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

The proteasome is a proteolytic enzyme complex essential for protein homeostasis in mammalian cells and protozoan parasites like Trichomonas vaginalis (Tv), the cause of the most common, non-viral sexually transmitted disease. Tv and other protozoan 20S proteasomes have been validated as druggable targets for antimicrobials. However, low yields and purity of the native proteasome have hindered studies of the Tv 20S proteasome (Tv20S). We address this challenge by creating a recombinant protozoan proteasome by expressing all seven α and seven ß subunits of Tv20S alongside the Ump-1 chaperone in insect cells. The recombinant Tv20S displays biochemical equivalence to its native counterpart, confirmed by various assays. Notably, the marizomib (MZB) inhibits all catalytic subunits of Tv20S, while the peptide inhibitor carmaphycin-17 (CP-17) specifically targets ß2 and ß5. Cryo-electron microscopy (cryo-EM) unveils the structures of Tv20S bound to MZB and CP-17 at 2.8 Å. These findings explain MZB's low specificity for Tv20S compared to the human proteasome and demonstrate CP-17's higher specificity. Overall, these data provide a structure-based strategy for the development of specific Tv20S inhibitors to treat trichomoniasis.

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

Background and Aims: Loss 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. Methods: Tamoxifen-inducible, intestinal epithelial cell-specific Ptpn2 knockout mice (Ptpn2 ΔIEC) and control Ptpn2 fl/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. Results: Ptpn2 ΔIEC mice exhibited increased mAIECred - but not K12 - bacterial load in the distal colon compared to infected Ptpn2 fl/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 ΔIEC vs. Ptpn2 fl/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 ΔIEC-K12 mice compared to their respective controls. This effect was further exacerbated in Ptpn2 ΔIEC mAIEC-infected mice. Further, Ptpn2 ΔIEC mice displayed lower IL-22, IL-6, IL-17A cytokine expression post mAIEC infection compared to Ptpn2 fl/fl controls. Recombinant IL-22 reversed the FD4 permeability defect and reduced bacterial burden in Ptpn2 ΔIEC mice post mAIEC challenge. Conclusion: Our 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.

Vaginal Tritrichomonas foetus infection in mice as an in vivo model for drug development against Trichomonas vaginalis.

Trichomonas vaginalis is the causative agent of the common sexually transmitted disease, trichomoniasis, which affects more than a hundred million people worldwide. Metronidazole and tinidazole, agents belonging to the 5-nitroheterocyclic class of antimicrobials, are most often used to treat infection, but increased resistance has been reported and adverse effects of these drugs can be significant. Consequently, an urgent need exists for the development of novel drug entities against trichomoniasis. Critical for antimicrobial drug development is the demonstration of in vivo efficacy. Murine models of vaginal T. vaginalis infection are unreliable for unknown reasons. Meanwhile, murine infections with the related bovine pathogen, Tritrichomonas foetus, tend to be more robust, although susceptibility to different antimicrobials might differ from T. vaginalis. Here, we explored the utility of T. foetus infection as a surrogate model for drug development against T. vaginalis. Four different T. foetus strains caused robust vaginal infection in young mice, while none of four diverse T. vaginalis strains did. Comparison of drug susceptibility profiles revealed that T. foetus and T. vaginalis were similarly susceptible to a range of 5-nitroheterocyclic and gold(I) compounds. By comparison, proteasome inhibitors were 10- to 15-fold less active against T. foetus than T. vaginalis, although one of the proteasome inhibitors, bortezomib, had low micromolar activity or better against multiple strains of both trichomonads. Different strains of T. foetus were used to demonstrate the utility of the murine vaginal infection models for in vivo efficacy testing, including for bortezomib and a gold(I) compound. The differences in susceptibility to proteasome inhibitors may be partially explained by differences in the proteasome subunit sequences between the two trichomonads, although the functional relevance of the proteasome was similar in both organisms. These findings indicate that T. foetus can serve as a reliable surrogate model for T. vaginalis in vitro and in murine infections in vivo, but caution must be exercised for specific drug classes with targets, such as the proteasome, that may display genetic divergence between the trichomonads.

Evolutionary dynamics of polyadenylation signals and their recognition strategies in protists.

The poly(A) signal, together with auxiliary elements, directs cleavage of a pre-mRNA and thus determines the 3' end of the mature transcript. In many species, including humans, the poly(A) signal is an AAUAAA hexamer, but we recently found that the deeply branching eukaryote Giardia lamblia uses a distinct hexamer (AGURAA) and lacks any known auxiliary elements. Our discovery prompted us to explore the evolutionary dynamics of poly(A) signals and auxiliary elements in the eukaryotic kingdom. We use direct RNA sequencing to determine poly(A) signals for four protists within the Metamonada clade (which also contains G. lamblia) and two outgroup protists. These experiments reveal that the AAUAAA hexamer serves as the poly(A) signal in at least four different eukaryotic clades, indicating that it is likely the ancestral signal, whereas the unusual Giardia version is derived. We find that the use and relative strengths of auxiliary elements are also plastic; in fact, within Metamonada, species like G. lamblia make use of a previously unrecognized auxiliary element where nucleotides flanking the poly(A) signal itself specify genuine cleavage sites. Thus, despite the fundamental nature of pre-mRNA cleavage for the expression of all protein-coding genes, the motifs controlling this process are dynamic on evolutionary timescales, providing motivation for future biochemical and structural studies as well as new therapeutic angles to target eukaryotic pathogens.

Mucosal vaccination in a murine gnotobiotic model of Giardia lamblia infection.

Giardia lamblia is an important protozoan cause of diarrheal disease worldwide, delayed development and cognitive impairment in children in low- and middle-income countries, and protracted post-infectious syndromes in developed regions. G. lamblia resides in the lumen and at the epithelial surface of the proximal small intestine but is not mucosa invasive. The protozoan parasite is genetically diverse with significant genome differences across strains and assemblages. Animal models, particularly murine models, have been instrumental in defining mechanisms of host defense against G. lamblia, but mice cannot be readily infected with most human pathogenic strains. Antibiotic pretreatment can increase susceptibility, suggesting that the normal microbiota plays a role in controlling G. lamblia infection in mice, but the broader implications on susceptibility to diverse strains are not known. Here, we have used gnotobiotic mice to demonstrate that robust intestinal infection can be achieved for a broad set of human-pathogenic strains of the genetic assemblages A and B. Furthermore, gnotobiotic mice were able to eradicate infection with a similar kinetics to conventional mice after trophozoite challenge. Germ-free mice could also be effectively immunized by the mucosal route with a protective antigen, α1-giardin, in a manner dependent on CD4 T cells. These results indicate that the gnotobiotic mouse model is powerful for investigating acquired host defenses in giardiasis, as the mice are broadly susceptible to diverse G. lamblia strains yet display no apparent defects in mucosal immunity needed for controlling and eradicating this lumen-dwelling pathogen.

Antigiardial and antiamebic activities of fexinidazole and its metabolites: new drug leads for giardiasis and amebiasis.

The intestinal parasites Giardia lamblia and Entamoeba histolytica are major causes of morbidity and mortality associated with diarrheal diseases. Metronidazole is the most common drug used to treat giardiasis and amebiasis. Despite its efficacy, treatment failures in giardiasis occur in up to 5%-40% of cases. Potential resistance of E. histolytica to metronidazole is an increasing concern. Therefore, it is critical to search for more effective drugs to treat giardiasis and amebiasis. We identified antigiardial and antiamebic activities of the rediscovered nitroimidazole compound, fexinidazole, and its sulfone and sulfoxide metabolites. Fexinidazole is equally active against E. histolytica and G. lamblia trophozoites, and both metabolites were 3- to 18-fold more active than the parent drug. Fexinidazole and its metabolites were also active against a metronidazole-resistant strain of G. lamblia. G. lamblia and E. histolytica cell extracts exhibited decreased residual nitroreductase activity when metabolites were used as substrates, indicating nitroreductase may be central to the mechanism of action of fexinidazole. In a cell invasion model, fexinidazole and its metabolites significantly reduced the invasiveness of E. histolytica trophozoites through basement membrane matrix. A q.d. oral dose of fexinidazole and its metabolites at 10 mg/kg for 3 days reduced G. lamblia infection significantly in mice compared to control. The newly discovered antigiardial and antiamebic activities of fexinidazole, combined with its FDA-approval and inclusion in the WHO Model List of Essential Medicines for the treatment of human African trypanosomiasis, offer decreased risk and a shortened development timeline toward clinical use of fexinidazole for treatment of giardiasis or amebiasis.

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 α and seven ß 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 ß1, ß2 and ß5 subunits, while CP-17 binds the ß2 and ß5 subunits. Next, we obtained cryo-EM structures of Tv20S in complex with these covalent inhibitors at 2.8Å 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.

Loss of cAMP Signaling in CD11c Immune Cells Protects Against Diet-Induced Obesity.

In obesity, CD11c+ innate immune cells are recruited to adipose tissue and create an inflammatory state that causes both insulin and catecholamine resistance. We found that ablation of Gnas, the gene that encodes Gαs, in CD11c expressing cells protects mice from obesity, glucose intolerance, and insulin resistance. Transplantation studies showed that the lean phenotype was conferred by bone marrow-derived cells and did not require adaptive immunity. Loss of cAMP signaling was associated with increased adipose tissue norepinephrine and cAMP signaling, and prevention of catecholamine resistance. The adipose tissue had reduced expression of catecholamine transport and degradation enzymes, suggesting that the elevated norepinephrine resulted from decreased catabolism. Collectively, our results identified an important role for cAMP signaling in CD11c+ innate immune cells in whole-body metabolism by controlling norepinephrine levels in white adipose tissue, modulating catecholamine-induced lipolysis and increasing thermogenesis, which, together, created a lean phenotype. ARTICLE HIGHLIGHTS: We undertook this study to understand how immune cells communicate with adipocytes, specifically, whether cAMP signaling in the immune cell and the adipocyte are connected. We identified a reciprocal interaction between CD11c+ innate immune cells and adipocytes in which high cAMP signaling in the immune cell compartment induces low cAMP signaling in adipocytes and vice versa. This interaction regulates lipolysis in adipocytes and inflammation in immune cells, resulting in either a lean, obesity-resistant, and insulin-sensitive phenotype, or an obese, insulin-resistant phenotype.

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 ß5 subunit of T. vaginalis is sufficient to kill the parasite, however, targeting of ß5 plus either ß1 or ß2 results in improved potency.

PTPN2 Is a Critical Regulator of Ileal Paneth Cell Viability and Function in Mice.

BACKGROUND & AIMS: Loss-of-function variants in the PTPN2 gene are associated with increased risk of inflammatory bowel disease. We recently showed that Ptpn2 is critical for intestinal epithelial cell (IEC) barrier maintenance, IEC-macrophage communication, and modulation of the gut microbiome in mice, restricting expansion of a small intestinal pathobiont associated with inflammatory bowel disease. Here, we aimed to identify how Ptpn2 loss affects ileal IEC subtypes and their function in vivo. METHODS: Constitutive Ptpn2 wild-type, heterozygous, and knockout (KO) mice, as well as mice with inducible deletion of Ptpn2 in IECs, were used in the study. Investigation was performed using imaging techniques, flow cytometry, enteroid culture, and analysis of gene and protein levels of IEC markers. RESULTS: Partial transcriptome analysis showed that expression of Paneth cell-associated antimicrobial peptides Lyz1, Pla2g2a, and Defa6 was down-regulated markedly in Ptpn2-KO mice compared with wild-type and heterozygous. In parallel, Paneth cell numbers were reduced, their endoplasmic reticulum architecture was disrupted, and the endoplasmic reticulum stress protein, C/EBP-homologous protein (CHOP), was increased in Ptpn2-KO mice. Despite reduced Paneth cell number, flow cytometry showed increased expression of the Paneth cell-stimulatory cytokines interleukin 22 and interferon γ+ in CD4+ T cells isolated from Ptpn2-KO ileum. Key findings in constitutive Ptpn2-KO mice were confirmed in epithelium-specific Ptpn2ΔIEC mice, which also showed impaired lysozyme protein levels in Paneth cells compared with Ptpn2fl/fl control mice. CONCLUSIONS: Constitutive Ptpn2 deficiency affects Paneth cell viability and compromises Paneth cell-specific antimicrobial peptide production. The observed effects may contribute to the increased susceptibility to intestinal infection and dysbiosis in these mice.

A Novel Hypomorphic Apex1 Mouse Model Implicates Apurinic/Apyrimidinic Endonuclease 1 in Oxidative DNA Damage Repair in Gastric Epithelial Cells.

Aims: Though best known for its role in oxidative DNA damage repair, apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein that regulates multiple host responses during oxidative stress, including the reductive activation of transcription factors. As knockout of the APE1-encoding gene, Apex1, is embryonically lethal, we sought to create a viable model with generalized inhibition of APE1 expression. Results: A hypomorphic (HM) mouse with decreased APE1 expression throughout the body was generated using a construct containing a neomycin resistance (NeoR) cassette knocked into the Apex1 site. Offspring were assessed for APE1 expression, breeding efficiency, and morphology with a focused examination of DNA damage in the stomach. Heterozygotic breeding pairs yielded 50% fewer HM mice than predicted by Mendelian genetics. APE1 expression was reduced up to 90% in the lungs, heart, stomach, and spleen. The HM offspring were typically smaller, and most had a malformed tail. Oxidative DNA damage was increased spontaneously in the stomachs of HM mice. Further, all changes were reversed when the NeoR cassette was removed. Primary gastric epithelial cells from HM mice differentiated more quickly and had more evidence of oxidative DNA damage after stimulation with Helicobacter pylori or a chemical carcinogen than control lines from wildtype mice. Innovation: A HM mouse with decreased APE1 expression throughout the body was generated and extensively characterized. Conclusion: The results suggest that HM mice enable studies of APE1's multiple functions throughout the body. The detailed characterization of the stomach showed that gastric epithelial cells from HM were more susceptible to DNA damage. Antioxid. Redox Signal. 38, 183-197.

Intestinal transgene delivery with native E. coli chassis allows persistent physiological changes.

Live bacterial therapeutics (LBTs) could reverse diseases 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) have difficulty in colonizing 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 bacteria isolated from the stool cultures of CR mice were modified to express functional genes. The reintroduction of these strains induces perpetual engraftment in the intestine. In addition, engineered native E. coli can induce functional changes that affect physiology of 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.

Conserved metabolic enzymes as vaccine antigens for giardiasis.

Giardia lamblia is a leading protozoal cause of diarrheal disease worldwide. Infection is associated with abdominal pain, malabsorption and weight loss, and protracted post-infectious syndromes. A human vaccine is not available against G. lamblia. Prior studies with human and murine immune sera have identified several parasite antigens, including surface proteins and metabolic enzymes with intracellular functions. While surface proteins have demonstrated vaccine potential, they can exhibit significant variation between G. lamblia strains. By comparison, metabolic enzymes show greater conservation but their vaccine potential has not been established. To determine whether such proteins can serve as vaccine candidates, we focused on two enzymes, α-enolase (ENO) and ornithine carbamoyl transferase (OCT), which are involved in glycolysis and arginine metabolism, respectively. We show in a cohort of patients with confirmed giardiasis that both enzymes are immunogenic. Intranasal immunization with either enzyme antigen in mice induced strong systemic IgG1 and IgG2b responses and modest mucosal IgA responses, and a marked 100- to 1,000-fold reduction in peak trophozoite load upon oral G. lamblia challenge. ENO immunization also reduced the extent and duration of cyst excretion. Examination of 44 cytokines showed only minimal intestinal changes in immunized mice, although a modest increase of CCL22 was observed in ENO-immunized mice. Spectral flow cytometry revealed increased numbers and activation state of CD4 T cells in the small intestine and an increase in α4ß7-expressing CD4 T cells in mesenteric lymph nodes of ENO-immunized mice. Consistent with a key role of CD4 T cells, immunization of CD4-deficient and Rag-2 deficient mice failed to induce protection, whereas mice lacking IgA were fully protected by immunization, indicating that immunity was CD4 T cell-dependent but IgA-independent. These results demonstrate that conserved metabolic enzymes can be effective vaccine antigens for protection against G. lamblia infection, thereby expanding the repertoire of candidate antigens beyond primary surface proteins.

Codelivery of Antigens and Adjuvant in Polymeric Nanoparticles Coated With Native Parasite Membranes Induces Protective Mucosal Immunity Against Giardia lamblia.

The protozoan pathogen Giardia lamblia is an important worldwide cause of diarrheal disease and malabsorption. Infection is managed with antimicrobials, although drug resistance and treatment failures are a clinical challenge. Prior infection provides significant protection, yet a human vaccine has not been realized. Individual antigens can elicit partial protection in experimental models, but protection is weaker than after prior infection. Here, we developed a multivalent nanovaccine by coating membranes derived from the parasite onto uniform and stable polymeric nanoparticles loaded with a mucosal adjuvant. Intranasal immunization with the nanovaccine induced adaptive immunity and effectively protected mice from G. lamblia infection.

Characterization of Metronidazole-Resistant Giardia intestinalis Lines by Comparative Transcriptomics and Proteomics.

Metronidazole (MTZ) is a clinically important antimicrobial agent that is active against both bacterial and protozoan organisms. MTZ has been used extensively for more than 60 years and until now resistance has been rare. However, a recent and dramatic increase in the number of MTZ resistant bacteria and protozoa is of great concern since there are few alternative drugs with a similarly broad activity spectrum. To identify key factors and mechanisms underlying MTZ resistance, we utilized the protozoan parasite Giardia intestinalis, which is commonly treated with MTZ. We characterized two in vitro selected, metronidazole resistant parasite lines, as well as one revertant, by analyzing fitness aspects associated with increased drug resistance and transcriptomes and proteomes. We also conducted a meta-analysis using already existing data from additional resistant G. intestinalis isolates. The combined data suggest that in vitro generated MTZ resistance has a substantial fitness cost to the parasite, which may partly explain why resistance is not widespread despite decades of heavy use. Mechanistically, MTZ resistance in Giardia is multifactorial and associated with complex changes, yet a core set of pathways involving oxidoreductases, oxidative stress responses and DNA repair proteins, is central to MTZ resistance in both bacteria and protozoa.

Olfactory receptor 2 in vascular macrophages drives atherosclerosis by NLRP3-dependent IL-1 production.

Atherosclerosis is an inflammatory disease of the artery walls and involves immune cells such as macrophages. Olfactory receptors (OLFRs) are G protein­coupled chemoreceptors that have a central role in detecting odorants and the sense of smell. We found that mouse vascular macrophages express the olfactory receptor Olfr2 and all associated trafficking and signaling molecules. Olfr2 detects the compound octanal, which activates the NLR family pyrin domain containing 3 (NLRP3) inflammasome and induces interleukin-1ß secretion in human and mouse macrophages. We found that human and mouse blood plasma contains octanal, a product of lipid peroxidation, at concentrations sufficient to activate Olfr2 and the human ortholog olfactory receptor 6A2 (OR6A2). Boosting octanal levels exacerbated atherosclerosis, whereas genetic targeting of Olfr2 in mice significantly reduced atherosclerotic plaques. Our findings suggest that inhibiting OR6A2 may provide a promising strategy to prevent and treat atherosclerosis.

Colesevelam ameliorates non-alcoholic steatohepatitis and obesity in mice.

BACKGROUND: Obesity, non-alcoholic fatty liver disease (NAFLD) and its more advanced form non-alcoholic steatohepatitis (NASH) are important causes of morbidity and mortality worldwide. Bile acid dysregulation is a pivotal part in their pathogenesis. The aim of this study was to evaluate the bile acid sequestrant colesevelam in a microbiome-humanized mouse model of diet-induced obesity and steatohepatitis. METHODS: Germ-free C57BL/6 mice were associated with stool from patients with NASH and subjected to 20 weeks of Western diet feeding with and without colesevelam. RESULTS: Colesevelam reduced Western diet-induced body and liver weight gain in microbiome-humanized mice compared with controls. It ameliorated Western diet-induced hepatic inflammation, steatosis, fibrosis and insulin resistance. Colesevelam increased de novo bile acid synthesis and decreased hepatic cholesterol content in microbiome-humanized mice fed a Western diet. It further induced the gene expression of the antimicrobials Reg3g and Reg3b in the distal small intestine and decreased plasma levels of LPS. CONCLUSIONS: Colesevelam ameliorates Western diet-induced steatohepatitis and obesity in microbiome-humanized mice.

The fecal mycobiome in non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Studies investigating the gut-liver axis have largely focused on bacteria, whereas little is known about commensal fungi. We characterized fecal fungi in patients with non-alcoholic fatty liver disease (NAFLD) and investigated their role in a fecal microbiome-humanized mouse model of Western diet-induced steatohepatitis. METHODS: We performed fungal internal transcribed spacer 2 sequencing using fecal samples from 78 patients with NAFLD, 16 controls and 73 patients with alcohol use disorder. Anti-Candida albicans (C. albicans) IgG was measured in blood samples from 17 controls and 79 patients with NAFLD. Songbird, a novel multinominal regression tool, was used to investigate mycobiome changes. Germ-free mice were colonized with feces from patients with non-alcoholic steatohepatitis (NASH), fed a Western diet for 20 weeks and treated with the antifungal amphotericin B. RESULTS: The presence of non-obese NASH or F2-F4 fibrosis was associated with a distinct fecal mycobiome signature. Changes were characterized by an increased log-ratio for Mucor sp./Saccharomyces cerevisiae (S. cerevisiae) in patients with NASH and F2-F4 fibrosis. The C. albicans/S. cerevisiae log-ratio was significantly higher in non-obese patients with NASH when compared with non-obese patients with NAFL or controls. We observed a different fecal mycobiome composition in patients with NAFLD and advanced fibrosis compared to those with alcohol use disorder and advanced fibrosis. Plasma anti-C. albicans IgG was increased in patients with NAFLD and advanced fibrosis. Gnotobiotic mice, colonized with human NASH feces and treated with amphotericin B were protected from Western diet-induced steatohepatitis. CONCLUSIONS: Non-obese patients with NAFLD and more advanced disease have a different fecal mycobiome composition to those with mild disease. Antifungal treatment ameliorates diet-induced steatohepatitis in mice. Intestinal fungi could be an attractive target to attenuate NASH. LAY SUMMARY: Non-alcoholic fatty liver disease is one of the most common chronic liver diseases and is associated with changes in the fecal bacterial microbiome. We show that patients with non-alcoholic fatty liver disease and more severe disease stages have a specific composition of fecal fungi and an increased systemic immune response to Candida albicans. In a fecal microbiome-humanized mouse model of Western diet-induced steatohepatitis, we show that treatment with antifungals reduces liver damage.

Class Ib MHC-Mediated Immune Interactions Play a Critical Role in Maintaining Mucosal Homeostasis in the Mammalian Large Intestine.

Lymphocytes within the intestinal epithelial layer (IEL) in mammals have unique composition compared with their counterparts in the lamina propria. Little is known about the role of some of the key colonic IEL subsets, such as TCRαß+CD8+ T cells, in inflammation. We have recently described liver-enriched innate-like TCRαß+CD8αα regulatory T cells, partly controlled by the non-classical MHC molecule, Qa-1b, that upon adoptive transfer protect from T cell-induced colitis. In this study, we found that TCRαß+CD8αα T cells are reduced among the colonic IEL during inflammation, and that their activation with an agonistic peptide leads to significant Qa-1b-dependent protection in an acute model of colitis. Cellular expression of Qa-1b during inflammation and corresponding dependency in peptide-mediated protection suggest that Batf3-dependent CD103+CD11b- type 1 conventional dendritic cells control the protective function of TCRαß+CD8αα T cells in the colonic epithelium. In the colitis model, expression of the potential barrier-protective gene, Muc2, is enhanced upon administration of a Qa-1b agonistic peptide. Notably, in steady state, the mucin metabolizing Akkermansia muciniphila was found in significantly lower abundance amid a dramatic change in overall microbiome and metabolome, increased IL-6 in explant culture, and enhanced sensitivity to dextran sulfate sodium in Qa-1b deficiency. Finally, in patients with inflammatory bowel disease, we found upregulation of HLA-E, a Qa-1b analog with inflammation and biologic non-response, in silico, suggesting the importance of this regulatory mechanism across species.

Comprehensive characterization of purine and pyrimidine transport activities in Trichomonas vaginalis and functional cloning of a trichomonad nucleoside transporter.

Trichomoniasis is a common and widespread sexually-transmitted infection, caused by the protozoan parasite Trichomonas vaginalis. T. vaginalis lacks the biosynthetic pathways for purines and pyrimidines, making nucleoside metabolism a drug target. Here we report the first comprehensive investigation into purine and pyrimidine uptake by T. vaginalis. Multiple carriers were identified and characterized with regard to substrate selectivity and affinity. For nucleobases, a high-affinity adenine transporter, a possible guanine transporter and a low affinity uracil transporter were found. Nucleoside transporters included two high affinity adenosine/guanosine/uridine/cytidine transporters distinguished by different affinities to inosine, a lower affinity adenosine transporter, and a thymidine transporter. Nine Equilibrative Nucleoside Transporter (ENT) genes were identified in the T. vaginalis genome. All were expressed equally in metronidazole-resistant and -sensitive strains. Only TvagENT2 was significantly upregulated in the presence of extracellular purines; expression was not affected by co-culture with human cervical epithelial cells. All TvagENTs were cloned and separately expressed in Trypanosoma brucei. We identified the main broad specificity nucleoside carrier, with high affinity for uridine and cytidine as well as purine nucleosides including inosine, as TvagENT3. The in-depth characterization of purine and pyrimidine transporters provides a critical foundation for the development of new anti-trichomonal nucleoside analogues.