A hallmark of enteroaggregative Escherichia coli (EAEC) infection is a formation of biofilm, which comprises a mucus layer with immersed bacteria in the intestines of patients. While studying the mucinolytic activity of Pic in an in vivo system, rat ileal loops, we surprisingly found that EAEC induced hypersecretion of mucus, which was accompanied by an increase in the number of mucus-containing goblet cells. Interestingly, an isogenic pic mutant (EAEC ⌬pic) was unable to cause this mucus hypersecretion. Furthermore, purified Pic was also able to induce intestinal mucus hypersecretion, and this effect was abolished when Pic was heat denatured. Site-directed mutagenesis of the serine protease catalytic residue of Pic showed that, unlike the mucinolytic activity, secretagogue activity did not depend on this catalytic serine protease motif. Other pathogens harboring the pic gene, such as Shigella flexneri and uropathogenic E. coli (UPEC), also showed results similar to those for EAEC, and construction of isogenic pic mutants of S. flexneri and UPEC confirmed this secretagogue activity. Thus, Pic mucinase is responsible for one of the pathophysiologic features of the diarrhea mediated by EAEC and the mucoid diarrhea induced by S. flexneri.

Many pathogenic bacteria subvert normal host cell processes by delivering effector proteins which mimic eukaryotic functions directly into target cells. EspF is a multifunctional protein injected into host cells by attaching and effacing pathogens, but its mechanism of action is not understood completely. In silico analyses of EspF revealed two key motifs: proline-rich domains and PDZ domain binding motifs. Such functional domains may allow EspF to act as an actin nucleation-promoting factor by mimicking host proteins. In agreement with these predictions, we found that EspF from rabbit enteropathogenic Escherichia coli (E22) participates in the regulation of actin polymerization by binding to a complex of proteins at the tight junctions (TJ). EspF bound to actin and profilin throughout the course of infection. However, after 2 h of infection, EspF also bound to the neural Wiskott-Aldrich syndrome protein and to the Arp2/3, zonula occludens-1 (ZO-1), and ZO-2 proteins. Moreover, EspF caused occludin, claudin, ZO-1, and ZO-2 redistribution and loss of transepithelial electrical resistance, suggesting that actin sequestration by EspF may cause local actin depolymerization leading to EspF-induced TJ disruption. Furthermore, EspF caused recruitment of these TJ proteins into the pedestals. An E22 strain lacking EspF did not cause TJ disruption and pedestals were smaller than those induced by the wild-type strain. Additionally, the pedestals were located mainly in the TJ. The overexpression of EspF caused bigger pedestals located along the length of the cells. Thus, actin sequestration by EspF allows the recruitment of junctional proteins into the pedestals, leading to the maturation of actin pedestals and the disruption of paracellular permeability.

Propolis is a resin that honey bees (Apis mellifera) produce by mixing wax, exudates collected from tree shoots, pollen, and enzymes. It has been used for its biological properties against pathogenic microorganisms including those of viral origin. In the present study, we demonstrate the antiviral effect of Mexican propolis, as well as of the three commercial flavonoids (quercetin, naringenin, and pinocembrin) present in its composition, in cell cultures infected with Canine Distemper Virus. The treatments were carried out with propolis, flavonoids individually, and a mixture of the three flavonoids at three different times. Antiviral activity was evaluated by the inhibition of the relative expression of the virus nucleoprotein gene (Real-Time qPCR) and by the determination of cellular viability (MTT assay). Propolis applied before infection decreased viral expression (0.72 versus 1.0, 1.65, and 1.75 relative expressions) and correlated with increased cell viability (0.314 versus 0.215, 0.259, and 0.237 absorbance units (AU)). The administration of a flavonoid mixture containing the three commercial flavonoids before infection induces a slight decrease in viral expression (0.93 versus 1, 1.42, and 1.82 relative expressions); however, it does not improve cellular viability (0.255 versus 0.247, 0.282, and 0.245 AU). Quercetin administrated at the same time of infection decreases viral expression (0.90 versus 1.0, 3.25, and 1.02 relative expressions) and improves cellular viability (0.294 versus 0.240, 0.250, and 0.245 AU). Pinocembrin and naringenin individually did not show any antiviral activity at the administration times evaluated in this study. The present work is the first in vitro study of the effect of propolis in Canine Distemper Virus and demonstrated the antiviral activity of Mexican propolis, in addition to the synergy that exists between the three flavonoids on cell viability and the expression of the nucleoprotein virus gene.

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