In Vivo Expression of Chicken Gut Anaerobes Identifies Carbohydrate- or Amino Acid-Utilising, Motile or Type VI Secretion System-Expressing Bacteria.

Complex gut microbiota increases chickens' resistance to enteric pathogens. However, the principles of this phenomenon are not understood in detail. One of the possibilities for how to decipher the role of gut microbiota in chickens' resistance to enteric pathogens is to systematically characterise the gene expression of individual gut microbiota members colonising the chicken caecum. To reach this aim, newly hatched chicks were inoculated with bacterial species whose whole genomic sequence was known. Total protein purified from the chicken caecum was analysed by mass spectrometry, and the obtained spectra were searched against strain-specific protein databases generated from known genomic sequences. Campylobacter jejuni, Phascolarctobacterium sp. and Sutterella massiliensis did not utilise carbohydrates when colonising the chicken caecum. On the other hand, Bacteroides, Mediterranea, Marseilla, Megamonas, Megasphaera, Bifidobacterium, Blautia, Escherichia coli and Succinatimonas fermented carbohydrates. C. jejuni was the only motile bacterium, and Bacteroides mediterraneensis expressed the type VI secretion system. Classification of in vivo expression is key for understanding the role of individual species in complex microbial populations colonising the intestinal tract. Knowledge of the expression of motility, the type VI secretion system, and preference for carbohydrate or amino acid fermentation is important for the selection of bacteria for defined competitive exclusion products.

Visualizing Arc protein dynamics and localization in the mammalian brain using AAV-mediated in situ gene labeling.

The activity-regulated cytoskeleton-associated (Arc) protein is essential for synaptic plasticity and memory formation. The Arc gene, which contains remnants of a structural GAG retrotransposon sequence, produces a protein that self-assembles into capsid-like structures harboring Arc mRNA. Arc capsids, released from neurons, have been proposed as a novel intercellular mechanism for mRNA transmission. Nevertheless, evidence for intercellular transport of Arc in the mammalian brain is still lacking. To enable the tracking of Arc molecules from individual neurons in vivo, we devised an adeno-associated virus (AAV) mediated approach to tag the N-terminal of the mouse Arc protein with a fluorescent reporter using CRISPR/Cas9 homologous independent targeted integration (HITI). We show that a sequence coding for mCherry can successfully be knocked in at the 5' end of the Arc open reading frame. While nine spCas9 gene editing sites surround the Arc start codon, the accuracy of the editing was highly sequence-dependent, with only a single target resulting in an in-frame reporter integration. When inducing long-term potentiation (LTP) in the hippocampus, we observed an increase of Arc protein highly correlated with an increase in fluorescent intensity and the number of mCherry-positive cells. By proximity ligation assay (PLA), we demonstrated that the mCherry-Arc fusion protein retains the Arc function by interacting with the transmembrane protein stargazin in postsynaptic spines. Finally, we recorded mCherry-Arc interaction with presynaptic protein Bassoon in mCherry-negative surrounding neurons at close proximity to mCherry-positive spines of edited neurons. This is the first study to provide support for inter-neuronal in vivo transfer of Arc in the mammalian brain.

Deconvolution of spatial sequencing provides accurate characterization of hESC-derived DA transplants in vivo.

Cell therapy for Parkinson's disease has experienced substantial growth in the past decades with several ongoing clinical trials. Despite increasing refinement of differentiation protocols and standardization of the transplanted neural precursors, the transcriptomic analysis of cells in the transplant after its full maturation in vivo has not been thoroughly investigated. Here, we present spatial transcriptomics analysis of fully differentiated grafts in their host tissue. Unlike earlier transcriptomics analyses using single-cell technologies, we observe that cells derived from human embryonic stem cells (hESCs) in the grafts adopt mature dopaminergic signatures. We show that the presence of phenotypic dopaminergic genes, which were found to be differentially expressed in the transplants, is concentrated toward the edges of the grafts, in agreement with the immunohistochemical analyses. Deconvolution shows dopamine neurons being the dominating cell type in many features beneath the graft area. These findings further support the preferred environmental niche of TH-positive cells and confirm their dopaminergic phenotype through the presence of multiple dopaminergic markers.

Host Species Adaptation of Obligate Gut Anaerobes Is Dependent on Their Environmental Survival.

The gut microbiota of warm-blooded vertebrates consists of bacterial species belonging to two main phyla; Firmicutes and Bacteroidetes. However, does it mean that the same bacterial species are found in humans and chickens? Here we show that the ability to survive in an aerobic environment is central for host species adaptation. Known bacterial species commonly found in humans, pigs, chickens and Antarctic gentoo penguins are those capable of extended survival under aerobic conditions, i.e., either spore-forming, aerotolerant or facultatively anaerobic bacteria. Such bacteria are ubiquitously distributed in the environment, which acts as the source of infection with similar probability in humans, pigs, chickens, penguins and likely any other warm-blooded omnivorous hosts. On the other hand, gut anaerobes with no specific adaptation for survival in an aerobic environment exhibit host adaptation. This is associated with their vertical transmission from mothers to offspring and long-term colonisation after administration of a single dose. This knowledge influences the design of next-generation probiotics. The origin of aerotolerant or spore-forming probiotic strains may not be that important. On the other hand, if Bacteroidetes and other host-adapted species are used as future probiotics, host preference should be considered.

Detoxification, Hydrogen Sulphide Metabolism and Wound Healing Are the Main Functions That Differentiate Caecum Protein Expression from Ileum of Week-Old Chicken.

Sections of chicken gut differ in many aspects, e.g., the passage of digesta (continuous vs. discontinuous), the concentration of oxygen, and the density of colonising microbiota. Using an unbiased LC-MS/MS protocol, we compared protein expression in 18 ileal and 57 caecal tissue samples that originated from 7-day old ISA brown chickens. We found that proteins specific to the ileum were either structural (e.g., 3 actin isoforms, villin, or myosin 1A), or those required for nutrient digestion (e.g., sucrose isomaltase, maltase-glucoamylase, peptidase D) and absorption (e.g., fatty acid-binding protein 2 and 6 or bile acid-CoA:amino acid N-acyltransferase). On the other hand, proteins characteristic of the caecum were involved in sensing and limiting the consequences of oxidative stress (e.g., thioredoxin, peroxiredoxin 6), cell adhesion, and motility associated with wound healing (e.g., fibronectin 1, desmoyokin). These mechanisms are coupled with the activation of mechanisms suppressing the inflammatory response (galectin 1). Rather prominent were also expressions of proteins linked to hydrogen sulphide metabolism in caecum represented by cystathionin beta synthase, selenium-binding protein 1, mercaptopyruvate sulphurtransferase, and thiosulphate sulphurtransferase. Higher mRNA expression of nuclear factor, erythroid 2-like 2, the main oxidative stress transcriptional factor in caecum, further supported our observations.

Characterization of chicken spleen transcriptome after infection with Salmonella enterica serovar Enteritidis.

In this study we were interested in identification of new markers of chicken response to Salmonella Enteritidis infection. To reach this aim, gene expression in the spleens of naive chickens and those intravenously infected with S. Enteritidis with or without previous oral vaccination was determined by 454 pyrosequencing of splenic mRNA/cDNA. Forty genes with increased expression at the level of transcription were identified. The most inducible genes encoded avidin (AVD), extracellular fatty acid binding protein (EXFABP), immune responsive gene 1 (IRG1), chemokine ah221 (AH221), trappin-6-like protein (TRAP6) and serum amyloid A (SAA). Using cDNA from sorted splenic B-lymphocytes, macrophages, CD4, CD8 and γδ T-lymphocytes, we found that the above mentioned genes were preferentially expressed in macrophages. AVD, EXFABP, IRG1, AH221, TRAP6 and SAA were induced also in the cecum of chickens orally infected with S. Enteritidis on day 1 of life or day 42 of life. Unusual results were obtained for the immunoglobulin encoding transcripts. Prior to the infection, transcripts coding for the constant parts of IgM, IgY, IgA and Ig light chain were detected in B-lymphocytes. However, after the infection, immunoglobulin encoding transcripts were expressed also by T-lymphocytes and macrophages. Expression of AVD, EXFABP, IRG1, AH221, TRAP6, SAA and all immunoglobulin genes can be therefore used for the characterization of the course of S. Enteritidis infection in chickens.

Characteristics and distribution of plasmids in a clonally diverse set of methicillin-resistant Staphylococcus aureus strains.

The aim of this study was to compare the plasmid contents of methicillin-resistant Staphylococcus aureus (MRSA) strains classified into different clonal clusters (CCs). The isolates were collected from 15 Czech hospitals in 2000-2008. Plasmid DNA was detected in 65 (89%) strains, and 33 of them harbored more than one plasmid type. Altogether 24 different types of plasmids were identified, ranging in size from 1.3 to 55 kb. Restriction endonuclease analysis, plasmid elimination, DNA hybridization, and sequencing were used for their further characterization. It has been found that the conjugative, erythromycin resistance and enterotoxin D encoding plasmids are harbored by strains from different CCs. On the other hand, chloramphenicol and tetracycline resistance plasmids, and most of the penicillinase and cryptic plasmids were only detected in certain CCs. Especially, the pUSA300-like plasmids were found exclusively in the USA300 clone strains. The high diversity in plasmid content detected in the study strains implies that plasmids play a major role in evolution of MRSA clonal lineages.