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The Type VI Secretion System Plays a Role in Type 1 Fimbria Expression and Pathogenesis of an Avian Pathogenic Escherichia coli Strain

ABSTRACT 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.

Characterization of IcmF of the type VI secretion system in an avian pathogenic Escherichia coli (APEC) strain

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.

Subpathotypes of Avian Pathogenic Escherichia coli (APEC) Exist as Defined by their Syndromes and Virulence Traits

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.

Influence of the major nitrite transporter NirC on the virulence of a Swollen Head Syndrome avian pathogenic E. coli (APEC) strain.

Avian Pathogenic Escherichia coli (APEC) strains are extra-intestinal E. coli that infect poultry and cause diseases. Nitrite is a central branch-point in bacterial nitrogen metabolism and is used as a cytotoxin by macrophages. Unlike nitric oxide (NO), nitrite cannot diffuse across bacterial membrane cells. The NirC protein acts as a specific channel to facilitate the transport of nitrite into Salmonella and E. coli cells for nitrogen metabolism and cytoplasmic detoxification. NirC is also required for the pathogenicity of Salmonella by downregulating the production of NO by the host macrophages. Based on an in vitro microarray that revealed the overexpression of the nirC gene in APEC strain SCI-07, we constructed a nirC-deficient SCI-07 strain (ΔnirC) and evaluated its virulence potential using in vivo and in vitro assays. The final cumulative mortalities caused by mutant and wild-type (WT) were similar; while the ΔnirC caused a gradual increase in the mortality rate during the seven days recorded, the WT caused mortality up to 24h post-infection (hpi). Counts of the ΔnirC cells in the spleen, lung and liver were higher than those of the WT after 48 hpi but similar at 24 hpi. Although similar number of ΔnirC and WT cells was observed in macrophages at 3 hpi, there was higher number of ΔnirC cells at 16 hpi. The cell adhesion ability of the ΔnirC strain was about half the WT level in the presence and absence of alpha-D-mannopyranoside. These results indicate that the nirC gene influences the pathogenicity of SCI-07 strain.

Green propolis modulates gut microbiota, reduces endotoxemia and expression of TLR4 pathway in mice fed a high-fat diet

Due to the various beneficial effects attributed to propolis, which include anti-inflammatory and anti-bacterial infection properties, the objective of the study was to evaluate the effect of propolis supplementation on the composition of the intestinal microbiota and its anti-inflammatory action. Forty male C57BL/6 mice were fed either a standard diet (control), a high-fat (HF) diet, or a high-fat diet supplemented with 0.2% crude propolis (HFP) for 2 or 5weeks prior to sacrifice. Blood samples were collected for the determination of lipopolysaccharide (LPS) and classical biochemical parameters. Expression of the TLR4 pathway in muscle, and DNA sequencing for the 16S rRNA of the gut microbiota were performed. The HF diet increased the proportion of the phylum Firmicutes and inflammatory biomarkers, while supplementation with propolis for five weeks rendered the microbiota profile nearly normal. Consistently with the above, the supplementation reduced levels of circulating LPS and down-regulated the TLR4 pathway and inflammatory cytokine expressions in muscle. Moreover, propolis improved such biochemical parameters as serum triacylglycerols and glucose levels. The data suggest that propolis supplementation reduces inflammatory response and endotoxemia by preventing dysbiosis in mice challenged with a high-fat diet.

Dietary whey proteins shield murine cecal microbiota from extensive disarray caused by a high-fat diet

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.

Ribotyping, biotyping and capsular typing of Haemophilus influenzae strains isolated from patients in Campinas, southeast Brazil

Forty-five Haemophilus influenzae strains isolated from patients were characterized based on biochemical characteristics. Their capsular types were determined by polymerase chain reaction (PCR); they were compared, using two molecular methods [ribotyping with a specific DNA probe amplified from the 16S rDNA region from H. influenzae and through restriction fragment length polymorphism (RLFP) of an amplified 16S DNA region]. The strains were better discriminated by the ribotyping technique that used the 16S probe and by the combination of both techniques. Biotypes I and IV were the most common, followed by biotypes VI, VIII and III. Biotypes II and VII were not found. Most of the capsular samples were nontypable (89%), with capsular types a and b found in 2 and 9% of the samples, respectively. We concluded that there is a very close genetic identity among pathogenic and non-pathogenic strains.

Prevalence of integrons in Shigella sonnei from Brazil

Diarrhea contributes to high morbidity and mortality rates in developing countries. Diarrhea from all causes is ranked as the fourth cause of death and the second cause of loss of years of productive life.1 Infection by Shigella spp. is characterized by a watery diarrhea that may progress to mucoid bloody diarrhea, also known as dysentery. Each year, over 163 million episodes of endemic shigellosis occur in DCs, whereas only 1.5 million occur in developed ones.1 The increasing number of travelers to DCs, which have become attractive tourist destinations, has also increased the number of diarrhea episodes among millions of persons who travel each year from industrialized countries to DCs.2 Shigella dysenteriae and Shigella flexneri are the predominant species in tropical areas, whereas Shigella sonnei is predominantly isolated in industrialized countries. Although the disease is often self-limiting, effective antimicrobial therapy reduces the duration and severity of the dysentery and can also prevent potentially lethal complications. Concomitantly, the excretion of the pathogen in stools is shortened significantly by antibiotic therapy, reducing the spread of infection.3 New research suggests that gene cassettes and integrons may be one mechanism involved in transmission of multidrug resistance genes in many Gram negative bacteria.4,5 In Shigella spp., the dissemination of resistance-inducing genes is mostly facilitated by the ability of the bacteria to acquire transposons or plasmids that might contain integrons and DNA sequences that consist of two conserved segments (5¢-CS and 3¢-CS) separated by a variable region that usually comprises one or more gene cassettes. The 5¢-CS region contains the integrase gene (intI), the integration site and a promoter region that allows expression of any number of gene cassettes inserted at the integration site site in a suitable orientation. The 3¢-CS region usually comprises qacE D1 (encoding resistance to quaternary ammonium compounds), sul (resistance to sulfonamides), followed by ORF5 (of unknown function) and/or tni genes (transposition functions).6 Detailed studies have been conducted on three main classes of integrons, namely integron 1 (Int1), integron 2 (Int2) and integron 3 (Int3), each one characterised by the presence of a distinct and specific intI.7 In the present study, 25 different strains of S. sonnei, obtained from sporadic cases of shigellosis, were isolated in hospitals from Brazil’s south eastern district of São Paulo (Campinas region), from six different Brazilian cities namely Campinas, Bragança Paulista, Vinhedo, Mogi Guacu, Limeria and Cosmopolis from 1997 to 20028 (Table 1). The research aim is to investigate whether the resistance for two or more antibiotics encountered in these strains is associated with the presence of Int1, Int2 and Int3. The strains were tested with seven antibiotics (chloramphenicol, trimethoprim-sulfamethoxazole, streptomycin, sulfamethoxazole (SUT), cephalothin, ampicillin and tetracycline (TT)) to determine the antibiotic resistance profile of each strain according to the Manual of the Clinical and Laboratory Standards Institute.9 For the cell DNA extraction, only the bacterial colonies of virulent strains of Shigella were selected for analysis, on the basis of the uptake of Congo red from growth media. DNA was extracted according to the Spin Column Protocol (DNeasy Blood and Tissue Hand Book, Qiagen, USA, 2006) using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA, 2006). The primers specific to Int1 (5¢-ATGCCCGTTCCATACAGAAG-3¢/ 5¢-CGGCCTTGCTGTTCTTCTAC-3¢), Int2 (5¢-AATGCGTTGCACTT CATTTG-3¢/5¢-ATGGGCAGTGAAGAGGTCAG-3¢) and Int3 (5¢-CCG GTTCAGTCTTTCCTCAA-3¢/5¢-GAGGCGTGTATCTGCCTCAT-3¢) were designed using Primer3 software (http://frodo.wi.mit.edu/). The National Center for Biotechnology Information database (http:// www.ncbi.nlm.nih.gov/nuccore/) for Int1, Int2 and Int3 are, respectively, Locus FJ501977.1, Locus EF560799.1 and Locus AY219651.1. The sequences obtained were used to design the primers, for which real-time PCR analysis was carried out on the samples for the amplification of the genes Int1, Int2 and Int3 and was on the basis of the work of Maguire et al.10 with a few adaptations. The DNA from PCR was purified using a DNA purification kit (Qiagen, USA, 2006) and later it was sent for sequencing at MWG (Eurofins MWG operon, www.operon.com) to verify and confirm the presence of Int1, Int2 and Int3. The amplification signals for Int1 were registered between cycles 20–25 and showed a clear consistent signal until the 50th cycle for all strains except strains CS7 and CS16C, which failed to register any

Helminth infection in mice improves insulin sensitivity via modulation of gut microbiota and fatty acid metabolism

&NA; Intestinal helminths are prevalent in individuals who live in rural areas of developing countries, where obesity, type 2 diabetes, and metabolic syndrome are rare. In the present study, we analyzed the modulation of the gut microbiota in mice infected with the helminth Strongyloides venezuelensis, and fed either a standard rodent chow diet or high‐fat diet (HFD). To investigate the effects of the microbiota modulation on the metabolism, we analyzed the expression of tight‐junction proteins present in the gut epithelium, inflammatory markers in the serum and tissue and quantified glucose tolerance and insulin sensitivity and resistance. Additionally, the levels of lipids related to inflammation were evaluated in the feces and serum. Our results show that infection with Strongyloides venezuelensis results in a modification of the gut microbiota, most notably by increasing Lactobacillus spp. These modifications in the microbiota alter the host metabolism by increasing the levels of anti‐inflammatory cytokines, switching macrophages from a M1 to M2 pattern in the adipose tissue, increasing the expression of tight junction proteins in the intestinal cells (thereby reducing the permeability) and decreasing LPS in the serum. Taken together, these changes correlate with improved insulin signaling and sensitivity, which could also be achieved with HFD mice treated with probiotics. Additionally, helminth infected mice produce higher levels of oleic acid, which participates in anti‐inflammatory pathways. These results suggest that modulation of the microbiota by helminth infection or probiotic treatment causes a reduction in subclinical inflammation, which has a positive effect on the glucose metabolism of the host. Graphical abstract Figure. No caption available.

Cloning and Purification of IpaC Antigen from Shigella flexneri: Proposal of a New Methodology

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.