Autophagy-associated immune dysregulation and hyperplasia in a patient with compound heterozygous mutations in ATG9A.

ABBREVIATIONS: BCL2: BCL2 apoptosis regulator; BCL10: BCL10 immune signaling adaptor; CARD11: caspase recruitment domain family member 11; CBM: CARD11-BCL10-MALT1; CR2: complement C3d receptor 2; EBNA: Epstein Barr nuclear antigen; EBV: Epstein-Barr virus; FCGR3A; Fc gamma receptor IIIa; GLILD: granulomatous-lymphocytic interstitial lung disease; HV: healthy volunteer; IKBKB/IKB kinase: inhibitor of nuclear factor kappa B kinase subunit beta; IL2RA: interleukin 2 receptor subunit alpha; MALT1: MALT1 paracaspase; MS4A1: membrane spanning 4-domain A1; MTOR: mechanistic target of rapamycin kinase; MYC: MYC proto-oncogene, bHLH: transcription factor; NCAM1: neural cell adhesion molecule 1; NFKB: nuclear factor kappa B; NIAID: National Institute of Allergy and Infectious Diseases; NK: natural killer; PTPRC: protein tyrosine phosphatase receptor type C; SELL: selectin L; PBMCs: peripheral blood mononuclear cells; TR: T cell receptor; Tregs: regulatory T cells; WT: wild-type.

Analysis and Correction of Inappropriate Image Duplication: the Molecular and Cellular Biology Experience.

We analyzed 960 papers published in Molecular and Cellular Biology (MCB) from 2009 to 2016 and found 59 (6.1%) to contain inappropriately duplicated images. The 59 instances of inappropriate image duplication led to 41 corrections, 5 retractions, and 13 instances in which no action was taken. Our experience suggests that the majority of inappropriate image duplications result from errors during figure preparation that can be remedied by correction. Nevertheless, ∼10% of papers with inappropriate image duplications in MCB were retracted (∼0.5% of total). If this proportion is representative, then as many as 35,000 papers in the literature are candidates for retraction due to inappropriate image duplication. The resolution of inappropriate image duplication concerns after publication required an average of 6 h of journal staff time per published paper. MCB instituted a pilot program to screen images of accepted papers prior to publication that identified 12 manuscripts (14.5% out of 83) with image concerns in 2 months. The screening and correction of papers before publication required an average of 30 min of staff time per problematic paper. Image screening can identify papers with problematic images prior to publication, reduces postpublication problems, and requires less staff time than the correction of problems after publication.

Human Epithelial Cells Discriminate between Commensal and Pathogenic Interactions with Candida albicans.

The commensal fungus, Candida albicans, can cause life-threatening infections in at risk individuals. C. albicans colonizes mucosal surfaces of most people, adhering to and interacting with epithelial cells. At low concentrations, C. albicans is not pathogenic nor does it cause epithelial cell damage in vitro; at high concentrations, C. albicans causes mucosal infections and kills epithelial cells in vitro. Here we show that while there are quantitative dose-dependent differences in exposed epithelial cell populations, these reflect a fundamental qualitative difference in host cell response to C. albicans. Using transcriptional profiling experiments and real time PCR, we found that wild-type C. albicans induce dose-dependent responses from a FaDu epithelial cell line. However, real time PCR and Western blot analysis using a high dose of various C. albicans strains demonstrated that these dose-dependent responses are associated with ability to promote host cell damage. Our studies support the idea that epithelial cells play a key role in the immune system by monitoring the microbial community at mucosal surfaces and initiating defensive responses when this community is dysfunctional. This places epithelial cells at a pivotal position in the interaction with C. albicans as epithelial cells themselves promote C. albicans stimulated damage.

Asparagine deprivation mediated by Salmonella asparaginase causes suppression of activation-induced T cell metabolic reprogramming.

Salmonellae are pathogenic bacteria that induce immunosuppression by mechanisms that remain largely unknown. Previously, we showed that a putative type II l-asparaginase produced by Salmonella Typhimurium inhibits T cell responses and mediates virulence in a murine model of infection. Here, we report that this putative L-asparaginase exhibits L-asparagine hydrolase activity required for Salmonella Typhimurium to inhibit T cells. We show that L-asparagine is a nutrient important for T cell activation and that L-asparagine deprivation, such as that mediated by the Salmonella Typhimurium L-asparaginase, causes suppression of activation-induced mammalian target of rapamycin signaling, autophagy, Myc expression, and L-lactate secretion. We also show that L-asparagine deprivation mediated by the Salmonella Typhimurium L-asparaginase causes suppression of cellular processes and pathways involved in protein synthesis, metabolism, and immune response. Our results advance knowledge of a mechanism used by Salmonella Typhimurium to inhibit T cell responses and mediate virulence, and provide new insights into the prerequisites of T cell activation. We propose a model in which l-asparagine deprivation inhibits T cell exit from quiescence by causing suppression of activation-induced metabolic reprogramming.

CD11b+ Ly6Chi Ly6G- immature myeloid cells recruited in response to Salmonella enterica serovar Typhimurium infection exhibit protective and immunosuppressive properties.

Immature myeloid cells in bone marrow are a heterogeneous population of cells that, under normal conditions, provide tissues with protective cell types such as granulocytes and macrophages. Under certain pathological conditions, myeloid cell homeostasis is altered and immature forms of these cells appear in tissues. Murine immature myeloid cells that express CD11b and Ly6C or Ly6G (two isoforms of Gr-1) have been associated with immunosuppression in cancer (in the form of myeloid-derived suppressor cells) and, more recently, infection. Here, we found that CD11b(+) Ly6C(hi) Ly6G(-) and CD11b(+) Ly6C(int) Ly6G(+) cells accumulated and persisted in tissues of mice infected with Salmonella enterica serovar Typhimurium (S. Typhimurium). Recruitment of CD11b(+) Ly6C(hi) Ly6G(-) but not CD11b(+) Ly6C(int) Ly6G(+) cells from bone marrow into infected tissues depended on chemokine receptor CCR2. The CD11b(+) Ly6C(hi) Ly6G(-) cells exhibited a mononuclear morphology, whereas the CD11b(+) Ly6C(int) Ly6G(+) cells exhibited a polymorphonuclear or band-shaped nuclear morphology. The CD11b(+) Ly6C(hi) Ly6G(-) cells differentiated into macrophage-like cells following ex vivo culture and could present antigen to T cells in vitro. However, significant proliferation of T cells was observed only when the ability of the CD11b(+) Ly6C(hi) Ly6G(-) cells to produce nitric oxide was blocked. CD11b(+) Ly6C(hi) Ly6G(-) cells recruited in response to S. Typhimurium infection could also present antigen to T cells in vivo, but increasing their numbers by adoptive transfer did not cause a corresponding increase in T cell response. Thus, CD11b(+) Ly6C(hi) Ly6G(-) immature myeloid cells recruited in response to S. Typhimurium infection exhibit protective and immunosuppressive properties that may influence the outcome of infection.

L-asparaginase II produced by Salmonella typhimurium inhibits T cell responses and mediates virulence.

Salmonella enterica serovar Typhimurium avoids clearance by the host immune system by suppressing T cell responses; however, the mechanisms that mediate this immunosuppression remain unknown. We show that S. Typhimurium inhibit T cell responses by producing L-Asparaginase II, which catalyzes the hydrolysis of L-asparagine to aspartic acid and ammonia. L-Asparaginase II is necessary and sufficient to suppress T cell blastogenesis, cytokine production, and proliferation and to downmodulate expression of the T cell receptor. Furthermore, S. Typhimurium-induced inhibition of T cells in vitro is prevented upon addition of L-asparagine. S. Typhimurium lacking the L-Asparaginase II gene (STM3106) are unable to inhibit T cell responses and exhibit attenuated virulence in vivo. L-Asparaginases are used to treat acute lymphoblastic leukemia through mechanisms that likely involve amino acid starvation of leukemic cells, and these findings indicate that pathogens similarly use L-asparagine deprivation to limit T cell responses.

Visible fluorescence detection of type III protein secretion from bacterial pathogens.

Type III protein secretion is essential for many gram-negative bacterial infections of host cells and an attractive target for new antibacterial drugs. Here, we describe a bacterial protein effector-carboxypeptidase G2 (CPG2) reporter system for fluorescence and visible detection of type III protein secretion in Salmonella typhimurium. This system provides a general method for measuring protein expression and secretion as well as a high-throughput and quantitative assay for analyzing type III protein secretion inhibitors.

Adaptation to environmental pH: integrating the Rim101 and calcineurin signal transduction pathways.

The ability to appropriately respond to environmental conditions is critical for the survival of simple microbes and for development of complex multicellular organisms. Sensing and responding to a given environmental condition requires the integration of numerous signals through one or more signal transduction pathways. This leads to changes in gene expression, and potentially post-translational modifications, that favour growth in the given environment. In the fungus Candida albicans, an important opportunistic pathogen, environmental pH has profound effects on morphology and proper adaptation to extracellular pH is critical for pathogenesis. Here, we demonstrate that the Rim101/PacC pH-sensing pathway acts in parallel to Crz1, via calcineurin, to adapt to alkaline pH. We also show that the Rim101 pathway acts in parallel to Crz2, independent of calcineurin, to adapt to high lithium concentrations and to repress filamentation at acidic pH. Our studies also revealed a novel requirement for Crz1, Crz2 and calcineurin for growth at acidic pH. From these studies, we propose that the Crz1 homologue Crz2 is calcineurin-independent, but like Crz1, acts in parallel to promote specific Rim101-dependent processes. These results establish and begin to dissect the complex interactions between important signal transduction pathways in C. albicans, which are critical for virulence.

Snf7p, a component of the ESCRT-III protein complex, is an upstream member of the RIM101 pathway in Candida albicans.

The success of Candida albicans as an opportunistic pathogen is based in part on its ability to adapt to diverse environments. The RIM101 pathway governs adaptation to neutral-alkaline environments and is required for virulence. Analysis of a genomic two-hybrid study conducted with Saccharomyces cerevisiae revealed that components involved in multivesicular bodies (MVB) transport may interact with RIM101 pathway members. Thus, we hypothesized that these proteins may function in the RIM101 pathway in C. albicans. We identified C. albicans homologs to S. cerevisiae Snf7p, Vps4p, and Bro1p and generated mutants in the cognate gene. We found that snf7Delta/Delta mutants, but not vps4Delta/Delta nor bro1Delta/Delta mutants, had phenotypes similar to, but more severe than, those of RIM101 pathway mutants. We found that the constitutively active RIM101-405 allele partially rescued snf7Delta/Delta mutant phenotypes. The vps4Delta/Delta mutant had subtle phenotypes, but these were not rescued by the RIM101-405 allele. Further, we found that the snf7Delta/Delta, vps4Delta/Delta, and bro1Delta/Delta mutants did not efficiently localize the vital dye FM4-64 to the vacuole and that it was often accumulated in an MVB-like compartment. This phenotype was not rescued by RIM101-405 or observed in RIM101 pathway mutants. These results suggest that Snf7p may serve two functions in the cell: one as a RIM101 pathway member and one for MVB transport to the vacuole.