Identification of bZIP interaction partners of viral proteins HBZ, MEQ, BZLF1, and K-bZIP using coiled-coil arrays

Basic-region leucine-zipper transcription factors (bZIPs) contain a segment rich in basic amino acids that can bind DNA, followed by a leucine zipper that can interact with other leucine zippers to form coiled-coil homo- or heterodimers. Several viruses encode proteins containing bZIP domains, including four that encode bZIPs lacking significant homology to any human protein. We investigated the interaction specificity of these four viral bZIPs by using coiled-coil arrays to assess self-associations as well as heterointeractions with 33 representative human bZIPs. The arrays recapitulated reported viral−human interactions and also uncovered new associations. MEQ and HBZ interacted with multiple human partners and had unique interaction profiles compared to any human bZIPs, whereas K-bZIP and BZLF1 displayed homospecificity. New interactions detected included HBZ with MAFB, MAFG, ATF2, CEBPG, and CREBZF and MEQ with NFIL3. These were confirmed in solution using circular dichroism. HBZ can heteroassociate with MAFB and MAFG in the presence of MARE-site DNA, and this interaction is dependent on the basic region of HBZ. NFIL3 and MEQ have different yet overlapping DNA-binding specificities and can form a heterocomplex with DNA. Computational design considering both affinity for MEQ and specificity with respect to other undesired bZIP-type interactions was used to generate a MEQ dimerization inhibitor. This peptide, anti-MEQ, bound MEQ both stably and specifically, as assayed using coiled-coil arrays and circular dichroism in solution. Anti-MEQ also inhibited MEQ binding to DNA. These studies can guide further investigation of the function of viral and human bZIP complexes.National Institutes of Health (U.S.) (NIH Award GM067681)National Science Foundation (U.S.) (NSF Award 0216437

Dogs Leaving the ICU Carry a Very Large Multi-Drug Resistant Enterococcal Population with Capacity for Biofilm Formation and Horizontal Gene Transfer

The enterococcal community from feces of seven dogs treated with antibiotics for 2–9 days in the veterinary intensive care unit (ICU) was characterized. Both, culture-based approach and culture-independent 16S rDNA amplicon 454 pyrosequencing, revealed an abnormally large enterococcal community: 1.4±0.8×108 CFU gram−1 of feces and 48.9±11.5% of the total 16,228 sequences, respectively. The diversity of the overall microbial community was very low which likely reflects a high selective antibiotic pressure. The enterococcal diversity based on 210 isolates was also low as represented by Enterococcus faecium (54.6%) and Enterococcus faecalis (45.4%). E. faecium was frequently resistant to enrofloxacin (97.3%), ampicillin (96.5%), tetracycline (84.1%), doxycycline (60.2%), erythromycin (53.1%), gentamicin (48.7%), streptomycin (42.5%), and nitrofurantoin (26.5%). In E. faecalis, resistance was common to tetracycline (59.6%), erythromycin (56.4%), doxycycline (53.2%), and enrofloxacin (31.9%). No resistance was detected to vancomycin, tigecycline, linezolid, and quinupristin/dalfopristin in either species. Many isolates carried virulence traits including gelatinase, aggregation substance, cytolysin, and enterococcal surface protein. All E. faecalis strains were biofilm formers in vitro and this phenotype correlated with the presence of gelE and/or esp. In vitro intra-species conjugation assays demonstrated that E. faecium were capable of transferring tetracycline, doxycycline, streptomycin, gentamicin, and erythromycin resistance traits to human clinical strains. Multi-locus variable number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE) of E. faecium strains showed very low genotypic diversity. Interestingly, three E. faecium clones were shared among four dogs suggesting their nosocomial origin. Furthermore, multi-locus sequence typing (MLST) of nine representative MLVA types revealed that six sequence types (STs) originating from five dogs were identical or closely related to STs of human clinical isolates and isolates from hospital outbreaks. It is recommended to restrict close physical contact between pets released from the ICU and their owners to avoid potential health risks

Critical Role of the Virus-Encoded MicroRNA-155 Ortholog in the Induction of Marek's Disease Lymphomas

Notwithstanding the well-characterised roles of a number of oncogenes in neoplastic transformation, microRNAs (miRNAs) are increasingly implicated in several human cancers. Discovery of miRNAs in several oncogenic herpesviruses such as KSHV has further highlighted the potential of virus-encoded miRNAs to contribute to their oncogenic capabilities. Nevertheless, despite the identification of several possible cancer-related genes as their targets, the direct in vivo role of virus-encoded miRNAs in neoplastic diseases such as those induced by KSHV is difficult to demonstrate in the absence of suitable models. However, excellent natural disease models of rapid-onset Marek's disease (MD) lymphomas in chickens allow examination of the oncogenic potential of virus-encoded miRNAs. Using viruses modified by reverse genetics of the infectious BAC clone of the oncogenic RB-1B strain of MDV, we show that the deletion of the six-miRNA cluster 1 from the viral genome abolished the oncogenicity of the virus. This loss of oncogenicity appeared to be primarily due to the single miRNA within the cluster, miR-M4, the ortholog of cellular miR-155, since its deletion or a 2-nucleotide mutation within its seed region was sufficient to inhibit the induction of lymphomas. The definitive role of this miR-155 ortholog in oncogenicity was further confirmed by the rescue of oncogenic phenotype by revertant viruses that expressed either the miR-M4 or the cellular homolog gga-miR-155. This is the first demonstration of the direct in vivo role of a virus-encoded miRNA in inducing tumors in a natural infection model. Furthermore, the use of viruses deleted in miRNAs as effective vaccines against virulent MDV challenge, enables the prospects of generating genetically defined attenuated vaccines

The Hoopoe's Uropygial Gland Hosts a Bacterial Community Influenced by the Living Conditions of the Bird

Molecular methods have revealed that symbiotic systems involving bacteria are mostly based on whole bacterial communities. Bacterial diversity in hoopoe uropygial gland secretion is known to be mainly composed of certain strains of enterococci, but this conclusion is based solely on culture-dependent techniques. This study, by using culture-independent techniques (based on the 16S rDNA and the ribosomal intergenic spacer region) shows that the bacterial community in the uropygial gland secretion is more complex than previously thought and its composition is affected by the living conditions of the bird. Besides the known enterococci, the uropygial gland hosts other facultative anaerobic species and several obligated anaerobic species (mostly clostridia). The bacterial assemblage of this community was largely invariable among study individuals, although differences were detected between captive and wild female hoopoes, with some strains showing significantly higher prevalence in wild birds. These results alter previous views on the hoopoe-bacteria symbiosis and open a new window to further explore this system, delving into the possible sources of symbiotic bacteria (e.g. nest environments, digestive tract, winter quarters) or the possible functions of different bacterial groups in different contexts of parasitism or predation of their hoopoe host.This work was supported by the Ministerio de Ciencia y Tecnología (projects CGL2005-06975/BOSFEDER; CGL2007-61251/BOSFEDER), the Ministerio de Ciencia e Innovación (projects CGL2009-14006/BOSFEDER; CGL2010-19233-C03-01/BOSFEDER; CGL2010-19233-C03-03/BOSFEDER), the Ministerio de Economía y Competitividad (projects CGL2013-48193-C3-1-P/BOSFEDER; CGL2013-48193-C3-2-P/BOSFEDER), and the Junta de Andalucía (RNM 345, P09-RNM-4557). SMRR received a grant from the Ministerio de Ciencia e Innovación (FPI program, BES-2011-047677)

Culture Enriched Molecular Profiling of the Cystic Fibrosis Airway Microbiome

The microbiome of the respiratory tract, including the nasopharyngeal and oropharyngeal microbiota, is a dynamic community of microorganisms that is highly diverse. The cystic fibrosis (CF) airway microbiome refers to the polymicrobial communities present in the lower airways of CF patients. It is comprised of chronic opportunistic pathogens (such as Pseudomonas aeruginosa) and a variety of organisms derived mostly from the normal microbiota of the upper respiratory tract. The complexity of these communities has been inferred primarily from culture independent molecular profiling. As with most microbial communities it is generally assumed that most of the organisms present are not readily cultured. Our culture collection generated using more extensive cultivation approaches, reveals a more complex microbial community than that obtained by conventional CF culture methods. To directly evaluate the cultivability of the airway microbiome, we examined six samples in depth using culture-enriched molecular profiling which combines culture-based methods with the molecular profiling methods of terminal restriction fragment length polymorphisms and 16S rRNA gene sequencing. We demonstrate that combining culture-dependent and culture-independent approaches enhances the sensitivity of either approach alone. Our techniques were able to cultivate 43 of the 48 families detected by deep sequencing; the five families recovered solely by culture-independent approaches were all present at very low abundance (<0.002% total reads). 46% of the molecular signatures detected by culture from the six patients were only identified in an anaerobic environment, suggesting that a large proportion of the cultured airway community is composed of obligate anaerobes. Most significantly, using 20 growth conditions per specimen, half of which included anaerobic cultivation and extended incubation times we demonstrate that the majority of bacteria present can be cultured

Composition and Diversity of the Fecal Microbiome and Inferred Fecal Metagenome Does Not Predict Subsequent Pneumonia Caused by <i>Rhodococcus equi</i> in Foals

<div><p>In equids, susceptibility to disease caused by <i>Rhodococcus equi</i> occurs almost exclusively in foals. This distribution might be attributable to the age-dependent maturation of immunity following birth undergone by mammalian neonates that renders them especially susceptible to infectious diseases. Expansion and diversification of the neonatal microbiome contribute to development of immunity in the gut. Moreover, diminished diversity of the gastrointestinal microbiome has been associated with risk of infections and immune dysregulation. We thus hypothesized that varying composition or reduced diversity of the intestinal microbiome of neonatal foals would contribute to increased susceptibility of their developing <i>R</i>. <i>equi</i> pneumonia. The composition and diversity indices of the fecal microbiota at 3 and 5 weeks of age were compared among 3 groups of foals: 1) foals that <u><i>subsequently</i></u> developed <i>R</i>. <i>equi</i> pneumonia after sampling; 2) foals that <u><i>subsequently</i></u> developed ultrasonographic evidence of pulmonary abscess formation or consolidation but <u><i>not</i></u> clinical signs (subclinical group); and, 3) foals that developed neither clinical signs nor ultrasonographic evidence of pulmonary abscess formation or consolidation. No significant differences were found among groups at either sampling time, indicating absence of evidence of an influence of composition or diversity of the fecal microbiome, or predicted fecal metagenome, on susceptibility to subsequent <i>R</i>. <i>equi</i> pneumonia. A marked and significant difference identified between a relatively short interval of time appeared to reflect ongoing adaptation to transition from a milk diet to a diet including available forage (including hay) and access to concentrate fed to the mare.</p></div

Prospective Evaluation of Laparoscopic Pancreatic Biopsies in 11 Healthy Cats

BackgroundDefinitive diagnosis of feline pancreatic disease is dependent on histologic examination of biopsies.HypothesisLaparoscopic punch biopsy of the pancreas does not significantly affect pancreatic health or clinical status of healthy cats, and provides an adequate biopsy sample for histopathology.AnimalsEleven healthy female domestic shorthair cats.MethodsEffects of laparoscopic pancreatic visualization alone in 5 cats compared with laparoscopic pancreatic visualization and punch biopsy in 6 cats were studied. Temperature, pulse, and respiratory rate, physical examination, and daily caloric intake were evaluated for 1 week before and 1 week after the procedure. Pain scores (simple descriptive score and dynamic interactive visual assessment score) were evaluated hourly during the 1st 6 hours postprocedure. Complete blood cell counts, serum biochemical profiles, serum feline pancreatic lipase immunoreactivity, and urine specific gravity were evaluated before the procedure and at 6, 24, and 72 hours postprocedure. One month postprocedure, during sterilization, the pancreas was reassessed visually in all cats, and microscopically in the biopsy group.ResultsFor all variables evaluated, there were no significant differences between biopsy and control cats. Re-evaluation of the pancreatic biopsy site 1 month later documented a normal tissue response to biopsy. The laparoscopic punch biopsy forceps provided high-quality pancreatic biopsy samples with an average size of 5 mm x 4 mm on 2-dimensional cut section.Conclusions and Clinical ImportanceLaparoscopic pancreatic biopsy is a useful and safe technique in healthy cats