Recognition of Yeast by Murine Macrophages Requires Mannan but Not Glucan

The first barrier against infection by Candida albicans involves fungal recognition and destruction by phagocytic cells of the innate immune system. It is well established that interactions between different phagocyte receptors and components of the fungal cell wall trigger phagocytosis and subsequent immune responses, but the fungal ligands mediating the initial stage of recognition have not been identified. Here, we describe a novel assay for fungal recognition and uptake by macrophages which monitors this early recognition step independently of other downstream events of phagocytosis. To analyze infection in live macrophages, we validated the neutrality of a codon-optimized red fluorescent protein (yEmRFP) biomarker in C. albicans; growth, hyphal formation, and virulence in infected mice and macrophages were unaffected by yEmRFP production. This permitted a new approach for studying phagocytosis by carrying out competition assays between red and green fluorescent yeast cells to measure the efficiency of yeast uptake by murine macrophages as a function of dimorphism or cell wall defects. These competition experiments demonstrate that, given a choice, macrophages display strong preferences for phagocytosis based on genus, species, and morphology. Candida glabrata and Saccharomyces cerevisiae are taken up by J774 macrophage cells more rapidly than C. albicans, and C. albicans yeast cells are favored over hyphal cells. Significantly, these preferences are mannan dependent. Mutations that affect mannan, but not those that affect glucan or chitin, reduce the uptake of yeast challenged with wild-type competitors by both J774 and primary murine macrophages. These results suggest that mannose side chains or mannosylated proteins are the ligands recognized by murine macrophages prior to fungal uptake.Candida albicans is an opportunistic fungus that normally resides in the human gut (26) and can cause mucosal infections. When host immune defenses are compromised or when anatomical breaches permit extreme fungal burdens, systemic and often lethal fungal colonization throughout the body can occur. In hospital-acquired bloodstream infections, the rate of mortality, hospital cost, and length of stay associated with disseminated candidiasis now outrank those associated with bacterial infections (37, 43). The most effective host barrier that limits Candida infections is microbial destruction by phagocytic cells of the innate immune system. In a healthy host, phagocytes—macrophages, neutrophils, and dendritic cells—recognize, ingest, and destroy the invading yeast by phagocytosis.The first step of a fungal infection requires the recognition of yeast by phagocytes. Despite the medical importance of this reaction, it remains poorly understood. As the interface between the yeast and its host, the fungal cell wall is crucial for recognition. The wall is a complex structure consisting of an elastic network of polysaccharides (glucans and chitin) that surrounds the plasma membrane and that in most yeast and fungi contains many different heavily mannosylated proteins (mannan) anchored to the wall in various ways (9, 27,–29). Three distinct layers that correspond to these three components can be seen by electron microscopy. The innermost layer is enriched with a small amount of chitin, the outermost layer consists of mannan, and in between these layers are flexible fibrils of β1,3-glucan. Another glucan (β1,6 linked) is relatively minor in amount but is important for maintaining wall structure because it cross-links β1,3-glucan to wall proteins and to chitin (24, 30). Yeast survival relies on the integrity of the cell wall because it shields the yeast from physical stress and osmotic shock. The wall also maintains cell shape, which is a precondition for growth and morphogenesis. The rapid switch between the yeast and hyphal forms that is essential for C. albicans virulence underscores the plasticity of the wall, whose composition, thickness, and structure vary tremendously in response to changes in the environment.Many phagocytic receptors implicated in fungal recognition have been identified. The interactions between these receptors and fungal wall components activate an array of host defense signaling pathways that promote actin cytoskeletal rearrangements and the membrane remodeling required for phagocytosis, production of toxic metabolites and hydrolytic enzymes within the phagosome that destroy the ingested yeast, and secretion of cytokines that are pro- or anti-inflammatory (for a review, see references 18, 31, and 36). These receptors are members of the C-type lectin receptor and Toll-like receptor families and include proteins that can recognize mannose, glucan, and, possibly, chitin or, possibly, multiligand combinations of these carbohydrates (for reviews, see references 22 and 49). Despite a wealth of information about the signaling cascades elicited by these host receptors, the identity of the fungal cell wall ligands that mediate the initial recognition event during host-fungal interactions remains unclear, in large part because good model systems for studying host-fungal interactions in the context of the live infective environment have been unavailable. Most current assays of fungal recognition rely on indirect readouts, for instance, virulence or cytokine production, which cannot distinguish the initial step of fungal recognition from other downstream events of phagocytosis. In addition, different experimental systems for studying fungal phagocytosis use different cell types that may display unique interactions with C. albicans and vice versa. Thus, there are conflicts in the literature about the contributions of fungal cell wall components to host recognition and phagocytosis.Here, we make use of a novel assay to help clarify discrepancies that currently exist in this field. We developed a biologically neutral red fungal fluorescent biomarker that can be stably introduced into most yeast and fungi to monitor C. albicans-host interactions during infection in live cells or animals. This permitted development of quantitative competition assays to measure uptake by macrophages of red fluorescent protein (RFP)- or green fluorescent protein (GFP)-labeled cells as a single isolated event within the complex process of phagocytosis in live cells. We apply this system to address two fundamental questions regarding fungal recognition by murine macrophages. First, do these macrophages display a preference toward yeast forms versus filamentous fungal forms, and second, how do the various fungal cell wall components contribute to this preference during the initial stage of fungal recognition? We demonstrate that, given a choice, J774 macrophages recognize and ingest yeast cells far more rapidly and efficiently than hyphal cells. Importantly, competitive fungal uptake by murine macrophages, both immortalized cell lines and primary cells, is markedly inhibited by reduction of cell wall mannan but not glucan or chitin. This points to a critical role for mannose side chains or mannosylated proteins as key fungal recognition ligands.     

Metformin inhibits mitochondrial adaptations to aerobic exercise training in older adults

Metformin and exercise independently improve insulin sensitivity and decrease the risk of diabetes. Metformin was also recently proposed as a potential therapy to slow aging. However, recent evidence indicates that adding metformin to exercise antagonizes the exercise‐induced improvement in insulin sensitivity and cardiorespiratory fitness. The purpose of this study was to test the hypothesis that metformin diminishes the improvement in insulin sensitivity and cardiorespiratory fitness after aerobic exercise training (AET) by inhibiting skeletal muscle mitochondrial respiration and protein synthesis in older adults (62 ± 1 years). In a double‐blinded fashion, participants were randomized to placebo (n = 26) or metformin (n = 27) treatment during 12 weeks of AET. Independent of treatment, AET decreased fat mass, HbA1c, fasting plasma insulin, 24‐hr ambulant mean glucose, and glycemic variability. However, metformin attenuated the increase in whole‐body insulin sensitivity and VO₂max after AET. In the metformin group, there was no overall change in whole‐body insulin sensitivity after AET due to positive and negative responders. Metformin also abrogated the exercise‐mediated increase in skeletal muscle mitochondrial respiration. The change in whole‐body insulin sensitivity was correlated to the change in mitochondrial respiration. Mitochondrial protein synthesis rates assessed during AET were not different between treatments. The influence of metformin on AET‐induced improvements in physiological function was highly variable and associated with the effect of metformin on the mitochondria. These data suggest that prior to prescribing metformin to slow aging, additional studies are needed to understand the mechanisms that elicit positive and negative responses to metformin with and without exercise.     

Isolation and characterization of gas vesicles from Microcyclus aquaticus

Intact gas vesicles of Microcyclus aquaticus S1 were isolated by using centrifugally accelerated flotation of vesicles and molecular sieve chromatography. Isolated gas vesicles were cylindrical organelles with biconical ends and measured 250×100 nm. The gas vesicle membrane was composed almost entirely of protein; neither lipid nor carbohydrate was detected, although one mole of phosphate per mole of protein was found. Amino acid analysis indicated that the protein contained 54.6% hydrophobic amino acid residues, lacked sulfur-containing amino acids, and had a low aromatic amino acid content. The protein subunit composition of the vesicles was determined by gel electrophoresis in (i) 0.1% sodium dodecyl sulfate at pH 9.0 and (ii) 5 M urea at pH 2.0. The membrane appeared to consist of one protein subunit of MW 50 000 daltons. Charge isomers of this subunit were not detected on urea gels. Antiserum prepared against purified gas vesicles of M. aquaticus S1 cross-reacted with the gas vesicles of all other gas vacuolate strains of M. aquaticus, as well as those of Prosthecomicrobium pneumaticum, Nostoc muscorum, and Anabaena flos-aquae, indicating that the gas vesicles of these widely divergent organisms have some antigenic determinants in common.Abbreviations SDS sodium dodecyl sulfate - MW molecular weight - Tris tris(hydroxymethyl)aminomethane - EDTA disodium ethylenediaminetetraacetic acid - BSA bovine serum albumin - TCA trichloroacetic acid - P _c pressure necessary to collapse gas vesicles     

Meeting Report: 1st International Functional Metagenomics Workshop May 7–8, 2012, St. Jacobs,Ontario, Canada.

This report summarizes the events of the 1^st International Functional Metagenomics Workshop. The workshop was held on May 7 and 8, 2012, in St. Jacobs, Ontario, Canada and was focused on building an international functional metagenomics community, exploring strategic research areas, and identifying opportunities for future collaboration and funding. The workshop was initiated by researchers at the University of Waterloo with support from the Ontario Genomics Institute (OGI), Natural Sciences and Engineering Research Council of Canada (NSERC) and the University of Waterloo.     

Unusual frequencies of certain alternating purine-pyrimidine runs in natural DNA sequences: relation to Z-DNA

Prokaryotic, eukaryotic and mitochondrial DNA sequences of total Length 300 000 nucleotides have been analyzed to find out whether stretches of alternating purines and pyrimidines are unusual in terms of occurrence, composition and base sequence. Alternating runs longer than 5 nucleotides are significantly under-represented in the natural sequences as compared to random ones. Octanucleotides are the most deficient, occurring at only 60% of the frequency expected in random sequences. An unexpectedly high proportion of these octamers consists of alternating tetramers with the repeat structure (PuPyPuPy)2 or (PyPuPyPu)2. DNA stretches containing such sequences can potentially form a S1 nuclease sensitive slippage (staggered loop) structure, which might serve as a locally unstacked intermediate in the B- to Z-DNA conformational transition.     

Iqg1p links spatial and secretion landmarks to polarity and cytokinesis

Cytokinesis requires the polarization of the actin cytoskeleton, the secretion machinery, and the correct positioning of the division axis. Budding yeast cells commit to their cytokinesis plane by choosing a bud site and polarizing their growth. Iqg1p (Cyk1p) was previously implicated in cytokinesis (Epp and Chant, 1997; Lippincott and Li, 1998; Osman and Cerione, 1998), as well as in the establishment of polarity and protein trafficking (Osman and Cerione, 1998). To better understand how Iqg1p influences these processes, we performed a two-hybrid screen and identified the spatial landmark Bud4p as a binding partner. Iqg1p can be coimmunoprecipitated with Bud4p, and Bud4p requires Iqg1p for its proper localization. Iqg1p also appears to specify axial bud-site selection and mediates the proper localization of the septin, Cdc12p, as well as binds and helps localize the secretion landmark, Sec3p. The double mutants iqg1Deltasec3Delta and bud4Deltasec3Delta display defects in polarity, budding pattern and cytokinesis, and electron microscopic studies reveal that these cells have aberrant septal deposition. Taken together, these findings suggest that Iqg1p recruits landmark proteins to form a targeting patch that coordinates axial budding with cytokinesis.     

Evaluation of methods to solubilize and analyze cell-associated ectoenzymes

A protocol for production, storage, and use of Shock 1 (Shk1) bioreporter cells for toxicity monitoring in wastewater treatment facilities was developed. Shk1 is a bioluminescent toxicity bioreporter for activated sludge previously constructed by the incorporation of lux genes into an activated sludge microorganism.

A number of factors affecting Shk1 growth and bioluminescence were examined including the growth medium, tetracycline concentration, storage conditions, and test media. Based on the results of these experiments, a toxicity testing protocol was developed that involved growth of cultures in nutrient broth with tetracycline, storage of cultures at 4 °C, cell activation by reinoculation into nutrient broth, and toxicity testing by cell injection into the test media. Effective use of this approach required standardized time intervals for cell growth, storage, activation and exposure in the test media.

Bioluminescence from Shk1 cells was measured in nutrient broth and influent wastewater and activated sludge mixed liquor from a municipal wastewater treatment plant. Using the Shk1 toxicity testing protocol, Zn EC₅₀ values for bioluminescence in nutrient broth, influent wastewater, and activated sludge mixed liquor were approximately 42, 7, and 32 mg/l, respectively. Zn concentrations as low as 1 mg/l could be detected in influent wastewater. The detection limit in influent wastewater is below the Zn concentrations typically reported to affect the activated sludge process.