Effects of lng Mutations on LngA Expression, Processing, and CS21 Assembly in Enterotoxigenic Escherichia coli E9034A

Enterotoxigenic Escherichia coli (ETEC) is a major cause of morbidity in children under 5 years of age in low- and middle-income countries and a leading cause of traveler's diarrhea worldwide. The ability of ETEC to colonize the intestinal epithelium is mediated by fimbrial adhesins, such as CS21 (Longus). This adhesin is a type IVb pilus involved in adherence to intestinal cells in vitro and bacterial self-aggregation. Fourteen open reading frames have been proposed to be involved in CS21 assembly, hitherto only the lngA and lngB genes, coding for the major (LngA) and minor (LngB) structural subunit, have been characterized. In this study, we investigated the role of the LngA, LngB, LngC, LngD, LngH, and LngP proteins in the assembly of CS21 in ETEC strain E9034A. The deletion of the lngA, lngB, lngC, lngD, lngH, or lngP genes, abolished CS21 assembly in ETEC strain E9034A and adherence to HT-29 cells was reduced 90%, compared to wild-type strain. Subcellular localization prediction of CS21 proteins was similar to other well-known type IV pili homologues. We showed that LngP is the prepilin peptidase of LngA, and that ETEC strain E9034A has another peptidase capable of processing LngA, although with less efficiency. Additionally, we present immuno-electron microscopy imagens to show that the LngB protein could be localized at the tip of CS21, and probably helps to control CS21 length. In conclusion, our results demonstrate that the LngA, LngB, LngC, LngD, LngH, and LngP proteins are essential for CS21 assembly, as well as for bacterial aggregation and adherence to HT-29 cells

Biogénesis y regulación de longus :una adhesina de escherichia coli enterotoxigénica

Ciencias Biomedica

The Assembly of Flagella in Enteropathogenic Escherichia coli Requires the Presence of a Functional Type III Secretion System

In enteropathogenic Escherichia coli (EPEC), the production of flagella and the type III secretion system (T3SS) is activated in the presence of host cultured epithelial cells. The goal of this study was to investigate the relationship between expression of flagella and the T3SS. Mutants deficient in assembling T3SS basal and translocon components (ΔespA, ΔespB, ΔespD, ΔescC, ΔescN, and ΔescV), and in secreting effector molecules (ΔsepD and ΔsepL) were tested for flagella production under several growth conditions. The ΔespA mutant did not produce flagella in any condition tested, although fliC was transcribed. The remaining mutants produced different levels of flagella upon growth in LB or in the presence of cells but were significantly diminished in flagella production after growth in Dulbecco’s minimal essential medium. We also investigated the role of virulence and global regulator genes in expression of flagella. The ΔqseB and ΔqseC mutants produced abundant flagella only when growing in LB and in the presence of HeLa cells, indicating that QseB and QseC act as negative regulators of fliC transcription. The ΔgrlR, ΔperA, Δler, Δhns, and Δfis mutants produced low levels of flagella, suggesting these regulators are activators of fliC expression. These data suggest that the presence of an intact T3SS is required for assembly of flagella highlighting the existence in EPEC of a cross-talk between these two virulence-associated T3SSs

The Transcription of Flagella of Enteropathogenic Escherichia coli O127:H6 Is Activated in Response to Environmental and Nutritional Signals

The flagella of enteropathogenic Escherichia coli (EPEC) O127:H6 E2348/69 mediate adherence to host proteins and epithelial cells. What environmental and nutritional signals trigger or down-regulate flagella expression in EPEC are largely unknown. In this study, we analyzed the influence of pH, oxygen tension, cationic and anionic salts (including bile salt), carbon and nitrogen sources, and catecholamines on the expression of the flagellin gene (fliC) of E2348/69. We found that sodium bicarbonate, which has been shown to induce the expression of type III secretion effectors, down-regulated flagella expression, explaining why E2348/69 shows reduced motility and flagellation when growing in Dulbecco’s Minimal Essential Medium (DMEM). Further, growth under a 5% carbon dioxide atmosphere, in DMEM adjusted to pH 8.2, in M9 minimal medium supplemented with 80 mM glucose or sucrose, and in DMEM containing 150 mM sodium chloride, 0.1% sodium deoxycholate, or 30 µM epinephrine significantly enhanced fliC transcription to different levels in comparison to growth in DMEM alone. When EPEC was grown in the presence of HeLa cells or in supernatants of cultured HeLa cells, high levels (4-fold increase) of fliC transcription were detected in comparison to growth in DMEM alone. Our data suggest that nutritional and host signals that EPEC may encounter in the intestinal niche activate fliC expression in order to favor motility and host colonization

The EcpD Tip Adhesin of the <i>Escherichia coli</i> Common Pilus Mediates Binding of Enteropathogenic <i>E. coli</i> to Extracellular Matrix Proteins

The attachment of enteropathogenic Escherichia coli (EPEC) to intestinal epithelial cells is facilitated by several adhesins; however, the individual host-cell receptors for pili-mediated adherence have not been fully characterized. In this study, we evaluated the hypothesis that the E. coli common pilus (ECP) tip adhesin protein EcpD mediates attachment of EPEC to several extracellular matrix (ECM) glycoproteins (fibronectin, laminin, collagens I and IV, and mucin). We found that the ΔecpA mutant, which lacks production of the EcpA filament but retains EcpD on the surface, adhered to these glycoproteins below the wild-type levels, while the ΔecpD mutant, which does not display EcpA or EcpD, bound significantly less to these host glycoproteins. In agreement, a purified recombinant EcpD subunit bound significantly more than EcpA to laminin, fibronectin, collagens I and IV, and mucin in a dose-dependent manner. These are compelling data that strongly suggest that ECP-producing EPEC may bind to host ECM glycoproteins and mucins through the tip adhesin protein EcpD. This study highlights the versatility of EPEC to bind to different host proteins and suggests that the interaction of ECP with the host’s ECM glycoproteins may facilitate colonization of the intestinal mucosal epithelium

Tracking Bioluminescent ETEC during In vivo BALB/c Mouse Colonization

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of diarrhea worldwide. Adhesion to the human intestinal tract is crucial for colonization. ETEC adhesive structures have been extensively studied; however, colonization dynamics remain uncharacterized. The aim of this study was to track bioluminescent ETEC during in vivo infection. The promoter region of dnaK was fused with the luc gene, resulting in the pRMkluc vector. E. coli K-12 and ETEC FMU073332 strains were electroporated with pRMkluc. E. coli K-12 pRMkluc was bioluminescent; in contrast, the E. coli K-12 control strain did not emit bioluminescence. The highest light emission was measured at 1.9 OD600 (9 h) and quantified over time. The signal was detected starting at time 0 and up to 12 h. Streptomycin-treated BALB/c mice were orogastrically inoculated with either ETEC FMU073332 pRMkluc or E. coli K-12 pRMkluc (control), and bacterial colonization was determined by measuring bacterial shedding in the feces. ETEC FMU073332 pRMkluc shedding started and stopped after inoculation of the control strain, indicating that mouse intestinal colonization by ETEC FMU073332 pRMkluc lasted longer than colonization by the control. The bioluminescence signal of ETEC FMU073332 pRMkluc was captured starting at the time of inoculation until 12 h after inoculation. The bioluminescent signal emitted by ETEC FMU073332 pRMkluc in the proximal mouse ileum was located, and the control signal was identified in the cecum. The detection of maximal light emission and bioluminescence duration allowed us to follow ETEC during in vivo infection. ETEC showed an enhanced colonization and tropism in the mouse intestine compared with those in the control strain. Here, we report the first study of ETEC colonization in the mouse intestine accompanied by in vivo imaging

Clinical Characteristics of Coronavirus Disease (COVID-19) in Mexican Children and Adolescents

Background: We analyzed the demographic, clinical, and diagnostic data of children and adolescents in Mexico, from the first case of coronavirus disease (COVID-19) to 28 February 2022. Methods: Using the open databases of the Ministry of Health and a tertiary pediatric hospital, we obtained demographic and clinical data from the beginning of the COVID-19 pandemic until 28 February 2022. In addition, quantitative reverse-transcription polymerase chain reaction outputs were used to determine the viral load, and structural protein-based serology was performed to evaluate IgG antibody levels. Results: Of the total 437,832 children and adolescents with COVID-19, 1187 died. Of these patients, 1349 were admitted to the Hospital Infantil de Mexico Federico Gómez, and 11 died. Obesity, asthma, and immunosuppression were the main comorbidities, and fever, cough, and headache were the main symptoms. In this population, many patients have a low viral load and IgG antibody levels. Conclusion: During the first 2 years of the COVID-19 pandemic in Mexico, children and adolescents had low incidence and mortality. They are a heterogeneous population, but many patients had comorbidities such as obesity, asthma, and immunosuppression; symptoms such as fever, cough, and headache; and low viral load and IgG antibodies