Lucas D. Bowler

Role of interspecies transfer of chromosomal genes in the evolution of penicillin resistance in pathogenic and commensal Neisseria species.

SummaryThe two pathogenic species of Neisseria, N. meningitidis and N. gonorrhoeae, have evolved resistance to penicillin by alterations in chromosomal genes encoding the high molecular weight penicillin-binding proteins, or PBPs. The PBP 2 gene (penA) has been sequenced from over 20 Neisseria isolates, including susceptible and resistant strains of the two pathogenic species, and five human commensal species. The genes from penicillin-susceptible strains of N. meningitidis and N. gonorrhoeae are very uniform, whereas those from penicillin-resistant strains consist of a mosaic of regions resembling those in susceptible strains of the same species, interspersed with regions resembling those in one, or in some cases, two of the commensal species. The mosaic structure is interpreted as having arisen from the horizontal transfer, by genetic transformation, of blocks of DNA, usually of a few hundred base pairs. The commensal species identified as donors in these interspecies recombinational events (N. flavescens and N. cinerea) are intrinsically more resistant to penicillin than typical isolates of the pathogenic species. Transformation has apparently provided N. meningitidis and N. gonorrhoeae with a mechanism by which they can obtain increased resistance to penicillin by replacing their penA genes (or the relevant parts of them) with the penA genes of related species that fortuitously produce forms of PBP 2 that are less susceptible to inhibition by the antibiotic. The ends of the diverged blocks of DNA in the penA genes of different penicillin-resistant strains are located at the same position more often than would be the case if they represent independent crossovers at random points along the gene. Some of these common crossover points may represent common ancestry, but reasons are given for thinking that some may represent independent events occurring at recombinational hotspots.

Linkage of inflammation and oxidative stress via release of glutathionylated peroxiredoxin-2, which acts as a danger signal

Significance Inflammation often complicates diseases associated with oxidative stress. This study shows that inflammatory macrophages release proteins with specific forms of cysteine oxidation to disulfides, particularly glutathionylation. Redox proteomics identified peroxiredoxin 2 (PRDX2) as a protein released in glutathionylated form by inflammation both in vivo and in vitro. Extracellular PRDX2 then triggers the production of TNF-α. These data indicate that redox-dependent mechanisms, in an oxidative cascade, can induce inflammation. The mechanism by which oxidative stress induces inflammation and vice versa is unclear but is of great importance, being apparently linked to many chronic inflammatory diseases. We show here that inflammatory stimuli induce release of oxidized peroxiredoxin-2 (PRDX2), a ubiquitous redox-active intracellular enzyme. Once released, the extracellular PRDX2 acts as a redox-dependent inflammatory mediator, triggering macrophages to produce and release TNF-α. The oxidative coupling of glutathione (GSH) to PRDX2 cysteine residues (i.e., protein glutathionylation) occurs before or during PRDX2 release, a process central to the regulation of immunity. We identified PRDX2 among the glutathionylated proteins released in vitro by LPS-stimulated macrophages using mass spectrometry proteomic methods. Consistent with being part of an inflammatory cascade, we find that PRDX2 then induces TNF-α release. Unlike classical inflammatory cytokines, PRDX2 release does not reflect LPS-mediated induction of mRNA or protein synthesis; instead, PRDX2 is constitutively present in macrophages, mainly in the reduced form, and is released in the oxidized form on LPS stimulation. Release of PRDX2 is also observed in human embryonic kidney cells treated with TNF-α. Importantly, the PRDX2 substrate thioredoxin (TRX) is also released along with PRDX2, enabling an oxidative cascade that can alter the –SH status of surface proteins and thereby facilitate activation via cytokine and Toll-like receptors. Thus, our findings suggest a model in which the release of PRDX2 and TRX from macrophages can modify the redox status of cell surface receptors and enable induction of inflammatory responses. This pathway warrants further exploration as a potential novel therapeutic target for chronic inflammatory diseases.

Interspecies recombination, and phylogenetic distortions, within the glutamine synthetase and shikimate dehydrogenase genes of Neisseria meningitidis and commensal Neisseria species

Visual inspection showed clear evidence of a history of intraspecies recombinational exchanges within the neighbouring meningococcal shikimate dehydrogenase (aroE ) and glutamine synthetase (glnA) genes, which was supported by the non‐congruence of the trees constructed from the sequences of these genes from different meningococcal strains, and by statistical tests for mosaic structure. Many examples were also found of highly localized interspecies recombinational exchanges between the meningococcal aroE and glnA genes and those of commensal Neisseria species. These exchanges appear to have inflated the sequence variation at these loci, and have resulted in major distortions of the phylogenetic trees constructed from the sequences of the aroE and glnA genes of human pathogenic and commensal Neisseria species. Statistical tests for sequence mosaicism, and for anomalies within the Neisseria species trees, strongly supported the view that frequent interspecies recombination has occurred within aroE and glnA. The high levels of sequence variation, and intra‐ and interspecies recombination, within aroE and glnA did not appear to be due to a ‘hitch‐hiking’ effect caused by positive selection for variation at a neighbouring gene. Our results suggest that interspecies recombinational exchanges with commensal Neisseria occur frequently in some meningococcal ‘housekeeping’ genes as they can be observed readily even when there appears to be no obvious selection for the recombinant phenotypes.

Membrane topology of penicillin‐binding protein 3 of Escherichia coli

The β‐lactamase fusion vector, pJBS633, has been used to analyse the organization of penicillin‐binding protein 3 (PBP3) in the cytoplasmic membrane of Escherichia coli. The fusion junctions in 84 in‐frame fusions of the coding region of mature TEM β‐lactamase to random positions within the PBP3 gene were determined. Fusions of β‐lactamase to 61 different positions in PBP3 were obtained. Fusions to positions within the first 31 residues of PBP3 resulted in enzymatically active fusion proteins which could not protect single cells of E. coli from killing by ampicillin, indicating that the β‐lactamase moieties of these fusion proteins were not translocated to the peri‐plasm. However, all fusions that contained: ≥36 residues of PBP3 provided single ceils of E coli with substantial levels of resistance to ampicillin, indicating that the β‐lactamase moieties of these fusion proteins were translocated to the periplasm. PBP3 therefore appeared to have a simple membrane topology with residues 36 to the carboxy‐terminus exposed on the periplasmic side of the cytoplasmic membrane. This topology was confirmed by showing that PBP3 was protected from proteolytic digestion at the cytoplasmic side of the inner membrane but was completely digested by proteolytic attack from the periplasmic side. PBP3 was only inserted in the cytoplasmic membrane at its amino terminus since replacement of its putative lipoprotein signal peptide with a normal signal peptide resulted in a water‐soluble, periplasmic form of the enzyme. The periplasmic form of PBP3 retained its penicillin‐binding activity and appeared to be truly water‐soluble since it fractionated, in the absence of detergents, with the expected molecular weight on Sephadex G‐100 and was not retarded by hydrophobic interaction chromatography on Phenyl‐Superose.

Genome signature-based dissection of human gut metagenomes to extract subliminal viral sequences

Bacterial viruses (bacteriophages) have a key role in shaping the development and functional outputs of host microbiomes. Although metagenomic approaches have greatly expanded our understanding of the prokaryotic virosphere, additional tools are required for the phage-oriented dissection of metagenomic data sets, and host-range affiliation of recovered sequences. Here we demonstrate the application of a genome signature-based approach to interrogate conventional whole-community metagenomes and access subliminal, phylogenetically targeted, phage sequences present within. We describe a portion of the biological dark matter extant in the human gut virome, and bring to light a population of potentially gut-specific Bacteroidales-like phage, poorly represented in existing virus like particle-derived viral metagenomes. These predominantly temperate phage were shown to encode functions of direct relevance to human health in the form of antibiotic resistance genes, and provided evidence for the existence of putative ‘viral-enterotypes’ among this fraction of the human gut virome.

Use of a β‐lactamase fusion vector to investigate the organization of penicillin‐binding protein 1B in the cytoplasmic membrane of Escherichia coli

The coding region for the mature form of TEM β–lactamase was fused to random positions within the coding region of the penicillin–binding protein 1B (PBP 1B) gene and the nucleotide sequences across the fusion junctions of 100 in–frame fusions were determined. All fusion proteins that contained at least the NH2–terminal 94 residues of PBP 1B provided individual cells of E. coli with substantial levels of ampicillin resistance, suggesting that the β–lactamase moiety had been translocated to the periplasm. Fusion proteins that contained ≤ 63 residues of PBP 1B possessed β–lactamase activity, but could not protect single cells of E. coli from ampicillin, indicating that the 3–lactamase moiety of these fusion proteins remained in the cytoplasm. The β–lactamase fusion approach suggested a model for the organization of PBP 1B in which the protein is embedded in the cytoplasmic membrane by a single hydrophobic trans–membrane segment (residues 64–87), with a short NH2–terminal domain (residues 1–63), and the remainder of the polypeptide (residues 68–844) exposed on the periplasmic side of the cytoplasmic membrane. The proposed model for the organization of PBP 1B was supported by experiments which showed that the protein was completely digested by proteinase K added from the periplasmic side of the cytoplasmic membrane but was only slightly reduced in size by protease attack from the cytoplasmic side of the membrane.

Representational difference analysis of cDNA for the detection of differential gene expression in bacteria: development using a model of iron-regulated gene expression in Neisseria meningitidis

Representational difference analysis of cDNA (cDNA RDA) provides a powerful technique for the identification of specific differences between two mRNA populations. The method has previously been used to analyse differential gene expression in eukaryotes, but until now has not been successfully applied to prokaryotes. A strain of Neisseria meningitidis with a deletion of the iron-regulated lactoferrin-binding protein A (IbpA) gene, grown under iron-replete conditions, and the isogenic parent strain, grown under iron limitation, were used as a model for developing cDNA RDA for use with bacteria. In this system, the technique should specifically detect the differential expression of the IbpA gene in the parent strain, along with other genes whose expression is switched on (or up-regulated) under iron-deficient conditions. Since cDNA RDA requires high-quality, representative mRNA, a variety of methods for the isolation of RNA were evaluated. A triisopropylnaphthalene sulphonic acid/ p-aminosalicylic acid-based technique was found to give the best results. cDNA was prepared from total RNA isolated from the two N. meningitidis strains and subjected to an adapted cDNA RDA procedure. The method resulted in the amplification of five major PCR products, which included fragments of the IbpA gene and the iron-regulated RTX-like toxin gene (frpC), thus validating the technique for use with bacteria.

Comparative (Meta)genomic Analysis and Ecological Profiling of Human Gut-Specific Bacteriophage φB124-14

Bacteriophage associated with the human gut microbiome are likely to have an important impact on community structure and function, and provide a wealth of biotechnological opportunities. Despite this, knowledge of the ecology and composition of bacteriophage in the gut bacterial community remains poor, with few well characterized gut-associated phage genomes currently available. Here we describe the identification and in-depth (meta)genomic, proteomic, and ecological analysis of a human gut-specific bacteriophage (designated φB124-14). In doing so we illuminate a fraction of the biological dark matter extant in this ecosystem and its surrounding eco-genomic landscape, identifying a novel and uncharted bacteriophage gene-space in this community. φB124-14 infects only a subset of closely related gut-associated Bacteroides fragilis strains, and the circular genome encodes functions previously found to be rare in viral genomes and human gut viral metagenome sequences, including those which potentially confer advantages upon phage and/or host bacteria. Comparative genomic analyses revealed φB124-14 is most closely related to φB40-8, the only other publically available Bacteroides sp. phage genome, whilst comparative metagenomic analysis of both phage failed to identify any homologous sequences in 136 non-human gut metagenomic datasets searched, supporting the human gut-specific nature of this phage. Moreover, a potential geographic variation in the carriage of these and related phage was revealed by analysis of their distribution and prevalence within 151 human gut microbiomes and viromes from Europe, America and Japan. Finally, ecological profiling of φB124-14 and φB40-8, using both gene-centric alignment-driven phylogenetic analyses, as well as alignment-free gene-independent approaches was undertaken. This not only verified the human gut-specific nature of both phage, but also indicated that these phage populate a distinct and unexplored ecological landscape within the human gut microbiome.

Redox proteomics of the inflammatory secretome identifies a common set of redoxins and other glutathionylated proteins released in inflammation, influenza virus infection and oxidative stress

Protein cysteines can form transient disulfides with glutathione (GSH), resulting in the production of glutathionylated proteins, and this process is regarded as a mechanism by which the redox state of the cell can regulate protein function. Most studies on redox regulation of immunity have focused on intracellular proteins. In this study we have used redox proteomics to identify those proteins released in glutathionylated form by macrophages stimulated with lipopolysaccharide (LPS) after pre-loading the cells with biotinylated GSH. Of the several proteins identified in the redox secretome, we have selected a number for validation. Proteomic analysis indicated that LPS stimulated the release of peroxiredoxin (PRDX) 1, PRDX2, vimentin (VIM), profilin1 (PFN1) and thioredoxin 1 (TXN1). For PRDX1 and TXN1, we were able to confirm that the released protein is glutathionylated. PRDX1, PRDX2 and TXN1 were also released by the human pulmonary epithelial cell line, A549, infected with influenza virus. The release of the proteins identified was inhibited by the anti-inflammatory glucocorticoid, dexamethasone (DEX), which also inhibited tumor necrosis factor (TNF)-α release, and by thiol antioxidants (N-butanoyl GSH derivative, GSH-C4, and N-acetylcysteine (NAC), which did not affect TNF-α production. The proteins identified could be useful as biomarkers of oxidative stress associated with inflammation, and further studies will be required to investigate if the extracellular forms of these proteins has immunoregulatory functions.

A machine learning approach to explore the spectra intensity pattern of peptides using tandem mass spectrometry data.

BackgroundA better understanding of the mechanisms involved in gas-phase fragmentation of peptides is essential for the development of more reliable algorithms for high-throughput protein identification using mass spectrometry (MS). Current methodologies depend predominantly on the use of derived m/z values of fragment ions, and, the knowledge provided by the intensity information present in MS/MS spectra has not been fully exploited. Indeed spectrum intensity information is very rarely utilized in the algorithms currently in use for high-throughput protein identification.ResultsIn this work, a Bayesian neural network approach is employed to analyze ion intensity information present in 13878 different MS/MS spectra. The influence of a library of 35 features on peptide fragmentation is examined under different proton mobility conditions. Useful rules involved in peptide fragmentation are found and subsets of features which have significant influence on fragmentation pathway of peptides are characterised. An intensity model is built based on the selected features and the model can make an accurate prediction of the intensity patterns for given MS/MS spectra. The predictions include not only the mean values of spectra intensity but also the variances that can be used to tolerate noises and system biases within experimental MS/MS spectra.ConclusionThe intensity patterns of fragmentation spectra are informative and can be used to analyze the influence of various characteristics of fragmented peptides on their fragmentation pathway. The features with significant influence can be used in turn to predict spectra intensities. Such information can help develop more reliable algorithms for peptide and protein identification.