. In contrast, Marilyn T cells showed greater proliferation and an inflammatory bias, in response to Ag presented by immature F1 BMDC in vitro. In summary, while pharmacological agents can skew DC to reinforce their immature tolerogenic phenotype, the outcome of presentation is ultimately an integrated response including T-cell-intrinsic components that can over-ride for immune activation.

MS4A4B is a GITR-associated membrane adapter, expressed by regulatory T cells, which modulates T cell activation.

In the aftermath of thymic negative selection, natural and adaptive regulatory T cells (Tregs) must acknowledge peripheral, "danger-free" self-Ag to ensure their sustained activity. In this paper, we show that natural and adaptive Tregs or T cells transduced with cDNA for Foxp3, just like Th1 cells, express members of the MS4A family of transmembrane molecules. Naive T cells transduced with MS4A4B become able to respond to lower levels of Ag. Using two family members, MS4A4B and MS4A6B, as baits in a yeast split-ubiquitin Treg library screen, we demonstrate their interaction with each other and with GITR, Orai1, and other surface receptors. Interaction of 4B with GITR augments GITR signaling and T cell IL-2 production in response to triggering with GITR ligand or anti-GITR Abs. This interaction provides a mechanism whereby MS4A family members, through lateral coassociation with costimulatory molecules, may amplify Ag signals. We propose that T cells preoccupied with immune defense use this MS4A family to enhance sensitivity to extrinsic Ag stimulation, ensuring its elimination, while Tregs use these adaptors to allow low level Ag signals to sustain regulatory function.

The structure of CrgA from Neisseria meningitidis reveals a new octameric assembly state for LysR transcriptional regulators.

LysR-type transcriptional regulators (LTTRs) form the largest family of bacterial regulators acting as both auto-repressors and activators of target promoters, controlling operons involved in a wide variety of cellular processes. The LTTR, CrgA, from the human pathogen Neisseria meningitidis, is upregulated during bacterial-host cell contact. Here, we report the crystal structures of both regulatory domain and full-length CrgA, the first of a novel subclass of LTTRs that form octameric rings. Non-denaturing mass spectrometry analysis and analytical ultracentrifugation established that the octameric form of CrgA is the predominant species in solution in both the presence and absence of an oligonucleotide encompassing the CrgA-binding sequence. Furthermore, analysis of the isolated CrgA-DNA complex by mass spectrometry showed stabilization of a double octamer species upon DNA binding. Based on the observed structure and the mass spectrometry findings, a model is proposed in which a hexadecameric array of two CrgA oligomers binds to its DNA target site.

CD4+CD25+ T(R) cells suppress innate immune pathology through cytokine-dependent mechanisms.

CD4(+)CD25(+) regulatory T (T(R)) cells can inhibit a variety of autoimmune and inflammatory diseases, but the precise mechanisms by which they suppress immune responses in vivo remain unresolved. Here, we have used Helicobacter hepaticus infection of T cell-reconstituted recombination-activating gene (RAG)(-/-) mice as a model to study the ability of CD4(+)CD25(+) T(R) cells to inhibit bacterially triggered intestinal inflammation. H. hepaticus infection elicited both T cell-mediated and T cell-independent intestinal inflammation, both of which were inhibited by adoptively transferred CD4(+)CD25(+) T(R) cells. T cell-independent pathology was accompanied by activation of the innate immune system that was also inhibited by CD4(+)CD25(+) T(R) cells. Suppression of innate immune pathology was dependent on T cell-derived interleukin 10 and also on the production of transforming growth factor beta. Thus, CD4(+)CD25(+) T(R) cells do not only suppress adaptive T cell responses, but are also able to control pathology mediated by innate immune mechanisms.

The structure of a reduced form of OxyR from Neisseria meningitidis.

BACKGROUND: Survival of the human pathogen, Neisseria meningitidis, requires an effective response to oxidative stress resulting from the release of hydrogen peroxide by cells of the human immune system. In N. meningitidis, expression of catalase, which is responsible for detoxifying hydrogen peroxide, is controlled by OxyR, a redox responsive LysR-type regulator. OxyR responds directly to intracellular hydrogen peroxide through the reversible formation of a disulphide bond between C199 and C208 in the regulatory domain of the protein. RESULTS: We report the first crystal structure of the regulatory domain of an OxyR protein (NMB0173 from N. meningitidis) in the reduced state i.e. with cysteines at positions 199 and 208. The protein was crystallized under reducing conditions and the structure determined to a resolution of 2.4 A. The overall fold of the Neisseria OxyR shows a high degree of similarity to the structure of a C199S mutant OxyR from E. coli, which cannot form the redox sensitive disulphide. In the neisserial structure, C199 is located at the start of helix alpha3, separated by 18 A from C208, which is positioned between helices alpha3 and alpha4. In common with other LysR-type regulators, full length OxyR proteins are known to assemble into tetramers. Modelling of the full length neisserial OxyR as a tetramer indicated that C199 and C208 are located close to the dimer-dimer interface in the assembled tetramer. The formation of the C199-C208 disulphide may thus affect the quaternary structure of the protein. CONCLUSION: Given the high level of structural similarity between OxyR from N. meningitidis and E. coli, we conclude that the redox response mechanism is likely to be similar in both species, involving the reversible formation of a disulphide between C199-C208. Modelling suggests that disulphide formation would directly affect the interface between regulatory domains in an OxyR tetramer which in turn may lead to an alteration in the spacing/orientation of the DNA-binding domains and hence the interaction of OxyR with its DNA binding sites.

Meningococcal genetic variation mechanisms viewed through comparative analysis of serogroup C strain FAM18.

The bacterium Neisseria meningitidis is commonly found harmlessly colonising the mucosal surfaces of the human nasopharynx. Occasionally strains can invade host tissues causing septicaemia and meningitis, making the bacterium a major cause of morbidity and mortality in both the developed and developing world. The species is known to be diverse in many ways, as a product of its natural transformability and of a range of recombination and mutation-based systems. Previous work on pathogenic Neisseria has identified several mechanisms for the generation of diversity of surface structures, including phase variation based on slippage-like mechanisms and sequence conversion of expressed genes using information from silent loci. Comparison of the genome sequences of two N. meningitidis strains, serogroup B MC58 and serogroup A Z2491, suggested further mechanisms of variation, including C-terminal exchange in specific genes and enhanced localised recombination and variation related to repeat arrays. We have sequenced the genome of N. meningitidis strain FAM18, a representative of the ST-11/ET-37 complex, providing the first genome sequence for the disease-causing serogroup C meningococci; it has 1,976 predicted genes, of which 60 do not have orthologues in the previously sequenced serogroup A or B strains. Through genome comparison with Z2491 and MC58 we have further characterised specific mechanisms of genetic variation in N. meningitidis, describing specialised loci for generation of cell surface protein variants and measuring the association between noncoding repeat arrays and sequence variation in flanking genes. Here we provide a detailed view of novel genetic diversification mechanisms in N. meningitidis. Our analysis provides evidence for the hypothesis that the noncoding repeat arrays in neisserial genomes (neisserial intergenic mosaic elements) provide a crucial mechanism for the generation of surface antigen variants. Such variation will have an impact on the interaction with the host tissues, and understanding these mechanisms is important to aid our understanding of the intimate and complex relationship between the human nasopharynx and the meningococcus.

Two-step assembly dynamics of the Bacillus subtilis divisome.

Cell division in bacteria is carried out by about a dozen proteins which assemble at midcell and form a complex known as the divisome. To study the dynamics and temporal hierarchy of divisome assembly in Bacillus subtilis, we have examined the in vivo localization pattern of a set of division proteins fused to green fluorescent protein in germinating spores and vegetative cells. Using time series and time-lapse microscopy, we show that the FtsZ ring assembles early and concomitantly with FtsA, ZapA, and EzrA. After a time delay of at least 20% of the cell cycle, a second set of division proteins, including GpsB, FtsL, DivIB, FtsW, Pbp2B, and DivIVA, are recruited to midcell. Together, our data provide in vivo evidence for two-step assembly of the divisome. Interestingly, overproduction of FtsZ advances the temporal assembly of EzrA but not of DivIVA, suggesting that a signal different from that of FtsZ polymerization drives the assembly of late divisome proteins. Microarray analysis shows that FtsZ depletion or overexpression does not significantly alter the transcription of division genes, supporting the hypothesis that cell division in B. subtilis is mainly regulated at the posttranscriptional level.

Expression of microRNAs in diffuse large B cell lymphoma is associated with immunophenotype, survival and transformation from follicular lymphoma.

MicroRNAs are naturally occurring small RNA species that regulate gene expression and are frequently abnormally expressed in cancers. However, the role of microRNAs in lymphoma is poorly understood. Therefore, we undertook a comprehensive study of microRNA expression in two of the most common lymphomas: diffuse large B-cell lymphoma (DLBCL) (n = 80) and follicular lymphoma (FCL) (n = 18) using microarrays containing probes for 464 human microRNAs. Unsupervised cluster analysis revealed distinct expression patterns between these two lymphomas and specific microRNA signatures (including members of the miR-17-92 cluster) were derived that correctly predicted lymphoma type in >95% of cases. Furthermore, we identified microRNAs in de novo DLBCL (n = 64) associated with germinal centre-like and non-germinal centre-like immunophenotypes, international prognostic index status and event-free survival in CHOP and rituximab (R)-CHOP treated patients. Despite the indolent nature of FCL a significant proportion of cases undergo high-grade transformation to more aggressive DLBCL. In order to see if transformation is associated with changes in microRNA expression we compared transformed DLBCL cases (n = 16) with de novo DLBCL, as well as FCL cases that underwent subsequent transformation (n = 7) with FCL cases that had not transformed at a median follow-up of 60 months (n = 11). Differential expression of 12 microRNAs correctly predicted >85% of transformed versus de novo DLBCL cases; six microRNAs (miR-223, 217, 222, 221 and let-7i and 7b) were found which could similarly predict or transformation in FCL (P < 0.05). These data suggest that microRNAs have potential as diagnostic and prognostic markers in these lymphomas and may be used to identify FCL patients at risk of high-grade transformation.

The structure of NMB1585, a MarR-family regulator from Neisseria meningitidis.

The structure of the MarR-family transcription factor NMB1585 from Neisseria meningitidis has been solved using data extending to a resolution of 2.1 A. Overall, the dimeric structure resembles those of other MarR proteins, with each subunit comprising a winged helix-turn-helix (wHtH) domain connected to an alpha-helical dimerization domain. The spacing of the recognition helices of the wHtH domain indicates that NMB1585 is pre-configured for DNA binding, with a putative inducer pocket that is largely occluded by the side chains of two aromatic residues (Tyr29 and Trp53). NMB1585 was shown to bind to its own promoter region in a gel-shift assay, indicating that the protein acts as an auto-repressor.

Structure of the cold-shock domain protein from Neisseria meningitidis reveals a strand-exchanged dimer.

The structure of the cold-shock domain protein from Neisseria meningitidis has been solved to 2.6 A resolution and shown to comprise a dimer formed by the exchange of two beta-strands between protein monomers. The overall fold of the monomer closely resembles those of other bacterial cold-shock proteins. The neisserial protein behaved as a monomer in solution and was shown to bind to a hexathymidine oligonucleotide with a stoichiometry of 1:1 and a K(d) of 1.25 microM.