Human GBP1 does not localize to pathogen vacuoles but restricts Toxoplasma gondii

Guanylate binding proteins (GBPs) are a family of large interferon‐inducible GTPases that are transcriptionally upregulated upon infection with intracellular pathogens. Murine GBPs (mGBPs) including mGBP1 and 2 localize to and disrupt pathogen‐containing vacuoles (PVs) resulting in the cell‐autonomous clearing or innate immune detection of PV‐resident pathogens. Human GBPs (hGBPs) are known to exert antiviral host defense and activate the NLRP3 inflammasome, but it is unclear whether hGBPs can directly recognize and control intravacuolar pathogens. Here, we report that endogenous or ectopically expressed hGBP1 fails to associate with PVs formed in human cells by the bacterial pathogens Chlamydia trachomatis or Salmonella typhimurium or the protozoan pathogen Toxoplasma gondii. While we find that hGBP1 expression has no discernible effect on intracellular replication of C. trachomatis and S. typhimurium, we observed enhanced early Toxoplasma replication in CRISPR hGBP1‐deleted human epithelial cells. We thus identified a novel role for hGBP1 in cell‐autonomous immunity that is independent of PV translocation, as observed for mGBPs. This study highlights fundamental differences between human and murine GBPs and underlines the need to study the functions of GBPs at cellular locations away from PVs

IRG and GBP host resistance factors target aberrant, ‘‘Non-self’’ vacuoles characterized by the missing of ‘‘Self’’ IRGM proteins

Interferon-inducible GTPases of the Immunity Related GTPase (IRG) and Guanylate Binding Protein (GBP) families provide resistance to intracellular pathogenic microbes. IRGs and GBPs stably associate with pathogen-containing vacuoles (PVs) and elicit immune pathways directed at the targeted vacuoles. Targeting of Interferon-inducible GTPases to PVs requires the formation of higher-order protein oligomers, a process negatively regulated by a subclass of IRG proteins called IRGMs. We found that the paralogous IRGM proteins Irgm1 and Irgm3 fail to robustly associate with ‘‘non-self’’ PVs containing either the bacterial pathogen Chlamydia trachomatis or the protozoan pathogen Toxoplasma gondii. Instead, Irgm1 and Irgm3 reside on ‘‘self’’ organelles including lipid droplets (LDs). Whereas IRGM-positive LDs are guarded against the stable association with other IRGs and GBPs, we demonstrate that IRGM-stripped LDs become high affinity binding substrates for IRG and GBP proteins. These data reveal that intracellular immune recognition of organelle-like structures by IRG and GBP proteins is partly dictated by the missing of ‘‘self’’ IRGM proteins from these structures.Fil: Haldar, Arun K.. University Of Duke; Estados UnidosFil: Saka, Hector Alex. University Of Duke; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Piro, Anthony S.. University Of Duke; Estados UnidosFil: Dunn, Joe Dan. University Of Duke; Estados UnidosFil: Henry, Stanley C.. University Of Duke; Estados Unidos. Veteran Affairs Medical Center; Estados UnidosFil: Taylor, Gregory A.. University Of Duke; Estados Unidos. Veteran Affairs Medical Center; Estados UnidosFil: Frickel, Eva M.. National Institute for Medical Research; Reino UnidoFil: Valdivia, Raphael H.. University Of Duke; Estados UnidosFil: Coers, Jörn. University Of Duke; Estados Unido

Class I major histocompatibility complexes loaded by a periodate trigger

Class I major histocompatibility complexes (MHCs) present peptide ligands on the cell surface for recognition by appropriate cytotoxic T cells. The unstable nature of unliganded MHC necessitates the production of recombinant class I complexes through in vitro refolding reactions in the presence of an added excess of peptides. This strategy is not amenable to high-throughput production of vast collections of class I complexes. To address this issue, we recently designed photocaged MHC ligands that can be cleaved by a UV light trigger in the MHC bound state under conditions that do not affect the integrity of the MHC structure. The results obtained with photocaged MHC ligands demonstrate that conditional MHC ligands can form a generally applicable concept for the creation of defined peptide−MHCs. However, the use of UV exposure to mediate ligand exchange is unsuited for a number of applications, due to the lack of UV penetration through cell culture systems and due to the transfer of heat upon UV irradiation, which can induce evaporation. To overcome these limitations, here, we provide proof-of-concept for the generation of defined peptide−MHCs by chemical trigger-induced ligand exchange. The crystal structure of the MHC with the novel chemosensitive ligand showcases that the ligand occupies the expected binding site, in a conformation where the hydroxyl groups should be reactive to periodate. We proceed to validate this technology by producing peptide−MHCs that can be used for T cell detection. The methodology that we describe here should allow loading of MHCs with defined peptides in cell culture devices, thereby permitting antigen-specific T cell expansion and purification for cell therapy. In addition, this technology will be useful to develop miniaturized assay systems for performing high-throughput screens for natural and unnatural MHC ligands

Researching underwater: a submerged study

This chapter explores the unknown territory of a lost project: an ethnography of a public swimming pool. The discussion is contextualised within my broader sociological theory of ‘nothing’, as a category of unmarked, negative social phenomena, including no-things, no-bodies, no-wheres, non-events and non-identities. These meaningful symbolic objects are constituted through social interaction, which can take two forms: acts of commission and acts of omission. I tell the story of how this project did not happen, through the things I did not do or that did not materialise, and how I consequently did not become a certain type of researcher. I identify three types of negative phenomena that I did not observe and document – invisible figures, silent voices and empty vessels – and, consequently, the knowledge I did not acquire. However, nothing is also productive, generating new symbolic objects as substitutes, alternatives and replacements: the somethings, somebodies and somewheres that are done or made instead. Thus finally, I reflect on how not doing this project led me to pursue others, cultivating a different research identity that would not otherwise have existed

Detection of Cytosolic Shigella flexneri via a C-Terminal Triple-Arginine Motif of GBP1 Inhibits Actin-Based Motility

ABSTRACT Dynamin-like guanylate binding proteins (GBPs) are gamma interferon (IFN-γ)-inducible host defense proteins that can associate with cytosol-invading bacterial pathogens. Mouse GBPs promote the lytic destruction of targeted bacteria in the host cell cytosol, but the antimicrobial function of human GBPs and the mechanism by which these proteins associate with cytosolic bacteria are poorly understood. Here, we demonstrate that human GBP1 is unique among the seven human GBP paralogs in its ability to associate with at least two cytosolic Gram-negative bacteria, Burkholderia thailandensis and Shigella flexneri. Rough lipopolysaccharide (LPS) mutants of S. flexneri colocalize with GBP1 less frequently than wild-type S. flexneri does, suggesting that host recognition of O antigen promotes GBP1 targeting to Gram-negative bacteria. The targeting of GBP1 to cytosolic bacteria, via a unique triple-arginine motif present in its C terminus, promotes the corecruitment of four additional GBP paralogs (GBP2, GBP3, GBP4, and GBP6). GBP1-decorated Shigella organisms replicate but fail to form actin tails, leading to their intracellular aggregation. Consequentially, the wild type but not the triple-arginine GBP1 mutant restricts S. flexneri cell-to-cell spread. Furthermore, human-adapted S. flexneri, through the action of one its secreted effectors, IpaH9.8, is more resistant to GBP1 targeting than the non-human-adapted bacillus B. thailandensis. These studies reveal that human GBP1 uniquely functions as an intracellular “glue trap,” inhibiting the cytosolic movement of normally actin-propelled Gram-negative bacteria. In response to this powerful human defense program, S. flexneri has evolved an effective counterdefense to restrict GBP1 recruitment

Identification of the Rheumatoid Arthritis Shared Epitope Binding Site on Calreticulin

Background: The rheumatoid arthritis (RA) shared epitope (SE), a major risk factor for severe disease, is a five amino acid motif in the third allelic hypervariable region of the HLA-DRb chain. The molecular mechanisms by which the SE affects susceptibility to – and severity of- RA are unknown. We have recently demonstrated that the SE acts as a ligand that interacts with cell surface calreticulin (CRT) and activates innate immune signaling. In order to better understand the molecular basis of SE-RA association, here we have undertaken to map the SE binding site on CRT. Principal Findings: Surface plasmon resonance (SPR) experiments with domain deletion mutants suggested that the SE binding site is located in the P-domain of CRT. The role of this domain as a SE-binding region was further confirmed by a sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azido-benzamido) hexanoamido] ethyl-1,3-dithiopropionate (sulfo-SBED) photoactive cross-linking method. In silico analysis of docking interactions between a conformationally intact SE ligand and the CRT P-domain predicted the region within amino acid residues 217–224 as a potential SE binding site. Site-directed mutagenesis demonstrated involvement of residues Glu 217 and Glu 223- and to a lesser extent residue Asp 220- in cell-free SPR-based binding and signal transduction assays. Significance: We have characterized here the molecular basis of a novel ligand-receptor interaction between the SE and CRT. The interaction represents a structurally and functionally well-defined example of cross talk between the adaptive an

Evidence-based activism: Patients' organisations, users' and activist's groups in knowledge

This article proposes the notion of ‘evidence-based activism’ to capture patients’ and health activists’ groups’ focus on knowledge production and knowledge mobilisation in the governance of health issues. It introduces empirical data and analysis on groups active in four countries (France, Ireland, Portugal and the United Kingdom), and in four condition-areas (rare diseases, Alzheimer’s disease, ADHD – Attention Deficit Hyperactivity Disorder and childbirth). It shows how these groups engage with, and articulate a variety of credentialed knowledge and ‘experiential knowledge’ with a view to explore concerned people’s situations, to make themselves part and parcel of the networks of expertise on their conditions in their national contexts, and to elaborate evidence on the issues they deem important to address both at an individual and at a collective level. This article argues that in contrast to health movements which contest institutions from the outside, patients’ and activists’ groups which embrace ‘evidence-based activism’ work ‘from within’ to imagine new epistemic and political appraisal of their causes and conditions. ‘Evidence-based activism’ entails a collective inquiry associating patients/activists and specialists/professionals in the conjoint fabrics of scientific statements and political claims. From a conceptual standpoint, ‘evidence-based activism’ sheds light on the ongoing co-production of matters of fact and matters of concern in contemporary technological democracies

Quality control of Bradyrhizobium inoculant strains: detection of nosZ and correlation of symbiotic efficiency with soybean leaf chlorophyll levels

Greenhouse gas emissions, such as N2O from excessive N-fertilizer use, are of concern. Symbiotic N2-fixation by pulses such as soybean might mitigate this issue, for which inoculants carrying locally adapted Bradyrhizobium strains are recommended. In the frame of this goal, enhancing the quality control of these inoculants is required on two key aspects: determining the presence of nosZ, for the strains being able to reduce N2O, and assessing N2-fixation potential. Previously it was demonstrated that, in soybean leaves, N-contents are well correlated with chlorophyll contents. However, no such correlations were made with either N obtained from N2-fixation or with nodules mass, which is an indicator of nodular activity. Here we aimed to leverage the correlation between N and chlorophyll levels to develop a simple and non-destructive laboratory method to be applied in quality control of inoculants, able to assess the N2-fixing capacity of rhizobial strains. To establish such correlations, we cultivated soybeans in vermiculite with N-free nutrient solution, and inoculated them with various Bradyrhizobium field isolates that displayed a range of symbiotic N2-fixing capacities. Subsequently, we measured chlorophyll with a portable chlorophyllometer, and correlated these measures with symbiotic parameters. Moreover, we tested for the presence of nosZ by PCR. We observed significant correlations between chlorophyll and shoot nitrogen obtained from N2-fixation and, in addition, we corroborated that chlorophyll contents were significantly correlated also with nodules mass. Two B. diazoefficiens strains stood out and possessed nosZ. In contrast, B. elkanii and B. japonicum isolates displayed lower chlorophyll and symbiotic performance, and lacked nosZ. Our findings highlight the potential of measuring chlorophyll contents and testing for the presence of nosZ as two straightforward techniques that may enhance laboratory tests for quality control, enabling selection of superior and safe locally isolated strains for soybean inoculants without increased production costs

Identification of the PDI-Family Member ERp90 as an Interaction Partner of ERFAD

In the endoplasmic reticulum (ER), members of the protein disulfide isomerase (PDI) family perform critical functions during protein maturation. Herein, we identify the previously uncharacterized PDI-family member ERp90. In cultured human cells, we find ERp90 to be a soluble ER-luminal glycoprotein that comprises five potential thioredoxin (Trx)-like domains. Mature ERp90 contains 10 cysteine residues, of which at least some form intramolecular disulfides. While none of the Trx domains contain a canonical Cys-Xaa-Xaa-Cys active-site motif, other conserved cysteines could endow the protein with redox activity. Importantly, we show that ERp90 co-immunoprecipitates with ERFAD, a flavoprotein involved in ER-associated degradation (ERAD), through what is most likely a direct interaction. We propose that the function of ERp90 is related to substrate recruitment or delivery to the ERAD retrotranslocation machinery by ERFAD