Plasmid encoded toxin (Pet) is a serine protease originally described in enteroaggregative Escherichia coli (EAEC) prototype strain 042 whose entire characterization was essentially obtained from studies performed with the purified toxin. Here we show that Pet is not exclusive to EAEC. Atypical enteropathogenic Escherichia coli (aEPEC) strains, isolated from diarrhea cases, express Pet and its detection in supernatants of infected HEp-2 cells coincides with the appearance of cell damage, which, in turn, were similar to those described with purified Pet. Pet secretion and the cytotoxic effects are time and culture medium dependent. In presence of DMEM supplemented with tryptone cell rounding and detachment were observed after just 5 h of incubation with the bacteria. In the absence of tryptone, the cytotoxic effects were detected only after 24 h of infection. We also show that, in addition to the prototype EAEC, other pet+ EAEC strains, also isolated from diarrhea cases, induce cellular damage in the same degree as the aEPEC. The cytotoxic effects of EAEC and aEPEC strains were significantly reduced in the presence of a serine protease inhibitor or anti-Pet IgG serum. Our results show a common aspect between the aEPEC and EAEC and provide the first evidence pointing to a role of Pet in aEPEC pathogenesis.

No abstract

Using clonal phylogenetic methods, it has been demonstrated that O111:H25 atypical enteropathogenic E. coli (aEPEC) strains belong to distinct clones, suggesting the possibility that their ability to interact with different hosts and abiotic surfaces can vary from one clone to another. Accordingly, the ability of O111:H25 aEPEC strains derived from human, cat and dogs to adhere to epithelial cells has been investigated, along with their ability to interact with macrophages and to form biofilms on polystyrene, a polymer used to make biomedical devices. The results demonstrated that all the strains analyzed were able to adhere to, and to form pedestals on, epithelial cells, mechanisms used by E. coli to become strongly attached to the host. The strains also show a Localized-Adherence-Like (LAL) pattern of adhesion on HEp-2 cells, a behavior associated with acute infantile diarrhea. In addition, the O111:H25 aEPEC strains derived either from human or domestic animals were able to form long filaments, a phenomenon used by some bacteria to avoid phagocytosis. O111:H25 aEPEC strains were also encountered inside vacuoles, a characteristic described for several bacterial strains as a way of protecting themselves against the environment. They were also able to induce TNF-α release via two routes, one dependent on TLR-4 and the other dependent on binding of Type I fimbriae. These O111:H25 strains were also able to form biofilms on polystyrene. In summary the results suggest that, regardless of their source (i.e. linked to human origin or otherwise), O111:H25 aEPEC strains carry the potential to cause human disease.

, muito obrigada pelos conselhos, amizade, torcida e ajuda. À minha querida amiga "Bibi" de Andrade, não tenho como agradecer tudo que tem feito, nesses anos de amizade, só posso retribuir. Te Adoro! Ao Silvio pela amizade, companheirismo e ajuda nas análises de estatísticas. À Tatiane Porangaba, pela amizade, risadas e ajuda, nos PCR. Aos amigos Chris Ozaki Marina dos Anjos, Diego Mafra, Chyntia (Model), Demétria e D. Maria, pelo companheirismo, ajuda, conselhos e carinho. Aos meus queridos amigos de bancada Lívia Corrêa, Daniel Christo, Bete Soares, Camila Barbosa e Taysi Mazur, vocês fizeram a diferença, com certeza devo muito do meu aprendizado a vocês! Aos amigos do Laboratório de Bacteriologia (Ludmila, Davi (nosso querido amigo "perueiro"

Atypical enteropathogenic Escherichia coli, an important cause of diarrhea in both children and adults is highly heterogenic regarding its virulence factors, which may explain its increasing findings in epidemiologic studies. The increase in the numbers of diarrhea cases caused by aEPEC denotes its adaptability and pathogenicity, though no reports exist regarding its actions on professional phagocytes. The objective of this study was to investigate the behavior of aEPEC during interaction with macrophages. The study of phagocytosis was performed with the strains LB7 (O55:H7), LB13 (O111:abH9) and BA487 (O55:H7). Phagocytosis assays with J774A1 enabled the analysis of intracellular bacterial survival, the production of immune mediators, intracellular location and the role of some surface receptors. aEPEC survives inside J774A1. The induction of nitric oxide (NO) production differs between strains. LB7 and BA487 induced NO, while LB13, which is less phagocytosed, did not, probably because it uses the arginase pathway. Survival of aEPEC within J77A1 may result from the prevention of the maturation of parasitophorus vacuoles, as neither Rab5 nor Rab7, which are markers of early and late vacuoles, respectively, are not detected in vacuoles containing bacteria. Fc receptor is not involved in the phagocytosis of aEPEC. Phagocytosis of opsonized bacteria did not increase, nor did phagocytosis decrease when Fc receptor was blocked. The analysis of the participation of TLR4 was performed using primary culture C3H/HeJ, a spontaneous mutant for the gene tlr4. Survival of LB7 and BA487 slightly decreased after 24h of infection. However, there was no survival of any of the strains EPECa in C57BL/6, which present TLR4, after 24h of infection. aEPEC do not induce NO in C3H/HeJ and C57BL/6, only elevated levels of TNF were detected. Survival within macrophages depends on the origin of the macrophage. As opposed to C57BL/6, aEPEC survives in J774A1, even in the presence of NO and appears to interfere with vacuole maturation, which might prevent lysosome fusion, explain survival of the bacteria. In the study of anti-phagocytosis, our goal was to advance in the characterization of the anti-phagocytic factor (AF) secreted by LB7 and previously described by our group. The results show that AF is of a peptidic nature and that its anti-phagocytic activity is not specific, since, after pre-incubation with the macrophages, it inhibited phagocytosis of Shigella flexneri and of latex particles. AF is secreted at 20 o C as well as at 37 o C. Due to the complexity of the TSB medium, AF could not be identified. For that reason, its secretion was evaluated in defined media, such as M9, supplemented M9, DMEM and DMEM without phenol. AF was detected after fractionation through SPE of the culture supernatants, in all tested media. However, secretion in M9 was low, which prevented detection and isolation by HPLC. On the other hand, HPLC fractions from DMEM media were cytotoxic, hindering isolation and identification of AF. For the identificatio...