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Terminal restriction fragment length polymorphism is a recent molecular approach that can assess subtle genetic differences between strains as well as provide insight into the structure and function of microbial communities. The technique has both high sensitivity and throughput making it ideal for comparative analyses.
The solution structure of bacteriocin AS-48, a 70-residue cyclic polypeptide from Enterococcus faecalis, consists of a globular arrangement of five alpha-helices enclosing a compact hydrophobic core. The head-to-tail union lies in the middle of helix 5, a fact that is shown to have a pronounced effect on the stability of the three-dimensional structure. Positive charges in the side chains of residues in helix 4 and in the turn linking helix 4 to helix 5 form a cluster that most probably determine its antibacterial activity by promoting pore formation in cell membranes. A similar five-helix structural motif has been found in the antimicrobial NK-lysin, an effector polypeptide of T and natural killer (NK) cells. Bacteriocin AS-48 lacks the three disulfide bridges characteristic of the saposin fold present in NK-lysin, and has no sequence homology with it. Nevertheless, the similar molecular architecture and high positive charge strongly suggest a common mechanism of antibacterial action.
The adenylyl and guanylyl cyclases catalyze the formation of 3', 5'-cyclic adenosine or guanosine monophosphate from the corresponding nucleoside 5'-triphosphate. The guanylyl cyclases, the mammalian adenylyl cyclases, and their microbial homologues function as pairs of homologous catalytic domains. The crystal structure of the rat type II adenylyl cyclase C2 catalytic domain was used to model by homology a mammalian adenylyl cyclase C1-C2 domain pair, a homodimeric adenylyl cyclase of Dictyostelium discoideum, a heterodimeric soluble guanylyl cyclase, and a homodimeric membrane guanylyl cyclase. Mg2+ATP or Mg2+GTP were docked into the active sites based on known stereochemical constraints on their conformation. The models are consistent with the activities of seven active-site mutants. Asp-310 and Glu-432 of type I adenylyl cyclase coordinate a Mg2+ ion. The D310S and D310A mutants have 10-fold reduced Vmax and altered [Mg2+] dependence. The NTP purine moieties bind in mostly hydrophobic pockets. Specificity is conferred by a Lys and an Asp in adenylyl cyclase, and a Glu, an Arg, and a Cys in guanylyl cyclase. The models predict that an Asp from one domain is a general base in the reaction, and that the transition state is stabilized by a conserved Asn-Arg pair on the other domain.
Mast cells are well known for their harmful role in IgE-mediated hypersensitivity reactions, but their physiological role remains a mystery. Several recent studies have reported that mast cells play a critical role in innate immunity in mice by releasing tumor necrosis factor alpha (TNF-alpha) to recruit neutrophils to sites of enterobacterial infection. In some cases, the mast cell TNF-alpha response was triggered when these cells directly bound FimH on the surface of Escherichia coli. We have identified CD48, a glycosylphosphatidylinositol-anchored molecule, to be the complementary FimH-binding moiety in rodent mast cell membrane fractions. We showed that (i) pretreatment of mast cell membranes with antibodies to CD48 or phospholipase C inhibited binding of FimH+ E. coli, (ii) FimH+ E. coli but not a FimH- derivative bound isolated CD48 in a mannose-inhibitable manner, (iii) binding of FimH+ bacteria to Chinese hamster ovary (CHO) cells was markedly increased when these cells were transfected with CD48 cDNA, and (iv) antibodies to CD48 specifically blocked the mast cell TNF-alpha response to FimH+ E. coli. Thus, CD48 is a functionally relevant microbial receptor on mast cells that plays a role in triggering inflammation.
Phagocyte activation is accompanied by assembly of an NADPH oxidase that reduces oxygen to form a number of reactive species. These oxygen radicals can eradicate invading microorganisms, regulate the function of other immune reactive cells, and cause damage to "innocent bystander" cells. It is generally assumed that the NADPH oxidase is activated exclusively in the plasma membrane. In neutrophils, this assumption does not fit with the subcellular localization of the membrane component of the oxidase, which is stored in granule compartments. It has now become increasingly evident that oxidants are also produced in an intracellular compartment that we identify as the specific granules. Myeloperoxidase is stored in another granule subset, the azurophil granules, and participates in the processing of the oxidative metabolites. We suggest that neutrophil activation is accompanied by fusion between azurophil and specific granules, allowing these peroxidase-dependent reactions to take place. The presented data suggest a requisite role for neutrophil oxidants complementing their function as microbial killing agents. Signaling capabilities of the oxidants, affecting for example, the state of protein phosphorylation, regulation of transcription factors, and induction of apoptosis, are discussed.
SUMMARY: Genome-centric analysis of metagenomic samples is a powerful method for understanding the function of microbial communities. Calculating read coverage is a central part of analysis, enabling differential coverage binning for recovery of genomes and estimation of microbial community composition. Coverage is determined by processing read alignments to reference sequences of either contigs or genomes. Per-reference coverage is typically calculated in an ad-hoc manner, with each software package providing its own implementation and specific definition of coverage. Here we present a unified software package CoverM which calculates several coverage statistics for contigs and genomes in an ergonomic and flexible manner. It uses "Mosdepth arrays" for computational efficiency and avoids unnecessary I/O overhead by calculating coverage statistics from streamed read alignment results. AVAILABILITY AND IMPLEMENTATION: CoverM is free software available at https://github.com/wwood/coverm. CoverM is implemented in Rust, with Python (https://github.com/apcamargo/pycoverm) and Julia (https://github.com/JuliaBinaryWrappers/CoverM_jll.jl) interfaces.
Lymphocytes bearing gamma delta T cell receptors (TCR) constitute a minor subpopulation of human peripheral blood lymphocytes. Their role and function during microbial infections are largely unknown. In 10 patients with Epstein-Barr virus-induced infectious mononucleosis, the gamma delta TCR-expressing T cell population expanded during the acute phase. These cells were largely delta TCS1-, CD4-, and CD8- but expressed activation antigens such as human leukocyte antigen-DR and CD38. The convalescent phase of infectious mononucleosis was characterized by a relative persistence of gamma delta T cells. Together these data suggest a possible role of gamma delta T cells in the control of primary Epstein-Barr virus infection in humans.
Group G streptococci that express M protein and resist phagocytosis in human blood (virulent strains) were compared with strains of groups G and A that are readily phagocytosed (avirulent). Virulent group G streptococci were less effective (P < .05) as activators of the alternative complement pathway (ACP) than were avirulent streptococci. In immunofluorescence studies, C3 bound more avidly to avirulent than to virulent group G streptococci. Resistance of virulent group G strains to ACP opsonization and to phagocytosis was markedly diminished by removal with pepsin of the type-specific portion of the M molecule. Preincubation with fibrinogen did not diminish ACP activation or C3 binding by virulent group G and A streptococci but did exert an antiphagocytic effect. Given the similarity of M proteins of groups G and A in structure and function, other microbial constituents are likely responsible for differences in the spectra of illnesses attributable to the two serogroups.
Xylose reductase has been purified to apparent homogeneity from cell extracts of the fungus Cryptococcus flavus grown on D-xylose as carbon source. The enzyme, the first of its kind from the phylum Basidiomycota, is a functional dimer composed of identical subunits of 35.3 kDa mass and requires NADP(H) for activity. Steady-state kinetic parameters for the reaction, D-xylose + NADPH + H(+)<--> xylitol + NADP(+), have been obtained at pH 7.0 and 25 degrees C. The catalytic efficiency for reduction of D-xylose is 150 times that for oxidation of xylitol. This and the 3-fold tighter binding of NADPH than NADP(+) indicate that the enzyme is primed for unidirectional metabolic function in microbial physiology. Kinetic analysis of enzymic reduction of aldehyde substrates differing in hydrophobic and hydrogen bonding capabilities with binary enzyme-NADPH complex has been used to characterize the substrate-binding pocket of xylose reductase. Total transition state stabilization energy derived from bonding with non-reacting sugar hydroxyls is approximately 15 kJ/mol, with a major contribution of 5-8 kJ/mol made by interactions with the C-2(R) hydroxy group. The aldehyde binding site is approximately 1.2 times more hydrophobic than n-octanol and can accommodate linear alkyl chains of <or=6 carbons. Hydrophobic interactions provide a total binding energy of approximately 10 kJ/mol. Specificity for the aldehyde substrate is achieved through large decreases in apparent K(m) ( approximately 100-fold) and smaller but significant increases in turnover number ( approximately 5-fold). We observed up to 250-fold preference of xylose reductase for reaction with pyridine carbaldehydes, 4-nitro-benzaldehyde, and alpha-oxo-aldehydes over reaction with D-xylose, perhaps reflecting a secondary role of this enzyme in detoxication metabolism of reactive endogenous aldehydes and compounds of xenobiotic origin.
Enhancement of Streptococcus faecalis infection and lowering of the complement level have been demonstrated in mice injected with a heat-treated suspension of baker's yeast (Saccharomyces cerevisiae). The leucocyte response to the infection was not affected. The yeast preparation showed, in vitro, an intense anti-complementary activity on mouse serum and interfered with the microbial killing function of the mouse peritoneal macrophages. No significant stimulation of the growth of S. faecalis in vitro in the presence of the yeast was observed. The enhancement of the infection in mice treated with the yeast seems to be mediated, mainly, by complement depletion.
Interactions between microbes shape the structure and function of microbial communities. While studying interactions is key to understanding microbial communities as a whole, gaining a detailed mechanistic view is challenging due to the scale of co-occurring interactions. The model nutrient approach enables the study of a subset of interactions involving a single nutrient class and can shed light on broader interaction mechanisms involving other nutrients. Here, we focus on cobamides, the cobalamin (vitamin B12) family of enzyme cofactors, to study nutrient competition and nutrient-sharing interactions in co-cultures and tri-cultures. We examined bacteria that were previously isolated from a grassland soil and were characterized as "dependents" (require cobamides but cannot synthesize them) or "producers" (synthesize cobamides). The outcome of competition between a pair of dependents was predictable based on monoculture growth characteristics, with the dominant microbe determined by its adaptation to a specific cobamide concentration range. Moreover, cobamide producers could support the cobamide-dependent growth of dependents in co-culture and influenced the outcome of competition between dependents in tri-culture. We analyzed the metabolic capacity encoded in the genomes of producers and dependents and found that cobamides are likely the main shared nutrient in our co- and tri-cultures. These results highlight the utility of the model nutrient approach to characterize and predict interactions in bacterial consortia of increasing complexity.
The present study was designed to establish the susceptibility of macrophage-mediated effector functions to tetanus toxin (TT). Using the murine macrophage cell line, GG2EE, generated in vitro by v-raf/v-myc oncogenes, we have previously provided evidence that TT selectively inhibits interferon gamma (IFN-gamma), but not basal, lysozyme activity. Here we show that while neither phagocytic nor candidacidal activities are affected by TT treatment, antitumoral activity is significantly impaired after exposure to TT. This phenomenon, which is dose-dependent, is fully ascribed to the holotoxin, as heat inactivated TT, C or A-B fragments result ineffective. Furthermore, C but not A-B fragment competes with TT in abrogating its inhibitory effects. Overall, these data indicate that TT is not a broad-spectrum, down-regulating signal on macrophage-mediated functions, thus implying that its toxic action is exerted on specific molecular targets.
The aim of this review is to interpret recent studies in which molecular methods were used to identify and characterize prokaryotes in lake sediments and related habitats. In the first part studies based on the phylogenetic diversity of prokaryotes found in lacustrine habitats are summarized. The application of various cultivation-independent methods for the characterization of distinct groups of sediment bacteria is exemplified with morphologically conspicuous, colorless sulfur bacteria in the second part of this review. Finally, traditional and recently developed methods are described which could be used for linking the function of microbial populations with their identification. The potential of these approaches for the study of lake sediments is discussed in order to give a perspective for future studies in this habitat.
The membrane integrity of a cell is a well-accepted criterion for characterizing viable (active or inactive) cells and distinguishing them from damaged and membrane-compromised cells. This information is of major importance in studies of the function of microbial assemblages in natural environments, in order to assign bulk activities measured by various methods to the very active cells that are effectively responsible for the observations. To achieve this task for bacteria in freshwater and marine waters, we propose a nucleic acid double-staining assay based on analytical flow cytometry, which allows us to distinguish viable from damaged and membrane-compromised bacteria and to sort out noise and detritus. This method is derived from the work of S. Barbesti et al. (Cytometry 40:214-218, 2000) which was conducted on cultured bacteria. The principle of this approach is to use simultaneously a permeant (SYBR Green; Molecular Probes) and an impermeant (propidium iodide) probe and to take advantage of the energy transfer which occurs between them when both probes are staining nucleic acids. A full quenching of the permeant probe fluorescence by the impermeant probe will point to cells with a compromised membrane, a partial quenching will indicate cells with a slightly damaged membrane, and a lack of quenching will characterize intact membrane cells identified as viable. In the present study, this approach has been adapted to bacteria in freshwater and marine waters of the Mediterranean region. It is fast and easy to use and shows that a large fraction of bacteria with low DNA content can be composed of viable cells. Admittedly, limitations stem from the unknown behavior of unidentified species present in natural environments which may depart from the established permeability properties with respect to the fluorescing dyes.
We take the view that adherence is not just a static process of holding hands but rather elicits a response in the targeted cell. From this point of view, adherence is an active process with an outcome. This outcome or fate is predictable only when several parameters of the host cell-adhesin interaction are known: is the adhesin acting alone or in series with other products, is the receptor up- or down-regulated at the time of ligation, which domain of the receptor is bound, and finally, which intracellular response circuits are connected to the receptor in the cell type targeted? Variations in these parameters are the basis for the ability of the adhesins of pathogens to orchestrate outcomes as disparate as simple address recognition versus actin nucleation, cytokine induction, activation of plasmin, derangement of leukocyte migration, or deposition of antibody on host cell membranes. The recognition of the relatedness of some eukaryotic and prokaryotic adhesive domains and the shared use of existing eukaryotic cell-cell interaction systems between host and pathogen suggest that the cellular interactions of interest in eukaryotic cell biology can be revealed by taking clues from the pathogens, which have studied and adapted to them the longest.
BACKGROUND: Dietary arginine, glutamine, and fish oil each have been shown to improve resistance to infection. The purpose of this study was to assess the potential benefit of different combinations and amounts of these components on bacterial translocation and related mortality during gut-derived sepsis. METHODS: Balb/c mice were fed for 10 days with an AIN-76A diet supplemented with different combinations and percentages of arginine, glutamine, glycine, fish oil, and medium-chain triglycerides. Controls were fed a complete AIN-76A diet or chow. After 10 days of feeding, all animals were transfused. On day 15, the animals were gavaged with 10(10) 111In-radiolabeled or unlabeled Escherichia coli and given a 30% burn injury. Animals gavaged with unlabeled bacteria were observed for survival (n = 317). Groups that showed the best survival as well as control groups were gavaged with labeled bacteria and killed 4 hours postburn (n = 60) for harvest of mesenteric lymph nodes, liver and spleen. RESULTS: Mice fed diets enriched with 5% fish oil + 2% arginine, 2% arginine + 2% glutamine, or 5% fish oil + 2% glutamine had higher survival than control groups. The animals fed fish oil+glutamine had significantly reduced translocation to the liver and spleen. Animals fed arginine+glutamine had an enhanced ability to kill translocated organisms in the liver compared with other groups. Fish oil+arginine improved both barrier function and microbial killing. CONCLUSIONS: Feeding with arginine+glutamine, fish oil+arginine, or fish oil+glutamine supplemented diets positively affects the outcome in a gut-derived sepsis model.
Toll-like receptors (TLR:) have recently been linked to the immunostimulatory function of microbial toxins in human and mice. TLR: signals activation of nuclear factor kappaB that leads to the production of a number of proinflammatory mediators. TLR:4 mediates the endotoxin-induced inflammatory response, whereas TLR:2 may be involved in the response to yeast and Gram-positive bacterial products. To better understand age-related changes in acute inflammatory response, we studied the ontogeny of TLR:2 and TLR:4 mRNA in murine fetal lung, liver, and placenta by quantitative reverse transcriptase-PCR. Different expression patterns were seen between the tissues and between the TLR: This is in accordance with the evidence that there are differences in the receptors for different microbial toxins and that the response is organ specific. We additionally show that the expression of TLR: was dependent on the stage of differentiation. In the liver, the levels of Tlr2 and Tlr4 were high regardless of the age. In the lung, Tlr2 and Tlr4 expression levels were barely detectable in immature fetus (d 14-15). Tlr2 and Tlr4 were increased several-fold during prenatal development and further increased after birth. The present results support the finding of a deficient inflammatory response of the immature lung to microbial toxins.
This review highlights the important roles played by magnesium in the growth and metabolic functions of microbial and animal cells, and therefore assigns a key role for magnesium ions in biotechnology. The fundamental biochemical and physiological actions of magnesium as a regulatory cation are outlined. Such actions are deemed to be relevant in an applied sense, because Mg2+ availability in cell culture and fermentation media can dramatically influence growth and metabolism of cells. Manipulation of extracellular and intracellular magnesium ions can thus be envisaged as a relatively simplistic, but nevertheless versatile, means of physiological cell engineering. In addition, biological antagonism between calcium and magnesium at the molecular level may have profound consequences for the optimization of biotechnological processes that exploit cells. In fermentation, for example, it is argued that the efficiency of microbial conversion of substrate to product may be improved by altering Mg:Ca concentration ratios in industrial feedstocks in a way that makes more magnesium available to the cells. With particular respect to yeast-based biotechnologies, magnesium availability is seen as being crucially important in governing central pathways of carbohydrate catabolism, especially ethanolic fermentation. It is proposed that such influences of magnesium ions are expressed at the combined levels of key enzyme activation and cell membrane stabilization. The former ensures optimum flow of substrate to ethanol and the latter acts to protect yeasts from physical and chemical stress.