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The misincorporation of 2′-deoxyribonucleotides (dNs) into RNA has important implications for the function of non-coding RNAs, the translational fidelity of coding RNAs and the mutagenic evolution of viral RNA genomes. However, quantitative appreciation for the degree to which dN misincorporation occurs is limited by the lack of analytical tools. Here, we report a method to hydrolyze RNA to release 2′-deoxyribonucleotide-ribonucleotide pairs (dNrN) that are then quantified by chromatography-coupled mass spectrometry (LC-MS). Using this platform, we found misincorporated dNs occurring at 1 per 10[superscript 3] to 10[superscript 5] ribonucleotide (nt) in mRNA, rRNAs and tRNA in human cells, Escherichia coli, Saccharomyces cerevisiae and, most abundantly, in the RNA genome of dengue virus. The frequency of dNs varied widely among organisms and sequence contexts, and partly reflected the in vitro discrimination efficiencies of different RNA polymerases against 2′-deoxyribonucleoside 5′-triphosphates (dNTPs). Further, we demonstrate a strong link between dN frequencies in RNA and the balance of dNTPs and ribonucleoside 5′-triphosphates (rNTPs) in the cellular pool, with significant stress-induced variation of dN incorporation. Potential implications of dNs in RNA are discussed, including the possibilities of dN incorporation in RNA as a contributing factor in viral evolution and human disease, and as a host immune defense mechanism against viral infections., National Institutes of Health (U.S.) (Grant ES022858), Singapore. National Research Foundation, Singapore-MIT Alliance for Research and Technology

Following the recent emergence of Zika virus (ZIKV), many murine and human neutralizing anti-ZIKV antibodies have been reported. Given the risk of virus escape mutants, engineering antibodies that target mutationally constrained epitopes with therapeutically relevant potencies can be valuable for combating future outbreaks. Here, we applied computational methods to engineer an antibody, ZAb_FLEP, that targets a highly networked and therefore mutationally constrained surface formed by the envelope protein dimer. ZAb_FLEP neutralized a breadth of ZIKV strains and protected mice in distinct in vivo models, including resolving vertical transmission and fetal mortality in infected pregnant mice. Serial passaging of ZIKV in the presence of ZAb_FLEP failed to generate viral escape mutants, suggesting that its epitope is indeed mutationally constrained. A single-particle cryo-EM reconstruction of the Fab-ZIKV complex validated the structural model and revealed insights into ZAb_FLEP's neutralization mechanism. ZAb_FLEP has potential as a therapeutic in future outbreaks. Tharakaraman et al. describe the engineering and validation of a neutralizing anti-Zika antibody (ZAb_FLEP) that targets a mutationally constrained surface epitope formed by the envelope protein. ZAb_FLEP neutralizes ZIKV strains in vitro and protects mice and unborn pups from Zika infection in vivo, indicating its potential as a therapeutic candidate., National Institutes of Health (U.S.) (Award 1R01AI111395), Singapore. National Research Foundation

Enterohemorrhagic E. coli (EHEC) is an important subset of Shiga toxin-producing (Stx-producing) E. coli (STEC), pathogens that have been implicated in outbreaks of food-borne illness and can cause intestinal and systemic disease, including severe renal damage. Upon attachment to intestinal epithelium, EHEC generates "attaching and effacing" (AE) lesions characterized by intimate attachment and actin rearrangement upon host cell binding. Stx produced in the gut transverses the intestinal epithelium, causing vascular damage that leads to systemic disease. Models of EHEC infection in conventional mice do not manifest key features of disease, such as AE lesions, intestinal damage, and systemic illness. In order to develop an infection model that better reflects the pathogenesis of this subset of STEC, we constructed an Stx-producing strain of Citrobacter rodentium, a murine AE pathogen that otherwise lacks Stx. Mice infected with Stx-producing C. rodentium developed AE lesions on the intestinal epithelium and Stx-dependent intestinal inflammatory damage. Further, the mice experienced lethal infection characterized by histopathological and functional kidney damage. The development of a murine model that encompasses AE lesion formation and Stx-mediated tissue damage will provide a new platform upon which to identify EHEC alterations of host epithelium that contribute to systemic disease.

Helicobacter cinaedi, a common human intestinal bacterium, has been implicated in various enteric and systemic diseases in normal and immunocompromised patients. Protection against oxidative stress is a crucial component of bacterium-host interactions. Alkyl hydroperoxide reductase C (AhpC) is an enzyme responsible for detoxification of peroxides and is important in protection from peroxide-induced stress. H. cinaedi possesses a single ahpC, which was investigated with respect to its role in bacterial survival during oxidative stress. The H. cinaedi ahpC mutant had diminished resistance to organic hydroperoxide toxicity but increased hydrogen peroxide resistance compared with the wild-type (WT) strain. The mutant also exhibited an oxygen-sensitive phenotype and was more susceptible to killing by macrophages than the WT strain. In vivo experiments in BALB/c and BALB/c interleukin-10 (IL-10)−/− mice revealed that the cecal colonizing ability of the ahpC mutant was significantly reduced. The mutant also had diminished ability to induce bacterium-specific immune responses in vivo, as shown by immunoglobulin (IgG2a and IgG1) serum levels. Collectively, these data suggest that H. cinaedi ahpC not only contributes to protecting the organism against oxidative stress but also alters its pathogenic properties in vivo., National Institutes of Health (U.S.) (grant R01CA067529), National Institutes of Health (U.S.) (grant R01DK052413), National Institutes of Health (U.S.) (grant R01RR032307), National Institutes of Health (U.S.) (grant P01CA26731), National Institutes of Health (U.S.) (grant P30ES002109)

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and may result in potentially fatal hemolytic uremia syndrome in humans. EHEC colonize the intestinal mucosa and promote the formation of actin-rich pedestals via translocated type III effectors. Two EHEC type III secreted effectors, Tir and EspFu/TccP, are key players for pedestal formation. We discovered that an EHEC effector protein called Non-LEE-encoded Ligase (NleL) is an E3 ubiquitin ligase. In vitro, we showed that the NleL C753 residue is critical for its E3 ligase activity. Functionally, we demonstrated that NleL E3 ubiquitin ligase activity is involved in modulating Tir-mediated pedestal formation. Surprisingly, EHEC mutant strain deficient in the E3 ligase activity induced more pedestals than the wild-type strain. The canonical EPEC strain E2348/69 normally lacks the nleL gene, and the ectopic expression of the wild-type EHEC nleL, but not the catalytically-deficient nleL(C753A) mutant, in this strain resulted in fewer actin-rich pedestals. Furthermore, we showed that the C. rodentium NleL homolog is a E3 ubiquitin ligase and is required for efficient infection of murine colonic epithelial cells in vivo. In summary, our study demonstrated that EHEC utilizes NleL E3 ubiquitin ligase activity to modulate Tir-mediated pedestal formation., National Institutes of Health (U.S.) (grant AI078092), National Institutes of Health (U.S.) (grant AI068655)

Background: Diagnosis of chronic intestinal inflammation, which characterizes inflammatory bowel disease (IBD), along with prediction of disease state is hindered by the availability of predictive serum biomarker. Serum biomarkers predictive of disease state will improve trials for therapeutic intervention, and disease monitoring, particularly in genetically susceptible individuals. Chronic inflammation during IBD is considered distinct from infectious intestinal inflammation thereby requiring biomarkers to provide differential diagnosis. To address whether differential serum biomarkers could be identified in murine models of colitis, immunological profiles from both chronic spontaneous and acute infectious colitis were compared and predictive serum biomarkers identified via multivariate modeling. Methodology/Principal Findings: Discriminatory multivariate modeling of 23 cytokines plus chlorotyrosine and nitrotyrosine (protein adducts from reactive nitrogen species and hypochlorite) in serum and tissue from two murine models of colitis was performed to identify disease-associated biomarkers. Acute C. rodentium-induced colitis in C57BL/6J mice and chronic spontaneous Helicobacter-dependent colitis in TLR4−/− x IL-10−/− mice were utilized for evaluation. Colon profiles of both colitis models were nearly identical with chemokines, neutrophil- and Th17-related factors highly associated with intestinal disease. In acute colitis, discriminatory disease-associated serum factors were not those identified in the colon. In contrast, the discriminatory predictive serum factors for chronic colitis were neutrophil- and Th17-related factors (KC, IL-12/23p40, IL-17, G-CSF, and chlorotyrosine) that were also elevated in colon tissue. Chronic colitis serum biomarkers were specific to chronic colitis as they were not discriminatory for acute colitis. Conclusions/Significance: Immunological profiling revealed strikingly similar colon profiles, yet distinctly different serum profiles for acute and chronic colitis. Neutrophil- and Th17-related factors were identified as predictive serum biomarkers of chronic colitis, but not acute colitis, despite their presence in colitic tissue of both diseases thereby demonstrating the utility of mathematical modeling for identifying disease-associated serum biomarkers., National Institutes of Health (U.S) (P01 CA026731), National Institutes of Health (U.S) (DK 55678), MIT-Singapore Alliance. Research and Technology-Infectious Diseases

Acute diarrheal illness is a global health problem that may be exacerbated by concurrent infection. Using Citrobacter rodentium , a murine model of attaching and effacing diarrheagenic Escherichia coli , we demonstrate that persistent Helicobacter hepaticus infection modulates host responses to diarrheal disease, resulting in delayed recovery from weight loss and from tissue damage. Chronic colitis in concurrently infected mice is characterized by macrophage and Foxp3 + regulatory T-cell accumulation. Prolonged disease is also associated with increased interleukin-17 expression, which may be due to suppression of gamma interferon during the acute phase of diarrheal infection. This new model of polymicrobial infection provides insight into the mechanism by which subclinical infection can exacerbate morbidity due to an unrelated self-limiting infection.

There have been considerable advances in our understanding of the molecular pathogenesis of enteropathogenic Escherichia coli and enterohemorrhagic E. coli infection. Given the difficulty of infecting laboratory mice with these diarrhea-causing pathogens, a growing number of studies have found the murine bacterial pathogen Citrobacter rodentium to provide a robust, relevant in-vivo model system.All inbred strains and outbred stocks of laboratory mice studied to date have been found to be susceptible to C. rodentium infection. The natural course of disease ranges from subclinical epithelial hyperplasia in the colon, to clinical diarrhea and colitis, to fatal infection, depending on the age, genetic background, and health status of the host. Infection is self-limiting, leading to disease resolution and protective immunity. Here we review recent discoveries related to bacterial virulence determinants, epithelial hyperplasia, innate and adaptive immune responses, and mechanisms of diarrhea.Infection of laboratory mice with C. rodentium provides a useful in-vivo model for studying the pathogenesis of infectious gastroenteritis and acute diarrheal illness, and for preclinical evaluation of candidate preventive and therapeutic agents.

Background & Aims The commensal microbiota is believed to have an important role in regulating immune responsiveness and preventing intestinal inflammation. Intestinal microbes produce signals that regulate inflammation via Toll-like receptor (TLR) signaling, but the mechanisms of this process are poorly understood. We investigated the role of the anti-inflammatory cytokine interleukin (IL)-10 in this signaling pathway using a mouse model of colitis. Methods Clinical, histopathologic, and functional parameters of intestinal inflammation were evaluated in TLR4 −/− , IL-10 −/− , and TLR4 −/− × IL-10 −/− mice that were free of specific pathogens and in TLR4 −/− × IL-10 −/− mice following eradication and reintroduction of Helicobacter hepaticus. Regulatory T-cell (Treg) function was evaluated by crossing each of the lines with transgenic mice that express green fluorescent protein under control of the endogenous regulatory elements of Foxp3 . Apoptotic cells in the colonic lamina propria were detected by a TUNEL assay. Results TLR4-mediated signals have 2 interrelated roles in promoting inflammation in TLR4 −/− × IL-10 −/− mice. In the absence of TLR4-mediated signals, secretion of proinflammatory and immunoregulatory cytokines is dysregulated. Tregs (Foxp3 + ) that secrete interferon-γ and IL-17 accumulate in the colonic lamina propria of TLR4 −/− × IL-10 −/− mice and do not prevent inflammation. Aberrant control of epithelial cell turnover results in the persistence of antigen-presenting cells that contain apoptotic epithelial fragments in the colonic lamina propria of Helicobacter- infected TLR4 −/− mice. Conclusions In mice that lack both IL-10- and TLR4-mediated signals, aberrant regulatory T-cell function and dysregulated control of epithelial homeostasis combine to exacerbate intestinal inflammation.