Paired guide RNA CRISPR-Cas9 screening for protein-coding genes and lncRNAs involved in transdifferentiation of human B-cells to macrophages.

CRISPR-Cas9 screening libraries have arisen as a powerful tool to identify protein-coding (pc) and non-coding genes playing a role along different processes. In particular, the usage of a nuclease active Cas9 coupled to a single gRNA has proven to efficiently impair the expression of pc-genes by generating deleterious frameshifts. Here, we first demonstrate that targeting the same gene simultaneously with two guide RNAs (paired guide RNAs, pgRNAs) synergistically enhances the capacity of the CRISPR-Cas9 system to knock out pc-genes. We next design a library to target, in parallel, pc-genes and lncRNAs known to change expression during the transdifferentiation from pre-B cells to macrophages. We show that this system is able to identify known players in this process, and also predicts 26 potential novel ones, of which we select four (two pc-genes and two lncRNAs) for deeper characterization. Our results suggest that in the case of the candidate lncRNAs, their impact in transdifferentiation may be actually mediated by enhancer regions at the targeted loci, rather than by the lncRNA transcripts themselves. The CRISPR-Cas9 coupled to a pgRNAs system is, therefore, a suitable tool to simultaneously target pc-genes and lncRNAs for genomic perturbation assays.

Galectin-3 Regulates γ-Herpesvirus Specific CD8 T Cell Immunity.

To gain insights into the molecular mechanisms and pathways involved in the activation of γ-herpesvirus (MHV68)-specific T cell receptor transnuclear (TN) CD8+ T cells, we performed a comprehensive transcriptomic analysis. Upon viral infection, we observed differential expression of several thousand transcripts encompassing various networks and pathways in activated TN cells compared with their naive counterparts. Activated cells highly upregulated galectin-3. We therefore explored the role of galectin-3 in influencing anti-MHV68 immunity. Galectin-3 was recruited at the immunological synapse during activation of CD8+ T cells and helped constrain their activation. The localization of galectin-3 to immune synapse was evident during the activation of both naive and memory CD8+ T cells. Galectin-3 knockout mice mounted a stronger MHV68-specific CD8+ T cell response to the majority of viral epitopes and led to better viral control. Targeting intracellular galectin-3 in CD8+ T cells may therefore serve to enhance response to efficiently control infections.

Rapid capture and labeling of cells on single domain antibodies-functionalized flow cell.

Current techniques to characterize leukocyte subgroups in blood require long sample preparation times and sizable sample volumes. A simplified method for leukocyte characterization using smaller blood volumes would thus be useful in diagnostic settings. Here we describe a flow system comprised of two functionalized graphene oxide (GO) surfaces that allow the capture of distinct leukocyte populations from small volumes blood using camelid single-domain antibodyfragments (VHHs) as capture agents. We used site-specifically labeled leukocytes to detect and identify cells exposed to fungal challenge. Combining the chemical and optical properties of GO with the versatility of the VHH scaffold in the context of a flow system provides a quick and efficient method for the capture and characterization of functional leukocytes.

Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans.

The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT) and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans.

Intestinal colonization by Candida albicans alters inflammatory responses in Bruton's tyrosine kinase-deficient mice.

The commensal yeast Candida albicans is part of the human intestinal microflora and is considered a "pathobiont", a resident microbe with pathogenic potential yet harmless under normal conditions. The aim of this study was to investigate the effect of C. albicans on inflammation of the intestinal tract and the role of Bruton's tyrosine kinase (Btk). Btk is an enzyme that modulates downstream signaling of multiple receptors involved in innate and adaptive immunity, including the major anti-fungal receptor Dectin-1. Colitis was induced in wild type and Btk-/- mice by treatment with dextran sodium sulfate (DSS) and the gastrointestinal tract of selected treatment groups were then colonized with C. albicans. Colonization by C. albicans neither dampened nor exacerbated inflammation in wild type mice, but colon length and spleen weight were improved in Btk-deficient mice colonized with C. albicans. Neutrophil infiltration was comparable between wild type and Btk-/- mice, but the knockout mice displayed severely reduced numbers of macrophages in the colon during both DSS and DSS/Candida treatment. Smaller numbers and reduced responsiveness of Btk-/- macrophages might partially explain the improved colon length of Btk-/- mice as a result of Candida colonization. Surprisingly, DSS/Candida-treated Btk-/- animals had higher levels of certain pro-inflammatory cytokines and levels of the anti-inflammatory cytokine TGF-ß were reduced compared to wild type. A clustering and correlation analysis showed that for wild type animals, spleen TGF-ß and colon IL-10 and for Btk-/- spleen and colon levels of IL-17A best correlated with the inflammatory parameters. We conclude that in Btk-/- immunocompromised animals, colonization of the gastrointestinal tract by the commensal yeast C. albicans alters inflammatory symptoms associated with colitis.

Bruton's Tyrosine Kinase (BTK) and Vav1 contribute to Dectin1-dependent phagocytosis of Candida albicans in macrophages.

Phagocytosis of the opportunistic fungal pathogen Candida albicans by cells of the innate immune system is vital to prevent infection. Dectin-1 is the major phagocytic receptor involved in anti-fungal immunity. We identify two new interacting proteins of Dectin-1 in macrophages, Bruton's Tyrosine Kinase (BTK) and Vav1. BTK and Vav1 are recruited to phagocytic cups containing C. albicans yeasts or hyphae but are absent from mature phagosomes. BTK and Vav1 localize to cuff regions surrounding the hyphae, while Dectin-1 lines the full length of the phagosome. BTK and Vav1 colocalize with the lipid PI(3,4,5)P3 and F-actin at the phagocytic cup, but not with diacylglycerol (DAG) which marks more mature phagosomal membranes. Using a selective BTK inhibitor, we show that BTK contributes to DAG synthesis at the phagocytic cup and the subsequent recruitment of PKCε. BTK- or Vav1-deficient peritoneal macrophages display a defect in both zymosan and C. albicans phagocytosis. Bone marrow-derived macrophages that lack BTK or Vav1 show reduced uptake of C. albicans, comparable to Dectin1-deficient cells. BTK- or Vav1-deficient mice are more susceptible to systemic C. albicans infection than wild type mice. This work identifies an important role for BTK and Vav1 in immune responses against C. albicans.

Fungal recognition is mediated by the association of dectin-1 and galectin-3 in macrophages.

Dectin-1, the major ß-glucan receptor in leukocytes, triggers an effective immune response upon fungal recognition. Here we use sortase-mediated transpeptidation, a technique that allows placement of a variety of probes on a polypeptide backbone, to monitor the behavior of labeled functional dectin-1 in live cells with and without fungal challenge. Installation of probes on dectin-1 by sortagging permitted highly specific visualization of functional protein on the cell surface and its subsequent internalization upon ligand presentation. Retrieval of sortagged dectin-1 expressed in macrophages uncovered a unique interaction between dectin-1 and galectin-3 that functions in the proinflammatory response of macrophages to pathogenic fungi. When macrophages expressing dectin-1 are exposed to Candida albicans mutants with increased exposure of ß-glucan, the loss of galectin-3 dramatically accentuates the failure to trigger an appropriate TNF-α response.

Utility of microsatellite markers and amplified fragment length polymorphism in the study of potentially ochratoxigenic black aspergilli.

Microsatellite markers and the results of amplified fragment length polymorphism (AFLP) were compared in the characterization of 68 Aspergillus carbonarius and A. niger aggregate strains of differing ochratoxin-producing ability and from different geographic areas, isolated mainly from grapes and soil. AFLP was applied to both A. carbonarius and A. niger aggregate strains, and it clearly differentiated these species. Microsatellite markers were only applied to A. niger aggregate strains because of the species-specific nature of these markers. Both AFLP and microsatellite marker analyses were able to divide A. niger aggregate strains into the two recognized internal transcribed spacer (ITS)-5.8S rDNA RFLP types, N and T. Clustering of A. niger aggregate strains was similar in both AFLP and microsatellite analyses, yielding an additional separation of N type strains into two groups. Both microsatellite marker and AFLP analyses showed high levels of polymorphism in the A. niger aggregate (index of discriminatory power 0.991 and 1.0, respectively). Of the two techniques, microsatellite marker analysis was quicker and more straightforward to perform. In addition, microsatellite marker analysis is more reproducible, and the results can be expressed as quantitative data, making microsatellite markers a good candidate for use in large-scale studies of genetic diversity in A. niger aggregate species.

Effects of temperature and incubation time on production of ochratoxin A by black aspergilli.

The effects of temperature (5-45 degrees C) on the growth and production of ochratoxin A (OTA) by eighteen strains of Aspergillus section Nigri, cultured on Czapek yeast autolysate agar (CYA) and on yeast extract sucrose agar (YES), were studied for an incubation period of 30 days. Isolates were selected to include different sources and different reported abilities to produce OTA. Temperature ranges for OTA production were more restrictive than those for growth and each strain tested differed in its optimum conditions for OTA production. Aspergillus niger aggregate strains achieved maximum OTA levels in YES medium mainly at 20-25 degrees C. The A. carbonarius strains produced the highest OTA levels in CYA medium at 15 or 20 degrees C. Significant amounts of OTA were produced after only five days of incubation. Due to their ability to produce OTA at a wide range of temperatures, OTA can be continuously produced in the field. This fact has to be taken into account in commodities such as grapes, raisins and wine, where A. carbonarius and members of the A. niger aggregate are considered to be the main sources of the OTA contamination.

Collaborative evaluation of optimal antifungal susceptibility testing conditions for dermatophytes.

A multicenter study was conducted to define the most suitable testing conditions for antifungal susceptibility of dermatophytes. Broth microdilution MICs of clotrimazole, itraconazole, and terbinafine were determined in three centers against 60 strains of dermatophytes. The effects of inoculum density (ca. 10(3) and 10(4) CFU/ml), incubation time (3, 7, and 14 days), endpoint criteria for MIC determination (complete [MIC-0] and prominent [MIC-2] growth inhibition), and incubation temperature (28 and 37 degrees C) on intra- and interlaboratory agreement were analyzed. The optimal testing conditions identified were an inoculum of 10(4) CFU/ml, a temperature of incubation of 28 degrees C, an incubation period of 7 days, and MIC-0.