Biology subjects

Eckmann, L.

Publications and source records attributed to Eckmann, L..

7 recordsLinked to original sources

bioRxiv2024

Intestinal Epithelial PTPN2 Limits Pathobiont Colonization by Immune-Directed Antimicrobial Responses

Background and AimsLoss of activity of the inflammatory bowel disease (IBD) susceptibility gene, protein tyrosine phosphatase non-receptor type 2 (PTPN2), is associated with altered microbiome composition in both human subjects and mice. Further, expansion of the bacterial pathobiont, adherent- invasive E. coli (AIEC), is strongly linked to IBD pathogenesis. The mechanism by which intestinal epithelial cells (IEC) maintain equilibrium between commensal microbiota and immune cells to restrict invading pathobionts is poorly understood. Here, we investigated the role of IEC-specific PTPN2 in regulating AIEC colonization. MethodsTamoxifen-inducible, intestinal epithelial cell-specific Ptpn2 knockout mice (Ptpn2{Delta}IEC) and control Ptpn2fl/fl mice were infected with either non-invasive E. coli K12, or fluorescent-tagged mAIEC (mAIECred) for four consecutive days or administered PBS. Subsequently, bacterial colonization in mouse tissues was quantified. mRNA and protein expression were assayed in intestinal epithelial cells (IECs) or whole tissue lysates by PCR and Western blot. Tissue cytokine expression was determined by ELISA. Intestinal barrier function was determined by in vivo administration of 4 kDa FITC-dextran (FD4) or 70kDa Rhodamine-B dextran (RD70) fluorescent probes. Confocal microscopy was used to determine the localization of tight-junction proteins. ResultsPtpn2{Delta}IEC mice exhibited increased mAIECred - but not K12 - bacterial load in the distal colon compared to infected Ptpn2fl/fl mice. The higher susceptibility to mAIECred infection was associated with altered levels of antimicrobial peptide (AMPs). Ileal RNA expression of the alpha-defensin AMPs, Defa5 and Defa6, as well as MMP7, was significantly lower in Ptpn2{Delta}IEC vs. Ptpn2fl/fl mice, after mAIECred but not K12 infection. Further, we observed increased tight junction-regulated permeability determined by elevated in vivo FD4 but not RD70 permeability in Ptpn2{Delta}IEC-K12 mice compared to their respective controls. This effect was further exacerbated in Ptpn2{Delta}IEC mAIEC-infected mice. Further, Ptpn2{Delta}IEC mice displayed lower IL-22, IL-6, IL-17A cytokine expression post mAIEC infection compared to Ptpn2fl/fl controls. Recombinant IL-22 reversed the FD4 permeability defect and reduced bacterial burden in Ptpn2{Delta}IEC mice post mAIEC challenge. ConclusionOur findings highlight that intestinal epithelial PTPN2 is crucial for mucosal immunity and gut homeostasis by promoting anti-bacterial defense mechanisms involving coordinated epithelial-immune responses to restrict pathobiont colonization.

immunology↗

bioRxiv2023

Structural elucidation of recombinant Trichomonas vaginalis 20S proteasome bound to covalent inhibitors

Proteasomes are essential for protein homeostasis in mammalian cells1-4 and in protozoan parasites such as Trichomonas vaginalis (Tv).5 Tv and other protozoan 20S proteasomes have been validated as druggable targets.6-8 However, in the case of Tv 20S proteasome (Tv20S), biochemical and structural studies were impeded by low yields and purity of the native proteasome. We successfully made recombinant Tv20S by expressing all seven and seven {beta} subunits together with the Ump-1 chaperone in insect cells. We isolated recombinant proteasome and showed that it was biochemically indistinguishable from the native enzyme. We confirmed that the recombinant Tv20S is inhibited by the natural product marizomib (MZB)9 and the recently developed peptide inhibitor carmaphycin-17 (CP-17)8,10. Specifically, MZB binds to the {beta}1, {beta}2 and {beta}5 subunits, while CP-17 binds the {beta}2 and {beta}5 subunits. Next, we obtained cryo-EM structures of Tv20S in complex with these covalent inhibitors at 2.8[A] resolution. The structures revealed the overall fold of the Tv20S and the binding mode of MZB and CP-17. Our work explains the low specificity of MZB and higher specificity of CP-17 towards Tv20S as compared to human proteasome and provides the platform for the development of Tv20S inhibitors for treatment of trichomoniasis.

biophysics↗

bioRxiv2023

Development of subunit selective substrates for Trichomonas vaginalis proteasome

The protozoan parasite, Trichomonas vaginalis (Tv) causes trichomoniasis, the most common, non-viral, sexually transmitted infection in the world. Only two closely related drugs are approved for its treatment. The accelerating emergence of resistance to these drugs and lack of alternative treatment options poses an increasing threat to public health. There is an urgent need for novel effective anti-parasitic compounds. The proteasome is a critical enzyme for T. vaginalis survival and was validated as a drug target to treat trichomoniasis. However, to develop potent inhibitors of the T. vaginalis proteasome, it is essential that we understand which subunits should be targeted. Previously, we identified two fluorogenic substrates that were cleaved by T. vaginalis proteasome, however after isolating the enzyme complex and performing an in-depth substrate specificity study, we have now designed three fluorogenic reporter substrates that are each specific for one catalytic subunit. We screened a library of peptide epoxyketone inhibitors against the live parasite and evaluated which subunits are targeted by the top hits. Together we show that targeting of the {beta}5 subunit of T. vaginalis is sufficient to kill the parasite, however, targeting of {beta}5 plus either {beta}1 or {beta}2 results in improved potency.

biochemistry↗

bioRxiv2022

Gnas ablation in CD11c+ cells prevents high-fat diet-induced obesity by elevating adipose tissue catecholamine levels and thermogenesis

CD11c+ immune cells are a potential therapeutic target for treatment of obesity-related insulin resistance and type 2 diabetes (T2D). In obesity, CD11c+ immune cells are recruited to white adipose tissue and create an inflammatory state that causes both insulin and catecholamine resistance. In this study, we found that ablation of Gnas, the gene that encodes Gas, in CD11c expressing cells protects mice from high-fat diet-induced obesity, glucose intolerance and insulin resistance. Gnas{Delta}CD11c mice (KO) had increased oxygen consumption, energy expenditure, and beigeing of white adipose tissue (WAT). Transplantation studies showed that the lean phenotype was conferred by bone marrow-derived cells and the absence of T and B cells by crossing the KO to a Rag1-/- background did not alter the phenotype. Notably, we observed elevated norepinephrine and elevated cAMP signaling in the WAT of KO mice. The KO adipose tissue also had reduced expression of catecholamine transport and degradation enzymes. Collectively, our results identified an important role of Gas in CD11c+ cells in whole body metabolism regulation by controlling norepinephrine levels in WAT, modulating catecholamine-induced lipolysis and increasing thermogenesis that together created a lean phenotype.

immunology↗

bioRxiv2021

Intestinal Transgene Delivery with Native E. coli Chassis Allows Persistent Physiological Changes

Live bacterial therapeutics (LBT) could reverse disease by engrafting in the gut and providing persistent beneficial functions in the host. However, attempts to functionally manipulate the gut microbiome of conventionally-raised (CR) hosts have been unsuccessful, because engineered microbial organisms (i.e., chassis) cannot colonize the hostile luminal environment. In this proof-of-concept study, we use native bacteria as chassis for transgene delivery to impact CR host physiology. Native Escherichia coli isolated from stool cultures of CR mice were modified to express functional bacterial (bile salt hydrolase) and eukaryotic (Interleukin-10) genes. Reintroduction of these strains induces perpetual engraftment in the intestine. In addition, engineered native E. coli can induce functional changes that affect host physiology and reverse pathology in CR hosts months after administration. Thus, using native bacteria as chassis to "knock-in" specific functions allows mechanistic studies of specific microbial activities in the microbiome of CR hosts, and enables LBT with curative intent.

synthetic biology↗

bioRxiv2020

Identification of a Novel Susceptibility Marker for SARS-CoV-2 Infection in Human Subjects and Risk Mitigation with a Clinically Approved JAK Inhibitor in Human/Mouse Cells

Coronavirus disease (COVID-19), caused by SARS-CoV-2, has affected over 65 million individuals and killed over 1.5 million persons (December 8, 2020; www.who.int)1. While fatality rates are higher among the elderly and those with underlying comorbidities2, host factors that promote susceptibility to SARS-CoV-2 infection and severe disease are poorly understood. Although individuals with certain autoimmune/inflammatory disorders show increased susceptibility to viral infections, there is incomplete knowledge of SARS-CoV-2 susceptibility in these diseases.3-7 We report that the autoimmune PTPN2 risk variant rs1893217 promotes expression of the SARS-CoV-2 receptor, ACE2, and increases cellular entry mediated by SARS-CoV-2 spike protein. Elevated ACE2 expression and viral entry were mediated by increased JAK-STAT signalling, and were reversed by the JAK inhibitor, tofacitinib. Collectively, our findings uncover a novel risk biomarker for increased expression of the SARS-CoV-2 receptor and viral entry, and identify a clinically approved therapeutic agent to mitigate this risk.

microbiology↗

bioRxiv2019

The compact genome of Giardia muris reveals important steps in the evolution of intestinal protozoan parasites

Diplomonad parasites of the genus Giardia have adapted to colonizing different hosts, most notably the intestinal tract of mammals. The human-pathogenic Giardia species, Giardia intestinalis, has been extensively studied at the genome and gene expression level, but no such information is available for other Giardia species. Comparative data would be particularly valuable for Giardia muris, which colonizes mice and is commonly used as a prototypic in vivo model for investigating host responses to intestinal parasitic infection. Here we report the draft-genome of G. muris. We discovered a highly streamlined genome, amongst the most densely encoded ever described for a nuclear eukaryotic genome. G. muris and G. intestinalis share many known or predicted virulence factors, including cysteine proteases and a large repertoire of cysteine-rich surface proteins involved in antigenic variation. Different to G. intestinalis, G. muris maintains tandem arrays of pseudogenized surface antigens at the telomeres, whereas intact surface antigens are present centrally in the chromosomes. The two classes of surface antigens engage in genetic exchange. Reconstruction of metabolic pathways from the G. muris genome suggest significant metabolic differences to G. intestinalis. Additionally, G. muris encodes proteins that might be used to modulate the prokaryotic microbiota. The responsible genes have been introduced in the Giardia genus via lateral gene transfer from prokaryotic sources. Our findings point to important evolutionary steps in the Giardia genus as it adapted to different hosts and it provides a powerful foundation for mechanistic exploration of host-pathogen interaction in the G. muris - mouse pathosystem.

evolutionary biology↗