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PubMed1999

Human milk lactoferrin binds ATP and dissociates into monomers.

The physiological role of lactoferrin (LF) is still unclear, but it has been suggested to be responsible for primary defence against microbial infections. Many different unique functions have been attributed to LF, including DNA and RNA binding, and transport into the nucleus, where LF binds to specific DNA sequences and activates transcription. Here we present evidence that in addition to the above (and below) mentioned functions LF binds ATP with a stoichiometry of 1 mole of nucleotide per mole of the protein and a Kd = 0.3 mM. The ATP-binding site is localized in the C-terminal domain of LF, in contrast to the antibacterial and polyanion-binding sites, which are located in the N-terminal domain. Binding of ATP by LF leads to dissociation of its oligomeric forms and to a change of the protein's interaction with polysaccharides, DNA and proteins.

Adenosine Triphosphate↗

PubMed1999

N-glycosylation of the carcinoembryonic antigen related cell adhesion molecule, C-CAM, from rat liver: detection of oversialylated bi- and triantennary structures.

Rat C-CAM is a ubiquitous, transmembrane and carcinoembryonic antigen related cell adhesion molecule. The human counterpart is known as biliary glycoprotein (BGP) or CD66a. It is involved in different cellular functions ranging from intercellular adhesion, microbial receptor activity, signaling and tumor suppression. In the present study N-glycosylation of C-CAM immunopurified from rat liver was analyzed in detail. The primary sequence of rat C-CAM contains 15 potential N-glycosylation sites. The N-glycans were enzymatically released from glycopeptides, fluorescently labeled with 2-aminobenzamide, and separated by two-dimensional HPLC. Oligosaccharide structures were characterized by enzymatic sequencing and MALDI-TOF-MS. Mainly bi- and triantennary complex structures were identified. The presence of type I and type II chains in the antennae of these glycans results in heterogeneous glycosylation of C-CAM. Sialylation of the sugars was found to be unusual; bi- and triantennary glycans contained three and four sialic acid residues, respectively, and this linkage seemed to be restricted to the type I chain in the antennae. Approximately 20% of the detected sugars contain these unusual numbers of sialic acids. C-CAM is the first transmembrane protein found to be oversialylated.

Adenosine Triphosphatases↗

PubMed2006

Pasteurella multocida toxin (PMT) activates RhoGTPases, induces actin polymerization and inhibits migration of human dendritic cells, but does not influence macropinocytosis.

Dendritic cells (DCs) are considered as one of the principal initiators of immune responses. In their immature state, they migrate into peripheral tissue in order to uptake antigen and to patrol for danger signals. Upon maturation, they acquire the ability to migrate to the lymph nodes and present the captured antigens to T cells in order to direct the development of specific immune responses. There is evidence that microbial compounds interfere with proper functions of DCs in order to block innate and specific immunity. Here we characterized the influence of Pasteurella multocida toxin (PMT) on monocyte-derived DCs. Using pull-down assays with recombinant rhotekin or p21-activated kinase, we demonstrated the activation of RhoGTPases by PMT in DCs. Moreover, PMT induced changes in DC morphology and actin polymerization, impaired chemotaxin-induced actin re-organization and inhibited their migration response. However, macropinocytosis was not influenced by PMT. In summary, these data indicate that PMT inhibits proper function of the motility machinery in DCs, which might limit the development of adaptive immune surveillance during infection with Pasteurella multocida.

Actins↗

PubMed2025

Function-based selection of synthetic communities enables mechanistic microbiome studies.

Understanding the complex interactions between microbes and their environment requires robust model systems such as synthetic communities (SynComs). We developed a functionally directed approach to generate SynComs by selecting strains that encode key functions identified in metagenomes. This approach enables the rapid construction of SynComs tailored to any ecosystem. To optimize community design, we implemented genome-scale metabolic models, providing in silico evidence for cooperative strain coexistence prior to experimental validation. Using this strategy, we designed multiple host-specific SynComs, including those for the rumen, mouse, and human microbiomes. By weighting functions differentially enriched in diseased versus healthy individuals, we constructed SynComs that capture complex host-microbe interactions. We designed an inflammatory bowel disease SynCom of 10 members that successfully induced colitis in gnotobiotic IL10-/- mice, demonstrating the potential of this method to model disease-associated microbiomes. Our study establishes a framework for designing functionally representative SynComs of any microbial ecosystem, facilitating mechanistic study.

Animals↗

PubMed2006

MBGD: a platform for microbial comparative genomics based on the automated construction of orthologous groups.

The microbial genome database for comparative analysis (MBGD) is a comprehensive platform for microbial comparative genomics. The central function of MBGD is to create orthologous groups among multiple genomes from precomputed all-against-all similarity relationships using the DomClust algorithm. The database now contains >300 published genomes and the number continues to grow. For researchers who are interested in ongoing genome projects, we have now started a new service called 'My MBGD,' which allows users to add their own genome sequences to MBGD for the purpose of identifying orthologs among both the new and the existing genomes. Furthermore, in order to make available the rapidly accumulating information on closely related genome sequences, we enhanced the interface for pairwise genome comparisons using the CGAT interface, which allows users to see nucleotide sequence alignments of non-coding as well as coding regions. MBGD is available at http://mbgd.genome.ad.jp/.

Algorithms↗

PubMed2003

Serum levels of mannan-binding lectin in chickens prior to and during experimental infection with avian infectious bronchitis virus.

Mannan-binding lectin (MBL) is a glycoprotein and a member of the C-type lectin super family, the collectin family, and the acute phase protein family. The MBL exerts its function by directly binding to microbial surfaces through its carbohydrate recognition domains, followed by direct opsonization or complement activation via MBL-associated serine proteases (MASP)-1 and -2. Thus, MBL plays a major role in the first-line innate defense against pathogens. We investigated the MBL concentrations in serum during experimental infectious bronchitis virus (IBV) infections in chickens. The results showed that the acute phase MBL response to infection with IBV was, to a degree (P < 0.0068), dependent on whether the chickens were inoculated after 12 h of rest (dark) or after 12 h of activity (light). The acute phase response in chickens challenged after 12 h of activity peaked after 4.6 d with an increase of 24%, whereas the acute phase response in chickens challenged after 12 h of rest peaked after 3.1 d with an increase of 51%. The specific antibody titer against IBV was also tested, and a difference (P < 0.0091) between the two experimental groups was found with peak titer values of 6,816 and 4,349. However, the highest value was found in chickens inoculated after 12 h of activity. Thus, an inverse relation exists between the MBL response and the IBV specific antibody response. The ability of MBL to activate the complement cascade was tested in a heterologous system by deposition of human C4 on the chicken MBL/MASP complex. The complement activation was directly associated with the concentration of MBL in serum, indicating neutralization of the virus before the humoral antibody response took over.

Acute-Phase Reaction↗

PubMed2005

Cecal ligation and puncture.

The model of cecal ligation and puncture (CLP) in rodents has been used extensively to investigate the clinical settings of sepsis and septic shock. This model produces a hyperdynamic, hypermetabolic state that can lead to a hypodynamic, hypometabolic stage, and eventual death. Blood cultures are positive for enteric organisms very early after CLP. The model has been widely used over the past 26 years and is highly versatile in adapting to a range of severity and testing objectives. It is inexpensive to prepare and technically straightforward. Aspects of sepsis research investigated using CLP include energetics, metabolism, resuscitation, antibiotic therapy, microbial factors, cardiovascular responses, immune function, mediator release, and cytokine expression patterns. The challenge of the small circulating blood volume in rodents can be overcome by using micromethods that enable analysis of small volumes, or alternatively, by using a large number of animals to obtain serial samples.

Animals↗

PubMed1989

Characteristics of oedema formation induced by N-formyl-methionyl-leucyl-phenylalanine in rabbit skin.

1. The characteristics of N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced oedema formation were investigated in vivo in rabbit skin. 2. FMLP injected intradermally alone induced a small increase in plasma leakage, but marked synergism with prostaglandin E2 (PGE2) in producing oedema responses was observed. In the presence of PGE2, FMLP was equiactive with C5a des Arg and 100-1000 times more active than histamine in terms of permeability-increasing activity. The response to FMLP was not dependent on endogenous histamine release. 3. FMLP-induced responses were of long duration (t1/2 approximately 40-50 min) when compared with bradykinin (t1/2 approximately 4-5 min). 4. The activity of a range of N-formyl peptides in increasing vascular permeability in skin correlated well with their activity as neutrophil stimulants in vitro. 5. Intravenous infusion of zymosan-activated plasma (ZAP) resulted in transient neutropenia and inhibition of oedema formation induced by FMLP and C5a des Arg in the skin. Responses to bradykinin were unaffected by the infusion of ZAP. 6. Intravenous injection of the non-steroidal antiinflammatory drug, ibuprofen, resulted in an inhibition of FMLP-induced, but not histamine-induced, oedema formation. This effect was independent of cyclo-oxygenase inhibition and the drug did not induced neutropenia. 7. Intravenous injection of the microtubule blocking agent colchicine inhibited FMLP-induced oedema formation. Responses to bradykinin were unaffected. When colchicine was administered after intradermal FMLP, subsequent plasma leakage was abolished. 8. The inference that receptors have evolved to bacterial secretions (i.e. FMLP) and products of the interaction of bacterial cell walls with tissue fluid (i.e. C5a des Arg), is consistent with the hypothesis that oedema formation is fundamentally a functional process concerned with regulating microbial lysis and opsonisation in an infected tissue.

Animals↗

PubMed1987

Adaptation of aquatic microbial communities to hg stress.

The mechanism of adaptation to Hg in four aquatic habitats was studied by correlating microbially mediated Hg volatilization with the adaptive state of the exposed communities. Community diversity, heterotrophic activity, and Hg resistance measurements indicated that adaptation of all four communities was stimulated by preexposure to Hg. In saline water communities, adaptation was associated with rapid volatilization after an initial lag period. This mechanism, however, did not promote adaptation in a freshwater sample, in which Hg was volatilized slowly, regardless of the resistance level of the microbial community. Distribution of the mer operon among representative colonies of the communities was not related to adaptation to Hg. Thus, although volatilization enabled some microbial communities to sustain their functions in Hg-stressed environments, it was not mediated by the genes that serve as a model system in molecular studies of bacterial resistance to mercurials.

Journal Article↗

PubMed2001

Development and evaluation of functional gene arrays for detection of selected genes in the environment.

To determine the potential of DNA array technology for assessing functional gene diversity and distribution, a prototype microarray was constructed with genes involved in nitrogen cycling: nitrite reductase (nirS and nirK) genes, ammonia mono-oxygenase (amoA) genes, and methane mono-oxygenase (pmoA) genes from pure cultures and those cloned from marine sediments. In experiments using glass slide microarrays, genes possessing less than 80 to 85% sequence identity were differentiated under hybridization conditions of high stringency (65 degrees C). The detection limit for nirS genes was approximately 1 ng of pure genomic DNA and 25 ng of soil community DNA using our optimized protocol. A linear quantitative relationship (r(2) = 0.89 to 0.94) was observed between signal intensity and target DNA concentration over a range of 1 to 100 ng for genomic DNA (or genomic DNA equivalent) from both pure cultures and mixed communities. However, the quantitative capacity of microarrays for measuring the relative abundance of targeted genes in complex environmental samples is less clear due to divergent target sequences. Sequence divergence and probe length affected hybridization signal intensity within a certain range of sequence identity and size, respectively. This prototype functional gene array did reveal differences in the apparent distribution of nir and amoA and pmoA gene families in sediment and soil samples. Our results indicate that glass-based microarray hybridization has potential as a tool for revealing functional gene composition in natural microbial communities; however, more work is needed to improve sensitivity and quantitation and to understand the associated issue of specificity.

Bacteria↗

PubMed2004

Large-scale identification of genes required for full virulence of Staphylococcus aureus.

Gene products required for in vivo growth and survival of microbial pathogens comprise a unique functional class and may represent new targets for antimicrobial chemotherapy, vaccine construction, or diagnostics. Although some factors governing Staphylococcus aureus pathogenicity have been identified and studied, a comprehensive genomic analysis of virulence functions will be a prerequisite for developing a global understanding of interactions between this pathogen and its human host. In this study, we describe a genetic screening strategy and demonstrate its use in screening a collection of 6,300 S. aureus insertion mutants for virulence attenuation in a murine model of systemic infection. Ninety-five attenuated mutants were identified, reassembled into new pools, and rescreened using the same murine model. This effort identified 24 highly attenuated mutants, each of which was further characterized for virulence attenuation in vivo and for growth phenotypes in vitro. Mutants were recovered in numbers up to 1,200-fold less than wild type in the spleens of systemically infected animals and up to 4,000-fold less than wild type in localized abscess infections. Genetic analysis of the mutants identified insertions in 23 unique genes. The largest gene classes represented by these mutants encoded enzymes involved in small-molecule biosynthesis and cell surface transmembrane proteins involved in small-molecule binding and transport. Additionally, three insertions defined two histidine kinase sensor-response regulator gene pairs important for S. aureus in vivo survival. Our findings extend the understanding of pathogenic mechanisms employed by S. aureus to ensure its successful growth and survival in vivo. Many of the gene products we have identified represent attractive new targets for antibacterial chemotherapy.

Animals↗

PubMed2005

Fate of heterotrophic microbes in pelagic habitats: focus on populations.

Major biogeochemical processes in the water columns of lakes and oceans are related to the activities of heterotrophic microbes, e.g., the mineralization of organic carbon from photosynthesis and allochthonous influx or its transport to the higher trophic levels. During the last 15 years, cultivation-independent molecular techniques have substantially contributed to our understanding of the diversity of the microbial communities in different aquatic systems. In parallel, the complexity of aquatic habitats at a microscale has inspired research on the ecophysiological properties of uncultured microorganisms that thrive in a continuum of dissolved to particulate organic matter. One possibility to link these two aspects is to adopt a"Gleasonian" perspective, i.e., to study aquatic microbial assemblages in situ at the population level rather than looking at microbial community structure, diversity, or function as a whole. This review compiles current knowledge about the role and fate of different populations of heterotrophic picoplankton in marine and inland waters. Specifically, we focus on a growing suite of techniques that link the analysis of bacterial identity with growth, morphology, and various physiological activities at the level of single cells. An overview is given of the potential and limitations of methodological approaches, and factors that might control the population sizes of different microbes in pelagic habitats are discussed.

Bacteria↗

PubMed1999

IV. Paneth cell antimicrobial peptides and the biology of the mucosal barrier.

The hypothesis that epithelial cells release preformed antibiotic peptides as components of mucosal innate immunity has gained experimental support in recent years. In the mammalian small intestine, Paneth cells secrete granules that are rich in alpha-defensins and additional antimicrobial peptides into the lumen of the crypt. The alpha-defensins are homologues of peptides that function as mediators of nonoxidative microbial cell killing in phagocytic leukocytes, and they are potent microbicidal agents in in vitro assays. Because certain mouse alpha-defensins stimulate cultured epithelial cells to secrete chloride ion, those peptides appear to be capable of interacting directly with the apical membranes of neighboring cells and perhaps influencing crypt physiology. In instances of crypt disruption or induced Paneth cell deficiency, crypt intermediate cells appear to compensate by accumulating and secreting Paneth cell antimicrobial peptides. Challenges for the future will be to understand the mechanisms of this epithelial plasticity and to show that Paneth cells contribute directly to innate immunity in the crypt microenvironment.

Animals↗

PubMed2003

Toll-like receptors in normal and cystic fibrosis airway epithelial cells.

Toll-like receptors (TLRs) mediate cellular responses to diverse microbial ligands. The distribution and function of TLRs in airway cells were studied to identify which are available to signal the presence of inhaled pathogens and to establish if differences in TLR expression are associated with the increased proinflammatory responses seen in cystic fibrosis (CF). Isogenic, polarized CF and control bronchial epithelial cell lines, human airway cells in primary culture, and cftr null and wild-type mice were compared. TLRs 1-10, MD2, and MyD88 were expressed in CF and normal cells. Only TLR2 transcription was modestly increased in CF as compared with normal epithelial cells following bacterial stimulation. TLR2 was predominantly at the apical surface of airway cells and was mobilized to cell surface in response to bacteria. TLR4 was present in a more basolateral distribution in airway cells, but appeared to have a limited role in epithelial responses. Lipopolysaccharide failed to activate nuclear factor-kappaB in these cells, and TLR2 dominant negative but not TLR4 dominant negative mutants inhibited activation by both Gram-negative and Gram-positive bacteria. Increased availability of TLR2 at the apical surfaces of CF epithelial cells is consistent with the increased proinflammatory responses seen in CF airways and suggests a selective participation of TLRs in the airway mucosa.

Animals↗

PubMed2002

Differential responses of mast cell Toll-like receptors 2 and 4 in allergy and innate immunity.

Toll-like receptor 2 (TLR2) and TLR4 play important roles in the early innate immune response to microbial challenge. To clarify the functional roles of TLRs 2 and 4 in mast cells, we examined bone marrow-derived mast cells (BMMCs) from TLR2 or TLR4 gene-targeted mice. Peptidoglycan (PGN) from Staphylococcus aureus stimulated mast cells in a TLR2-dependent manner to produce TNF-alpha, IL-4, IL-5, IL-6, and IL-13, but not IL-1beta. In contrast, LPS from Escherichia coli stimulated mast cells in a TLR4-dependent manner to produce TNF-alpha, IL-1beta, IL-6, and IL-13, but not IL-4 nor IL-5. Furthermore, TLR2- but not TLR4-dependent mast cell stimulation resulted in mast cell degranulation and Ca2+ mobilization. In a mast cell-dependent model of acute sepsis, TLR4 deficiency of BMMCs in mice resulted in significantly higher mortality because of defective neutrophil recruitment and production of proinflammatory cytokines in the peritoneal cavity. Intradermal injection of PGN led to increased vasodilatation and inflammation through TLR2-dependent activation of mast cells in the skin. Taken together, these results suggest that direct activation of mast cells via TLR2 or TLR4 by respective microligands contributes to innate and allergic immune responses.

Animals↗

PubMed2006

Innate immunity mediated by TLR9 modulates pathogenicity in an animal model of multiple sclerosis.

Inflammatory diseases of the CNS, such as MS and its animal model EAE, are characterized by infiltration of activated lymphocytes and phagocytes into the CNS. Within the CNS, activation of resident cells initiates an inflammatory cascade, leading to tissue destruction, demyelination, and neurologic deficit. TLRs recognize microbes and are pivotal mediators of innate immunity. Within the CNS, augmented TLR expression during EAE is observed, even in the absence of any apparent microbial involvement. To determine the functional relevance of this phenomenon during sterile autoimmunity, we studied the role of different TLRs as well as their common signaling adaptor MyD88 in the development of EAE. We found that MyD88 mice were completely EAE resistant. Surprisingly, this protection is partly due to engagement of the CpG receptor TLR9. Restricting the MyD88 or TLR9 mutation to host radio-resistant cells, including the cells within the CNS, revealed that engagement of radio-resistant cells modulated the disease course and histopathological changes. Our data clearly demonstrate that both TLR9 and MyD88 are essential modulators of the autoimmune process during the effector phase of disease and suggest that endogenous "danger signals" modulate the disease pathogenesis.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Synoviocyte-packaged Chlamydia trachomatis induces a chronic aseptic arthritis.

The basic mechanisms underlying reactive arthritis and specifically the joint injury that follows intra-articular Chlamydia trachomatis infection have not been defined. The present study addresses this question through the development of an experimental model. Stable cell lines were generated from synoviocytes harvested from the knee joints of Lewis rats. The synoviocytes were cocultivated with C. trachomatis to allow invasion by the microbe and were then transferred by intra-articular injection into the knee joints of Lewis rats. The ensuing arthritis could be subdivided into an early phase (</= 14 d) and a late phase. The early phase was characterized by intense, primarily neutrophilic, synovitis; accelerated cartilage injury; dissemination of Chlamydia to liver and spleen; and viable Chlamydia in the joints. The late phase was marked by mixed mononuclear lymphocyte infiltration in the joint; dysplastic cartilage injury and repair; absence of viable organisms; and development of a distinctive humoral response. Western blot analysis comparing reactive arthritis patients to the experimental model indicates that candidate arthritogenic chlamydial antigens are comparable between the two. This model demonstrates that an intense synovitis can be induced by this intracellular pathogen, and that chronic inflammation can persist well beyond the culture-positive phase. Furthermore, these data show that the synoviocyte is a suitable host cell for C. trachomatis and can function as a reservoir of microbial antigens sufficient to perpetuate joint injury.

Animals↗

PubMed2026

Association between the gut microbiome and plasma metabolites linked to vocalization-based temperament in Merino sheep.

BACKGROUND: Temperament, as a determinant of behavioural and emotional responses, has a substantial adaptive value in different environments. This study aims to investigate the association between the gut microbiota and temperament plasticity, and clarify the potential metabolic mechanism that underpins that association by running a multi-omics study in sheep. METHODS: The TrackSheep research cohort was generated using 200 healthy juvenile Merino ewes, and the rumen microbiota, plasma metabolome, and temperament phenotype was measured. RESULTS: Rumen metagenomic analysis identified 25 microbial species and 16 MetaCyc pathways that explained 37.5% and 11.1%, respectively, of the variation in temperament as estimated using the vocal reactivity to stress. Among these, the γ-aminobutyric acid (GABA) shunt and allantoin degradation pathways showed the strongest associations with vocal behaviour. Multi-omic integration linked these microbial pathways to plasma metabolites that are involved in neurotransmission, antioxidant defense, and energy metabolism, including acetyl-L-carnitine (ALCAR) and urocortisone, which partially mediated the effects of microbial pathways on vocalisations. Notably, functional genomic and mediation analyses indicated that the abundance of Cryptobacteroides sp902761655 was associated with the activity of GABA shunt pathway, where GABA co-occurred with succinate production, in turn correlating with reduced inhibitory effects of ALCAR on stress-susceptible temperament. Although plasma metabolite shifts observed immediately after behavioural tests reflected stress exposure, their associations with rumen microbiota highlight microbiome-metabolite interplay that could underly behavioural variation. CONCLUSIONS: Our study provides the first large-scale multi-omics evidence linking the rumen microbiome to a dimension of emotional reactivity in livestock, while underscoring the need for longitudinal and experimental validation to establish causal mechanisms. Video Abstract.

Animals↗