Infliximab (IFX), a monoclonal antibody targeting tumour necrosis factor-α, is utilised as a rescue therapy in acute severe ulcerative colitis (ASUC). The relationships between IFX concentrations in serum and colonic mucosa and the pharmacodynamic changes underlying mucosal healing are not well defined. We conducted an open-label, prospective, observational study in hospitalised patients with ASUC requiring rescue IFX to characterise the pharmacokinetics (PK) of IFX in serum and colonic mucosa, and to assess the relationships between IFX exposure, molecular signatures, and treatment response. Patients were followed for up to 22 weeks (wks), with longitudinal collection of serum samples (wks 0-22) and biopsies (day 2 and between wk 1622). Serum and tissue IFX concentrations were compared between endoscopic remitters (ERs; Mayo endoscopic subscore of 0 or 1 at wk 22 without an interim UC-related hospitalisation or colectomy) and non-remitters (NRs). RNA sequencing was performed on colonic biopsies to analyse differential gene expression and pathways between ERs and NRs. 14 patients (7 ERs and 7 NRs) completed the study. PK analysis demonstrated that ERs maintained significantly higher median dose-normalised serum IFX area under the curve than NRs during early induction (wk 0-2: 0.943 vs 0.402 µg*day/mL/mg, p = 0.007), induction (wk 014: 2.053 vs 1.223, p = 0.007), and maintenance therapy (wk 15-22: 0.823 vs 0.188, p = 0.007) (Figure 1A). In contrast, tissue IFX concentrations were similar, regardless of inflammation status or response outcome (Figure 1B). Transcriptomic profiling showed similar gene expression on day 2 between ERs and NRs. However, at end of study, profound transcriptional changes were observed in ERs, with 382 genes upregulated and 2,536 genes downregulated (Figure 2A). NRs showed only 1 end-of-study differentially expressed gene (DEG) compared to day 2 (Figure 2B). Pathway analysis of the DEGs in ERs indicated a significant decrease in both innate and adaptive immune responses. This was accompanied by a concomitant upregulation of metabolic processes, reflecting the molecular hallmarks associated with mucosal healing. Endoscopic remission in ASUC was strongly associated with high systemic IFX exposure across all treatment phases. Transcriptomic profiling showed a profound molecular differentiation, characterised by widespread suppression of inflammatory pathways and upregulation of metabolism in ERs, despite similar tissue IFX concentrations to NRs. Our study was the first to demonstrate the tissue transcriptional pharmacodynamics of IFX in ASUC while controlling for colonic drug exposure. Conflict of interest: Battat, Robert: Speaker/consulting/moderator: Bristol Myers Squibb, Johnson and Johnson Innovative Medicine, AbbVie, Takeda, Ferring, Celltrion, Pfizer, Merck, Eli Lilly Advisory boards: Celltrion, AbbVie, Pfizer, Janssen, Bristol Myers Squibb, Takeda, Merck, Eli Lilly Educational grants and educational sponsorships: Abbvie, Johnson and Johnson Innovative Medicine, Pfizer, Eli Lilly, Amgen, Pendopharm, Kye Pharma, Merck, Celltrion, Organon, Takeda Travel support: Pfizer, Celltrion, Johnson and Johnson Innovative Medicine, Abbvie. Massimino, Luca: Employee of Alimentiv Inc. Lefevre, Pavine: Employee of Alimentiv Inc. Ahmed, Waseem: No conflict of interest Charilaou, Paris: Advisory board fees from AbbVie and consulting fees from Prometheus Biosciences Boland, Brigid: consulting fees and participation on advisory board from Celltrion, Sanofi, Abbvie, Merck and has grant funding from Merck, Mirador, Prometheus, SRT Therapeutics and Gilead. Singh, Siddharth: No conflict of interest Dulai, Parambir: Grant: Takeda, Bristol Meyer Squibb, Merck, Genentech, Geneoscopy Personal Fees: Abbvie, Abivax, Alimentiv, Bristol Meyer Squibb, Boehringer Ingelheim, Celltrion, Cristcot, Genentech, Geneoscopy, Janssen, Lilly, Merck, Pfizer, Sanofi, Takeda Duijvestein, Marjolijn: Speaking fee from Bristol Meyers Squibb, Takeda, Galapagos, Janssen, Dr. Falk advisory board fees from Abbvie, Bristol Meyers Squibb, Celltrion, Galapagos/Alfasigma, Janssen, Takeda and grant/research support: Pfizer, Bristol Meyers Squibb, Galapagos Alfasigma, Janssen, MSD, Eli Lilly Eckmann, Lars: No conflict of interest Silverberg, Mark: Speaker for Abbvie, Janssen, Takeda, Pfizer, Ferring, Novartis, and Lilly. Advisor for Abbvie, Janssen, Takeda, Pfizer, Ferring, Gilead, Amgen, Merck, and Lilly. Research for Abbvie, Janssen, Takeda, Pfizer, and Gilead. Consults for Abbvie, Janssen, Takeda, Pfizer, and Lilly. Longman, Randy: Grant: Grant support from Boeringer Ingelheim. Personal Fees: Consultant for Pfizer, Sanofi, CJ Biosciences, Ancilia, Lukin, Dana: Consulting: Abbvie, Altrubio, Boehringer Ingelheim, Eli Lilly, Johnson & Johnson, Palatin, Pfizer, Prime, PSI, Takeda, Vedanta. Grants: Boehringer Ingelheim, Johnson & Johnson Speaking: Abbvie, Johnson & Johnson Scherl, Ellen: Grant/research support from Abbott (AbbVie), AstraZeneca, CCFA, Janssen Research &Development, Johns Hopkins University, National Institute of Diabetes and Digestive and Kidney (NIDDK), National Institute of Health (NIH), New York Crohn’s Foundation, Pfizer, UCB, UCSF–CCFA Clinical Research Alliance, Genentech, Seres Therapeutics, Celgene Corporation. Consulting fees are reported for AbbVie, Crohn’s and Colitis Foundation of America (CCFA), Entera Health, Evidera, GI Health Foundation, Janssen, Protagonist Therapeutics, Seres Health, Takeda Pharmaceuticals, Bristol Myers Squibb. Stock shareholder of Gilead, and honoraria of GIHealth Foundation for non-branded speaker’s bureau, Janssen for non-branded speaker’s bureau. Rueffer, Matthew: Employee of Alimentiv Inc. Smith, Michelle: Employee of Alimentiv Inc. Filice, Melissa: Employee of Alimentiv Inc. Teft, Wendy: Employee of Alimentiv Inc Feagan, Brian G.: Consulting Fees: AbbVie, Abivax, Adaxion, Adiso, AgomAB Therapeutics, Akros, Alira Health, Ally Bridge Group, Apini Therapeutics, Argenx, Attovia Tx, Avoro Capital Advisors, Belmore Law, Biora Therapeutics, Blackbird Laboratories, Boehringer-Ingelheim, BMS, Boxer Capital, Celgene/BMS, Clarivate, Connect Biopharm, EcoR1, Eli Lilly, Ensho Therapeutics, Equillium, Evida, Enveda, Evommune Inc. Faes Farma, First Wave, Forbion, Galapagos, Genentech/Roche, General Atlantic, Genesis Therapeutics, Gerson Lehrman Group, Gilead, Guidepoint, Imhotex, ImiDomics, Immunic Therapeutics, Janssen, Japan Tobacco Inc., LifeMine Therapeutics, Mage Biologics, Merck, Mirador Therapeutics, Mobius Care, Monte Rosa Tx, Morphic Therapeutics, Nimbus Therapeutics, Novartis, Nxera, OncoC3, Palisade Bio, Pendopharm, Pfizer,Q32 Bio, REDX, Roche, Sanofi, Sobi, Sorriso, Spyre Therapeutics, Sun Pharma, Surrozen Inc., Synedgen, Takeda, Tegus Inc., Teva, Tillotts, Trex Bio, TR1X Inc. TVM Lifesciences, Ventyx Biosciences, Versant Ventures, Vida Ventures, Ysios Capital, Zagbio Stock Shareholder: Connect Biopharm, EnGene, Evida, SRT, Imidomics, Enveda Sandborn, William: Grant: Abbvie, Abivax, Arena Pharmaceuticals, Boehringer Ingelheim, Celgene, Genentech, Gilead Sciences, Glaxo Smith Kline, Janssen, Lilly, Pfizer, Prometheus Biosciences, Seres Therapeutics, Shire, Takeda, Theravance Biopharma Personal Fees: Abbvie, Abivax, Admirx, Alfasigma, Alimentiv (Robarts Clinical Trials, owned by Health Academic Research Trust [HART]), Alivio Therapeutics, Allakos, Amgen, Applied Molecular Transport, Arena Pharmaceuticals, Bausch Health (Salix), Beigene, Bellatrix Pharmaceuticals, Boehringer Ingelheim, Boston Pharmaceuticals, Bristol Meyers Squibb, Celgene, Celltrion, Cellularity, Cosmo Pharmaceuticals, Escalier Biosciences, Equillium, Forbion, Genentech/Roche, Gilead Sciences, Glenmark Pharmaceuticals, Gossamer Bio, Immunic (Vital Therapies), Index Pharmaceuticals, Intact Therapeutics, Janssen, Kyverna Therapeutics, Landos Biopharma, Lilly, Oppilan Pharma, Otsuka, Pandion Therapeutics, Pfizer, Progenity, Prometheus Biosciences, Protagonists Therapeutics, Provention Bio, Reistone Biopharma, Seres Therapeutics, Shanghai Pharma Biotherapeutics, Shire, Shoreline Biosciences, Sublimity Therapeutics, Surrozen, Takeda, Theravance Biopharma, Thetis Pharmaceuticals, Tillotts Pharma, UCB, Vendata Biosciences, Ventyx Biosciences, Vimalan Biosciences, Vivelix Pharmaceuticals, Vivreon Biosciences, Zealand Pharma Other: Allakos, BeiGene, Gossamer Bio, Oppilan Pharma, Prometheus Biosciences, Progenity, Shoreline Biosciences, Ventyx Biosciences, Vimalan Biosciences, Vivreon Biosciences Jairath, Vipul: Consulting Fees: Abbvie, Alimentiv, Amgen, Anaptys Bio, Asahi Kasei, Asieris, Astra Zeneca, Attovia, Blackbird Labs, BMS, Boehringer Ingleheim, Biomebank, Caldera, Calluna, Catalytic Health, Celltrion, Ensho, Enthera, Exeliome Biosciences, Ferring, Fresenius Kabi, Gilead, Granite Bio, GSK, Janssen, Lilly, Merck, Mountainfield, MRM Health, Nxera, Organon, OSE Immunotherapeutics, Pendopharm, Pioneering Medicine, Pfizer, Prometheus, Roche/Genentech, Sanofi, SCOPE, Shattuck Labs, Sorriso, Spyre, Synedgen, Takeda, Teva, Tillotts, Union Therapeutics, Ventus, Ventyx, Vividion, Xencor, Zealand Pharma. Vande Casteele, Niels: Grant: Takeda, UCB Pharma, R-Biopharm Personal Fees: Janssen, Pfizer, Boehringer Ingelheim, Progenity, Prometheus, Takeda, UCB Pharma

BACKGROUND:Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein that regulates host responses to oxidative stress. We investigated its role in controlling DNA damage following infection with a genotoxic intestinal microbe. METHODS:Mice rendered hypomorphic for APE1 were crossed with tamoxifen (TMX)-inducible or constitutive Vil1ACre mice to yield intestinal epithelial cell-targeted knockout mice (Apex1iΔIEC or Apex1cΔIEC). Mice were infected with a genotoxic strain of E. coli (pks + E. coli NC101) to determine the APE1-sensitive effects on the accumulation of reactive oxygen species (ROS) and oxidative DNA damage. Murine and human primary colonic epithelial cell lines genetically altered to reduce APE1 expression were infected with a pks-deficient, the genotoxic E. coli strain or its synthetic colibactin genotoxin. RESULTS:APE1 expression was markedly diminished in enterocytes of Apex1iΔIEC mice, which was accompanied by increased constitutive epithelial cell DNA damage comparable to that observed in APE1 hypomorphic mice. The DNA damage, as indicated by γH2AX levels, was attenuated in germ-free Apex1iΔIEC mice and in APE1-hypomorphic mice treated with broad-spectrum antibiotics, indicating that commensal bacteria exert genotoxic effects that are unmasked by the loss of APE1 function. Levels of ROS and oxidative DNA damage in APE1-deficient primary colonic epithelial cells were increased significantly by pks + E. coli NC101 or its synthetic colibactin 742 genotoxin and this damage was prevented by prior treatment with an antioxidant. CONCLUSIONS:APE1 protects epithelial cells by inhibiting the accumulation of ROS and oxidative DNA damage induced by intestinal bacteria, including the colibactin produced by E. coli NC101.

ABSTRACT Trichomonas vaginalis is the causative agent of the most prevalent, non-viral sexually transmitted infectious disease, yet treatment options are limited to three nitroheterocyclic antimicrobials of the same drug class, and resistance to these agents is a manifest clinical problem. The 20S proteasome is a validated new drug target against T. vaginalis, but inhibitors of the proteasome complex have so far only shown modest selectivity over human cells. We screened a library of 373 diverse peptide inhibitors with different reactive warheads against diverse T. vaginalis strains in a growth and survival assay and identified several compounds with potencies in the 10–20 nM range. Notably, these compounds were up to 200-fold selective for T. vaginalis over mammalian cells and could overcome metronidazole resistance. Removal of the epoxide or the adjacent methyl group in the inhibitors carrying an epoxyketone functionality abolished activity, underlining the functional importance of this warhead. Biochemical and whole-cell assays of inhibitory activity against each of the three catalytically active subunits (β1, β2, and β5) of the proteasome showed that inhibition of β5 was sufficient to mediate activity against T. vaginalis . Furthermore, the high selectivity of the best compounds came from their preferential inhibition of T. vaginalis β5 over human β5. Docking calculations of the most selective inhibitor to the T. vaginalis substrate-binding pocket revealed the formation of two stabilizing hydrogen bonds with a threonine residue that is absent in the human proteasome. These results encourage the therapeutic development of highly potent and selective proteasome inhibitors against T. vaginalis .

Alcohol-associated liver disease represents a significant global health challenge, with gut microbial dysbiosis and bacterial translocation playing a critical role in its pathogenesis. Patients with alcohol-associated hepatitis had increased fecal abundance of mammalian viruses, including retroviruses. This study investigated the role of endogenous retroviruses (ERVs) in the development of alcohol-associated liver disease. Transcriptomic analysis of duodenal and liver biopsies revealed increased expression of several human ERVs, including HERV-K and HERV-H, in patients with alcohol-associated liver disease compared with individuals acting as controls. Chronic-binge ethanol feeding markedly induced ERV abundance in intestinal epithelial cells but not the livers of mice. Ethanol increased ERV expression and activated the Z-DNA binding protein 1 (Zbp1)-mixed lineage kinase domain-like pseudokinase (Mlkl) signaling pathways to induce necroptosis in intestinal epithelial cells. Antiretroviral treatment reduced ethanol-induced intestinal ERV expression, stabilized the gut barrier, and decreased liver disease in microbiota-humanized mice. Furthermore, mice with an intestine-specific deletion of Zbp1 were protected against bacterial translocation and ethanol-induced steatohepatitis. These findings indicate that ethanol exploits this pathway by inducing ERVs and promoting innate immune responses, which results in the death of intestinal epithelial cells, gut barrier dysfunction, and liver disease. Targeting the ERV/Zbp1 pathway may offer new therapies for patients with alcohol-associated liver disease.

BACKGROUND & AIMS:Coronavirus disease (COVID-19), caused by severe acquired respiratory syndrome-Coronavirus-2 (SARS-CoV-2), triggered a global pandemic with severe medical and socioeconomic consequences. Although fatality rates are higher among the elderly and those with underlying comorbidities, 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. The aim of our study was to investigate whether the autoimmunity risk gene, PTPN2, which also confers elevated risk to develop inflammatory bowel disease, affects susceptibility to SARS-CoV-2 viral uptake. METHODS:Using samples from PTPN2 genotyped patients with inflammatory bowel disease, PTPN2-deficient mice, and human intestinal and lung epithelial cell lines, we investigated how PTPN2 affects expression of the SARS-CoV-2 receptor angiotensin converting enzyme 2 (ACE2), and uptake of virus-like particles expressing the SARS-CoV2 spike protein and live SARS-CoV-2 virus. RESULTS:We report that the autoimmune PTPN2 loss-of-function risk variant rs1893217 promotes expression of the SARS-CoV-2 receptor, ACE2, and increases cellular entry of SARS-CoV-2 spike protein and live virus. Elevated ACE2 expression and viral entry were mediated by increased Janus kinase-signal transducers and activators of transcription signaling and were reversed by the Janus kinase inhibitor, tofacitinib. CONCLUSION: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.

Alcohol-associated liver disease poses a global health burden with high mortality. Imbalances in the gut microbiota are important for disease progression. Using metagenomic sequencing of fecal samples from a multicenter, international cohort of patients with alcohol-associated hepatitis, we found that the presence of virulence factor KpsM, encoded in the genome of Escherichia coli (E. coli), correlated with patient mortality. Functional studies using gnotobiotic mouse models and genetic manipulation of bacteria demonstrated that kpsM-positive E. coli exacerbate ethanol-induced liver disease. The kpsM gene mediates the translocation of capsular polysaccharides to the cell surface. This enables kpsM-positive E. coli to evade phagocytosis by the scavenger receptor Marco on Kupffer cells in the liver, leading to bacterial spread. Importantly, inhibiting kpsM-dependent capsules with the small molecule 2-(4-phenylphenyl)benzo[g]quinoline-4-carboxylic acid (C7) attenuated ethanol-induced liver disease in mice. We show that precision targeting of the virulence factor KpsM is a promising approach to improve outcomes of patients with alcohol-associated hepatitis.

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. Furthermore, 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. Tamoxifen-inducible, intestinal epithelial cell-specific Ptpn2 knockout mice (Ptpn2∆IEC) and control Ptpn2fl/fl mice were infected with either noninvasive 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. Ptpn2∆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∆IEC vs. Ptpn2fl/fl mice, after mAIECred but not K12 infection. Furthermore, we observed an increased tight junction-regulated permeability determined by elevated in vivo FD4 but not RD70 permeability in Ptpn2∆IEC-K12 mice compared to their respective controls. This effect was further exacerbated in Ptpn2∆IEC mAIEC-infected mice. Further, Ptpn2∆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∆IEC mice post mAIEC challenge. Our findings highlight that the 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.

Sialic acids (Sias) are a diverse family of nine-carbon backbone monosaccharides occupying terminal positions on cell surface and secreted glycans and are abundant at mucosal surfaces. Sias can be modified with O-acetyl esters on the side chain (C7 to C9) hydroxyls. Structural analysis and functional studies of these modifications are challenging due to chemical lability and variable resistance to sialidases. For in-depth analysis of the expression and functions of O-acetyl Sia modifications, we used a unique set of sialoglycan-recognizing probes, HPLC analysis of DMB-derivatized Sias and mice lacking the first known sialate O-acetyltransferase, CASD1. C7/C9-O-acetylated Sias are most abundant in the colon, with lower levels in the heart, brain, and spleen, and minimal levels in other digestive organs of wild-type mice. CASD1 deficiency led to a marked loss of C9/C7-O-acetylated Sias in the colon and other tissues. No differences were observed in colonic O-acetylated Sias from conventional and germ-free wild-type mice, indicating that Sia O-acetylation is independent of the commensal microbiota. Nonetheless, CASD1 deficiency caused subtle changes in microbial gene repertoire consistent with potential exploitation of Sias by subsets of intestinal microbes. Furthermore, CASD1-deficient mice exhibited more severe inflammation and ulceration upon colitis induction compared to controls. Reduced Sia O-acetylation was observed in mice during acute colitis and in colon biopsies from patients with inflammatory bowel disease. Together, our findings suggest CASD1 is the primary physiologically relevant enzyme to add C7/C8/C9-O-acetyl ester groups to Sias and that these Sia modifications exert important gut-protective functions, perhaps by preventing microbial Sia release and metabolism.

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.

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.

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 alpha and seven beta 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 beta 2 and beta 5. Cryo-electron microscopy (cryo-EM) unveils the structures of Tv20S bound to MZB and CP-17 at 2.8 angstrom. 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.

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, alpha 1-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.

The protozoan parasite Trichomonas vaginalis (Tv) causes trichomoniasis, the most common non-viral sexually transmitted infection in the world. Although Tv has been linked to significant health complications, only two closely related 5-nitroimidazole drugs are approved for its treatment. The emergence of resistance to these drugs and lack of alternative treatment options poses an increasing threat to public health, making development of novel anti- Trichomonas compounds an urgent need. The proteasome, a critical enzyme complex found in all eukaryotes has three catalytic subunits, β1, β2, and β5 and has been validated as a drug target to treat trichomoniasis. With the goal of developing tools to study the Tv proteasome, we isolated the enzyme complex and identified inhibitors that preferentially inactivate either one or two of the three catalytic subunits. Using a mass spectrometry-based peptide digestion assay, these inhibitors were used to define the substrate preferences of the β1, β2 and β5 subunits. Subsequently, three model fluorogenic substrates were designed, each specific for one of the catalytic subunits. This novel substrate profiling methodology will allow for individual subunit characterization of other proteasomes of interest. Using the new substrates, we screened a library of 284 peptide epoxyketone inhibitors against Tv and determined the subunits targeted by the most active compounds. The data show that inhibition of the Tv β5 subunit alone is toxic to the parasite. Taken together, the optimized proteasome subunit substrates will be instrumental for understanding the molecular determinants of proteasome specificity and for accelerating drug development against trichomoniasis.

Abstract Background Transcriptomic analysis has the potential to facilitate drug development and clinical decision making in inflammatory bowel disease. Current understanding of pharmacodynamic response to vedolizumab is limited by a lack of publicly available mucosal biopsy RNA samples/sequencing data and corresponding clinical information. We aimed to identify gene signatures associated with response to vedolizumab in ulcerative colitis (UC) patients by pooling data from 3 international sites. Methods Patients receiving vedolizumab for the treatment of moderate-to-severe UC at Hospital Clínic de Barcelona (IDIBAPS), Mount Sinai Hospital (MSH), and University of California, San Diego (UCSD) with baseline and post-treatment (week 14 [± 4 weeks]) mucosal biopsy RNA-sequencing data and Mayo Clinic endoscopic subscores (MCES) were eligible for inclusion. Endoscopic response was defined as an MCES <1. Demographic and clinical data were collected. Differential expressed genes (DEGs) between baseline and week 14 were identified using linear mixed models with a >1.5 or <-1.5-fold change (adjusted P-value <.05). Enrichment was performed using Reactome pathway analysis and a previously published UC gene signature (adjusted P-value <.05).1 Analyses were performed using R version 4.3.0. Results Twenty-three patients were included (IDIBAPS: n=12; MSH: n=8; UCSD: n=3). Site-specific differences in sex, surgical history, and corticosteroid use were observed (Table). Analysing data by site yielded a relatively small number of DEGs between baseline and week 14 (IDIBAPS=57 [27 up-regulated, 30 down-regulated]; MSH=0; UCSD=0) among endoscopic responders (IDIBAPS: n=5; MSH: n=7; UCSD: n=2). Combining data from the 3 sites increased the number of DEGs among endoscopic responders at week 14 (n=14) to 1162 (662 up-regulated and 500 down-regulated). The down-regulated DEGs were enriched for genes involved in 37 of 103 up-regulated Reactome pathways in UC, including neutrophil degranulation, interleukin-4 and interleukin-13 signalling, interleukin-10 signalling, and integrin cell surface interactions. The up-regulated DEGs were enriched for genes involved in 1 of 39 down-regulated Reactome pathways in UC (Drug ADME). Conclusion We defined a pharmacodynamic signature using mucosal biopsies from UC patients who had endoscopic response to vedolizumab at week 14. The signature was enriched for several, but not all, pathways involved in UC. In addition to providing insight into vedolizumab’s mechanism of action, we underscored the utility of multi-site collaboration to access data, increase statistical power, and enhance the generalizability of research findings. Reference: 1. Linggi et al. Sci Rep 2021;11:18243.

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.

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 Gas, 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 WAT, modulating catecholamine-induced lipolysis, and increasing thermogenesis, which together created a lean phenotype.

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.

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 β5 subunit of T. vaginalis is sufficient to kill the parasite, however, targeting of β5 plus either β1 or β2 results in improved potency.

ABSTRACTProteasomes are essential for protein homeostasis in mammalian cells1-4and in protozoan parasites such asTrichomonas vaginalis (Tv).5Tvand other protozoan 20S proteasomes have been validated as druggable targets.6-8However, in the case ofTv20S proteasome (Tv20S), biochemical and structural studies were impeded by low yields and purity of the native proteasome. We successfully made recombinantTv20S by expressing all seven α and seven β 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 recombinantTv20S is inhibited by the natural product marizomib (MZB)9and the recently developed peptide inhibitor carmaphycin-17 (CP-17)8,10. Specifically, MZB binds to the β1, β2 and β5 subunits, while CP-17 binds the β2 and β5 subunits. Next, we obtained cryo-EM structures ofTv20S in complex with these covalent inhibitors at 2.8Å resolution. The structures revealed the overall fold of theTv20S and the binding mode of MZB and CP-17. Our work explains the low specificity of MZB and higher specificity of CP-17 towardsTv20S as compared to human proteasome and provides the platform for the development ofTv20S inhibitors for treatment of trichomoniasis.

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 β 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 β1, β2 and β5 subunits, while CP-17 binds the β2 and β5 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.