VirK Is a Periplasmic Protein Required for Efficient Secretion of Plasmid-Encoded Toxin from Enteroaggregative Escherichia coli

Despite the autotransporter (AT) moniker, AT secretion appears to involve the function of periplasmic chaperones. We identified four periplasmic proteins that specifically bound to plasmid-encoded toxin (Pet), an AT produced by enteroaggregative Escherichia coli (EAEC). These proteins include the 17-kDa Skp chaperone and the 37-kDa VirK protein. We found that the virK gene is present in different Enterobacteriaceae. VirK bound to misfolded conformations of the Pet passenger domain, but it did not bind to the folded passenger domain or to the beta domain of Pet. Assays with an EAEC Delta virK mutant and its complemented version showed that, in the absence of VirK, Pet was not secreted but was instead retained in the periplasm as proteolytic fragments. In contrast, Pet was secreted from a Delta skp mutant. VirK was not required for the insertion of porin proteins into the outer membrane but assisted with insertion of the Pet beta domain into the outer membrane. Loss of VirK function blocked the EAEC-mediated cytotoxic effect against HEp-2 cells. Thus, VirK facilitates the secretion of the AT Pet by maintaining the passenger domain in a conformation that both avoids periplasmic proteolysis and facilitates beta-domain insertion into the outer membrane

New Perspectives on the Application of Chito-Oligosaccharides Derived from Chitin and Chitosan: A Review

The study of chitin and chitosan has stood out for many years due to their potential application in various areas such as the food industry, where they are either used as additives, prebiotics, or bio-conservatives; as to biomedical and pharmaceutical industries, where they function to treat diseases. Besides, in the agriculture field, it is known that they can cause a positive effect on the development of plants and optimize nitrogen fixation. In recent years, attention has been paid to their derivatives, chito-oligosaccharides which, unlike chitin and chitosan, they have different chemical characteristics, like their solubility, a characteristic that facilitates their use, contrary to chitin and chitosan. Moreover, the small size of chito-oligosaccharides can facilitate their entry into the cell. This review covers recent studies on the biological functions of chito-oligosaccharides and their impact on a priority area such as agriculture, where these compounds could be used to substitute the demand for chemical compounds that, until now, have generated serious health issues as well as environmental pollution

Pathogenic lifestyles of E. coli pathotypes in a standarized epithelial cell model influence inflammatory signaling pathways and cytokines secretion

Inflammatory response is key for the host defense against diarrheagenic E. coli and contributes to the pathogenesis of the disease but there is not a comparative study among different diarrheagenic pathotypes. We analyzed the inflammatory response induced by five diarrheagenic pathotypes in a HT-29 cell infection model. The model was unified to reproduce the pathogenesis of each pathotype. To compare the inflammatory responses we evaluated: (i) nuclear NF-κB and ERK1/2 translocation by confocal microscopy; (ii) kinetics of activation by each pathway detecting p65 and ERK1/2 phosphorylation by Western blotting; (iii) pathways modulation through bacterial infections with or without co-stimulation with TNF-α or EGF; (iv) cytokine profile induced by each pathotype with and without inhibitors of each pathway. EHEC but mainly EPEC inhibited translocation and activation of p65 and ERK1/2 pathways, as well as cytokines secretion; inhibition of p65 and ERK1/2 phosphorylation prevailed in the presence of TNF-α and EGF, respectively. Intracellular strains, EIEC/S. flexneri, caused a strong translocation, activation and cytokines secretion but they could not inhibit TNF-α and EGF stimulation. ETEC and mainly EAEC caused a moderate translocation, but a differential activation, and high cytokines secretion; interestingly TNF-α and EGF stimulation did no modify p65 and ERK1/2 activation. The use of inhibitors of NF-κB and/or ERK1/2 showed that NF-κB is crucial for cytokine induction by the different pathotypes; only partially depended on ERK1/2 activation. Thus, in spite of their differences, the pathotypes can also be divided in three groups according to their inflammatory response as those (i) that inject effectors to cause A/E lesion, which are able to inhibit NF-κB and ERK1/2 pathways, and cytokine secretion; (ii) with fimbrial adherence and toxin secretion with a moderate inhibition of both pathways but high cytokines secretion through autocrine cytokine regulation; and (iii) the intracellular bacteria that induce the highest pathways activation and cytokines secretion by using different activation mechanisms. This study provides a comprehensive analysis of how the different pathogenesis schemes of E. coli pathotypes manipulate inflammatory signaling pathways, which leads to a specific proinflammatory cytokine secretion in a cell model infection that reproduce the hallmarks of infection of each pathotype

Actin Cytoskeleton Manipulation by Effector Proteins Secreted by Diarrheagenic Escherichia coli Pathotypes

The actin cytoskeleton is a dynamic structure necessary for cell and tissue organization, including the maintenance of epithelial barriers. Disruption of the epithelial barrier coincides with alterations of the actin cytoskeleton in several disease states. These disruptions primarily affect the paracellular space, which is normally regulated by tight junctions. Thereby, the actin cytoskeleton is a common and recurring target of bacterial virulence factors. In order to manipulate the actin cytoskeleton, bacteria secrete and inject toxins and effectors to hijack the host cell machinery, which interferes with host-cell pathways and with a number of actin binding proteins. An interesting model to study actin manipulation by bacterial effectors is Escherichia coli since due to its genome plasticity it has acquired diverse genetic mobile elements, which allow having different E. coli varieties in one bacterial species. These E. coli pathotypes, including intracellular and extracellular bacteria, interact with epithelial cells, and their interactions depend on a specific combination of virulence factors. In this paper we focus on E. coli effectors that mimic host cell proteins to manipulate the actin cytoskeleton. The study of bacterial effector-cytoskeleton interaction will contribute not only to the comprehension of the molecular causes of infectious diseases but also to increase our knowledge of cell biology

Antigiardial Activity of Podophyllotoxin-Type Lignans from Bursera fagaroides var. fagaroides

Giardiasis, a diarrheal disease, is highly prevalent in developing countries. Several drugs are available for the treatment of this parasitosis; unfortunately, all of them have variable efficacies and adverse effects. Bursera fagaroides has been known for its anti-inflammatory and antidiarrheal properties in Mexican traditional medicine. We investigated the in vitro anti-giardial activities of four podophyllotoxin-type lignans from Bursera fagaroides var. fagaroides, namely, 5′-desmethoxy-β-peltatin-A-methylether (5-DES), acetylpodophyllotoxin (APOD), burseranin (BUR), and podophyllotoxin (POD). All lignans affected the Giardia adhesion and electron microscopy images revealed morphological alterations in the caudal region, ventral disk, membrane, and flagella, to different extents. Only 5-DES, APOD, and POD caused growth inhibition. Using the Caco-2 human cell line as a model of the intestinal epithelium, we demonstrated that APOD displayed direct antigiardial killing activity and low toxicity on Caco-2 cells. This finding makes it an attractive potential starting point for new antigiardial drugs

Drug Susceptibility Testing and Synergistic Antibacterial Activity of Curcumin with Antibiotics against Enterotoxigenic Escherichia coli

Aim: This study investigated the susceptibility of Enterotoxigenic Escherichia coli to curcumin, as well as its synergistic effect with 12 antimicrobial drugs. Methods and Results: Our study shows that curcumin did not affect bacterial growth. The antimicrobial susceptibility of curcumin and antibiotic synergy were identified using disc diffusion on Mueller-Hinton agar. The strain of Enterotoxigenic Escherichia coli used was resistant to Ampicillin, Amoxicillin/Clavulanic acid, Ampicillin/Sulbactam, Ciprofloxacin, and Cefazolin. There was synergy between curcumin and the majority of antibiotics tested. Maximum synergy was observed with combinations of 330 µg/mL curcumin and Ceftazidime, followed by Cefotaxime, Amoxicillin/Clavulanic acid, Ampicillin, Aztreonam, Trimethoprim, Ciprofloxacin, Ceftriaxone, Cefazolin, Tetracycline, and Imipenem. Conclusion: Our findings indicated that curcumin might be useful as a combinatorial strategy to combat the antibiotic resistance of Enterotoxigenic Escherichia coli