Enterotoxigenic Escherichia coli heat-labile toxin drives enteropathic changes in small intestinal epithelia.

Enterotoxigenic E. coli (ETEC) produce heat-labile (LT) and/or heat-stable (ST) enterotoxins, and commonly cause diarrhea in resource-poor regions. ETEC have been linked repeatedly to sequelae in children including enteropathy, malnutrition, and growth impairment. Although cellular actions of ETEC enterotoxins leading to diarrhea are well-established, their contributions to sequelae remain unclear. LT increases cellular cAMP to activate protein kinase A (PKA) that phosphorylates ion channels driving intestinal export of salt and water resulting in diarrhea. As PKA also modulates transcription of many genes, we interrogated transcriptional profiles of LT-treated intestinal epithelia. Here we show that LT significantly alters intestinal epithelial gene expression directing biogenesis of the brush border, the major site for nutrient absorption, suppresses transcription factors HNF4 and SMAD4 critical to enterocyte differentiation, and profoundly disrupts microvillus architecture and essential nutrient transport. In addition, ETEC-challenged neonatal mice exhibit substantial brush border derangement that is prevented by maternal vaccination with LT. Finally, mice repeatedly challenged with toxigenic ETEC exhibit impaired growth recapitulating the multiplicative impact of recurring ETEC infections in children. These findings highlight impacts of ETEC enterotoxins beyond acute diarrheal illness and may inform approaches to prevent major sequelae of these common infections including malnutrition that impact millions of children.

Enterotoxigenic Escherichia coli Degrades the Host MUC2 Mucin Barrier To Facilitate Critical Pathogen-Enterocyte Interactions in Human Small Intestine.

Enterotoxigenic Escherichia coli (ETEC) isolates are genetically diverse pathological variants of E. coli defined by the production of heat-labile (LT) and/or heat-stable (ST) toxins. ETEC strains are estimated to cause hundreds of millions of cases of diarrheal illness annually. However, it is not clear that all strains are equally equipped to cause disease, and asymptomatic colonization with ETEC is common in low- to middle-income regions lacking basic sanitation and clean water where ETEC are ubiquitous. Recent molecular epidemiology studies have revealed a significant association between strains that produce EatA, a secreted autotransporter protein, and the development of symptomatic infection. Here, we demonstrate that LT stimulates production of MUC2 mucin by goblet cells in human small intestine, enhancing the protective barrier between pathogens and enterocytes. In contrast, using explants of human small intestine as well as small intestinal enteroids, we show that EatA counters this host defense by engaging and degrading the MUC2 mucin barrier to promote bacterial access to target enterocytes and ultimately toxin delivery, suggesting that EatA plays a crucial role in the molecular pathogenesis of ETEC. These findings may inform novel approaches to prevention of acute diarrheal illness as well as the sequelae associated with ETEC and other pathogens that rely on EatA and similar proteases for efficient interaction with their human hosts.

CEACAMs serve as toxin-stimulated receptors for enterotoxigenic Escherichia coli.

The enterotoxigenic Escherichia coli (ETEC) are among the most common causes of diarrheal illness and death due to diarrhea among young children in low-/middle-income countries (LMICs). ETEC have also been associated with important sequelae including malnutrition and stunting, placing children at further risk of death from diarrhea and other infections. Our understanding of the molecular pathogenesis of acute diarrheal disease as well as the sequelae linked to ETEC are still evolving. It has long been known that ETEC heat-labile toxin (LT) activates production of cAMP in the cell, signaling the modulation of cellular ion channels that results in a net efflux of salt and water into the intestinal lumen, culminating in watery diarrhea. However, as LT also promotes ETEC adhesion to intestinal epithelial cells, we postulated that increases in cAMP, a critical cellular "second messenger," may be linked to changes in cellular architecture that favor pathogen-host interactions. Indeed, here we show that ETEC use LT to up-regulate carcinoembryonic antigenrelated cell adhesion molecules (CEACAMs) on the surface of small intestinal epithelia, where they serve as critical bacterial receptors. Moreover, we show that bacteria are specifically recruited to areas of CEACAM expression, in particular CEACAM6, and that deletion of this CEACAM abrogates both bacterial adhesion and toxin delivery. Collectively, these results provide a paradigm for the molecular pathogenesis of ETEC in which the bacteria use toxin to drive up-regulation of cellular targets that enhances subsequent pathogen-host interactions.

Conservation and global distribution of non-canonical antigens in Enterotoxigenic Escherichia coli.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) cause significant diarrheal morbidity and mortality in children of resource-limited regions, warranting development of effective vaccine strategies. Genetic diversity of the ETEC pathovar has impeded development of broadly protective vaccines centered on the classical canonical antigens, the colonization factors and heat-labile toxin. Two non-canonical ETEC antigens, the EtpA adhesin, and the EatA mucinase are immunogenic in humans and protective in animal models. To foster rational vaccine design that complements existing strategies, we examined the distribution and molecular conservation of these antigens in a diverse population of ETEC isolates. METHODS: Geographically diverse ETEC isolates (n = 1159) were interrogated by PCR, immunoblotting, and/or whole genome sequencing (n = 46) to examine antigen conservation. The most divergent proteins were purified and their core functions assessed in vitro. RESULTS: EatA and EtpA or their coding sequences were present in 57.0% and 51.5% of the ETEC isolates overall, respectively; and were globally dispersed without significant regional differences in antigen distribution. These antigens also exhibited >93% amino acid sequence identity with even the most divergent proteins retaining the core adhesin and mucinase activity assigned to the prototype molecules. CONCLUSIONS: EtpA and EatA are well-conserved molecules in the ETEC pathovar, suggesting that they serve important roles in virulence and that they could be exploited for rational vaccine design.

Interrogation of a live-attenuated enterotoxigenic Escherichia coli vaccine highlights features unique to wild-type infection.

Enterotoxigenic Escherichia coli (ETEC) infections are a common cause of severe diarrheal illness in low- and middle-income countries. The live-attenuated ACE527 ETEC vaccine, adjuvanted with double mutant heat-labile toxin (dmLT), affords clear but partial protection against ETEC challenge in human volunteers. Comparatively, initial wild-type ETEC challenge completely protects against severe diarrhea on homologous re-challenge. To investigate determinants of protection, vaccine antigen content was compared to wild-type ETEC, and proteome microarrays were used to assess immune responses following vaccination and ETEC challenge. Although molecular interrogation of the vaccine confirmed expression of targeted canonical antigens, relative to wild-type ETEC, vaccine strains were deficient in production of flagellar antigens, immotile, and lacked production of the EtpA adhesin. Similarly, vaccination ± dmLT elicited responses to targeted canonical antigens, but relative to wild-type challenge, vaccine responses to some potentially protective non-canonical antigens including EtpA and the YghJ metalloprotease were diminished or absent. These studies highlight important differences in vaccine and wild-type ETEC antigen content and call attention to distinct immunologic signatures that could inform investigation of correlates of protection, and guide vaccine antigen selection for these pathogens of global importance.

Molecular Determinants of Enterotoxigenic Escherichia coli Heat-Stable Toxin Secretion and Delivery.

Enterotoxigenic Escherichia coli (ETEC), a heterogeneous diarrheal pathovar defined by production of heat-labile (LT) and/or heat-stable (ST) toxins, causes substantial morbidity among young children in the developing world. Studies demonstrating a major burden of ST-producing ETEC have focused interest on ST toxoids for ETEC vaccines. We examined fundamental aspects of ST biology using ETEC strain H10407, which carries estH and estP genes encoding STh and STp, respectively, in addition to eltAB genes responsible for LT. Here, we found that deletion of estH significantly diminished cyclic GMP (cGMP) activation in target epithelia, while deletion of estP had a surprisingly modest impact, and a dual estH estP mutant was not appreciably different from the estH mutant. However, we noted that either STh or STp recombinant peptides stimulated cGMP production and that the loss of estP was compensated by enhanced estH transcription. We also found that the TolC efflux protein was essential for toxin secretion and delivery, providing a potential avenue for efflux inhibitors in treatment of acute diarrheal illness. In addition, we demonstrated that the EtpA adhesin is required for optimal delivery of ST and that antibodies against either the adhesin or STh significantly impaired toxin delivery and cGMP activation in target T84 cells. Finally, we used FLAG epitope fusions to demonstrate that the STh propeptide sequence is secreted by ETEC, potentially providing additional epitopes for antibody neutralization. These studies collectively extend our understanding of ETEC pathogenesis and potentially inform additional avenues to mitigate disease by these common diarrheal pathogens.

Human Experimental Challenge With Enterotoxigenic Escherichia coli Elicits Immune Responses to Canonical and Novel Antigens Relevant to Vaccine Development.

Background: Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrheal illness in the developing world. Enterotoxigenic E coli vaccinology has been challenged by genetic diversity and heterogeneity of canonical antigens. Examination of the antigenic breadth of immune responses associated with protective immunity could afford new avenues for vaccine development. Methods: Antibody lymphocyte supernatants (ALS) and sera from 20 naive human volunteers challenged with ETEC strain H10407 and from 10 volunteers rechallenged 4-6 weeks later with the same strain (9 of whom were completely protected on rechallenge) were tested against ETEC proteome microarrays containing 957 antigens. Results: Enterotoxigenic E coli challenge stimulated robust serum and mucosal (ALS) responses to canonical vaccine antigens (CFA/I, and the B subunit of LT) as well as a small number of antigens not presently targeted in ETEC vaccines. These included pathovar-specific secreted proteins (EtpA, EatA) as well as highly conserved E coli antigens including YghJ, flagellin, and pertactin-like autotransporter proteins, all of which have previously afforded protection against ETEC infection in preclinical studies. Conclusions: Taken together, studies reported here suggest that immune responses after ETEC infection involve traditional vaccine targets as well as a select number of more recently identified protein antigens that could offer additional avenues for vaccine development for these pathogens.

Enterotoxigenic Escherichia coli-blood group A interactions intensify diarrheal severity.

Enterotoxigenic Escherichia coli (ETEC) infections are highly prevalent in developing countries, where clinical presentations range from asymptomatic colonization to severe cholera-like illness. The molecular basis for these varied presentations, which may involve strain-specific virulence features as well as host factors, has not been elucidated. We demonstrate that, when challenged with ETEC strain H10407, originally isolated from a case of cholera-like illness, blood group A human volunteers developed severe diarrhea more frequently than individuals from other blood groups. Interestingly, a diverse population of ETEC strains, including H10407, secrete the EtpA adhesin molecule. As many bacterial adhesins also agglutinate red blood cells, we combined the use of glycan arrays, biolayer inferometry, and noncanonical amino acid labeling with hemagglutination studies to demonstrate that EtpA is a dominant ETEC blood group A-specific lectin/hemagglutinin. Importantly, we have also shown that EtpA interacts specifically with glycans expressed on intestinal epithelial cells from blood group A individuals and that EtpA-mediated bacterial-host interactions accelerate bacterial adhesion and effective delivery of both the heat-labile and heat-stable toxins of ETEC. Collectively, these data provide additional insight into the complex molecular basis of severe ETEC diarrheal illness that may inform rational design of vaccines to protect those at highest risk.

A Role for Salivary Peptides in the Innate Defense Against Enterotoxigenic Escherichia coli.

Background: Diarrheal disease from enterotoxigenic Escherichia coli (ETEC) causes significant worldwide morbidity and mortality in young children residing in endemic countries and is the leading cause of traveler's diarrhea. As ETEC enters the body through the oral cavity and cotransits the digestive tract with salivary components, we hypothesized that the antimicrobial activity of salivary proteins might extend beyond the oropharynx into the proximal digestive tract. Results: Here, we show that the salivary peptide histatin-5 binds colonization factor antigen I pili, thereby blocking adhesion of ETEC to intestinal epithelial cells. Mechanistically, we demonstrate that histatin-5 stiffens the typically dynamic pili, abolishing their ability to function as spring-like shock absorbers, thereby inhibiting colonization within the turbulent vortices of chyme in the gastrointestinal tract. Conclusions: Our data represent the first report of a salivary component exerting specific antimicrobial activity against an enteric pathogen and suggest that histatin-5 and related peptides might be exploited for prophylactic and/or therapeutic uses. Numerous viruses, bacteria, and fungi traverse the oropharynx to cause disease, so there is considerable opportunity for various salivary components to neutralize these pathogens prior to arrival at their target organ. Identification of additional salivary components with unexpectedly broad antimicrobial spectra should be a priority.

Immunogenicity and Protective Efficacy against Enterotoxigenic Escherichia coli Colonization following Intradermal, Sublingual, or Oral Vaccination with EtpA Adhesin.

Enterotoxigenic Escherichia coli (ETEC) strains are a common cause of diarrhea. Extraordinary antigenic diversity has prompted a search for conserved antigens to complement canonical approaches to ETEC vaccine development. EtpA, an immunogenic extracellular ETEC adhesin relatively conserved in the ETEC pathovar, has previously been shown to be a protective antigen following intranasal immunization. These studies were undertaken to explore alternative routes of EtpA vaccination that would permit use of a double mutant (R192G L211A) heat-labile toxin (dmLT) adjuvant. Here, oral vaccination with EtpA adjuvanted with dmLT afforded significant protection against small intestinal colonization, and the degree of protection correlated with fecal IgG, IgA, or total fecal antibody responses to EtpA. Sublingual vaccination yielded compartmentalized mucosal immune responses with significant increases in anti-EtpA fecal IgG and IgA, and mice vaccinated via this route were also protected against colonization. In contrast, while intradermal (i.d.) vaccination achieved high levels of both serum and fecal antibodies against both EtpA and dmLT, mice vaccinated via the i.d. route were not protected against subsequent colonization and the avidity of serum IgG and IgA EtpA-specific antibodies was significantly lower after i.d. immunization compared to other routes. Finally, we demonstrate that antiserum from vaccinated mice significantly impairs binding of LT to cognate GM1 receptors and shows near complete neutralization of toxin delivery by ETEC in vitro Collectively, these data provide further evidence that EtpA could complement future vaccine strategies but also suggest that additional effort will be required to optimize its use as a protective immunogen.

EatA, an immunogenic protective antigen of enterotoxigenic Escherichia coli, degrades intestinal mucin.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of morbidity and mortality due to infectious diarrhea in developing countries for which there is presently no effective vaccine. A central challenge in ETEC vaccinology has been the identification of conserved surface antigens to formulate a broadly protective vaccine. Here, we demonstrate that EatA, an immunogenic secreted serine protease of ETEC, contributes to virulence by degrading MUC2, the major protein present in the small intestinal mucous layer, and that removal of this barrier in vitro accelerates toxin access to the enterocyte surface. In addition, we demonstrate that vaccination with the recombinant secreted passenger domain of EatA (rEatAp) elicits high titers of antibody and is protective against intestinal infection with ETEC. These findings may have significant implications for development of both subunit and live-attenuated vaccines against ETEC and other enteric pathogens, including Shigella flexneri, that express similar proteins.

Enterotoxigenic Escherichia coli secretes a highly conserved mucin-degrading metalloprotease to effectively engage intestinal epithelial cells.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of death due to diarrheal illness among young children in developing countries, and there is currently no effective vaccine. Many elements of ETEC pathogenesis are still poorly defined. Here we demonstrate that YghJ, a secreted ETEC antigen identified in immunoproteomic studies using convalescent patient sera, is required for efficient access to small intestinal enterocytes and for the optimal delivery of heat-labile toxin (LT). Furthermore, YghJ is a highly conserved metalloprotease that influences intestinal colonization of ETEC by degrading the major mucins in the small intestine, MUC2 and MUC3. Genes encoding YghJ and its cognate type II secretion system (T2SS), which also secretes LT, are highly conserved in ETEC and exist in other enteric pathogens, including other diarrheagenic E. coli and Vibrio cholerae bacteria, suggesting that this mucin-degrading enzyme may represent a shared virulence feature of these important pathogens.

Folate metabolic pathways in Leishmania.

Trypanosomatid parasitic protozoans of the genus Leishmania are autotrophic for both folate and unconjugated pteridines. Leishmania salvage these metabolites from their mammalian hosts and insect vectors through multiple transporters. Within the parasite, folates are reduced by a bifunctional DHFR (dihydrofolate reductase)-TS (thymidylate synthase) and by a novel PTR1 (pteridine reductase 1), which reduces both folates and unconjugated pteridines. PTR1 can act as a metabolic bypass of DHFR inhibition, reducing the effectiveness of existing antifolate drugs. Leishmania possess a reduced set of folate-dependent metabolic reactions and can salvage many of the key products of folate metabolism from their hosts. For example, they lack purine synthesis, which normally requires 10-formyltetrahydrofolate, and instead rely on a network of purine salvage enzymes. Leishmania elaborate at least three pathways for the synthesis of the key metabolite 5,10-methylene-tetrahydrofolate, required for the synthesis of thymidylate, and for 10-formyltetrahydrofolate, whose presumptive function is for methionyl-tRNAMet formylation required for mitochondrial protein synthesis. Genetic studies have shown that the synthesis of methionine using 5-methyltetrahydrofolate is dispensable, as is the activity of the glycine cleavage complex, probably due to redundancy with serine hydroxymethyltransferase. Although not always essential, the loss of several folate metabolic enzymes results in attenuation or loss of virulence in animal models, and a null DHFR-TS mutant has been used to induce protective immunity. The folate metabolic pathway provides numerous opportunities for targeted chemotherapy, with strong potential for 'repurposing' of compounds developed originally for treatment of human cancers or other infectious agents.

Wikipedia: a key tool for global public health promotion.

The Internet has become an important health information resource for patients and the general public. Wikipedia, a collaboratively written Web-based encyclopedia, has become the dominant online reference work. It is usually among the top results of search engine queries, including when medical information is sought. Since April 2004, editors have formed a group called WikiProject Medicine to coordinate and discuss the English-language Wikipedia's medical content. This paper, written by members of the WikiProject Medicine, discusses the intricacies, strengths, and weaknesses of Wikipedia as a source of health information and compares it with other medical wikis. Medical professionals, their societies, patient groups, and institutions can help improve Wikipedia's health-related entries. Several examples of partnerships already show that there is enthusiasm to strengthen Wikipedia's biomedical content. Given its unique global reach, we believe its possibilities for use as a tool for worldwide health promotion are underestimated. We invite the medical community to join in editing Wikipedia, with the goal of providing people with free access to reliable, understandable, and up-to-date health information.

A role for tetrahydrofolates in the metabolism of iron-sulfur clusters in all domains of life.

Iron-sulfur (Fe/S) cluster enzymes are crucial to life. Their assembly requires a suite of proteins, some of which are specific for particular subsets of Fe/S enzymes. One such protein is yeast Iba57p, which aconitase and certain radical S-adenosylmethionine enzymes require for activity. Iba57p homologs occur in all domains of life; they belong to the COG0354 protein family and are structurally similar to various folate-dependent enzymes. We therefore investigated the possible relationship between folates and Fe/S cluster enzymes using the Escherichia coli Iba57p homolog, YgfZ. NMR analysis confirmed that purified YgfZ showed stereoselective folate binding. Inactivating ygfZ reduced the activities of the Fe/S tRNA modification enzyme MiaB and certain other Fe/S enzymes, although not aconitase. When successive steps in folate biosynthesis were ablated, folE (lacking pterins and folates) and folP (lacking folates) mutants mimicked the ygfZ mutant in having low MiaB activities, whereas folE thyA mutants supplemented with 5-formyltetrahydrofolate (lacking pterins and depleted in dihydrofolate) and gcvP glyA mutants (lacking one-carbon tetrahydrofolates) had intermediate MiaB activities. These data indicate that YgfZ requires a folate, most probably tetrahydrofolate. Importantly, the ygfZ mutant was hypersensitive to oxidative stress and grew poorly on minimal media. COG0354 genes of bacterial, archaeal, fungal, protistan, animal, or plant origin complemented one or both of these growth phenotypes as well as the MiaB activity phenotype. Comparative genomic analysis indicated widespread functional associations between COG0354 proteins and Fe/S cluster metabolism. Thus COG0354 proteins have an ancient, conserved, folate-dependent function in the activity of certain Fe/S cluster enzymes.

Identification of transport-critical residues in a folate transporter from the folate-biopterin transporter (FBT) family.

The Synechocystis Slr0642 protein and its plastidial Arabidopsis (Arabidopsis thaliana) ortholog At2g32040 belong to the folate-biopterin transporter (FBT) family within the major facilitator superfamily. Both proteins transport folates when expressed in Escherichia coli. Because the structural requirements for transport activity are not known for any FBT protein, we applied mutational analysis to identify residues that are critical to transport and interpreted the results using a comparative structural model based on E. coli lactose permease. Folate transport was assessed via the growth of an E. coli pabA abgT strain, which cannot synthesize or take up folates or p-aminobenzoylglutamate. In total, 47 residues were replaced with Cys or Ala. Mutations at 22 positions abolished folate uptake without affecting Slr0642 expression in membranes, whereas other mutations had no effect. Residues important for function mostly line the predicted central cavity and are concentrated in the core alpha-helices H1, H4, H7, and H10. The essential residue locations are consistent with a folate-binding site lying roughly equidistant from both faces of the transporter. Arabidopsis has eight FBT proteins besides At2g32040, often lacking conserved critical residues. When six of these proteins were expressed in E. coli or in Leishmania folate or pterin transporter mutants, none showed evidence of folate or pterin transport activity, and only At2g32040 was isolated by functional screening of Arabidopsis cDNA libraries in E. coli. Such negative data could reflect roles in transport of other substrates. These studies provide the first insights into the native structure and catalytic mechanism of FBT family carriers.

The enzymes of the 10-formyl-tetrahydrofolate synthetic pathway are found exclusively in the cytosol of the trypanosomatid parasite Leishmania major.

In most organisms 10-formyl-tetrahydrofolate (10-CHO-THF) participates in the synthesis of purines in the cytosol and formylation of mitochondrial initiator methionyl-tRNA(Met). Here we studied 10-CHO-THF biosynthesis in the protozoan parasite Leishmania major, a purine auxotroph. Two distinct synthetic enzymes are known, a bifunctional methylene-tetrahydrofolate dehydrogenase/cyclohydrolase (DHCH) or formyl-tetrahydrofolate ligase (FTL), and phylogenomic profiling revealed considerable diversity for these in trypanosomatids. All species surveyed contain a DHCH1, which was shown recently to be essential in L. major. A second DHCH2 occurred only in L. infantum, L. mexicana and T. cruzi, and as a pseudogene in L. major. DHCH2s bear N-terminal extensions and we showed a LiDHCH2-GFP fusion was targeted to the mitochondrion. FTLs were found in all species except Trypanosoma brucei. L. major ftl(-) null mutants were phenotypically normal in growth, differentiation, animal infectivity and sensitivity to a panel of pteridine analogs, but grew more slowly when starved for serine or glycine, as expected for amino acids that are substrates in C1-folate metabolism. Cell fractionation and western blotting showed that both L. major DHCH1 and FTL were localized to the cytosol and not the mitochondrion. These localization data predict that in L. major cytosolic 10-formyl-tetrahydrofolate must be transported into the mitochondrion to support methionyl-tRNA(Met) formylation. The retention in all the trypanosomatids of at least one enzyme involved in 10-formyl-tetrahydrofolate biosynthesis, and the essentiality of this metabolite in L. major, suggests that this pathway represents a promising new area for chemotherapeutic attack in these parasites.

Seeking health information online: does Wikipedia matter?

OBJECTIVE To determine the significance of the English Wikipedia as a source of online health information. DESIGN The authors measured Wikipedia's ranking on general Internet search engines by entering keywords from MedlinePlus, NHS Direct Online, and the National Organization of Rare Diseases as queries into search engine optimization software. We assessed whether article quality influenced this ranking. The authors tested whether traffic to Wikipedia coincided with epidemiological trends and news of emerging health concerns, and how it compares to MedlinePlus. MEASUREMENTS Cumulative incidence and average position of Wikipedia compared to other Web sites among the first 20 results on general Internet search engines (Google, Google UK, Yahoo, and MSN, and page view statistics for selected Wikipedia articles and MedlinePlus pages. RESULTS Wikipedia ranked among the first ten results in 71-85% of search engines and keywords tested. Wikipedia surpassed MedlinePlus and NHS Direct Online (except for queries from the latter on Google UK), and ranked higher with quality articles. Wikipedia ranked highest for rare diseases, although its incidence in several categories decreased. Page views increased parallel to the occurrence of 20 seasonal disorders and news of three emerging health concerns. Wikipedia articles were viewed more often than MedlinePlus Topic (p = 0.001) but for MedlinePlus Encyclopedia pages, the trend was not significant (p = 0.07-0.10). CONCLUSIONS Based on its search engine ranking and page view statistics, the English Wikipedia is a prominent source of online health information compared to the other online health information providers studied.

PTR1-dependent synthesis of tetrahydrobiopterin contributes to oxidant susceptibility in the trypanosomatid protozoan parasite Leishmania major.

Leishmania must survive oxidative stress, but lack many classical antioxidant enzymes and rely heavily on trypanothione-dependent pathways. We used forward genetic screens to recover loci mediating oxidant resistance via overexpression in Leishmania major, which identified pteridine reductase 1 (PTR1). Comparisons of isogenic lines showed ptr1 (-) null mutants were 18-fold more sensitive to H(2)O(2) than PTR1-overproducing lines, and significant three- to fivefold differences were seen with a broad panel of oxidant-inducing agents. The toxicities of simple nitric oxide generators and other drug classes (except antifolates) were unaffected by PTR1 levels. H(2)O(2) susceptibility could be modulated by exogenous biopterin but not folate, in a PTR1- but not dihydrofolate reductase-dependent manner, implicating H(4)B metabolism specifically. Neither H(2)O(2) consumption nor the level of intracellular oxidative stress was affected by PTR1 levels. Coupled with the fact that reduced pteridines are at least 100-fold less abundant than cellular thiols, these data argue strongly that reduced pteridines act through a mechanism other than scavenging. The ability of unconjugated pteridines to counter oxidative stress has implications to infectivity and response to chemotherapy. Since the intracellular pteridine levels of Leishmania can be readily manipulated, these organisms offer a powerful setting for the dissection of pteridine-dependent oxidant susceptibility in higher eukaryotes.