RESUMO
Avian Pathogenic Escherichia coli is responsible for significant economic losses in the poultry industry by causing a range of systemic or localized diseases collectively termed colibacillosis. The virulence mechanisms of these strains that are pathogenic in poultry and possibly pathogenic in humans have not yet been fully elucidated. This work was developed to study if over-expressed genes in a microarray assay could be potentially involved in the pathogenicity of an Avian Pathogenic Escherichia coli strain isolated from a swollen head syndrome case. For this study, five over-expressed genes were selected for the construction of null mutants [flgE (flagellar hook), tyrR (transcriptional regulator), potF (putrescine transporter), yehD (putative adhesin) and bfr (bacterioferritin)]. The constructed mutants were evaluated for their capacity for the adhesion and invasion of in vitro cultured cells, their motility capacity, and their pathogenic potential in one-day-old chickens compared with the wild-type strain (WT). The Δbfr strain showed a decreased adhesion capacity on avian fibroblasts compared with WT, in the presence and absence of alpha-D-mannopyranoside, and the ΔpotF strain showed decreased adhesion only in the absence of alpha-D-mannopyranoside. The ΔtyrR mutant had a reduced ability to invade Hep-2 cells. No mutant showed changes in invading CEC-32 cells. The mutants ΔflgE and ΔtyrR showed a decreased ability to survive in HD-11 cells. The motility of the mutant strains Δbfr, ΔyehD and ΔpotF was increased, while the ΔtyrR mutant showed reduction, and the ΔflgE became non-motile. No mutant strain caused the same mortality of the WT in one-day-old chickens, showing attenuation to different degrees.
Assuntos
Galinhas/microbiologia , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Doenças das Aves Domésticas/microbiologia , Animais , Aderência Bacteriana/genética , Proteínas de Bactérias/genética , Linhagem Celular , Embrião de Galinha , Grupo dos Citocromos b/genética , Escherichia coli/isolamento & purificação , Escherichia coli/patogenicidade , Infecções por Escherichia coli/microbiologia , Feminino , Ferritinas/genética , Perfilação da Expressão Gênica/veterinária , Humanos , Mutação , Análise de Sequência com Séries de Oligonucleotídeos/veterinária , Regulação para Cima , Virulência , Fatores de Virulência/genéticaRESUMO
High-fat diets are used to induce adverse alterations in the intestinal microbiota, or dysbiosis, generalized inflammation and metabolic stress, which ultimately may lead to obesity. The influence of dietary whey proteins, whether intact or hydrolyzed, has been reported to improve glucose homeostasis and reduce stress. Therefore, the purpose of this work was to test if dietary milk-whey proteins, both in the intact form and hydrolyzed, could have an effect on the compositional changes of the cecal microbiota that can be induced in mice when receiving a high-fat diet in combination with the standard casein. Male C57BL/6 mice were fed a control casein diet (AIN 93-G); high-fat-casein (HFCAS); high-fat-whey protein concentrate (HFWPC) and high-fat whey-protein hydrolysate (HFWPH) for 9weeks. The intestinal microbiota composition was analyzed by 16S-rRNA of the invariant (V1-V3) gene, potentially endotoxemic lipopolysaccharide (LPS) release was determined colorimetrically, and liver fat infiltration assessed by light microscopy. The high-fat diet proved to induce dysbiosis in the animals by inverting the dominance of the phylum Firmicutes over Bacteroidetes, promoted the increase of LPS and resulted in liver fat infiltration. The whey proteins, whether intact or hydrolyzed, resisted the installation of dysbiosis, prevented the surge of circulating LPS and prevented fat infiltration in the liver. It is concluded that dietary whey proteins exert metabolic actions that tend to preserve the normal microbiota profile, while mitigating liver fat deposition in mice consuming a high-fat diet for nine weeks. Such beneficial effects were not seen when casein was the dietary protein. The hydrolyzed whey protein still differed from the normal whey protein by selectively protecting the Bacteroidetes phylum.
RESUMO
Shigella flexneri is a Gram-negative bacillus that is responsible for a severe form of dysentery called Shigellosis, which mainly affects children and the elderly in both underdeveloped and developed countries. Pathogenic S. flexneri strains possess a large virulence plasmid that codes for effector proteins that are required for the entry and spread of the bacteria into colonocytes. Among these proteins is the translocator IpaC, which plays an important role in the invasion process; IpaC is implicated in pore formation in the host cell membrane and induces cytoskeletal rearrangements in macrophages and epithelial cells, thereby promoting bacterial entry. The ability of IpaC to insert onto the plasma membrane is due to a large nonpolar region of the protein structure. This characteristic also renders difficulties in recovery and purification when the protein is expressed in E. coli. Several works have considered different methodologies for the improved production and purification of IpaC. Herein, we propose an alternative method that is based on changes in the induction temperature and extraction buffer to facilitate the accumulation of high yields of soluble proteins for their further processing and ultimate use in biotechnological approaches.
Assuntos
Antígenos de Bactérias/metabolismo , Shigella flexneri/metabolismo , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Cromatografia de Afinidade , Clonagem Molecular , Disenteria Bacilar/imunologia , Disenteria Bacilar/microbiologia , Ensaio de Imunoadsorção Enzimática , Shigella flexneri/imunologiaRESUMO
Avian pathogenic Escherichia coli (APEC) strains cause different types of systemic extraintestinal infections in poultry, collectively termed colibacillosis, which can cause significant economic losses in the poultry industry. To date, there have been no descriptions of genes or characteristics that allow for the classification of avian strains pathotypes responsible for causing specific diseases in their hosts. In this study we aimed to characterize avian E. coli strains representing 4 groups, including one of commensal strains (AFEC - Avian Fecal Escherichia coli) and 3 groups of APEC strains, where each group is responsible for causing a different disease syndrome in their respective hosts (septicemia, omphalitis and swollen head syndrome). We chose to examine several biological characteristics of these strains including: adhesion to eukaryotic cells, pathogenicity levels according to the lethal dose (50%) assay, phylogenetic group and virulence gene profiles. The comparison of strains based on these genotypic and phenotypic traits, using multivariate statisticals tools and complex networks, allowed us to infer information about the population structure of the studied groups. Our results indicate that APEC strains do not constitute a unique homogeneous group, but rather a structured set of subgroups, where each one is associated with a specific infectious syndrome which can possibly be used to define pathotypes or subpathotypes within APEC strains. These results offer new possibilities with which to study the genes responsible for various pathogenetic processes within APEC strains, and for vaccine development. It may be important to consider these subgroups when developing a vaccine in an effort for obtain cross protection, which has not yet been successfully accomplished when working with APEC strains.
RESUMO
The intracellular multiplication factor (IcmF) protein is a component of the recently described type VI secretion system (T6SS). IcmF has been shown to be required for intra-macrophage replication and inhibition of phagosome-lysosome fusion in Legionella pneumophila. In Vibrio cholerae it is involved in motility, adherence and conjugation. Given that we previously reported that two T6SS genes (hcp and clpV) contribute to the pathogenesis of a septicaemic strain (SEPT362) of avian pathogenic Escherichia coli (APEC), we investigated the function of IcmF in this strain. Further elucidation of the virulence mechanisms of APEC is important because this pathogen is responsible for financial losses in the poultry industry, and is closely related to human extraintestinal pathogenic E. coli (ExPEC) strains, representing a potential zoonotic risk, as well as serving as a reservoir of virulence genes. Here we show that an APEC icmF mutant has decreased adherence to and invasion of epithelial cells, as well as decreased intra-macrophage survival. The icmF mutant is also defective for biofilm formation on abiotic surfaces. Additionally, expression of the flagella operon is decreased in the icmF mutant, leading to decreased motility. The combination of these phenotypes culminates in this mutant being altered for infection in chicks. These results suggest that IcmF in APEC may play a role in disease, and potentially also in the epidemiological spread of this pathogen through enhancement of biofilm formation.
Assuntos
Sistemas de Secreção Bacterianos , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Doenças das Aves Domésticas/microbiologia , Animais , Aderência Bacteriana , Biofilmes , Linhagem Celular , Galinhas , Escherichia coli/genética , Escherichia coli/patogenicidade , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Humanos , VirulênciaRESUMO
Avian pathogenic Escherichia coli (APEC) strains frequently cause extraintestinal infections and are responsible for significant economic losses in the poultry industry worldwide. APEC isolates are closely related to human extraintestinal pathogenic E. coli (ExPEC) strains and may also act as pathogens for humans. Known APEC virulence factors include adhesins such as type 1 fimbriae and curli, iron acquisition systems, and cytotoxins. Here we show that APEC strain SEPT362, isolated from a septicemic hen, expresses a type VI secretion system (T6SS); causes cytoskeleton rearrangements; and invades epithelial cells, replicates within macrophages, and causes lethal disease in chicks. To assess the contribution of the T6SS to SEPT362 pathogenesis, we generated two mutants, hcp (which encodes a protein suggested to be both secreted and a structural component of the T6SS) and clpV (encoding the T6SS ATPase). Both mutants showed decreased adherence and actin rearrangement on epithelial cells. However, only the hcp mutant presented a mild decrease in its ability to invade epithelial cells, and none of these mutants were defective for intramacrophage replication. Transcriptome studies showed that the level of expression of type 1 fimbriae was decreased in these mutants, which may account for the diminished adhesion and invasion of epithelial cells. The T6SS seems to be important for the disease process, given that both mutants were attenuated for infection in chicks. These results suggest that the T6SS influences the expression of type 1 fimbriae and contributes to APEC pathogenesis.