Dynamic interactions of a conserved enterotoxigenic Escherichia coli adhesin with intestinal mucins govern epithelium engagement and toxin delivery

At present, there is no vaccine for enterotoxigenic Escherichia coli (ETEC), an important cause of diarrheal illness. Nevertheless, recent microbial pathogenesis studies have identified a number of molecules produced by ETEC that contribute to its virulence and are novel antigenic targets to complement canonical vaccine approaches. EtpA is a secreted two-partner adhesin that is conserved within the ETEC pathovar. EtpA interacts with the tips of ETEC flagella to promote bacterial adhesion, toxin delivery, and intestinal colonization by forming molecular bridges between the bacteria and the epithelial surface. However, the nature of EtpA interactions with the intestinal epithelium remains poorly defined. Here, we demonstrate that EtpA interacts with glycans presented by transmembrane and secreted intestinal mucins at epithelial surfaces to facilitate pathogen-host interactions that culminate in toxin delivery. Moreover, we found that a major effector molecule of ETEC, the heat-labile enterotoxin (LT), may enhance these interactions by stimulating the production of the gel-forming mucin MUC2. Our studies suggest, however, that EtpA participates in complex and dynamic interactions between ETEC and the gastrointestinal mucosae in which host glycoproteins promote bacterial attachment while simultaneously limiting the epithelial engagement required for effective toxin delivery. Collectively, these data provide additional insight into the intricate nature of ETEC interactions with the intestinal epithelium that have potential implications for rational approaches to vaccine design

Blood Group O-Dependent cellular responses to cholera toxin: Parallel clinical and epidemiological links to severe cholera

Because O blood group has been associated with more severe cholera infections, it has been hypothesized that cholera toxin (CT) may bind non-O blood group antigens of the intestinal mucosae, thereby preventing efficient interaction with target GM1 gangliosides required for uptake of the toxin and activation of cyclic adenosine monophosphate (cAMP) signaling in target epithelia. Herein, we show that after exposure to CT, human enteroids expressing O blood group exhibited marked increase in cAMP relative to cells derived from blood group A individuals. Likewise, using CRISPR/Cas9 engineering, a functional group O line (HT-29-A(−/−)) was generated from a parent group A HT-29 line. CT stimulated robust cAMP responses in HT-29-A(−/−) cells relative to HT-29 cells. These findings provide a direct molecular link between blood group O expression and differential cellular responses to CT, recapitulating clinical and epidemiologic observations

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 \u3e93% 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

Inositol phosphorylceramide synthase null Leishmania are viable and virulent in animal infections where salvage of host sphingomyelin predominates

Many pathogens synthesize inositol phosphorylceramide (IPC) as the major sphingolipid (SL), differing from the mammalian host where sphingomyelin (SM) or more complex SLs predominate. The divergence between IPC synthase and mammalian SL synthases has prompted interest as a potential drug target. However, in the trypanosomatid protozoan Leishmania, cultured insect stage promastigotes lack de novo SL synthesis (Δspt

De novo meningitis caused by Propionibacterium acnes in a patient with metastatic melanoma

Propionibacterium acnes is a known cause of postneurosurgical meningitis; however, it is rarely implicated in de novo meningitis. Herein we report a case of a 49-year-old male with de novo meningitis caused by P. acnes with metastatic melanoma as the only identified risk factor for his infection

Choline, DHA, and diarrheal disease associated with growth faltering in a case-control study

BACKGROUND: Children with recurrent infectious diarrhea are susceptible to growth faltering. DHA and choline may play a role in this relationship due to their involvement in lipid metabolism, gut immunity, and inflammatory pathways. OBJECTIVES: This study aimed to characterize the contributions made by DHA and choline status and enteric damage in young children in the association between diarrheal illness and child growth. METHODS: A longitudinal case-control study was conducted among children aged 6-36 mo ( RESULTS: At baseline, mean plasma DHA concentrations (1.03 µg/mL; 95% CI: 0.91, 1.15) were not significantly different between cases and controls, nor was there a difference in mean plasma choline concentrations (4.5 µg/mL; 95% CI: 3.8, 5.1). Mean plasma I-FABP concentrations were significantly higher at follow-up in cases (3.34; 95% CI: 3.28, 3.40) than controls (3.20; 95% CI: 3.13, 3.27; CONCLUSIONS: I-FABP concentrations were significantly higher in cases as compared with controls at follow-up, suggesting ongoing enteric damage and increased risk for malnutrition. Plasma DHA and I-FABP may have a role in childhood growth outcomes

The sphingolipids ceramide and inositol phosphorylceramide protect the Leishmania major membrane from sterol-specific toxins

The accessibility of sterols in mammalian cells to exogenous sterol-binding agents has been well-described previously, but sterol accessibility in distantly related protozoa is unclear. The human pathogen Leishmania major uses sterols and sphingolipids distinct from those used in mammals. Sterols in mammalian cells can be sheltered from sterol-binding agents by membrane components, including sphingolipids, but the surface exposure of ergosterol in Leishmania remains unknown. Here, we used flow cytometry to test the ability of the L. major sphingolipids inositol phosphorylceramide (IPC) and ceramide to shelter ergosterol by preventing binding of the sterol-specific toxins streptolysin O and perfringolysin O and subsequent cytotoxicity. In contrast to mammalian systems, we found that Leishmania sphingolipids did not preclude toxin binding to sterols in the membrane. However, we show that IPC reduced cytotoxicity and that ceramide reduced perfringolysin O- but not streptolysin O-mediated cytotoxicity in cells. Furthermore, we demonstrate ceramide sensing was controlled by the toxin L3 loop, and that ceramide was sufficient to protect L. major promastigotes from the anti-leishmaniasis drug amphotericin B. Based on these results, we propose a mechanism whereby pore-forming toxins engage additional lipids like ceramide to determine the optimal environment to sustain pore formation. Thus, L. major could serve as a genetically tractable protozoan model organism for understanding toxin-membrane interactions

Prevalence of diarrheagenic Escherichia coli and impact on child health in Cap-Haitien, Haiti

BACKGROUND: Diarrheagenic Escherichia coli (DEC) are common pathogens infecting children during their growth and development. Determining the epidemiology and the impact of DEC on child anthropometric measures informs prioritization of prevention efforts. These relationships were evaluated in a novel setting, Cap-Haitien, Haiti. METHODS: We performed pre-specified secondary analysis of a case-control study of community-dwelling children, 6-36 months of age, enrolled 96 cases with diarrhea and 99 asymptomatic controls. Assessments were performed at enrollment and one month later at follow-up. Established endpoint PCR methodologies targeted DEC gDNA isolated from fecal swabs. The association between DEC and anthropometric z-scores at enrollment was determined using multivariate linear regression. Lastly, we assessed the association between specific biomarkers, choline and docosahexaenoic acid (DHA) and diarrheal burden. RESULTS: Enterotoxigenic Escherichia coli (ETEC) was identified in 21.9% of cases vs. 16.1% of controls with heat-stable producing ETEC significantly associated with symptomatic disease. Enteroaggregative E. coli (EAEC) was found in 30.2% of cases vs. 27.3% of controls, and typical enteropathogenic E. coli in 6.3% vs. 4.0% of cases and controls, respectively. Multivariate linear regression, controlled for case or control status, demonstrated ETEC and EAEC were significantly associated with reduced weight-age z-score (WAZ) and height-age z-score (HAZ) after adjusting for confounders. An interaction between ETEC and EAEC was observed. Choline and DHA were not associated with diarrheal burden. CONCLUSIONS: DEC are prevalent in north Haitian children. ETEC, EAEC, household environment, and diet are associated with unfavorable anthropometric measures, with possible synergistic interactions between ETEC and EAEC. Further studies with longer follow up may quantify the contribution of individual pathogens to adverse health outcomes

Grandi Byen-supporting child growth and development through integrated, responsive parenting, nutrition and hygiene: Study protocol for a randomized controlled trial

BACKGROUND: Poor child growth and development outcomes stem from complex relationships encompassing biological, behavioral, social, and environmental conditions. However, there is a dearth of research on integrated approaches targeting these interwoven factors. The Grandi Byen study seeks to fill this research gap through a three-arm longitudinal randomized controlled trial which will evaluate the impact of an integrated nutrition, responsive parenting, and WASH (water, sanitation and hygiene) intervention on holistic child growth and development. METHODS: We will recruit 600 mother-infant dyads living in Cap-Haitien, Haiti and randomize them equally into one of the following groups: 1) standard well-baby care; 2) nutritional intervention (one egg per day for 6 months); and 3) multicomponent Grandi Byen intervention (responsive parenting, nutrition, WASH + one egg per day for 6 months). Primary outcomes include child growth as well as cognitive, language, motor, and social-emotional development. The study also assesses other indicators of child health (bone maturation, brain growth, diarrheal morbidity and allergies, dietary intake, nutrient biomarkers) along with responsive parenting as mediating factors influencing the primary outcomes. An economic evaluation will assess the feasibility of large-scale implementation of the interventions. DISCUSSION: This study builds on research highlighting the importance of responsive parenting interventions on overall child health, as well as evidence demonstrating that providing an egg daily to infants during the complementary feeding period can prevent stunted growth. The multicomponent Grandi Byen intervention may provide evidence of synergistic or mediating effects of an egg intervention with instruction on psychoeducational parenting and WASH on child growth and development. Grandi Byen presents key innovations with implications for the well-being of children living in poverty globally. TRIAL REGISTRATION: NCT04785352 . Registered March 5, 2021 at https://clinicaltrials.gov/