Immune Evasion,Stress Resistance,and Efficient Nutrient Acquisition Are Crucial for Intracellular Survival of Candida glabrata within Macrophages

Candida glabrata is both a human fungal commensal and an opportunistic pathogen which can withstand activities of the immune system. For example, C. glabrata can survive phagocytosis and replicates within macrophages. However, the mechanisms underlying intracellular survival remain unclear. In this work, we used a functional genomic approach to identify C. glabrata determinants necessary for survival within human monocyte-derived macrophages by screening a set of 433 deletion mutants. We identified 23 genes which are required to resist killing by macrophages. Based on homologies to Saccharomyces cerevisiae orthologs, these genes are putatively involved in cell wall biosynthesis, calcium homeostasis, nutritional and stress response, protein glycosylation, or iron homeostasis. Mutants were further characterized using a series of in vitro assays to elucidate the genes'' functions in survival. We investigated different parameters of C. glabrata-phagocyte interactions: uptake by macrophages, replication within macrophages, phagosomal pH, and recognition of mutant cells by macrophages as indicated by production of reactive oxygen species and tumor necrosis factor alpha (TNF-α). We further studied the cell surface integrity of mutant cells, their ability to grow under nutrient-limited conditions, and their susceptibility to stress conditions mirroring the harsh environment inside a phagosome. Additionally, resistance to killing by neutrophils was analyzed. Our data support the view that immune evasion is a key aspect of C. glabrata virulence and that increased immune recognition causes increased antifungal activities by macrophages. Furthermore, stress resistance and efficient nutrient acquisition, in particular, iron uptake, are crucial for intraphagosomal survival of C. glabrata.     

The importance of scale in comparative microbiome research: New insights from the gut and glands of captive and wild lemurs

Research on animal microbiomes is increasingly aimed at determining the evolutionary and ecological factors that govern host–microbiome dynamics, which are invariably intertwined and potentially synergistic. We present three empirical studies related to this topic, each of which relies on the diversity of Malagasy lemurs (representing a total of 19 species) and the comparative approach applied across scales of analysis. In Study 1, we compare gut microbial membership across 14 species in the wild to test the relative importance of host phylogeny and feeding strategy in mediating microbiome structure. Whereas host phylogeny strongly predicted community composition, the same feeding strategies shared by distant relatives did not produce convergent microbial consortia, but rather shaped microbiomes in host lineage‐specific ways, particularly in folivores. In Study 2, we compare 14 species of wild and captive folivores, frugivores, and omnivores, to highlight the importance of captive populations for advancing gut microbiome research. We show that the perturbational effect of captivity is mediated by host feeding strategy and can be mitigated, in part, by modified animal management. In Study 3, we examine various scent‐gland microbiomes across three species in the wild or captivity and show them to vary by host species, sex, body site, and a proxy of social status. These rare data provide support for the bacterial fermentation hypothesis in olfactory signal production and implicate steroid hormones as mediators of microbial community structure. We conclude by discussing the role of scale in comparative microbial studies, the links between feeding strategy and host–microbiome coadaptation, the underappreciated benefits of captive populations for advancing conservation research, and the need to consider the entirety of an animal's microbiota. Ultimately, these studies will help move the field from exploratory to hypothesis‐driven research.     

Properties of a mini 9R-lipoxygenase from Nostoc sp. PCC 7120 and its mutant forms

Lipoxygenases (LOXs) consist of a class of enzymes that catalyze the regio- and stereospecific dioxygenation of polyunsaturated fatty acids. Current reports propose that a conserved glycine residue in the active site of R-lipoxygenases and an alanine residue at the corresponding position in S-lipoxygenases play a crucial role in determining the stereochemistry of the product. Recently, a bifunctional lipoxygenase with a linoleate diol synthase activity from Nostoc sp. PCC7120 with R stereospecificity and the so far unique feature of carrying an alanine instead of the conserved glycine in the position of the sequence determinant for chiral specificity was identified. The recombinant carboxy-terminal domain was purified after expression in Escherichia coli. The ability of the enzyme to use linoleic acid esterified to a bulky phosphatidylcholine molecule as a substrate suggested a tail-fist binding orientation of the substrate. Site directed mutagenesis of the alanine to glycine did not cause alterations in the stereospecificity of the products, while mutation of the alanine to valine or isoleucine modified both regio- and enantioselectivity of the enzyme. Kinetic measurements revealed that substitution of Ala by Gly or Val did not significantly influence the reaction characteristics, while the A162I mutant showed a reduced vmax. Based on the mutagenesis data obtained, we suggest that the existing model for stereocontrol of the lipoxygenase reaction may be expanded to include enzymes that seem to have in general a smaller amino acid in R and a bulkier one in S lipoxygenases at the position that controls stereospecificity.     

Influence of a Probiotic Strain of Enterococcus faecium on Salmonella enterica Serovar Typhimurium DT104 Infection in a Porcine Animal Infection Model

The beneficial effects of probiotic Enterococcus spp. in different hosts, such as mice and humans, have previously been reported in several studies. However, studies of large domestic animals, as well as challenge studies with pathogenic microorganisms, are very rare. Here, we investigated the influence of oral treatment of pigs with the probiotic bacterium Enterococcus faecium NCIMB 10415 on Salmonella enterica serovar Typhimurium DT104 infections in weaning piglets. Clinical symptoms, fecal excretion, the organ distribution of Salmonella, and the humoral immune response (immunoglobulin G [IgG], IgM, and IgA levels) in serum were examined. A pool of 89 piglets was randomly divided into probiotic and control groups. The probiotic group received a feed supplement containing E. faecium starting on day 14 postpartum prior to challenge with Salmonella serovar Typhimurium DT104 at 28 days postpartum. After challenge with Salmonella serovar Typhimurium DT104, piglets in both groups showed no severe clinical signs of salmonellosis. However, fecal excretion and colonization of Salmonella in organs were significantly greater in piglets fed E. faecium. Likewise, the humoral immune response against Salmonella (serum IgM and IgA levels) was significantly greater in the probiotic group animals than in control animals. The results of this study suggest that E. faecium NCIMB 10415 treatment enhanced the course of infection in weaning piglets challenged with Salmonella serovar Typhimurium DT104. However, the probiotic treatment also appeared to result in greater production of specific antibodies against Salmonella serovar Typhimurium DT104.The problem of increasing microbial resistance to antibiotics resulting from years of overuse and the resulting ban on the use of antibiotics in animal production have led to increased interest in alternatives to antibiotics in animal production. In recent years, probiotic bacteria have been considered as an alternative means of reducing pathogen loads in animal breeding and production units. However, while a number of studies have focused on the mode of action of probiotics, the mode of action these bacteria is not fully understood yet.A recent interdisciplinary research study of the modes of action of probiotics in swine showed that Enterococcus faecium NCIMB 10415 reduced the pathogenic bacterial load of healthy piglets (20, 26, 30, 36). In vitro studies further demonstrated that this E. faecium probiotic strain decreased the rate of invasion of a porcine intestinal epithelial cell line by Salmonella enterica serovar Typhimurium. To determine whether probiotics also provide a measure of protection during infections, experimental challenge studies with pathogenic bacteria at a defined infectious dose and under comparable conditions seem to be necessary. Field studies could be more representative of the real situation; however, the infection pressure is too low and difficult to define, and systematic sampling cannot be done.Studies of larger domestic and production animals are rare. Most such studies deal with the mode of action of probiotics in the healthy host, and only a few studies have investigated the mode of action in the context of infections with pathogenic bacteria, such as Salmonella.In a related study, weaned piglets were fed a mixture of five probiotic strains (one Pediococcus strain and four Lactobacillus strains) and challenged with Salmonella serovar Typhimurium (7). In that study, reduced incidence, severity, and duration of diarrhea and a reduced microbiological load of Salmonella were observed. Fedorka-Cray et al. (11) observed reduced numbers of Salmonella bacteria in cecal contents and at the ileocolic junction in S. enterica serovar Choleraesuis-challenged weaning piglets fed a competitive exclusion culture. In vitro investigations showed that Enterococcus strains have inhibitory effects on the growth of S. enterica serovar Enteritidis, and these effects were explained by both enterotoxin and nonenterotoxin factors (37). Other studies showed that E. faecium may be beneficial to the adhesion and colonization of Clostridium jejuni in the canine intestine (29) and reduced the rate of carryover infections with obligate intracellular pathogens from infected sows in piglets (26). E. faecium has also been shown to influence the composition of the bacterial community in the avian, swine, and canine gastrointestinal tracts (25, 29, 36).Infections with S. enterica are some of the most important sources of human gastroenteritis (39). In Germany, 52,563 human salmonellosis cases were reported in 2006 (http://www3.rki.de/SurvStat). The consumption of contaminated pork and pork products was found to be associated with 20% of human salmonellosis cases in Germany (33), indicating the importance of meat or meat products as a potential source of infection for consumers. Salmonella serovar Typhimurium, especially phage type DT104, is the Salmonella serotype most frequently isolated from pork (27), and it is of particular concern because of its acquisition of multiple antibiotic resistance (1, 38).In this study, we investigated the effect of E. faecium NCIMB 10415 on the infection dynamics of Salmonella serovar Typhimurium DT104, fecal shedding, and the patterns of Salmonella distribution in internal organs, as well as on the humoral immune response to Salmonella in weaning piglets. To the best of our knowledge, this is the first experimental study of the mode of action of a probiotic strain of E. faecium in which dissemination to different internal organs was investigated using weaned piglets experimentally infected with Salmonella.     

Are Natural Killer Cells Protecting the Metabolically Healthy Obese Patient?

With the emerging obesity pandemic, identifying those who appear to be protected from adverse consequences such as type 2 diabetes and certain malignancies will become important. We propose that the circulating immune system plays a role in the development of these comorbidities. Clinical data and blood samples were collected from 52 patients with severe obesity attending a hospital weight‐management clinic and 11 lean healthy controls. Patients were classified into metabolically “healthy obese” (n = 26; mean age 42.6 years, mean BMI 46.8 kg/m²) or “unhealthy obese” (n = 26; mean age 45 years, mean BMI 47.5 kg/m²) groups, based upon standard cutoff points for blood pressure, lipid profile, and fasting glucose. Circulating lymphoid populations and phenotypes were assessed by flow cytometry. Obese patients had significantly less circulating natural killer (NK) and cytotoxic T lymphocytes (CTL) compared to lean controls. There were significantly higher levels of NK cells and CTLs in the healthy obese group compared to the unhealthy obese group (NK: 11.7% vs. 6.5%, P < 0.0001, CD8 13.4% vs. 9.3%, P = 0.04), independent of age and BMI and these NK cells were also less activated in the healthy compared to the unhealthy group (CD69, 4.1% vs. 11.8%, P = 0.03). This is the first time that quantitative differences in the circulating immune system of obese patients with similar BMI but different metabolic profiles have been described. The significantly higher levels of CTLs and NK cells, which express fewer inhibitory molecules, could protect against malignancy, infection, and metabolic disease seen in obesity.     

Co-occurrence patterns of Bornean vertebrates suggest competitive exclusion is strongest among distantly related species

Assessing the importance of deterministic processes in structuring ecological communities is a central focus of community ecology. Typically, community ecologists study a single taxonomic group, which precludes detection of potentially important biotic interactions between distantly related species, and inherently assumes competition is strongest between closely related species. We examined distribution patterns of vertebrate species across the island of Borneo in Southeast Asia to assess the extent to which inter-specific competition may have shaped ecological communities on the island and whether the intensity of inter-specific competition in present-day communities varies as a function of evolutionary relatedness. We investigated the relative extent of competition within and between species of primates, birds, bats and squirrels using species presence–absence and attribute data compiled for 21 forested sites across Borneo. We calculated for each species pair the checkerboard unit value (CU), a statistic that is often interpreted as indicating the importance of interspecific competition. The percentage of species pairs with significant CUs was lowest in within-taxon comparisons. Moreover, for invertebrate-eating species the percentage of significantly checkerboarded species pairs was highest in comparisons between primates and other taxa, particularly birds and squirrels. Our results are consistent with the interpretation that competitive interactions between distantly related species may have shaped the distribution of species and thus the composition of Bornean vertebrate communities. This research highlights the importance of taking into account the broad mammalian and avian communities in which species occur for understanding the factors that structure biodiversity.     

Diferrocenes containing thiadiazole connectivities

2,5-Diferrocenyl-1,3,4-thiadiazole, 2,5-Fc₂-^cC₂N₂S, (3) has been synthesized by a two-fold Negishi ferrocenylation of dibromothiadiazole (1) with FcZnCl (2) (Fc = Fe(η⁵-C₅H₄)(η⁵-C₅H₅)) in presence of [Pd(Ph₃P)₄] as catalyst. Additional spacer units between the ferrocenyls and the ^cC₂N₂S core could be introduced by using the Sonogashira C,C cross-coupling protocol. Reaction of 2,5-Br₂-^cC₂N₂S (1) or 2,5-(C₆H₄-4′-I)₂-^cC₂N₂S (6) with FcCCH (4) using [PdCl₂(Ph₃P)₂] and [CuI] as catalyst produced the appropriate organometallics 2,5-(FcCC)₂-^cC₂N₂S (5) or 2,5-(C₆H₄-4′-CCFc)₂-^cC₂N₂S (7). The electronic and structural properties of 3, 5, and 7 were investigated with UV-Vis spectroscopy and single crystal X-ray diffraction (3). Complex 3 adopts a solid state structure with none of the ferrocenyl substituents being coplanar with the thiadiazole ring. Cyclic, square wave, linear sweep voltammetry and in-situ NIR spectro-electrochemistry highlight the electrochemical properties of 3. In dichloromethane (0.1 mol L⁻¹ [N(ⁿBu)₄][B(C₆F₅)₄]), compound 3 displays two well resolved electrochemical reversible one-electron events with formal reduction potentials of 0.192 and 0.338 V versus FcH/FcH⁺. In contrast, in presence of [N(ⁿBu)₄][PF₆], the thiadiazoles 3 (E⁰ = 0.22 V), 5 (E⁰ = 0.18 V) and 7 (E⁰ = 0.09 V) show simultaneously oxidation of the two ferrocenyl termini versus FcH/FcH⁺. Spectro-electrochemical studies, performed in a dichloromethane solution of 0.2 mol L⁻¹ [N(ⁿBu)₄][B(C₆F₅)₄], also show that 3 can successively be oxidized via 3⁺ to 3²⁺. A weak IVCT absorption (ε ca. 300 L mol⁻¹ cm⁻¹) at 1560 nm was found and is consistent with appreciable interactions between neutral ferrocenyl and positively charged ferrocenium mixed valent intermediates. Mixed-valent compound 3⁺ corresponds to a class II molecule according to Robin and Day.     

Alternative splice variants of alpha 7 beta 1 integrin selectively recognize different laminin isoforms.

The integrin alpha(7)beta(1) occurs in several cytoplasmic (alpha(7A), alpha(7B)) and extracellular splice variants (alpha(7X1), alpha(7X2)), which are differentially expressed during development of skeletal and heart muscle. The extracellular variants result from the alternative splicing of exons X1 and X2, corresponding to a segment within the putative ligand binding domain. To study the specificity and affinity of the X1/X2 variants to different laminin isoforms, soluble alpha(7)beta(1) complexes were prepared by recombinant coexpression of the extracellular domains of the alpha- and beta-subunits. The binding of these complexes to purified ligands was measured by solid phase binding assays. Surprisingly, the alternative splice variants revealed different and specific affinities to different laminin isoforms. While the alpha(7X2) variant bound much more strongly to laminin-1 than the alpha(7X1) variant, the latter showed a high affinity binding to laminins-8 and -10/11. Laminin-2, the major laminin isoform in skeletal muscle, was recognized by both variants, whereas none of the two variants were able to interact with laminin-5. A specific blocking antibody inhibited the binding of both variants to all laminins tested, indicating the involvement of common epitopes in alpha(7X1)beta(1) and alpha(7X2)beta(1). Because laminin-8 and -10/11 as well as alpha(7X1) are expressed in developing skeletal and cardiac muscle, these findings suggest that alpha(7X1)beta(1) may represent a physiological receptor with novel specificities for laminin-8 and -10.