The mechanism of two-phase motility in the spirochete Leptospira: Swimming and crawling.

Many species of bacteria are motile, but their migration mechanisms are considerably diverse. Whatever mechanism is used, being motile allows bacteria to search for more optimal environments for growth, and motility is a crucial virulence factor for pathogenic species. The spirochete Leptospira, having two flagella in the periplasmic space, swims in liquid but has also been previously shown to crawl over solid surfaces. The present motility assays show that the spirochete movements both in liquid and on surfaces involve a rotation of the helical cell body. Direct observations of cell-surface movement with amino-specific fluorescent dye and antibody-coated microbeads suggest that the spirochete attaches to the surface via mobile, adhesive outer membrane components, and the cell body rotation propels the cell relative to the anchoring points. Our results provide models of how the spirochete switches its motility mode from swimming to crawling.

Leptospiral flagellar sheath protein FcpA interacts with FlaA2 and FlaB1 in Leptospira biflexa.

Leptospira spp. are spirochete bacteria that possess periplasmic flagella (PFs) underneath the outer membrane; each flagellum is attached to each end of the protoplasmic cylinder. PFs of Leptospira have a coiled shape that bends the end of the cell body. However, the molecular mechanism by which multiple flagellar proteins organize to form the distinctively curled PF of Leptospira remains unclear. Here we obtained a slow-motility mutant of L. biflexa MD4-3 by random insertion mutagenesis using a Himar1 transposon. In MD4-3, the gene encoding the flagellar sheath protein, flagellar-coiling protein A (FcpA), which was recently identified in L. interrogans, was inactivated. As with L. interrogans ΔfcpA strains, the L. biflexa ΔfcpA strain lacked a distinct curvature at both ends of the cell body, and its motility was significantly reduced as compared with that of the wild-type strain. PFs isolated from the ΔfcpA strain were straight and were thinner than those isolated from the wild-type strain. Western blot analysis revealed that flagellar proteins FlaA1, FlaA2, FlaB1, and FlaB2 were expressed in the ΔfcpA strain but the flagellar proteins, except for FlaB2 were not incorporated in its PFs. Immunoprecipitation assay using anti-FcpA antiserum demonstrated that FcpA associates with FlaA2 and FlaB1. The association between FcpA and FlaA2 was also verified using pull-down assay. The regions of FlaA2 and FlaB1 interacting with FcpA were determined using a bacterial two-hybrid assay. These results suggest that FcpA together with FlaA2, produces coiling of PF of the Leptospira, and the interaction between the sheath and core filament may be mediated by FcpA and FlaB1.

Microproteomics with microfluidic-based cell sorting: Application to 1000 and 100 immune cells.

Ultimately, cell biology seeks to define molecular mechanisms underlying cellular functions. However, heterogeneity within cell populations must be considered for optimal assay design and data interpretation. Although single-cell analyses are desirable for addressing this issue, practical considerations, including assay sensitivity, limit their broad application. Therefore, omics studies on small numbers of cells in defined subpopulations represent a viable alternative for elucidating cell functions at the molecular level. MS-based proteomics allows in-depth proteome exploration, although analyses of small numbers of cells have not been pursued due to loss during the multistep procedure involved. Thus, optimization of the proteomics workflow to facilitate the analysis of rare cells would be useful. Here, we report a microproteomics workflow for limited numbers of immune cells using non-damaging, microfluidic chip-based cell sorting and MS-based proteomics. Samples of 1000 or 100 THP-1 cells were sorted, and after enzymatic digestion, peptide mixtures were subjected to nano-LC-MS analysis. We achieved reasonable proteome coverage from as few as 100-sorted cells, and the data obtained from 1000-sorted cells were as comprehensive as those obtained using 1 µg of whole cell lysate. With further refinement, our approach could be useful for studying cell subpopulations or limited samples, such as clinical specimens.

Bioanalytical insights into mediator lipidomics.

The importance of lipids in health and disease has been widely acknowledged. Lipids are well known to undergo enzymatic and/or non-enzymatic conversions to lipid mediators (LMs), which demonstrate potent actions in various biological events, such as the regulation of cellular signaling pathways and the promotion and resolution of inflammation. LMs activate G-protein-coupled receptors (GPCRs) to exert various functions. Monitoring these mediators in disease is essential to uncover the mechanisms of pathogenesis for many diseases, such as asthma, rheumatoid arthritis, Alzheimer's disease, and cancer. Along with technical developments in mass spectrometry, highly sensitive and multiplexed analyses of LMs in the human periphery and other tissues have become available. These advancements enable the temporal and spatial profiling of LMs; therefore, the findings obtained from LM profiling are expected to decode pathology. As trace amounts of LMs can exert functions, the development of a highly sensitive, accurate, and robust analytical method is necessary. Although not mandatory, mediator lipidomics validation is becoming popular and remains challenging. Because LMs already exist in biological matrices, evaluations of the matrix effect and extraction efficiencies are important issues. Thus, more careful analyses are required. In this review, we focus on mediator lipidomics, including polyunsaturated fatty acids (PUFAs), such as omega-3 and omega-6 fatty acids, and LMs derived from PUFAs, such as eicosanoids, lipoxins and resolvins. In addition to the recent progress in human mediator lipidomics, bioanalytical insights derived from this field (i.e., effective sample preparation from biological matrices and evaluation of the matrix effect) are described herein.

Genomic, proteomic, morphological, and phylogenetic analyses of vB_EcoP_SU10, a podoviridae phage with C3 morphology.

A recently isolated phage, vB_EcoP_SU10 (SU10), with the unusual elongated C3 morphotype, can infect a wide range of Escherichia coli strains. We have sequenced the genome of this phage and characterized it further by mass spectrometry based proteomics, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and ultra-thin section electron microscopy. The genome size is 77,327 base pairs and its genes, and genome architecture, show high similarity to the phiEco32 phage genes and genome. The TEM images reveal that SU10 have a quite long tail for being a Podoviridae phage, and that the tail also changes conformation upon infection. The ultra-thin section electron microscopy images of phages at the stage of replication within the host cell show that the phages form a honeycomb-like structure under packaging of genomes and assembly of mature capsids. This implies a tight link between the replication and cutting of the concatemeric genome, genome packaging, and capsid assembly. We have also performed a phylogenetic analysis of the structural genes common between Podoviridae phages of the C1 and C3 morphotypes. The result shows that the structural genes have coevolved, and that they form two distinct groups linked to their morphotypes. The structural genes of C1 and C3 phages appear to have diverged around 280 million years ago applying a molecular clock calibrated according to the presumed split between the Escherichia - Salmonella genera.

Ontogeny, distribution and potential roles of 5-hydroxymethylcytosine in human liver function.

BACKGROUND: Interindividual differences in liver functions such as protein synthesis, lipid and carbohydrate metabolism and drug metabolism are influenced by epigenetic factors. The role of the epigenetic machinery in such processes has, however, been barely investigated. 5-hydroxymethylcytosine (5hmC) is a recently re-discovered epigenetic DNA modification that plays an important role in the control of gene expression. RESULTS: In this study, we investigate 5hmC occurrence and genomic distribution in 8 fetal and 7 adult human liver samples in relation to ontogeny and function. LC-MS analysis shows that in the adult liver samples 5hmC comprises up to 1% of the total cytosine content, whereas in all fetal livers it is below 0.125%. Immunohistostaining of liver sections with a polyclonal anti-5hmC antibody shows that 5hmC is detected in most of the hepatocytes. Genome-wide mapping of the distribution of 5hmC in human liver samples by next-generation sequencing shows significant differences between fetal and adult livers. In adult livers, 5hmC occupancy is overrepresented in genes involved in active catabolic and metabolic processes, whereas 5hmC elements which are found in genes exclusively in fetal livers and disappear in the adult state, are more specific to pathways for differentiation and development. CONCLUSIONS: Our findings suggest that 5-hydroxymethylcytosine plays an important role in the development and function of the human liver and might be an important determinant for development of liver diseases as well as of the interindividual differences in drug metabolism and toxicity.

Comprehensive analysis of MHC ligands in clinical material by immunoaffinity-mass spectrometry.

Major histocompatibility complexes (MHC) are expressed on antigen-presenting cells (APC) that display peptide antigens. This is a crucial step to activate a T-cell response. Since immunogenic ligand of MHC is closely related with autoimmunity, inflammatory diseases, and cancer, comprehensive analysis of MHC ligands (the so-called Ligandome) is essential to unveil disease pathogenesis. Recently, immunotherapies such as vaccination have been focused on as new therapies of cancer, HIV, and infectious diseases. Therefore, the importance of comprehensive analysis of MHC ligands is increasing. Mass spectrometry has been the core technology of ligand identification since the 1990s. The sensitivity of mass spectrometers has been improved dramatically in recent years; thus, it enables to identify MHC ligands in clinical materials. This chapter lays out the workflow of MHC ligand identification in clinical materials, especially human bronchoalveolar (BAL) cells. MHC-ligand complexes are enriched by immunoaffinity extraction and captured ligand peptides are identified by LC-MS/MS. MHC class II ligand in BAL cells is described in this text; however, this approach is applicable to MHC class I and other clinical materials such as tissues.

Quantitative intact proteomics investigations of alveolar macrophages in sarcoidosis.

Alveolar macrophages are important for granuloma formation, which is the histological hallmark in sarcoidosis. Their function as antigen-presenting cells in sarcoidosis is also believed to be relevant to the outcome of disease, resulting either in remission or a prolonged chronic inflammation in the lungs. Our aim was to study alterations in the expression levels of the soluble proteome of alveolar macrophages in pulmonary sarcoidosis as compared with healthy controls, with the goal of identifying specific proteins and pathways important for the mechanisms of disease and/or disease phenotype. Quantitative proteomics approach using two-dimensional difference gel electrophoresis coupled to mass spectrometry was applied. Data was evaluated using multivariate modelling and pathway analyses. 69 protein spots were found to be significantly altered between sarcoidosis and healthy controls. Among these, 25 unique proteins were identified. Several of the identified proteins were related to key alveolar macrophage functionality, including the Fcγ-mediated phagocytosis and clathrin-mediated endocytosis pathways. Global proteomics analysis provided identification of alterations of a subset of proteins not previously reported in sarcoidosis. These alterations primarily affect biological pathways related to phagocytic macrophage functionality. These findings provide important insights into the role of macrophages in sarcoidosis pathogenesis.

Maresins: novel macrophage mediators with potent antiinflammatory and proresolving actions.

The endogenous cellular and molecular mechanisms that control acute inflammation and its resolution are of wide interest. Using self-resolving inflammatory exudates and lipidomics, we have identified a new pathway involving biosynthesis of potent antiinflammatory and proresolving mediators from the essential fatty acid docosahexaenoic acid (DHA) by macrophages (MPhis). During the resolution of mouse peritonitis, exudates accumulated both 17-hydroxydocosahexaenoic acid, a known marker of 17S-D series resolvin (Rv) and protectin biosynthesis, and 14S-hydroxydocosa-4Z,7Z,10Z,12E,16Z,19Z-hexaenoic acid from endogenous DHA. Addition of either DHA or 14S-hydroperoxydocosa-4Z,7Z,10Z,12E,16Z,19Z-hexaenoic acid to activated MPhis converted these substrates to novel dihydroxy-containing products that possessed potent antiinflammatory and proresolving activity with a potency similar to resolvin E1, 5S,12R,18R-trihydroxyeicosa-6Z,8E,10E,14Z,16E-pentaenoic acid, and protectin D1, 10R,17S-dihydroxydocosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid. Stable isotope incorporation, intermediate trapping, and characterization of physical and biological properties of the products demonstrated a novel 14-lipoxygenase pathway, generating bioactive 7,14-dihydroxydocosa-4Z,8,10,12,16Z,19Z-hexaenoic acid, coined MPhi mediator in resolving inflammation (maresin), which enhances resolution. These findings suggest that maresins and this new metabolome may be involved in some of the beneficial actions of DHA and MPhis in tissue homeostasis, inflammation resolution, wound healing, and host defense.

Rapid appearance of resolvin precursors in inflammatory exudates: novel mechanisms in resolution.

Resolution of inflammation is essential. Although supplementation of omega-3 fatty acids is widely used, their availability at sites of inflammation is not known. To this end, a multidisciplinary approach was taken to determine the relationship of circulating omega-3 to inflammatory exudates and the generation of resolution signals. In this study, we monitored resolvin precursors in evolving exudates, which initially paralleled increases in edema and infiltrating neutrophils. We also prepared novel microfluidic chambers to capture neutrophils from a drop of blood within minutes that permitted single-cell monitoring. In these, docosahexaenoic acid-derived resolvin D1 rapidly stopped neutrophil migration, whereas precursor docosahexaenoic acid did not. In second organ injury via ischemia-reperfusion, resolvin metabolically stable analogues were potent organ protectors reducing neutrophils. Together, these results indicate that circulating omega-3 fatty acids rapidly appear in inflammatory sites that require conversion to resolvins that control excessive neutrophil infiltration, protect organs, and foster resolution.

Anesthetics impact the resolution of inflammation.

BACKGROUND: Local and volatile anesthetics are widely used for surgery. It is not known whether anesthetics impinge on the orchestrated events in spontaneous resolution of acute inflammation. Here we investigated whether a commonly used local anesthetic (lidocaine) and a widely used inhaled anesthetic (isoflurane) impact the active process of resolution of inflammation. METHODS AND FINDINGS: Using murine peritonitis induced by zymosan and a systems approach, we report that lidocaine delayed and blocked key events in resolution of inflammation. Lidocaine inhibited both PMN apoptosis and macrophage uptake of apoptotic PMN, events that contributed to impaired PMN removal from exudates and thereby delayed the onset of resolution of acute inflammation and return to homeostasis. Lidocaine did not alter the levels of specific lipid mediators, including pro-inflammatory leukotriene B(4), prostaglandin E(2) and anti-inflammatory lipoxin A(4), in the cell-free peritoneal lavages. Addition of a lipoxin A(4) stable analog, partially rescued lidocaine-delayed resolution of inflammation. To identify protein components underlying lidocaine's actions in resolution, systematic proteomics was carried out using nanospray-liquid chromatography-tandem mass spectrometry. Lidocaine selectively up-regulated pro-inflammatory proteins including S100A8/9 and CRAMP/LL-37, and down-regulated anti-inflammatory and some pro-resolution peptides and proteins including IL-4, IL-13, TGF-â and Galectin-1. In contrast, the volatile anesthetic isoflurane promoted resolution in this system, diminishing the amplitude of PMN infiltration and shortening the resolution interval (Ri) approximately 50%. In addition, isoflurane down-regulated a panel of pro-inflammatory chemokines and cytokines, as well as proteins known to be active in cell migration and chemotaxis (i.e., CRAMP and cofilin-1). The distinct impact of lidocaine and isoflurane on selective molecules may underlie their opposite actions in resolution of inflammation, namely lidocaine delayed the onset of resolution (T(max)), while isoflurane shortened resolution interval (Ri). CONCLUSIONS: Taken together, both local and volatile anesthetics impact endogenous resolution program(s), altering specific resolution indices and selective cellular/molecular components in inflammation-resolution. Isoflurane enhances whereas lidocaine impairs timely resolution of acute inflammation.

A nonenzymatic modification of the amino-terminal domain of histone H3 by bile acid acyl adenylate.

Although it has been proposed that the secondary bile acids, deoxycholic acid and lithocholic acid, increase the number of aberrant crypt foci in the colon and may act as colon tumor promoters, there is little evidence detailing their mechanism of action. Histones play an important role in controlling gene expression, and the posttranslational modification of histones plays a role in regulation of intracellular signal transduction. In particular, the amino-terminal tail domain of histone H3 is sensitive to several posttranslational modifications, and acetylation of this domain changes its electrostatic environment and results in the loss of native folding. Therefore, we studied the modification of epsilon-amino groups on human histone H3 by deoxycholyl adenylate, which is an active intermediate in deoxycholyl thioester biosynthesis. After incubation of recombinant human histone H3 with a smaller amount of acyl adenylate, followed by enzymatic digestion, the peptide fragment mixtures were analyzed by matrix-assisted laser desorption ionization mass spectrometry. These data showed the formation of only one adduct fragment, which corresponded to amino acids 3-8 with a deoxycholate adduct, suggesting that the epsilon-amino group of Lys(4) had the highest reactivity. This novel modification, formation of a bile acid adduct on the histone H3 amino-terminal tail domain through an active acyl adenylate, may relate to the carcinogenesis-promoting effects of secondary bile acids.

Exopeptidase degradation for the analysis of phosphorylation site in a mono-phosphorylated peptide with matrix-assisted laser desorption/ionization mass spectrometry.

The utility of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) coupled with a peptide ladder sequencing method employing exopeptidase degradation for the analysis of phosphorylation site in a mono-phosphorylated peptide is investigated. MALDI-TOFMS analysis of time-dependent exopeptidase digestion using carboxypeptidase W and aminopeptidase M of the mono-phosphorylated 33-48 fragment isolated from a beta-casein tryptic digestion mixture allowed for the sequencing analysis from both the C-terminus and N-terminus. Negative ion detection MALDI-TOFMS made it possible to clearly measure the peptide ladder of mono-phosphorylated peptide by the strong negative charge localized at the phosphoric acid group. Since exopeptidase activity was suppressed by the existence of a phosphorylated amino acid residue, the termination exopeptidase degradation therefore suggested the existence of a phosphorylated amino acid residue at that site. This peptide ladder sequencing method using exopeptidases was effective for the identification of the site of a phosphorylated amino acid residue by a simple MALDI-TOFMS analysis in the negative ion detection mode.