RESUMEN
This study examined the effect of exposure of small and large intestinal epithelial cells to the bacterial lipopolysaccharide (LPS) on uptake of free form of vitamin B1, i.e., thiamin. The intestinal tract encounters two sources of thiamin: diet and the gut microbiota. Absorption of thiamin in both the small and large intestine occurs via a carrier-mediated process that involves thiamin transporters-1 & -2 (THTR-1 & -2). Complementary in vitro (human duodenal epithelial HuTu-80 cells and human colonic epithelial NCM460 cells), in vivo (mice), and ex vivo (human primary differentiated enteroid and colonoid monolayers) models were used. The results showed that exposure to LPS causes a significant inhibition in carrier-mediated [3H]-thiamin uptake by small and large intestinal epithelia, with no change in levels of expression of THTR-1& -2 mRNAs and their total cellular proteins. However, a significant decrease in the fractions of the THTR-1& -2 proteins that are expressed at the cell membranes of these epithelial cells was observed. These effects of LPS appeared to involve a protein kinase A (PKA) signaling pathway as activating this pathway caused a reversal in the inhibition of thiamin uptake and level of expression of its transporters at the cell membrane. These findings demonstrate that exposure of gut epithelia to LPS (a situation that occurs under different pathological conditions) leads to inhibition in thiamin uptake due to a decrease in level of expression of its transporters at the cell membrane that is likely mediated via a PKA-signaling pathway.
RESUMEN
Humans and mammals obtain vitamin B1 from dietary and gut microbiota sources. A considerable amount of the microbiota-generated vitamin exists in the form of thiamine pyrophosphate (TPP), and colonocytes are capable of absorbing TPP via a specific carrier-mediated process that involves the colonic TPP transporter (cTPPT encoded by SLC44A4). Little is known about the relative contribution of the SLC44A4 transporter toward total colonic carrier-mediated TPP uptake and its role in colon physiology. To address these issues, we generated an Slc44a4 knockout (KO) mouse model (by Cre-Lox recombination) and found a near-complete inhibition in colonic carrier-mediated [3H]TPP uptake in the Slc44a4 KO compared with wild-type (WT) littermates. We also observed a significant reduction in KO mice's body weight and a shortening of their colon compared with WT. Using RNAseq and Ingenuity pathway analysis (IPA) approaches, we found that knocking out the colonic Slc44a4 led to changes in the level of expression of many genes, including upregulation in those associated with intestinal inflammation and colitis. Finally, we found that the Slc44a4 KO mice were more susceptible to the effect of the colitogenic dextran sodium sulfate (DSS) compared with WT animals, a finding that lends support to the recent prediction by multiple genome-wide association studies (GWAS) that SLC44A4 is a possible colitis susceptibility gene. In summary, the results of these investigations show that Slc44a4 is the predominant or only transporter involved in the colonic uptake of TPP, that the transporter is important for colon physiology, and that its deletion increases susceptibility to inflammation.NEW & NOTEWORTHY This study shows that Slc44a4 is the predominant or only transport system involved in the uptake of the gut microbiota-generated thiamine pyrophosphate (TPP) in the colon and that its deletion affects colon physiology and increases its susceptibility to inflammation.
Asunto(s)
Colon , Microbioma Gastrointestinal , Ratones Noqueados , Tiamina Pirofosfato , Animales , Humanos , Masculino , Ratones , Transporte Biológico , Colitis/metabolismo , Colitis/microbiología , Colitis/genética , Colitis/inducido químicamente , Colon/metabolismo , Colon/microbiología , Microbioma Gastrointestinal/fisiología , Absorción Intestinal , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Ratones Endogámicos C57BL , Tiamina Pirofosfato/metabolismoRESUMEN
This study investigated the effect of the bacterial endotoxin lipopolysaccharide (LPS) on colonic uptake of thiamin pyrophosphate (TPP), the biologically active form of vitamin B1 that is generated by gut microbiota. We used three complementary models in our study: in vitro (human-derived colonic epithelial NCM460), ex vivo (human differentiated colonoid monolayers), and in vivo (mouse colonic tissue). The results showed that exposure of NCM460 cells to LPS leads to a significant inhibition of carrier-mediated TPP uptake as well as in decreased expression of the colonic TPP transporter (cTPPT) protein, mRNA, and heterologous nuclear RNA (hnRNA) compared with untreated controls. Similarly, exposure of human differentiated colonoid monolayers and mice to LPS caused significant inhibition in colonic carrier-mediated TPP uptake and in cTPPT protein, mRNA, and hnRNA expression. The effect of LPS on colonic TPP uptake and cTTPT expression was also found to be associated with a significant reduction in activity of the SLC44A4 promoter as well as in decreased expression of the nuclear factor Elf-3 (E74-like ETS transcription factor 3), which is needed for promoter activity. Finally, we found that knocking down the Toll-like receptor 4 (TLR4) and blocking the nuclear factor kappa B (NF-κB), JNK, and p38 signaling pathways with the use of pharmacological inhibitors lead to significant abrogation in the degree of LPS-mediated inhibition in TPP uptake and cTPPT expression. These results demonstrated that exposure of colonic epithelia to LPS inhibits colonic TPP uptake via transcriptional mechanism(s) and that the effect is mediated via TLR4 receptor and NF-κB/p38/JNK signaling pathways.NEW & NOTEWORTHY This study examined the effect of the bacterial lipopolysaccharide (LPS) on the colonic uptake of thiamin pyrophosphate (TPP), the biologically active form of vitamin B1. Three complementary models were used: in vitro (human NCM460 cells), ex vivo (human colonoids), and in vivo (mice). The results showed LPS to significantly suppress TPP uptake and the expression of its transporter, and that these effects are mediated via the membrane TLR4 receptor, and involve the NF-κB/p38/JNK signaling pathways.
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FN-kappa B , Tiamina Pirofosfato , Humanos , Ratones , Animales , Tiamina Pirofosfato/metabolismo , FN-kappa B/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Lipopolisacáridos/farmacología , Difosfatos , Sistema de Señalización de MAP Quinasas , ARN Nuclear Heterogéneo/metabolismo , Línea Celular , Tiamina/metabolismo , ARN Mensajero/metabolismoRESUMEN
Hypoxia exerts profound effects on cell physiology, but its effect on colonic uptake of the microbiota-generated forms of vitamin B1 (i.e., thiamin pyrophosphate [TPP] and free thiamine) has not been described. Here, we used human colonic epithelial NCM460 cells and human differentiated colonoid monolayers as in vitro and ex vivo models, respectively, and were subjected to either chamber (1% O2, 5% CO2, and 94% N2) or chemically (desferrioxamine; 250 µM)-induced hypoxia followed by determination of different physiological-molecular parameters. We showed that hypoxia causes significant inhibition in TPP and free thiamin uptake by colonic NCM460 cells and colonoid monolayers; it also caused a significant reduction in the expression of TPP (SLC44A4) and free thiamin (SLC19A2 and SLC19A3) transporters and in activity of their gene promoters. Furthermore, hypoxia caused a significant induction in levels of hypoxia-inducible transcription factor (HIF)-1α but not HIF-2α. Knocking down HIF-1α using gene-specific siRNAs in NCM460 cells maintained under hypoxic conditions, on the other hand, led to a significant reversal in the inhibitory effect of hypoxia on TPP and free thiamin uptake as well as on the expression of their transporters. Finally, a marked reduction in level of expression of the nuclear factors cAMP responsive element-binding protein 1 and gut-enriched Krüppel-like factor 4 (required for activity of SLC44A4 and SLC19A2 promoters, respectively) was observed under hypoxic conditions. In summary, hypoxia causes severe inhibition in colonic TPP and free thiamin uptake that is mediated at least in part via HIF-1α-mediated transcriptional mechanisms affecting their respective transporters.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia , Microbiota , Tiamina , Transporte Biológico , Hipoxia de la Célula/fisiología , Humanos , Hipoxia/metabolismo , Hipoxia/microbiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Tiamina/metabolismo , Tiamina Pirofosfato/metabolismoRESUMEN
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.
Asunto(s)
Antígenos CD/metabolismo , Moléculas de Adhesión Celular/metabolismo , Escherichia coli Enterotoxigénica/metabolismo , Infecciones por Escherichia coli/metabolismo , Adhesinas Bacterianas/metabolismo , Antígenos CD/genética , Toxinas Bacterianas/metabolismo , Células CACO-2 , Moléculas de Adhesión Celular/genética , Diarrea/microbiología , Células Epiteliales/metabolismo , Infecciones por Escherichia coli/microbiología , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Mucosa Intestinal/metabolismo , TranscriptomaRESUMEN
The aim of this study was to examine the effect of TNFα (i.e., a predominant proinflammatory cytokine produced during chronic gut inflammation) on colonic uptake of thiamin pyrophosphate (TPP) and free thiamin, forms of vitamin B1 that are produced by the gut microbiota and are absorbed via distinct carrier-mediated systems. We utilized human-derived colonic epithelial CCD841 and NCM460 cells, human differentiated colonoid monolayers, and mouse intact colonic tissue preparations together with an array of cellular/molecular approaches in our investigation. The results showed that exposure of colonic epithelial cells to TNFα leads to a significant inhibition in TPP and free thiamin uptake. This inhibition was associated with: 1) a significant suppression in the level of expression of the colonic TPP transporter (cTPPT; encoded by SLC44A4), as well as thiamin transporters-1 & 2 (THTR-1 & -2; encoded by SLC19A2 & SLC19A3, respectively); 2) marked inhibition in activity of the SLC44A4, SLC19A2, and SLC19A3 promoters; and 3) significant suppression in level of expression of nuclear factors that are needed for activity of these promoters (i.e., CREB-1, Elf-3, NF-1A, SP-1). Furthermore, the inhibitory effects were found to be mediated via JNK and ERK1/2 signaling pathways. We also examined the level of expression of cTPPT and THTR-1 & -2 in colonic tissues of patients with active ulcerative colitis and found the levels to be significantly lower than in healthy controls. These findings demonstrate that exposure of colonocytes to TNFα suppresses TPP and free thiamin uptake at the transcriptional level via JNK- and Erk1/2-mediated pathways.
Asunto(s)
Tiamina Pirofosfato , Factor de Necrosis Tumoral alfa , Humanos , Ratones , Animales , Tiamina Pirofosfato/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Células Acinares/metabolismo , Tiamina/metabolismo , Tiamina/farmacología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismoRESUMEN
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.
Asunto(s)
Toxinas Bacterianas , Escherichia coli Enterotoxigénica , Infecciones por Escherichia coli , Proteínas de Escherichia coli , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Diarrea , Enterocitos , Escherichia coli Enterotoxigénica/metabolismo , Enterotoxinas/metabolismo , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Intestino Delgado , Mucina 2/genética , Mucina 2/metabolismo , Mucinas/metabolismoRESUMEN
Enterotoxigenic Escherichia coli (ETEC) are ubiquitous diarrheal pathogens that thrive in areas lacking basic human needs of clean water and sanitation. These genetically plastic organisms cause tremendous morbidity among disadvantaged young children, in the form of both acute diarrheal illness and sequelae of malnutrition and growth impairment. The recent discovery of additional plasmid-encoded virulence factors and elucidation of their critical role in the molecular pathogenesis of ETEC may inform new approaches to the development of broadly protective vaccines. Although the pathogens have been closely linked epidemiologically with nondiarrheal sequelae, these conditions remain very poorly understood. Similarly, while canonical effects of ETEC toxins on cellular signaling promoting diarrhea are clear, emerging data suggest that these toxins may also drive changes in intestinal architecture and associated sequelae. Elucidation of molecular events underlying these changes could inform optimal approaches to vaccines that prevent acute diarrhea and ETEC-associated sequelae.
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Diarrea/prevención & control , Escherichia coli Enterotoxigénica/inmunología , Infecciones por Escherichia coli/prevención & control , Proteínas de Escherichia coli , Vacunas contra Escherichia coli , Toxinas Bacterianas , Niño , Preescolar , Escherichia coli Enterotoxigénica/genética , Enterotoxinas , Humanos , Desnutrición , PlásmidosRESUMEN
Although Bordetella hinzii coccobacilli is most commonly identified in respiratory tracts of birds and rodents, this organism has occasionally been isolated in human infections. We describe a case of B. hinzii spontaneous bacterial peritonitis in Missouri, USA. Whole-genome sequencing of blood and peritoneal fluid isolates confirmed B. hinzii infection.
Asunto(s)
Infecciones por Bordetella , Bordetella , Peritonitis , Bordetella/genética , Infecciones por Bordetella/diagnóstico , Humanos , Missouri , Peritonitis/diagnósticoRESUMEN
Vitamin B7 (biotin) is essential for normal health and its deficiency/suboptimal levels occur in a variety of conditions including chronic alcoholism. Mammals, including humans, obtain biotin from diet and gut-microbiota via absorption along the intestinal tract. The absorption process is carrier mediated and involves the sodium-dependent multivitamin transporter (SMVT; SLC5A6). We have previously shown that chronic alcohol exposure significantly inhibits intestinal/colonic biotin uptake via suppression of Slc5a6 transcription in animal and cell line models. However, little is known about the transcriptional/epigenetic factors that mediate this suppression. In addition, the effect of alcohol metabolites (generated via alcohol metabolism by gut microbiota and host tissues) on biotin uptake is still unknown. To address these questions, we first demonstrated that chronic alcohol exposure inhibits small intestinal and colonic biotin uptake and SMVT expression in human differentiated enteroid and colonoid monolayers. We then showed that chronic alcohol exposures of both, Caco-2 cells and mice, are associated with a significant suppression in expression of the nuclear factor KLF-4 (needed for Slc5a6 promoter activity), as well as with epigenetic alterations (histone modifications). We also found that chronic exposure of NCM460 human colonic epithelial cells as well as human differentiated colonoid monolayers, to alcohol metabolites (acetaldehyde, ethyl palmitate, ethyl oleate) significantly inhibited biotin uptake and SMVT expression. These findings shed light onto the molecular/epigenetic mechanisms that mediate the inhibitory effect of chronic alcohol exposure on intestinal biotin uptake. They further show that alcohol metabolites are also capable of inhibiting biotin uptake in the gut.NEW & NOTEWORTHY Using complementary models, including human differentiated enteroid and colonoid monolayers, this study shows the involvement of molecular and epigenetic mechanisms in mediating the inhibitory effect of chronic alcohol exposure on biotin uptake along the intestinal tract. The study also shows that alcohol metabolites (generated by gut microbiota and host tissues) cause inhibition in gut biotin uptake.
Asunto(s)
Biotina/metabolismo , Metilación de ADN , Epigénesis Genética , Etanol/farmacología , Mucosa Intestinal/efectos de los fármacos , Acetaldehído/farmacología , Animales , Células CACO-2 , Células Cultivadas , Etanol/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Ratones Endogámicos C57BL , Ácidos Oléicos/farmacología , Ácidos Palmíticos/farmacología , Simportadores/genética , Simportadores/metabolismoRESUMEN
The primary objective of this cross-sectional study was to examine the associations of self-reported health, happiness, marital happiness, frequency of sexual activity, and number of partners from a multinational survey of individuals who are consensually non-monogamous (CNM) or open to being CNM, completed in 2012 with 4062 respondents. We compared data from this survey with the 2010-2014 US General Social Surveys (GSS). This study explored these variables and their predictors by gender (including 612 non-binary-gendered CNM individuals), marital status, number of partners, sexual frequency, age, education, and income and were broken down by behavioral sexual orientation, marital status, and other relevant categories. Respondents in our CNM sample generally reported being as healthy (sometimes healthier; e.g., all respondents M-W Z = 7.66, p < .001, η2 = 0.007), happy (frequently happier; e.g., multiple-partnered Z = 15.43, p < .001, η2 = 0.069), happy in their marriages (in some cases happier; e.g., multiple-partnered females Z = 2.61, p = .009, η2 = 0.067), and reported having more frequent sexual activity (e.g., all Z = 29.54, p < .001, η2 = 0.094) with more partners (e.g., all Z = 60.75, p < .001, η2 = 0.393) compared to corresponding individuals within the GSS. This study contributes to knowledge about commonalities and differences between the general population and those who are CNM regarding health, happiness, and happiness in marriage, including differences in optimal number of sexual partners and sexual frequency.
Asunto(s)
Matrimonio , Parejas Sexuales , Adulto , Estudios Transversales , Femenino , Felicidad , Humanos , Masculino , Estado Civil , Autoinforme , Conducta SexualRESUMEN
Modeling host-pathogen interactions with human intestinal epithelia using enteroid monolayers on permeable supports (such as Transwells) represents an alternative to animal studies or use of colon cancer-derived cell lines. However, the static monolayer model does not expose epithelial cells to mechanical forces normally present in the intestine, including luminal flow and serosal blood flow (shear force) or peristaltic forces. To determine the contribution of mechanical forces in the functional response of human small intestine to a virulence factor of a pathogenic intestinal bacterium, human jejunal enteroids were cultured as monolayers in microengineered fluidic-based Organ-Chips (Intestine-Chips) exposed to enterotoxigenic Escherichia coli heat-stable enterotoxin A (ST) and evaluated under conditions of static fluid, apical and basolateral flow, and flow plus repetitive stretch. Application of flow increased epithelial cell height and apical and basolateral secretion of cyclic GMP (cGMP) under baseline, unstimulated conditions. Addition of ST under flow conditions increased apical and basolateral secretion of cGMP relative to the level under static conditions but did not enhance intracellular cGMP accumulation. Cyclic stretch did not have any significant effect beyond that contributed by flow. This study demonstrates that fluid flow application initiates changes in intestinal epithelial cell characteristics relative to those of static culture conditions under both baseline conditions and with exposure to ST enterotoxin and suggests that further investigations of the application of these mechanical forces will provide insights into physiology and pathophysiology that more closely resemble intact intestine than study under static conditions.
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GMP Cíclico/fisiología , Escherichia coli Enterotoxigénica/fisiología , Enterotoxinas/fisiología , Infecciones por Escherichia coli/fisiopatología , Proteínas de Escherichia coli/fisiología , Intestino Delgado/fisiología , Transducción de Señal/fisiología , Estrés Mecánico , Toxinas Bacterianas , Humanos , Yeyuno/citología , Factores de Virulencia/fisiologíaRESUMEN
The third E. coli and the Mucosal Immune System (ECMIS) meeting was held at Ghent University in Belgium from 2 to 5 June 2019. It brought together an international group of scientists interested in mechanisms of colonization, host response, and vaccine development. ECMIS distinguishes itself from related meetings on these enteropathogens by providing a greater emphasis on animal health and disease and covering a broad range of pathotypes, including enterohemorrhagic, enteropathogenic, enterotoxigenic, enteroaggregative, and extraintestinal pathogenic Escherichia coli As it is well established that the genus Shigella represents a subspecies of E. coli, these organisms along with related enteroinvasive E. coli are also included. In addition, Tannerella forsythia, a periodontal pathogen, was presented as an example of a pathogen which uses its surface glycans for mucosal interaction. This review summarizes several highlights from the 2019 meeting and major advances to our understanding of the biology of these pathogens and their impact on the host.
Asunto(s)
Infecciones por Escherichia coli/inmunología , Escherichia coli/fisiología , Inmunidad Mucosa , Infecciones por Bacterias Gramnegativas/inmunología , Tannerella forsythia/fisiologíaRESUMEN
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.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Escherichia coli Enterotoxigénica/efectos de los fármacos , Inmunidad Innata , Proteínas y Péptidos Salivales/metabolismo , Péptidos Catiónicos Antimicrobianos/química , Células CACO-2 , Proteínas Fimbrias/metabolismo , HumanosRESUMEN
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.
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Antígenos Bacterianos/inmunología , Escherichia coli Enterotoxigénica/inmunología , Infecciones por Escherichia coli/inmunología , Vacunas contra Escherichia coli/inmunología , Anticuerpos Antibacterianos/inmunología , Proteínas Portadoras/inmunología , Proteínas de Escherichia coli/inmunología , Humanos , Glicoproteínas de Membrana/inmunología , Péptido HidrolasasRESUMEN
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.
Asunto(s)
Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Escherichia coli Enterotoxigénica/genética , Escherichia coli Enterotoxigénica/metabolismo , Enterotoxinas/genética , Enterotoxinas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Línea Celular , GMP Cíclico/metabolismo , Células Epiteliales/microbiología , Células Epiteliales/patología , Eliminación de Gen , Humanos , Proteínas de Transporte de Membrana/metabolismoRESUMEN
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.
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Adhesinas Bacterianas/metabolismo , Escherichia coli Enterotoxigénica/fisiología , Mucosa Intestinal/microbiología , Mucinas/metabolismo , Acetilgalactosamina/metabolismo , Adhesinas Bacterianas/genética , Animales , Células CACO-2 , Escherichia coli Enterotoxigénica/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Células HT29 , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mucina 2/genética , Mucina 2/metabolismo , Interferencia de ARNRESUMEN
Adhesion to epithelial cells and flagella-mediated motility are critical virulence traits for many Gram-negative pathogens, including enterotoxigenic Escherichia coli (ETEC), a major cause of diarrhoea in travellers and children in developing countries. Many flagellated pathogens export putative adhesins belonging to the two-partner secretion (TPS) family. However, the actual function of these adhesins remains largely undefined. Here we demonstrate that EtpA, a TPS exoprotein adhesin of enterotoxigenic E. coli, mimics and interacts with highly conserved regions of flagellin, the major subunit of flagella, and that these interactions are critical for adherence and intestinal colonization. Although conserved regions of flagellin are mostly buried in the flagellar shaft, our results suggest that they are at least transiently exposed at the tips of flagella where they capture EtpA adhesin molecules for presentation to eukaryotic receptors. Similarity of EtpA to molecules encoded by other motile pathogens suggests a potential common pattern for bacterial adhesion, whereas participation of conserved regions of flagellin in adherence has implications for development of vaccines for Gram-negative pathogens.
Asunto(s)
Adhesión Bacteriana , Escherichia coli Enterotoxigénica/citología , Escherichia coli Enterotoxigénica/metabolismo , Células Epiteliales/microbiología , Proteínas de Escherichia coli/metabolismo , Flagelos/metabolismo , Interacciones Huésped-Patógeno , Glicoproteínas de Membrana/metabolismo , Animales , Vacunas Bacterianas/inmunología , Línea Celular , Secuencia Conservada , Escherichia coli Enterotoxigénica/patogenicidad , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/prevención & control , Flagelos/química , Flagelina/química , Flagelina/inmunología , Flagelina/metabolismo , Intestino Delgado/citología , Intestino Delgado/microbiología , Ratones , Unión ProteicaRESUMEN
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.
Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas Portadoras/metabolismo , Escherichia coli Enterotoxigénica/enzimología , Escherichia coli Enterotoxigénica/metabolismo , Proteínas de Escherichia coli/metabolismo , Mucina 2/metabolismo , Factores de Virulencia/metabolismo , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Proteínas Portadoras/inmunología , Modelos Animales de Enfermedad , Escherichia coli Enterotoxigénica/inmunología , Infecciones por Escherichia coli/prevención & control , Proteínas de Escherichia coli/inmunología , Vacunas contra Escherichia coli/administración & dosificación , Vacunas contra Escherichia coli/inmunología , Humanos , Hidrólisis , Ratones , Péptido Hidrolasas , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/inmunología , Factores de Virulencia/inmunologíaRESUMEN
Enterotoxigenic Escherichia coli (ETEC) strains are among the most common causes of diarrheal illness worldwide. These pathogens disproportionately afflict children in developing countries, where they cause substantial morbidity and are responsible for hundreds of thousands of deaths each year. Although these organisms are important targets for enteric vaccines, most development efforts to date have centered on a subset of plasmid-encoded fimbrial adhesins known as colonization factors and heat-labile toxin (LT). Emerging data suggest that ETEC undergoes considerable changes in its surface architecture, sequentially deploying a number of putative adhesins during its interactions with the host. We demonstrate here that one putative highly conserved, chromosomally encoded adhesin, EaeH, engages the surfaces of intestinal epithelial cells and contributes to bacterial adhesion, LT delivery, and colonization of the small intestine.