Factors related to Campylobacter spp. carriage in client-owned dogs visiting veterinary clinics in a region of Ontario, Canada.

From July 2008 until May 2009, 240 client-owned pet dogs from seven veterinary clinics in the Region of Waterloo, Ontario, Canada participated in a study to determine pet-related management factors that may be associated with the presence of Campylobacter spp. in dogs. The prevalence of Campylobacter spp. carriage in our study population of pet dogs was 22%, with 19% of the dogs positive for C. upsaliensis, and 3% positive for C. jejuni. A significant risk factor from multivariable logistic regression models for both Campylobacter spp. and C. upsaliensis carriage was having homemade cooked food as the dog's diet or added to its diet, and a significant sparing factor for both models was treatment with antibiotics in the previous month. Increasing age of the dog decreased the odds of Campylobacter spp. and C. upsaliensis carriage. Based on the high prevalence of Campylobacter, and specifically C. upsaliensis, further research concerning pet dogs as a risk factor for campylobacteriosis in humans is warranted.

Targeted localized degradation of Paired protein in Drosophila development.

BACKGROUND: Selective spatial regulation of gene expression lies at the core of pattern formation in the embryo. In the fruit fly Drosophila, localized transcriptional regulation accounts for much of the embryonic pattern. RESULTS: We identified a gene, partner of paired (ppa), whose properties suggest that localized receptors for protein degradation are integrated into regulatory networks of transcription factors to ensure robust spatial regulation of gene expression. We found that the Ppa protein interacts with the Pax transcription factor Paired (Prd) and contains an F-box, a motif found in receptors for ubiquitin-mediated protein degradation. In normal development, Prd functions only in cells in which ppa mRNA expression has been repressed by another segmentation protein, Even-skipped (Eve). When ppa was expressed ectopically in these cells, Prd protein, but not mRNA, levels diminished. When ppa function was removed from cells that express prd mRNA, Prd protein levels increased. CONCLUSIONS: Ppa co-ordinates Prd degradation and is important for expression of Prd to be correctly localized. In the presence of Ppa, Prd protein is targeted for degradation at sites where its mis-expression would disrupt development. In the absence of Ppa, Prd is longer-lived and regulates downstream target genes.

Differential repression of GAL4 and adjacent transcription activators by operators in the yeast GAL upstream activating sequence.

The upstream activating sequence of the adjacent and divergently transcribed GAL1 and GAL10 genes of Saccharomyces cerevisiae (UASG) contains at least three distinct classes of overlapping transcriptional control sites. The transcription activator GAL4 binds to four related sites in UASG and induces expression of GAL1 and GAL10 when galactose is available. We showed that UASG contains two additional positive control sites, designated GAL4/galactose-independent activating elements (GAEs), which reside at positions adjacent to or overlapping the GAL4-binding sites. When separated from neighboring sequences in UASG, the GAEs activate transcription independently of GAL4 with no requirement for galactose. In the intact GAL1-GAL10 divergent promoter region, their activity is ordinarily repressed by multiple negative control elements, the GAL operators. When galactose is available, GAL4 overcomes the activity of the GAL operators, while the putative GAE-binding proteins stay repressed. Combined, these results imply that distinct activators (GAL4 and GAE proteins) bound at adjacent or overlapping sites in UASG are differentially regulated by putative repressor proteins simultaneously bound at adjacent GAL operators. We surmise that GAE1 and GAE2 may have a physiological function other than regulation of galactose catabolism per se and discuss three hypotheses to account for their presence in UASG.

Opposing regulatory functions of positive and negative elements in UASG control transcription of the yeast GAL genes.

The yeast GAL1 and GAL10 genes are transcribed at a remarkably low basal level when galactose is unavailable and are induced by over 4 orders of magnitude when it becomes available. Approximately six negative control elements (designated GAL operators GALO1 to GALO6) are located adjacent to or overlapping four binding sites for the transcription activator GAL4 in the GAL upstream activating sequence UASG. The negative control elements contribute to the broad range of inducibility of GAL1 and GAL10 by inhibiting two GAL4/galactose-independent activating elements (GAE1 and GAE2) in UASG. In turn, multiple GAL4-binding sites in UASG are necessary for GAL4 to overcome repression by the negative control elements under fully inducing conditions. When glucose in addition to galactose is available (repressing conditions), the ability of GAL4 to activate transcription is diminished as a result of its reduced affinity for DNA and the reduced availability of inducer. Under these conditions, the negative control elements inhibit transcriptional activation from the glucose-attenuated GAL4 sites, thus accounting at least in part for glucose repression acting in cis. A normal part of transcriptional regulation of the GAL1 and GAL10 genes, therefore, appears to involve a balance between the opposing functions of positive and negative control elements.

The Protein Interactome of Streptococcus pneumoniae and Bacterial Meta-interactomes Improve Function Predictions.

The functions of roughly a third of all proteins in Streptococcus pneumoniae, a significant human-pathogenic bacterium, are unknown. Using a yeast two-hybrid approach, we have determined more than 2,000 novel protein interactions in this organism. We augmented this network with meta-interactome data that we defined as the pool of all interactions between evolutionarily conserved proteins in other bacteria. We found that such interactions significantly improved our ability to predict a protein's function, allowing us to provide functional predictions for 299 S. pneumoniae proteins with previously unknown functions. IMPORTANCE Identification of protein interactions in bacterial species can help define the individual roles that proteins play in cellular pathways and pathogenesis. Very few protein interactions have been identified for the important human pathogen S. pneumoniae. We used an experimental approach to identify over 2,000 new protein interactions for S. pneumoniae, the most extensive interactome data for this bacterium to date. To predict protein function, we used our interactome data augmented with interactions from other closely related bacteria. The combination of the experimental data and meta-interactome data significantly improved the prediction results, allowing us to assign possible functions to a large number of poorly characterized proteins.

Progress and potential of Drosophila protein interaction maps.

Protein-protein interactions mediate many important cellular processes and are central to the mechanisms by which most proteins function. Charting the interactions among the proteins involved in a process has been an essential step in characterising the function of proteins and pathways. The yeast two-hybrid system is one approach to detecting protein interactions that can now be scaled-up to assay large sets of proteins systematically, such as those being identified from genome sequencing efforts. The system has already been extensively used to acquire data that have enabled construction of large protein interaction maps (PIMs). When combined with other data, including data being generated by other functional genomics approaches, PIMs help assign function to new proteins and delineate functional networks. Hypotheses generated in such a manner often must be tested by additional experimentation, preferably in vivo. The model organism Drosophila melanogaster has a wealth of genetic and bioinformatic tools available for such analyses. The proteome predicted from the recently sequenced Drosophila genome indicates that humans have more genes in common with Drosophila than with any other invertebrate model organism characterised to date. Thus, the construction and characterisation of Drosophila PIMs will help define the functions of many conserved genes and pathways, and will provide the pharmaceutical research industry with invaluable data to assist with drug target identification and validation.

Canadian integrated program for antimicrobial resistance surveillance: Retail food highlights, 2003-2012.

BACKGROUND: The Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) is a collaborative, integrated program designed to track antimicrobial resistance (AMR) among enteric bacteria isolated from various livestock commodities along the food-producing continuum ("farm to fork") and in humans. OBJECTIVE: To provide a summary of the prevalence and trends in AMR among select bacteria isolated from raw, fresh chicken, pork, and beef in 2012 at the retail food level and to link these data with other findings from CIPARS. METHODS: Meat samples were collected from randomly selected geographic areas across Canada weighted by population for subsequent isolation of bacteria and interpretation of the associated AMR profiles. Salmonella, Campylobacter and generic Escherichia coli (E. coli) were tested in chicken, and E. coli was tested in beef and pork. Data were analyzed for 2012 and temporal and regional trends were examined between 2003 and 2012 by province/region. RESULTS: Overall, resistance levels to Salmonella in retail chicken varied widely by region and year. For example, ceftiofur resistance to Salmonella in retail chicken was significantly lower in 2012 than in 2004 in Ontario and in Québec; however, among all regions sampled, resistance was significantly higher in 2012 compared to 2006. Across all regions sampled, resistance to Campylobacter in retail chicken was relatively low in 2012 (<16%) with the exception of tetracycline resistance. In 2012, ciprofloxacin resistance to Campylobacter in chicken declined in British Columbia but significantly increased in Ontario, compared to 2011. In 2012, ß-lactam resistance to E. coli in retail beef remained low (≤1%) and was also relatively low comparable to previous years in pork. CONCLUSION: In Canada, as is the case worldwide, there is evidence of resistance to medically important antimicrobials among bacteria from retail meats. Resistance among organisms isolated from poultry, beef, and pork at the retail food level is characterized by wide variation over time and across different regions.

A cross-sectional study examining Campylobacter and other zoonotic enteric pathogens in dogs that frequent dog parks in three cities in south-western Ontario and risk factors for shedding of Campylobacter spp.

An estimated 6 million pet dogs live in Canadian households with the potential to transmit zoonotic pathogens to humans. Dogs have been identified as carriers of Salmonella, Giardia and Campylobacter spp., particularly Campylobacter upsaliensis, but little is known about the prevalence and risk factors for these pathogens in pet dogs that visit dog parks. This study examined the prevalence of these organisms in the faeces of dogs visiting dog parks in three cities in south-western Ontario, as well as risk factors for shedding Campylobacter spp. and C. upsaliensis. From May to August 2009, canine faecal samples were collected at ten dog parks in the cities of Guelph and Kitchener-Waterloo, Ontario, Canada. Owners were asked to complete a questionnaire related to pet characteristics and management factors including age, diet and activities in which the dog participates. Faecal samples were collected from 251 dogs, and 189 questionnaires were completed. Salmonella, Giardia and Campylobacter spp. were present in 1.2%, 6.4% and 43.0% of faecal samples, respectively. Of the Campylobacter spp. detected, 86.1% were C. upsaliensis, 13% were C. jejuni and 0.9% were C. coli. Statistically significant sparing factors associated with the shedding of Campylobacter spp. included the feeding of a commercial dry diet and the dog's exposure to compost. Age of dog had a quadratic effect, with young dogs and senior dogs having an increased probability of shedding Campylobacter spp. compared with adult dogs. The only statistically significant risk factor for shedding C. upsaliensis was outdoor water access including lakes and ditches, while dogs >1 year old were at a lower risk than young dogs. Understanding the pet-related risk factors for Campylobacter spp. and C. upsaliensis shedding in dogs may help in the development of awareness and management strategies to potentially reduce the risk of transmitting this pathogen from dogs to humans.

A cross-sectional study examining the prevalence and risk factors for anti-microbial-resistant generic Escherichia coli in domestic dogs that frequent dog parks in three cities in south-western Ontario, Canada.

Anti-microbial resistance can threaten health by limiting treatment options and increasing the risk of hospitalization and severity of infection. Companion animals can shed anti-microbial-resistant bacteria that may result in the exposure of other dogs and humans to anti-microbial-resistant genes. The prevalence of anti-microbial-resistant generic Escherichia coli in the faeces of dogs that visited dog parks in south-western Ontario was examined and risk factors for shedding anti-microbial-resistant generic E. coli identified. From May to August 2009, canine faecal samples were collected at ten dog parks in three cities in south-western Ontario, Canada. Owners completed a questionnaire related to pet characteristics and management factors including recent treatment with antibiotics. Faecal samples were collected from 251 dogs, and 189 surveys were completed. Generic E. coli was isolated from 237 of the faecal samples, and up to three isolates per sample were tested for anti-microbial susceptibility. Eighty-nine percent of isolates were pan-susceptible; 82.3% of dogs shed isolates that were pan-susceptible. Multiclass resistance was detected in 7.2% of the isolates from 10.1% of the dogs. Based on multilevel multivariable logistic regression, a risk factor for the shedding of generic E. coli resistant to ampicillin was attending dog day care. Risk factors for the shedding of E. coli resistant to at least one anti-microbial included attending dog day care and being a large mixed breed dog, whereas consumption of commercial dry and home cooked diets was protective factor. In a multilevel multivariable model for the shedding of multiclass-resistant E. coli, exposure to compost and being a large mixed breed dog were risk factors, while consumption of a commercial dry diet was a sparing factor. Pet dogs are a potential reservoir of anti-microbial-resistant generic E. coli; some dog characteristics and management factors are associated with the prevalence of anti-microbial-resistant generic E. coli in dogs.

Evaluation of pet-related management factors and the risk of Salmonella spp. carriage in pet dogs from volunteer households in Ontario (2005-2006).

The purpose of this study was to determine pet-related management factors that may be associated with the presence of Salmonella spp. in feces of pet dogs from volunteer households. From October 2005 until May 2006, 138 dogs from 84 households in Ontario were recruited to participate in a cross-sectional study. Five consecutive daily fecal samples were collected from each dog and enrichment culture for Salmonella spp. was performed. A higher than expected number of the dogs (23.2%; 32/138) had at least one fecal sample positive for Salmonella, and 25% (21/84) of the households had at least one dog shedding Salmonella. Twelve serotypes of Salmonella enterica subsp. enterica were identified, with the predominant serotypes being Typhimurium (33.3%; 13/39), Kentucky (15.4%; 6/39), Brandenburg (15.4%; 6/39) and Heidelberg (12.8%; 5/39). Univariable logistic regression models were created with a random effect for household to account for clustering. Statistically significant risk factors for a dog testing positive included having contact with livestock, receiving a probiotic in the previous 30 days, feeding a commercial or homemade raw food diet, feeding raw meat and eggs, feeding a homemade cooked diet, and having more than one dog in the household. In two-variable models that controlled for feeding raw food, the non-dietary variables were no longer statistically significant. These results highlight the potential public health risk of including raw animal products in canine diets.

A role for cyclin J in the rapid nuclear division cycles of early Drosophila embryogenesis.

The nuclear division cycles of early Drosophila embryogenesis have a number of unique features that distinguish them from later cell cycles. These features include the lack of some checkpoints that operate in later cell cycles, the absence of gap phases, and very rapid DNA synthesis phases. The molecular mechanisms that control these rapid nuclear division cycles are poorly understood. Here we describe analysis of cyclin J, a previously uncharacterized cyclin which has an RNA expression pattern that suggests a possible role in early embryogenesis. We show that the cyclin J protein is present in early embryos where it forms active kinase complexes with cyclin-dependent kinase (Cdk) 2. To determine whether cyclin J plays a role in controlling the early nuclear cycles we isolated peptide aptamers that specifically bind to cyclin J and inhibit its ability to activate Cdks. We injected the inhibitory aptamers into syncytial Drosophila embryos and demonstrated that they caused defects in chromosome segregation and progression through mitosis. We obtained similar results by injecting cyclin J antibodies into embryos. Our results suggest that a cyclin J-associated kinase activity is required for the early embryonic division cycles.

Interaction mating reveals binary and ternary connections between Drosophila cell cycle regulators.

We characterized interactions between Drosophila melanogaster cell cycle regulatory proteins by a yeast interaction-mating technique. The results were displayed as two-dimensional matrices that revealed individual binary interactions between proteins. Each protein (Cdi, cyclin-dependent kinase interactor) interacted with a distinct spectrum of cyclin-dependent kinases (Cdk) from Drosophila and other organisms. Some Cdis interacted with other Cdis, indicating that these proteins may form trimeric complexes that include Cdks. Similar analysis of interaction matrices may be generally useful in detecting other multiprotein complexes and in establishing connectivity between individual complex members. Moreover, such analysis may also help assign function to newly identified proteins, identify domains involved in protein-protein interactions, and aid the dissection of genetic regulatory networks.

Targeting cyclin-dependent kinases in Drosophila with peptide aptamers.

Two-hybrid technology provides a simple way to isolate small peptide aptamers that specifically recognize and strongly bind to a protein of interest. These aptamers have the potential to dominantly interfere with specific activities of their target proteins and, therefore, could be used as in vivo inhibitors. Here we explore the ability to use peptide aptamers as in vivo inhibitors by expressing aptamers directed against cell cycle regulators in Drosophila. We expressed two peptide aptamers, each of which specifically recognizes one of the two essential cyclin-dependent kinases (Cdks), DmCdk1 and DmCdk2, in Drosophila. Expression of each Cdk aptamer during organogenesis caused adult eye defects typical of those caused by cell cycle inhibition. Co-overexpression of DmCdk1 or DmCdk2 resulted in suppression of the eye phenotypes, indicating that each aptamer interacts with a Cdk target in vivo and suggesting that these peptides disrupt normal eye development by inhibiting Cdk function. Moreover, the specificity of each aptamer for one of the two Cdks as determined in two-hybrid assays was retained in Drosophila. Combined, our results demonstrate that peptide aptamers generated by yeast two-hybrid methods can serve as inhibitory reagents to target specific proteins in vivo.

Understanding gene and allele function with two-hybrid methods.

Two-hybrid schemes for detecting protein-protein interactions have deepened our understanding of biology by allowing scientists to identify individual important proteins. Recent developments will allow biologists to chart regulatory networks and to rapidly generate hypotheses for the function of genes, allelic variants, and the connections between proteins that make up these networks. Future developments will allow biologists to test inferences about the function of network elements, and allow global approaches to questions of biological function.

Human procathepsin B interacts with the annexin II tetramer on the surface of tumor cells.

To study potential roles of plasma membrane-associated extracellular cathepsin B in tumor cell invasion and metastasis, we used the yeast two-hybrid system to screen for proteins that interact with human procathepsin B. The annexin II light chain (p11), one of the two subunits of the annexin II tetramer, was one of the proteins identified. We have confirmed that recombinant human procathepsin B interacts with p11 as well as with the annexin II tetramer in vitro. Furthermore, procathepsin B could interact with the annexin II tetramer in vivo as demonstrated by coimmunoprecipitation. Cathepsin B and the annexin II tetramer were shown by immunofluorescent staining to colocalize on the surface of human breast carcinoma and glioma cells. Taken together, our results indicate that the annexin II tetramer can serve as a binding protein for procathepsin B on the surface of tumor cells, an interaction that may facilitate tumor invasion and metastasis.

Identification of 12-lipoxygenase interaction with cellular proteins by yeast two-hybrid screening.

The platelet isoform of 12-lipoxygenase (12-LOX) is expressed in a variety of human tumors. 12-LOX metabolizes arachidonic acid to 12(S)-hydroxyeicosateraenoic acid (12(S)-HETE), which induces a number of cellular responses associated with tumor progression and metastasis. Little is known about 12-LOX regulation and no direct regulators of 12-LOX activity have been identified. To identify potential regulators of 12-LOX, we isolated cDNAs encoding 12-LOX interacting proteins using the yeast two-hybrid system. We screened a yeast two-hybrid interaction library from human epidermoid carcinoma A431 cells and identified four cellular proteins that interact specifically with 12-LOX. We identified type II keratin 5, lamin A, the cytoplasmic domain of integrin beta4 subunit and a phosphoprotein C8FW as 12-LOX interacting proteins. Here, we demonstrated that keratin 5, a 58 kD protein required for formation of 8 nm intermediate filaments, binds to 12-LOX in human tumor cells and may contribute to the regulated trafficking of 12-LOX. We also showed that lamin A binds 12-LOX in human tumor cells. These proteins provide the first candidate regulators of 12-LOX.

Isolation of Drosophila cyclin D, a protein expressed in the morphogenetic furrow before entry into S phase.

During Drosophila development, nuclear and cell divisions are coordinated in response to developmental signals. In yeast and mammalian cells, signals that control cell division regulate the activity of cyclin-dependent kinases (Cdks) through proteins such as cyclins that interact with the Cdks. Here we describe two Drosophila cyclins identified from a set of Cdk-interacting proteins. One, cyclin J, is of a distinctive sequence type; its exclusive maternal expression pattern suggests that it may regulate oogenesis or the early nuclear divisions of embryogenesis. The other belongs to the D class of cyclins, previously identified in mammalian cells. We show that Drosophila cyclin D is expressed in early embryos and in imaginal disc cells in a pattern that anticipates cell divisions. Expression in the developing eye disc at the anterior edge of the morphogenetic furrow suggests that cyclin D acts early, prior to cyclin E, in inducing G1-arrested cells to enter S phase. Our results also suggest that, although cyclin D may be necessary, its expression alone is not sufficient to initiate the events leading to S phase.

Groucho is required for Drosophila neurogenesis, segmentation, and sex determination and interacts directly with hairy-related bHLH proteins.

We have used the interaction trap, a yeast two-hybrid system, to identify proteins interacting with hairy, a basic-helix-loop-helix (bHLH) protein that represses transcription during Drosophila embryonic segmentation. We find that the groucho (gro) protein binds specifically to hairy and also to hairy-related bHLH proteins encoded by deadpan and the Enhancer of split complex. The C-terminal WRPW motif present in all these bHLH proteins is essential for this interaction. We demonstrate that these associations reflect in vivo maternal requirements for gro during neurogenesis, segmentation, and sex determination, three processes regulated by the above bHLH proteins, and we propose that gro is a transcriptional corepressor recruited to specific target promoters by hairy-related bHLH proteins.