Host-derived CEACAM-laden vesicles engage enterotoxigenic Escherichia coli for elimination and toxin neutralization.

Enterotoxigenic Escherichia coli (ETEC) cause hundreds of millions of diarrheal illnesses annually ranging from mildly symptomatic cases to severe, life-threatening cholera-like diarrhea. Although ETEC are associated with long-term sequelae including malnutrition, the acute diarrheal illness is largely self-limited. Recent studies indicate that in addition to causing diarrhea, the ETEC heat-labile toxin (LT) modulates the expression of many genes in intestinal epithelia, including carcinoembryonic cell adhesion molecules (CEACAMs) which ETEC exploit as receptors, enabling toxin delivery. Here, however, we demonstrate that LT also enhances the expression of CEACAMs on extracellular vesicles (EV) shed by intestinal epithelia and that CEACAM-laden EV increase in abundance during human infections, mitigate pathogen-host interactions, scavenge free ETEC toxins, and accelerate ETEC clearance from the gastrointestinal tract. Collectively, these findings indicate that CEACAMs play a multifaceted role in ETEC pathogen-host interactions, transiently favoring the pathogen, but ultimately contributing to innate responses that extinguish these common infections.

Application of DNA aptamers to block enterotoxigenic Escherichia coli toxicity in a Galleria mellonella larval model.

Enterotoxigenic Escherichia coli (ETEC) is the major bacterial cause of diarrheal diseases in pigs, particularly at young ages, resulting in significant costs to swine farming. The pathogenicity of ETEC is largely dependent on the presence of fimbriae and the ability to produce toxins. Fimbriae are responsible for their initial adhesion to the intestinal epithelial cells, leading to the onset of infection. In particular, the F4 type (K88) fimbriae are often attributed to neonatal infections and have also been associated with post-weaning diarrheal infections. This disease is traditionally prevented or treated with antibiotics, but their use is being severely restricted due to the emergence of resistant bacteria and their impact on human health. Emerging approaches such as aptamers that target the F4-type fimbriae and block the initial ETEC adhesion are a promising alternative. The aim of this study is to assess the effectiveness of two aptamers, Apt31 and Apt37, in controlling ETEC infection in the G. mellonella in vivo model. Initially, the dissociation constant (KD) of each aptamer against ETEC was established using real-time quantitative PCR methodology. Subsequently, different concentrations of the aptamers were injected into Galleria mellonella to study their toxicity. Afterwards, the anti-ETEC potential of Apt31 and Apt37 was assessed in the larvae model. The determined KD was 81.79 nM (95% CI: 31.21-199.4 nM) and 50.71 nM (95% CI: 26.52-96.15 nM) for the Apt31 and Apt37, respectively, showing no statistical difference. No toxicity was observed in G. mellonella following injection with both aptamers at any concentration. However, the administration of Apt31 together with ETEC-F4+ in G. mellonella resulted in a significant improvement of approximately 30% in both larvae survival and health index compared to ETEC-F4+ alone. These findings suggest that aptamers have promising inhibitory effect against ETEC infections and pave the way for additional in vivo studies.

The genetic resistance of sows to Escherichia coli F4 adhesion reduces their response to a vaccine containing F4 fimbriae but does not affect the preweaning performance of their susceptible piglets.

INTRODUCTION: Pigs without intestinal receptors for F4 fimbriae are congenitally resistant to F4 fimbriae-bearing enterotoxigenic Escherichia coli (ETEC F4). In general, 50 % and 100 % of piglets born to resistant (RR) sows crossed with hetero- or homozygous susceptible (SR, SS) boars, respectively, are susceptible but do not receive colostral antibodies against F4 fimbriae unless the sows have been vaccinated. The question arises as to whether resistant sows produce protective amounts of F4 antifimbrial antibodies after vaccination. The serum and colostrum antibody titres of 12 resistant and 12 susceptible vaccinated gilts were compared. The effect of the receptor status of the dam and sire on the preweaning performance of 5027 piglets was evaluated using Agroscope's recordings. The sows of the experimental herd, where ETEC F4 was circulating, were vaccinated against ETEC twice during the first pregnancy and once during each following pregnancy. The log2 transformed F4 antibody titres in the serum obtained after the second vaccine injection as well as in the colostrum of the 12 resistant animals were lower than the titres of the susceptible animals (serum: F4ab 11,19 ± 1,44 vs. 12,18 ± 1,33, P = 0,096; F4ac 10,03 ± 1,58 vs. 11,59 ± 1,43, P = 0,019; colostrum: F4ab 12,20 ± 2,41 vs. 14,02 ± 1,31, P = 0,033; F4ac 10,93 ± 2,46 vs. 13,03 ± 5,21, P = 0,006). The heat labile enterotoxin (LT) antibody titres after vaccination did not differ between susceptible and resistant animals (p > 0,10). Preweaning mortality in the offspring of RR sows × SS boars was slightly lower than in the offspring of SS sows × RR boars (P = 0,04), suggesting that the disease risk of susceptible piglets born to vaccinated resistant sows was not increased, even though they received colostrum with a slightly reduced content of antibody against F4 fimbriae.

INTRODUCTION: Les porcs dépourvus de récepteurs intestinaux pour les fimbriae F4 sont congénitalement résistants aux Escherichia coli entérotoxinogènes porteurs de fimbriae F4 (ETEC F4). En général, 50 % et 100 % des porcelets nés de truies résistantes (RR) croisées avec des verrats hétéro- ou homozygotes sensibles (SR, SS), respectivement, sont sensibles mais ne reçoivent pas d'anticorps colostraux contre les fimbriae F4, à moins que les truies n'aient été vaccinées. La question se pose de savoir si les truies résistantes produisent des quantités protectrices d'anticorps antifimbriae F4 après la vaccination. Les titres d'anticorps dans le sérum et le colostrum de 12 truies reproductrices vaccinées résistantes et de 12 truies reproductrices vaccinées sensibles ont été comparés et l'effet du statut récepteur de la mère et du père sur les performances avant sevrage de 5027 porcelets a été évalué. Les truies du troupeau expérimental, où circulait ETEC F4, ont été vaccinées deux fois au cours de la première gestation et une fois au cours de chaque gestation suivante contre ETEC. Les titres d'anticorps F4 transformés en log2 dans le sérum obtenu après la deuxième injection de vaccin ainsi que dans le colostrum des 12 animaux résistants étaient inférieurs aux titres des animaux sensibles (sérum : F4ab 11,19 ± 1,44 vs. 12,18 ± 1,33, P = 0,096 ; F4ac 10,03 ± 1,58 vs. 11,59 ± 1,43, P = 0,019 ; colostrum : F4ab 12,20 ± 2,41 vs. 14,02 ± 1,31, P = 0,033 ; F4ac 10,93 ± 2,46 vs. 13,03 ± 5,21, P = 0,006). Les titres d'anticorps contre l'entérotoxine thermolabile (LT) après la vaccination ne différaient pas entre les animaux sensibles et résistants (p > 0,10). La mortalité avant sevrage dans la progéniture des truies RR × verrats SS était légèrement inférieure à celle de la progéniture des truies SS × verrats RR (P = 0,04), ce qui suggère que le risque de maladie des porcelets sensibles nés de truies résistantes vaccinées n'a pas été augmenté, même s'ils ont reçu du colostrum avec une teneur légèrement réduite en anticorps contre les fimbriae F4.

Enterotoxigenic Escherichia coli heat labile enterotoxin affects neutrophil effector functions via cAMP/PKA/ERK signaling.

Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrheal illness in humans and animals, induced by enterotoxins produced by these pathogens. Despite the crucial role of neutrophils in combatting bacterial infections, our understanding of how enterotoxins impact neutrophil function is limited. To address this knowledge gap, we used heat-labile enterotoxin (LT) and heat-stable enterotoxin a (STa) to investigate their impact on the effector functions of neutrophils. Our study reveals that pSTa does not exert any discernible effect on the function of neutrophils. In contrast, LT altered the migration and phagocytosis of neutrophils and induced the production of inflammatory factors via activation of cAMP/PKA and ERK1/2 signaling. LT also attenuated the release of neutrophil extracellular traps by neutrophils via the PKA signaling pathway. Our findings provide novel insights into the impact of LT on neutrophil function, shedding light on the underlying mechanisms that govern its immunoregulatory effects. This might help ETEC in subverting the immune system and establishing infection.

Coupling enterotoxigenic Escherichia coli heat-stable peptide toxin with 8-arm PEG enhances immunogenicity.

Enterotoxigenic Escherichia coli (ETEC) strains, which produce the heat-stable enterotoxin (ST) either alone or in combination with the heat-labile enterotoxin, contribute to the bulk of the burden of child diarrheal disease in resource-limited countries and are associated with mortality. Developing an effective vaccine targeting ST presents challenges due to its potent enterotoxicity, non-immunogenicity, and the risk of autoimmune reaction stemming from its structural similarity to the human endogenous ligands, guanylin, and uroguanylin. This study aimed to assess a novel synthetic vaccine carrier platform employing a single chemical coupling step for making human ST (STh) immunogenic. Specifically, the method involved cross-linking STh to an 8-arm N-hydroxysuccinimide (NHS) ester-activated PEG cross-linker. A conjugate of STh with 8-arm structure was prepared, and its formation was confirmed through immunoblotting analysis. The impact of conjugation on STh epitopes was assessed using ELISAs with polyclonal and monoclonal antibodies targeting various epitopes of STh. Immunization of mice with the conjugate induced the production of anti-STh antibodies, exhibiting neutralizing activity against STh.

Host-derived CEACAM-laden vesicles engage enterotoxigenic E. coli for elimination and toxin neutralization.

Enterotoxigenic Escherichia coli (ETEC) cause hundreds of millions of diarrheal illnesses annually ranging from mildly symptomatic cases to severe, life-threatening cholera-like diarrhea. Although ETEC are associated with long-term sequelae including malnutrition, the acute diarrheal illness is largely self-limited. Recent studies indicate that in addition to causing diarrhea, the ETEC heat-labile toxin (LT) modulates the expression of many genes in intestinal epithelia, including carcinoembryonic cell adhesion molecules (CEACAMs) which ETEC exploit as receptors, enabling toxin delivery. Here however, we demonstrate that LT also enhances the expression of CEACAMs on extracellular vesicles (EV) shed by intestinal epithelia and that CEACAM-laden EV increase in abundance during human infections, mitigate pathogen-host interactions, scavenge free ETEC toxins, and accelerate ETEC clearance from the gastrointestinal tract. Collectively, these findings indicate that CEACAMs play a multifaceted role in ETEC pathogen-host interactions, transiently favoring the pathogen, but ultimately contributing to innate responses that extinguish these common infections.

Host species shapes genotype, antimicrobial resistance, and virulence profiles of enterotoxigenic Escherichia coli (ETEC) from livestock in the United States.

Enterotoxigenic Escherichia coli (ETEC) are significant pathogen in both cattle and pigs, causing diarrhea in these animals and leading to economic losses in the livestock industry. Understanding the dissimilarity in genotype, antimicrobial resistance (AMR), and virulence between bovine and swine ETEC is crucial for development of targeted preventive and therapeutic approaches for livestock. However, a comprehensive study on this area remains lacking. Here, we performed whole-genome sequencing-based analyses of bovine (n = 554) and swine (n = 623) ETEC collected in the United States over a 53-year period. We identified distinct ETEC genotypes (fimH type, O antigen, H antigen, sequence type) in cattle and pigs. Furthermore, specific AMR and virulence profiles were associated with bovine and swine ETEC. Compared to swine ETEC, bovine ETEC were less diverse in genotypes and had a significantly (P < 0.001) lower number of AMR genes per isolate but higher co-occurrence of Shiga toxin and enterotoxin genes. Our results provide an overview of the key genomic differences between bovine and swine ETEC in the United States, which might be attributed to host adaptation and antibiotic usage practice. Ongoing surveillance and research are essential to monitor the genetic diversity and AMR patterns of ETEC in different host species. IMPORTANCE: Enterotoxigenic Escherichia coli (ETEC)-associated diarrhea represent one of the most economically important diseases in the livestock industry. By analyzing over a thousand livestock-derived ETEC samples in the United States, our study unveiled a clear distinction in ETEC's genetic traits (i.e., genotypes, antimicrobial resistance [AMR], and virulence profiles) that might be tied to the different use of antibiotics in cattle and pigs, and the bacteria's adaptation to their specific animal hosts. This understanding is crucial for tailoring preventive and therapeutic strategies. It also highlights the significance of ongoing surveillance and research into the evolution of bacterial pathogens like ETEC in livestock by using advanced techniques such as whole-genome sequencing.

Metallo-Glycodendrimeric Materials against Enterotoxigenic Escherichia coli.

Conjugation of carbohydrates to nanomaterials has been extensively studied and recognized as an alternative in the biomedical field. Dendrimers synthesized with mannose at the end group and with entrapped zero-valent copper/silver could be a potential candidate against bacterial proliferation. This study is aimed at investigating the bactericidal activity of metal-glycodendrimers. The Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction was used to synthesize a new mannosylated dendrimer containing 12 mannopyranoside residues in the periphery. The enterotoxigenic Escherichia coli fimbriae 4 (ETEC:F4) viability, measured at 600 nm, showed the half-inhibitory concentration (IC50) of metal-free glycodendrimers (D), copper-loaded glycodendrimers (D:Cu) and silver-loaded glycodendrimers (D:Ag) closed to 4.5 × 101, 3.5 × 101 and to 1.0 × 10-2 µg/mL, respectively, and minimum inhibitory concentration (MIC) of D, D:Cu and D:Ag of 2.0, 1.5 and 1.0 × 10-4 µg/mL, respectively. The release of bacteria contents onto broth and the inhibition of ETEC:F4 biofilm formation increased with the number of metallo-glycodendrimer materials, with a special interest in silver-containing nanomaterial, which had the highest activity, suggesting that glycodendrimer-based materials interfered with bacteria-bacteria or bacteria-polystyrene interactions, with bacteria metabolism and can disrupt bacteria cell walls. Our findings identify metal-mannose-dendrimers as potent bactericidal agents and emphasize the effect of entrapped zero-valent metal against ETEC:F4.

A Novel Multiepitope Fusion Antigen as a Vaccine Candidate for the Prevention of Enterotoxigenic E. coli-Induced Calf Diarrhea.

Calf diarrhea caused by enterotoxigenic E. coli (ETEC) poses an enormous economic challenge in the cattle industry. Fimbriae and enterotoxin are crucial virulence factors and vaccine targets of ETEC. Since these proteins have complicated components with large molecular masses, the development of vaccines by directly expressing these potential targets is cumbersome Therefore, this study aimed to develop a multiepitope fusion antigen designated as MEFA by integrating major epitopes of FanC and Fim41a subunits and a toxoid epitope of STa into the F17G framework. The 3D modeling predicted that the MEFA protein displayed the epitopes from these four antigens on its surface, demonstrating the desired structural characteristics. Then, the MEFA protein was subsequently expressed and purified for mouse immunization. Following that, our homemade ELISA showed that the mouse antiserum had a consistent increase in polyclonal antibody levels with the highest titer of 1:217 to MEFA. Furthermore, the western blot assay demonstrated that this anti-MEFA serum could react with all four antigens. Further, this antiserum exhibited inhibition on ETEC adhesion to HCT-8 cells with inhibitory rates of 92.8%, 84.3%, and 87.9% against F17+, F5+, and F41+ ETEC strains, respectively. Additionally, the stimulatory effect of STa toxin on HCT-8 cells was decreased by approximately 75.3% by anti-MEFA serum. This study demonstrates that the MEFA protein would be an antigen candidate for novel subunit vaccines for preventing ETEC-induced diarrhea in cattle.

Construction of the ETECFinder database for the characterization of enterotoxigenic Escherichia coli (ETEC) and revision of the VirulenceFinder web tool at the CGE website.

The identification of pathogens is essential for effective surveillance and outbreak detection, which lately has been facilitated by the decreasing cost of whole-genome sequencing (WGS). However, extracting relevant virulence genes from WGS data remains a challenge. In this study, we developed a web-based tool to predict virulence-associated genes in enterotoxigenic Escherichia coli (ETEC), which is a major concern for human and animal health. The database includes genes encoding the heat-labile toxin (LT) (eltA and eltB), heat-stable toxin (ST) (est), colonization factors CS1 through 30, F4, F5, F6, F17, F18, and F41, as well as toxigenic invasion and adherence loci (tia, tibAC, etpBAC, eatA, yghJ, and tleA). To construct the database, we revised the existing ETEC nomenclature and used the VirulenceFinder webtool at the CGE website [VirulenceFinder 2.0 (dtu.dk)]. The database was tested on 1,083 preassembled ETEC genomes, two BioProjects (PRJNA421191 with 305 and PRJNA416134 with 134 sequences), and the ETEC reference genome H10407. In total, 455 new virulence gene alleles were added, 50 alleles were replaced or renamed, and two were removed. Overall, our tool has the potential to greatly facilitate ETEC identification and improve the accuracy of WGS analysis. It can also help identify potential new virulence genes in ETEC. The revised nomenclature and expanded gene repertoire provide a better understanding of the genetic diversity of ETEC. Additionally, the user-friendly interface makes it accessible to users with limited bioinformatics experience. IMPORTANCE: Detecting colonization factors in enterotoxigenic Escherichia coli (ETEC) is challenging due to their large number, heterogeneity, and lack of standardized tests. Therefore, it is important to include these ETEC-related genes in a more comprehensive VirulenceFinder database in order to obtain a more complete coverage of the virulence gene repertoire of pathogenic types of E. coli. ETEC vaccines are of great importance due to the severity of the infections, primarily in children. A tool such as this could assist in the surveillance of ETEC in order to determine the prevalence of relevant types in different parts of the world, allowing vaccine developers to target the most prevalent types and, thus, a more effective vaccine.