Bifunctional Immunity Proteins Protect Bacteria against FtsZ-Targeting ADP-Ribosylating Toxins.

ADP-ribosylation of proteins can profoundly impact their function and serves as an effective mechanism by which bacterial toxins impair eukaryotic cell processes. Here, we report the discovery that bacteria also employ ADP-ribosylating toxins against each other during interspecies competition. We demonstrate that one such toxin from Serratia proteamaculans interrupts the division of competing cells by modifying the essential bacterial tubulin-like protein, FtsZ, adjacent to its protomer interface, blocking its capacity to polymerize. The structure of the toxin in complex with its immunity determinant revealed two distinct modes of inhibition: active site occlusion and enzymatic removal of ADP-ribose modifications. We show that each is sufficient to support toxin immunity; however, the latter additionally provides unprecedented broad protection against non-cognate ADP-ribosylating effectors. Our findings reveal how an interbacterial arms race has produced a unique solution for safeguarding the integrity of bacterial cell division machinery against inactivating post-translational modifications.

A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing.

Bacterial toxins represent a vast reservoir of biochemical diversity that can be repurposed for biomedical applications. Such proteins include a group of predicted interbacterial toxins of the deaminase superfamily, members of which have found application in gene-editing techniques1,2. Because previously described cytidine deaminases operate on single-stranded nucleic acids3, their use in base editing requires the unwinding of double-stranded DNA (dsDNA)-for example by a CRISPR-Cas9 system. Base editing within mitochondrial DNA (mtDNA), however, has thus far been hindered by challenges associated with the delivery of guide RNA into the mitochondria4. As a consequence, manipulation of mtDNA to date has been limited to the targeted destruction of the mitochondrial genome by designer nucleases9,10.Here we describe an interbacterial toxin, which we name DddA, that catalyses the deamination of cytidines within dsDNA. We engineered split-DddA halves that are non-toxic and inactive until brought together on target DNA by adjacently bound programmable DNA-binding proteins. Fusions of the split-DddA halves, transcription activator-like effector array proteins, and a uracil glycosylase inhibitor resulted in RNA-free DddA-derived cytosine base editors (DdCBEs) that catalyse C•G-to-T•A conversions in human mtDNA with high target specificity and product purity. We used DdCBEs to model a disease-associated mtDNA mutation in human cells, resulting in changes in respiration rates and oxidative phosphorylation. CRISPR-free DdCBEs enable the precise manipulation of mtDNA, rather than the elimination of mtDNA copies that results from its cleavage by targeted nucleases, with broad implications for the study and potential treatment of mitochondrial disorders.

Self-activating G protein α subunits engage seven-transmembrane regulator of G protein signaling (RGS) proteins and a Rho guanine nucleotide exchange factor effector in the amoeba Naegleria fowleri.

The free-living amoeba Naegleria fowleri is a causative agent of primary amoebic meningoencephalitis and is highly resistant to current therapies, resulting in mortality rates >97%. As many therapeutics target G protein-centered signal transduction pathways, further understanding the functional significance of G protein signaling within N. fowleri should aid future drug discovery against this pathogen. Here, we report that the N. fowleri genome encodes numerous transcribed G protein signaling components, including G protein-coupled receptors, heterotrimeric G protein subunits, regulator of G protein signaling (RGS) proteins, and candidate Gα effector proteins. We found N. fowleri Gα subunits have diverse nucleotide cycling kinetics; Nf Gα5 and Gα7 exhibit more rapid nucleotide exchange than GTP hydrolysis (i.e., "self-activating" behavior). A crystal structure of Nf Gα7 highlights the stability of its nucleotide-free state, consistent with its rapid nucleotide exchange. Variations in the phosphate binding loop also contribute to nucleotide cycling differences among Gα subunits. Similar to plant G protein signaling pathways, N. fowleri Gα subunits selectively engage members of a large seven-transmembrane RGS protein family, resulting in acceleration of GTP hydrolysis. We show Nf Gα2 and Gα3 directly interact with a candidate Gα effector protein, RGS-RhoGEF, similar to mammalian Gα12/13 signaling pathways. We demonstrate Nf Gα2 and Gα3 each engage RGS-RhoGEF through a canonical Gα/RGS domain interface, suggesting a shared evolutionary origin with G protein signaling in the enteric pathogen Entamoeba histolytica. These findings further illuminate the evolution of G protein signaling and identify potential targets of pharmacological manipulation in N. fowleri.

Helicobacter pylori Exposure in Nausea and Vomiting of Pregnancy Increases Risk of Preterm Delivery.

Background: Hyperemesis gravidarum (HG), a severe form of nausea and vomiting in pregnancy (NVP), is a leading indication for hospitalization in the first trimester. NVP and HG are associated with Helicobacter pylori (HP) infection in non-United States cohorts. How HP exposure and NVP interact to affect metabolic disturbance and pregnancy outcomes is not known. Materials and Methods: We designed a retrospective cohort study relating HP and NVP to serum electrolyte laboratory results, preterm delivery, and infant birth weight. Single academic institution discovery and independent multi-institutional validation cohorts included pregnant subjects with an HP test result. Associations of HP, NVP, and pregnancy outcomes were assessed with odds ratio calculations, Student's t-tests, and multivariate logistic regression. Results: Among subjects with positive HP test results, the prevalence of hyperemesis gravidarum (HG) was 0.025 (66 of 2671) and NVP was 0.27 (710 of 2671). Subjects with negative HP had prevalence of HG 0.015 (165 of 10,960) and NVP 0.22 (2392 of 10,960). History of HP exposure increased risk of NVP, including HG (odds ratio 1.3, 95% CI 1.1-1.4). Patients with HP exposure had lower serum potassium (mean difference 0.1 mEq/L) and bicarbonate (mean difference 0.3 mEq/L) during pregnancy than HP-negative patients (p < 0.01). Serum potassium was lowest in subjects with both NVP and HP exposure (mean 3.5 mEq/L [3.4-3.6], p < 0.0001). HP exposure alone carried increased risk for preterm delivery (OR 1.3 [1.1-1.4]). NVP alone increased risk of preterm delivery (OR 2.8 [2.5-3.1]) including second trimester delivery (OR 2.2 [1.7-2.8]). In multivariate analysis, HP exposure in the setting of NVP further increased risk of preterm delivery (adjusted OR 1.4 [1.0-1.9], p = 0.03). Conclusions: H. pylori exposure and diagnosis of NVP are individually associated with metabolic disturbances and adverse pregnancy outcomes such as preterm labor and delivery, and their combination further increases risk in US populations.

Centrizonal hepatocyte dropout in allograft liver biopsies: a clinicopathological study.

AIMS: Centrizonal hepatocyte dropout has been described in diverse liver pathologies, including viral hepatitis, venous outflow obstruction, and allograft cellular rejection. However, its clinical significance remains uncertain. METHODS AND RESULTS: We designed a clinicopathological study of 206 allograft liver biopsies with centrizonal hepatocyte dropout. Centrizonal hepatocyte dropout was associated most frequently with cellular rejection (n = 62), asymptomatic/protocol biopsies (n = 56), immediate post-transplantation biopsies (n = 21), biliary obstruction (n = 14), and viral hepatitis (n = 13). The differential diagnosis is informed by timing post-transplantation, biliary imaging and laboratory test results. 'Cholestatic' and 'hepatocytic' laboratory patterns were associated with biliary obstruction and cellular rejection, respectively. A mixed pattern peaking after biopsy was observed in viral hepatitis cases. In the context of cellular rejection, dropout was not associated with the time interval to normalisation of serum alanine aminotransferase (ALT), but was associated with shorter transplant-free survival (hazard ratio 4, P = 0.01) than that of histological severity-matched controls. In time zero allograft biopsies, time to ALT normalisation was prolonged (median, 15 versus 11 days, P = 0.002) in allografts with centrizonal dropout, with no effect on retransplant-free survival. CONCLUSIONS: Centrizonal hepatocyte dropout has low clinicopathological diagnostic specificity. However, it correlates with adverse clinical outcomes in allograft cellular rejection and time zero biopsies.

Neutrophilic inflammation in gallbladder carcinoma correlates with patient survival: A case-control study.

Gallbladder carcinoma is an uncommon malignancy with an overall 5-year survival of less than 5%. Gallbladder carcinoma has been strongly linked with cholelithiasis and chronic inflammation. Case reports and series have described cholecystitis with acute (neutrophilic) inflammation in association with gallbladder carcinoma, although a clear relationship to patient outcome has not been established. Our series included 8 cases of gallbladder carcinoma with high tumor-associated neutrophils (>25 per high power field) that were associated with shorter patient survival (Cox regression coefficient 6.2, p = 0.004) than age- and stage-matched controls. High tumor-associated neutrophils were not associated with gallbladder rupture/perforation or increased bacterial load measured by 16S PCR. Neutrophilic inflammation with gallbladder carcinoma correlates to shorter survival, independent of patient age and stage of carcinoma. The findings suggest that the degree of neutrophilic inflammation may have prognostic significance in specimens from patients with gallbladder carcinoma after cholecystectomy. Further studies with larger case numbers are needed to confirm and generalize these findings.

Elevated White Blood Cell Count Does Not Predict Clostridium difficile Nucleic Acid Testing Results.

BACKGROUND: An elevated white blood cell count (WBC; >15 000/µL) is an established prognostic marker in patients with Clostridium difficile infection (CDI). Small observational studies have suggested that a markedly elevated WBC should prompt consideration of CDI. However, there is limited evidence correlating WBC elevation with the results of C. difficile nucleic acid amplification testing (NAAT). METHODS: Retrospective review of laboratory testing, outcomes, and treatment of 16 568 consecutive patients presenting to 4 hospitals over 4 years with NAAT and WBC testing on the same day. RESULTS: No significant relationship between C. difficile NAAT results and concurrent WBC in the inpatient setting was observed. Although an elevated WBC did predict NAAT results in the outpatient and emergency department populations (P < .001), accuracy was poor, with receiver-operator areas under the curve of 0.59 and 0.56, respectively. An elevated WBC (>15 000/µL) in CDI was associated with a longer median hospital length of stay (15.5 vs 11.0 days; P < .01), consistent with leukocytosis as a prognostic marker in CDI. NAAT-positive inpatients with elevated WBC were more likely to be treated with metronidazole and/or vancomycin (relative ratio, 1.2; 95% confidence interval [CI], 1.1-1.3) and die in the hospital (relative ratio, 2.9; 95% CI, 2.0-4.3). CONCLUSIONS: Although WBC is an important prognostic indicator in patients with CDI, an isolated WBC elevation has low sensitivity and specificity as a predictor of fecal C. difficile NAAT positivity in the inpatient setting. A high or rising WBC in isolation is not a sufficient indication for CDI testing.

Quantification of Human Epidermal Growth Factor Receptor 2 by Immunopeptide Enrichment and Targeted Mass Spectrometry in Formalin-Fixed Paraffin-Embedded and Frozen Breast Cancer Tissues.

BACKGROUND: Conventional HER2-targeting therapies improve outcomes for patients with HER2-positive breast cancer (BC), defined as tumors showing HER2 protein overexpression by immunohistochemistry and/or ERBB2 gene amplification determined by in situ hybridization (ISH). Emerging HER2-targeting compounds show benefit in some patients with neither HER2 protein overexpression nor ERBB2 gene amplification, creating a need for new assays to select HER2-low tumors for treatment with these compounds. We evaluated the analytical performance of a targeted mass spectrometry-based assay for quantifying HER2 protein in formalin-fixed paraffin-embedded (FFPE) and frozen BC biopsies. METHODS: We used immunoaffinity-enrichment coupled to multiple reaction monitoring-mass spectrometry (immuno-MRM-MS) to quantify HER2 protein (as peptide GLQSLPTHDPSPLQR) in 96 frozen and 119 FFPE BC biopsies. We characterized linearity, lower limit of quantification (LLOQ), and intra- and inter-day variation of the assay in frozen and FFPE tissue matrices. We determined concordance between HER2 immuno-MRM-MS and predicate immunohistochemistry and ISH assays and examined the benefit of multiplexing the assay to include proteins expressed in tumor subcompartments (e.g., stroma, adipose, lymphocytes, epithelium) to account for tissue heterogeneity. RESULTS: HER2 immuno-MRM-MS assay linearity was ≥103, assay coefficient of variation was 7.8% (FFPE) and 5.9% (frozen) for spiked-in analyte, and 7.7% (FFPE) and 7.9% (frozen) for endogenous measurements. Immuno-MRM-MS-based HER2 measurements strongly correlated with predicate assay HER2 determinations, and concordance was improved by normalizing to glyceraldehyde-3-phosphate dehydrogenase. HER2 was quantified above the LLOQ in all tumors. CONCLUSIONS: Immuno-MRM-MS can be used to quantify HER2 in FFPE and frozen BC biopsies, even at low HER2 expression levels.

Loss of nectin-3 expression as a marker of tumor aggressiveness in pancreatic neuroendocrine tumor.

Pancreatic neuroendocrine tumors (PanNETs) are rare, and prediction of aggressive characteristics, such as recurrence and metastasis and prognosis of PanNETs remain difficult. Nectins are cell adhesion molecules that regulate the formation of adherens and tight junctions. In this study, we investigated the clinicopathological significance of nectin-3 expression in patients with PanNETs. Immunohistochemical analysis of nectin-3 expression was performed on 78 cases of PanNET. Low nectin-3 expression in the membrane (positive ratio ≤25%) was observed in 62 cases (79.5%) and was significantly correlated with larger tumor size (>20 mm; P = 0.003), G2/G3 tumors (P = 0.025), higher Ki67 labeling index (≥3%; P = 0.009), lymphatic involvement (P = 0.047), advanced pT-factor (T2-T4; P = 0.003), lymph node metastasis (P = 0.006), advanced Union for International Cancer Control/American Joint Committee on Cancer-stage (Stage II-IV; P = 0.001), advanced ENETS stage (Stage IIa-IV; P = 0.001), nonfunctioning tumors (P = 0.002), and a shorter disease-free survival (P = 0.019). However, there was no significant correlation between nectin-3 expression in the membrane and/or cytoplasm and the clinicopathological parameters. The present results suggest that decreased nectin-3 expression in the membrane is associated with increased tumor aggressiveness of PanNETs. Clinically, immunohistochemical analysis of nectin-3 may help predict tumor aggressiveness for PanNETs.

Gallbladder carcinoma and epithelial dysplasia: Appropriate sampling for histopathology.

Gallbladder carcinoma (GC) is an uncommon malignancy with an overall 5-year survival of <5%. Due to overlap of clinical presentation with the more common cholecystitis, an estimated 50-65% of all GCs are found incidentally. Epithelial dysplasia is identified in ~50% of specimens with invasive carcinoma. Recent expert panel guidelines have recommended histologic examination of the entire gallbladder in cases where initial sampling reveals dysplasia. 89 cases of GC, 34 high grade dysplasia (HGD), and 60 low grade dysplasia (LGD) were identified in cholecystectomy specimens assessed at our institution over the last 15 years. Pre-operative imaging (either ultrasound or CT) only identified 52% of mass lesions in GC cases. Among gallbladder specimens with epithelial dysplasia only at initial sampling, additional sectioning was performed in 59% of HGD and 55% of LGD. Additional sectioning of gallbladder specimens with HGD had a higher yield (10%) for identifying invasive carcinoma than those with LGD (0 of 28). The diagnostic yield of additional sectioning is significantly higher in the setting of high grade as compared to low grade dysplasia, suggesting that sampling at the discretion of the pathologist may be sufficient for the latter.

Entamoeba histolytica RacC selectively engages p21-activated kinase effectors.

Rho family GTPases modulate actin cytoskeleton dynamics by signaling through multiple effectors, including the p21-activated kinases (PAKs). The intestinal parasite Entamoeba histolytica expresses ∼20 Rho family GTPases and seven isoforms of PAK, two of which have been implicated in pathogenesis-related processes such as amoebic motility and invasion and host cell phagocytosis. Here, we describe two previously unstudied PAK isoforms, EhPAK4 and EhPAK5, as highly specific effectors of EhRacC. A structural model based on 2.35 Å X-ray crystallographic data of a complex between EhRacC(Q65L)·GTP and the EhPAK4 p21 binding domain (PBD) reveals a fairly well-conserved Rho/effector interface despite deviation of the PBD α-helix. A structural comparison with EhRho1 in complex with EhFormin1 suggests likely determinants of Rho family GTPase signaling specificity in E. histolytica. These findings suggest a high degree of Rho family GTPase diversity and specificity in the single-cell parasite E. histolytica. Because PAKs regulate pathogenesis-related processes in E. histolytica, they may be valid pharmacologic targets for anti-amoebiasis drugs.

Structural determinants of ubiquitin conjugation in Entamoeba histolytica.

Ubiquitination is important for numerous cellular processes in most eukaryotic organisms, including cellular proliferation, development, and protein turnover by the proteasome. The intestinal parasite Entamoeba histolytica harbors an extensive ubiquitin-proteasome system. Proteasome inhibitors are known to impair parasite proliferation and encystation, suggesting the ubiquitin-proteasome pathway as a viable therapeutic target. However, no functional studies of the E. histolytica ubiquitination enzymes have yet emerged. Here, we have cloned and characterized multiple E. histolytica ubiquitination components, spanning ubiquitin and its activating (E1), conjugating (E2), and ligating (E3) enzymes. Crystal structures of EhUbiquitin reveal a clustering of unique residues on the α1 helix surface, including an eighth surface lysine not found in other organisms, which may allow for a unique polyubiquitin linkage in E. histolytica. EhUbiquitin is activated by and forms a thioester bond with EhUba1 (E1) in vitro, in an ATP- and magnesium-dependent fashion. EhUba1 exhibits a greater maximal initial velocity of pyrophosphate:ATP exchange than its human homolog, suggesting different kinetics of ubiquitin activation in E. histolytica. EhUba1 engages the E2 enzyme EhUbc5 through its ubiquitin-fold domain to transfer the EhUbiquitin thioester. However, EhUbc5 has a >10-fold preference for EhUba1∼Ub compared with unconjugated EhUba1. A crystal structure of EhUbc5 allowed prediction of a noncovalent "backside" interaction with EhUbiquitin and E3 enzymes. EhUbc5 selectively engages EhRING1 (E3) to the exclusion of two HECT family E3 ligases, and mutagenesis indicates a conserved mode of E2/RING-E3 interaction in E. histolytica.

Heterotrimeric G-protein signaling is critical to pathogenic processes in Entamoeba histolytica.

Heterotrimeric G-protein signaling pathways are vital components of physiology, and many are amenable to pharmacologic manipulation. Here, we identify functional heterotrimeric G-protein subunits in Entamoeba histolytica, the causative agent of amoebic colitis. The E. histolytica Gα subunit EhGα1 exhibits conventional nucleotide cycling properties and is seen to interact with EhGßγ dimers and a candidate effector, EhRGS-RhoGEF, in typical, nucleotide-state-selective fashions. In contrast, a crystal structure of EhGα1 highlights unique features and classification outside of conventional mammalian Gα subfamilies. E. histolytica trophozoites overexpressing wildtype EhGα1 in an inducible manner exhibit an enhanced ability to kill host cells that may be wholly or partially due to enhanced host cell attachment. EhGα1-overexpressing trophozoites also display enhanced transmigration across a Matrigel barrier, an effect that may result from altered baseline migration. Inducible expression of a dominant negative EhGα1 variant engenders the converse phenotypes. Transcriptomic studies reveal that modulation of pathogenesis-related trophozoite behaviors by perturbed heterotrimeric G-protein expression includes transcriptional regulation of virulence factors and altered trafficking of cysteine proteases. Collectively, our studies suggest that E. histolytica possesses a divergent heterotrimeric G-protein signaling axis that modulates key aspects of cellular processes related to the pathogenesis of this infectious organism.

A P-loop mutation in Gα subunits prevents transition to the active state: implications for G-protein signaling in fungal pathogenesis.

Heterotrimeric G-proteins are molecular switches integral to a panoply of different physiological responses that many organisms make to environmental cues. The switch from inactive to active Gαßγ heterotrimer relies on nucleotide cycling by the Gα subunit: exchange of GTP for GDP activates Gα, whereas its intrinsic enzymatic activity catalyzes GTP hydrolysis to GDP and inorganic phosphate, thereby reverting Gα to its inactive state. In several genetic studies of filamentous fungi, such as the rice blast fungus Magnaporthe oryzae, a G42R mutation in the phosphate-binding loop of Gα subunits is assumed to be GTPase-deficient and thus constitutively active. Here, we demonstrate that Gα(G42R) mutants are not GTPase deficient, but rather incapable of achieving the activated conformation. Two crystal structure models suggest that Arg-42 prevents a typical switch region conformational change upon Gα(i1)(G42R) binding to GDP·AlF(4)(-) or GTP, but rotameric flexibility at this locus allows for unperturbed GTP hydrolysis. Gα(G42R) mutants do not engage the active state-selective peptide KB-1753 nor RGS domains with high affinity, but instead favor interaction with Gßγ and GoLoco motifs in any nucleotide state. The corresponding Gα(q)(G48R) mutant is not constitutively active in cells and responds poorly to aluminum tetrafluoride activation. Comparative analyses of M. oryzae strains harboring either G42R or GTPase-deficient Q/L mutations in the Gα subunits MagA or MagB illustrate functional differences in environmental cue processing and intracellular signaling outcomes between these two Gα mutants, thus demonstrating the in vivo functional divergence of G42R and activating G-protein mutants.

Regulators of G-protein signaling and their Gα substrates: promises and challenges in their use as drug discovery targets.

Because G-protein coupled receptors (GPCRs) continue to represent excellent targets for the discovery and development of small-molecule therapeutics, it is posited that additional protein components of the signal transduction pathways emanating from activated GPCRs themselves are attractive as drug discovery targets. This review considers the drug discovery potential of two such components: members of the "regulators of G-protein signaling" (RGS protein) superfamily, as well as their substrates, the heterotrimeric G-protein α subunits. Highlighted are recent advances, stemming from mouse knockout studies and the use of "RGS-insensitivity" and fast-hydrolysis mutations to Gα, in our understanding of how RGS proteins selectively act in (patho)physiologic conditions controlled by GPCR signaling and how they act on the nucleotide cycling of heterotrimeric G-proteins in shaping the kinetics and sensitivity of GPCR signaling. Progress is documented regarding recent activities along the path to devising screening assays and chemical probes for the RGS protein target, not only in pursuits of inhibitors of RGS domain-mediated acceleration of Gα GTP hydrolysis but also to embrace the potential of finding allosteric activators of this RGS protein action. The review concludes in considering the Gα subunit itself as a drug target, as brought to focus by recent reports of activating mutations to GNAQ and GNA11 in ocular (uveal) melanoma. We consider the likelihood of several strategies for antagonizing the function of these oncogene alleles and their gene products, including the use of RGS proteins with Gα(q) selectivity.

Modulation of diabetic wound healing using carbon monoxide gas-entrapping materials.

Diabetic wound healing is uniquely challenging to manage due to chronic inflammation and heightened microbial growth from elevated interstitial glucose. Carbon monoxide (CO), widely acknowledged as a toxic gas, is also known to provide unique therapeutic immune modulating effects. To facilitate delivery of CO, we have designed hyaluronic acid-based CO-gas-entrapping materials (CO-GEMs) for topical and prolonged gas delivery to the wound bed. We demonstrate that CO-GEMs promote the healing response in murine diabetic wound models (full-thickness wounds and pressure ulcers) compared to N2-GEMs and untreated controls.

Regulator of G-protein signaling-21 (RGS21) is an inhibitor of bitter gustatory signaling found in lingual and airway epithelia.

The gustatory system detects tastants and transmits signals to the brain regarding ingested substances and nutrients. Although tastant receptors and taste signaling pathways have been identified, little is known about their regulation. Because bitter, sweet, and umami taste receptors are G protein-coupled receptors (GPCRs), we hypothesized that regulators of G protein signaling (RGS) proteins may be involved. The recent cloning of RGS21 from taste bud cells has implicated this protein in the regulation of taste signaling; however, the exact role of RGS21 has not been precisely defined. Here, we sought to determine the role of RGS21 in tastant responsiveness. Biochemical analyses confirmed in silico predictions that RGS21 acts as a GTPase-accelerating protein (GAP) for multiple G protein α subunits, including adenylyl cyclase-inhibitory (Gα(i)) subunits and those thought to be involved in tastant signal transduction. Using a combination of in situ hybridization, RT-PCR, immunohistochemistry, and immunofluorescence, we demonstrate that RGS21 is not only endogenously expressed in mouse taste buds but also in lung airway epithelial cells, which have previously been shown to express components of the taste signaling cascade. Furthermore, as shown by reverse transcription-PCR, the immortalized human airway cell line 16HBE was found to express transcripts for tastant receptors, RGS21, and downstream taste signaling components. Over- and underexpression of RGS21 in 16HBE cells confirmed that RGS21 acts to oppose bitter tastant signaling to cAMP and calcium second messenger changes. Our data collectively suggests that RGS21 modulates bitter taste signal transduction.

Structural disruption of Ntox15 nuclease effector domains by immunity proteins protects against type VI secretion system intoxication in Bacteroidales.

Bacteroidales use type VI secretion systems (T6SS) to competitively colonize and persist in the colon. We identify a horizontally transferred T6SS with Ntox15 family nuclease effector (Tde1) that mediates interbacterial antagonism among Bacteroidales, including several derived from a single human donor. Expression of cognate (Tdi1) or orphan immunity proteins in acquired interbacterial defense systems protects against Tde1-dependent attack. We find that immunity protein interaction induces a large effector conformational change in Tde nucleases, disrupting the active site and altering the DNA-binding site. Crystallographic snapshots of isolated Tde1, the Tde1/Tdi1 complex, and homologs from Phocaeicola vulgatus (Tde2/Tdi2) illustrate a conserved mechanism of immunity inserting into the central core of Tde, splitting the nuclease fold into two subdomains. The Tde/Tdi interface and immunity mechanism are distinct from all other polymorphic toxin-immunity interactions of known structure. Bacteroidales abundance has been linked to inflammatory bowel disease activity in prior studies, and we demonstrate that Tde and T6SS structural genes are each enriched in fecal metagenomes from ulcerative colitis subjects. Genetically mobile Tde1-encoding T6SS in Bacteroidales mediate competitive growth and may be involved in inflammatory bowel disease. Broad immunity is conferred by Tdi1 homologs through a fold-disrupting mechanism unique among polymorphic effector-immunity pairs of known structure. IMPORTANCE Bacteroidales are related to inflammatory bowel disease severity and progression. We identify type VI secretion system (T6SS) nuclease effectors (Tde) which are enriched in ulcerative colitis and horizontally transferred on mobile genetic elements. Tde-encoding T6SSs mediate interbacterial competition. Orphan and cognate immunity proteins (Tdi) prevent intoxication by multiple Tde through a new mechanism among polymorphic toxin systems. Tdi inserts into the effector central core, splitting Ntox15 into two subdomains and disrupting the active site. This mechanism may allow for evolutionary diversification of the Tde/Tdi interface as observed in colicin nuclease-immunity interactions, promoting broad neutralization of Tde by orphan Tdi. Tde-dependent T6SS interbacterial antagonism may contribute to Bacteroidales diversity in the context of ulcerative colitis.