Generation of an arginine-tRNA-adapted Saccharomyces cerevisiae strain for effective heterologous protein expression.

The tRNA population reflects the codon bias of the organism and affects the translation of heterologous target mRNA molecules. In this study, Saccharomyces cerevisiae strains with modified levels of rare tRNA were engineered, that allowed efficient generation of recombinant proteins with unfavorable codon usage. We established a novel synthetic tRNA expression cassette and verified functional nonsense suppressor tRNAGlnSCUA generation in a stop codon read-through assay with a modified ß-galactosidase reporter gene. Correlation between altered tRNA and protein level was shown by survival of copper sensitive S. cerevisiae cells in the presence of copper ions by an increased transcription of tRNAArgCCG molecules, recognizing rare codons in a modified CUP1 gene. Genome integration of tRNA expression cassette led to the generation of arginine-tRNA-adapted S. cerevisiae strains, which showed elevated tRNA levels (tRNAArgCCG, tRNAArgGCG and tRNAArgUCG) pairing to rare codons. The modified strain MNY3 revealed a considerably improved monitoring of protein-protein interaction from Aspergillus fumigatus bait and prey sequences in yeast two-hybrid experiments. In future, this principle to overcome limited recombinant protein expression by tRNA adaption of expression strains instead of codon adaption might provide new designer yeast cells for an efficient protein production and for improved genome-wide protein-protein interaction analyses.

Characterization of avian γδ T-cell subsets after Salmonella enterica serovar Typhimurium infection of chicks.

Avian γδ T lymphocytes are frequently found in blood and organs and are assumed to be crucial to the immune defense against Salmonella infections of chicks. To elucidate the so-far-unknown immunological features of subpopulations of avian γδ T cells in the course of infection, day-old chicks were infected orally with Salmonella enterica serovar Typhimurium. Until 11 days after infection, the occurrence as well as transcription of the CD8 antigen and immunologically relevant protein genes of CD8α(-) and CD8α(+high) (CD8αα(+) CD8αß(+)) γδ cells were analyzed using flow cytometry and quantitative real-time reverse transcription-PCR (RT-PCR) with blood, spleen, thymus, and cecum samples. After infection, an increased percentage of CD8α(+high) γδ T lymphocytes was found in blood, in spleen, and, with the highest values and most rapidly, in cecum. Within the CD8α(+high) subset, a significant rise in the number of CD8αα(+) cells was accompanied by enhanced CD8α antigen expression and reduced gene transcription of the CD8ß chain. CD8αα(+) and CD8αß(+) cells showed elevated transcription for Fas, Fas ligand (FasL), interleukin-2 receptor α (IL-2Rα), and gamma interferon (IFN-γ). While the highest fold changes in mRNA levels were observed in CD8αß(+) cells, the mRNA expression rates of CD8αß(+) cells never significantly exceeded those of the CD8αα(+) cells. In conclusion, both CD8α(+high) γδ T-cell subpopulations (CD8αα(+) and CD8αß(+)) might be a potential source of IFN-γ in Salmonella-infected chicks. However, due to their prominent frequency in blood and organs after infection, the avian CD8αα(+) γδ T-cell subset seems to be unique and of importance in the course of Salmonella Typhimurium infection of very young chicks.

Heterogeneity of avian gammadelta T cells.

gammadelta T cells are distinct with respect to tissue localisation, phenotype and biological functions and similarities between species are not very apparent. To elucidate local and functional heterogeneity of non-stimulated avian gammadelta T cells, the CD8-characterised gammadelta T cell subsets [CD8alpha(+high) (CD8alphaalpha(+) and CD8alphabeta(+)); CD8alpha(+dim); CD8(-)] of blood, spleen and caecum were flow cytometrically quantified and analysed for proliferation state as well as sorted for determination of immune-relevant gene expression by quantitative real-time RT-PCR. The number of avian CD8-characterised gammadelta T cell subsets differed in dependence on tissue and age of bird. Compared to blood and spleen, caecum showed the highest percentage of gammadelta T cells as well as of the CD8alpha(+high) gammadelta T cell subset in 7-week-old birds. Generally, the CD8alphabeta(+) cells significantly outnumbered the CD8alphaalpha(+) lymphocytes within the CD8alpha(+high) gammadelta T cell population of all organs. Additionally, the splenic CD8alphabeta(+) subpopulation revealed the highest proliferation activity. By RT-PCR, mRNA expression of immune-relevant genes was proved in non-stimulated gammadelta T cell subsets, but on different levels. Generally, both CD8alpha(+high) cell subsets (CD8alphaalpha(+) and CD8alphabeta(+)) of blood and spleen showed elevated expression levels for Fas ligand (FasL), XCL1 (lymphotactin) and interferon-gamma (IFNgamma) compared to the CD8alpha(-) gammadelta T cell subset. In contrast, all caecal gammadelta T cell subsets showed similar high levels of these transcripts. Notably, the CD8alphaalpha(+) cells of all locations showed unique expression of TLR4 and interleukin (IL)-2. The results demonstrated that avian gammadelta T cells are not only heterogeneous concerning their CD8 antigen characteristics and tissue localisation, but also with regard to functional features such as proliferation and mRNA expression.

Chicken cecum immune response to Salmonella enterica serovars of different levels of invasiveness.

Day-old chicks are very susceptible to infections with Salmonella enterica subspecies. The gut mucosa is the initial site of host invasion and provides the first line of defense against the bacteria. To study the potential of different S. enterica serovars to invade the gut mucosa and trigger an immune response, day-old chicks were infected orally with Salmonella enterica serovar Enteritidis, S. enterica serovar Typhimurium, S. enterica serovar Hadar, or S. enterica serovar Infantis, respectively. The localization of Salmonella organisms in gut mucosa and the number of immune cells in cecum were determined by immunohistochemistry in the period between 4 h and 9 days after infection. Using quantitative real-time reverse transcription (RT)-PCR, mRNA expression of various cytokines, chemokines, and inducible nitric oxide synthase (iNOS) was examined in cecum. As a result, all S. enterica serovars were able to infect epithelial cells and the lamina propria. Notably, serovar Enteritidis showed the highest invasiveness of lamina propria tissue, whereas serovars Typhimurium and Hadar displayed moderate invasiveness and serovar Infantis hardly any invasion capabilities. Only a limited number of bacteria of all serovars were found within intestinal macrophages. Elevated numbers of granulocytes, CD8+ cells, and TCR1+ cells and mRNA expression rates for interleukin 12 (IL-12), IL-18, tumor necrosis factor alpha factor, and iNOS in cecum correlated well with the invasiveness of serovars in the lamina propria. In contrast, changes in numbers of TCR2+ and CD4+ cells and IL-2 mRNA expression seemed to be more dependent on infection of epithelial cells. The data indicate that the capability of Salmonella serovars to enter the cecal mucosa and invade lower regions affects both the level and character of the immune response in tissue.

In vitro and in vivo generation of heterophil extracellular traps after Salmonella exposure.

The release of extracellular traps (ETs) by granulocytes is a unique strategy to stop the dissemination of microbial pathogens. This study was undertaken to elucidate the potential of avian granulocytes (heterophils) to form ETs that can arrest and kill Salmonella organisms. After in vitro exposure of isolated heterophils and in vivo infection of day-old chicks with Salmonella enterica subsp. enterica serovars Infantis (SI) or Enteritidis (SE), the generation of ETs as well as the trapping and survivability of Salmonella organisms in the ET meshwork were determined by means of microscopy and spectrophotometry. In vitro, heterophils were able to form ETs within 15min after SE and SI inoculation. At 120min and with a multiplicity of infection of 1 and 5, SI induced significantly more ETs and DNA release than SE. Both SE and SI were found to be associated with the ET structures. Live-dead staining showed most of the microorganisms within the extracellular scaffold alive. In vivo, heterophils were detected in cecal lumen of SE-, but not SI-infected chicks. In cecum of the SE-exposed chicks, ET formations were scarcely detected whereas intact heterophils with phagocytosed bacteria were frequently found. The results evidence the capability of heterophils to generate ETs after SE and SI exposure in vitro. However, an infection of chicks with Salmonella did not significantly induce the formation of ET structures in cecum. Thus, the process to form ETs (ETosis) seems not to be of special relevance for Salmonella defense within the cecal lumen of young chicks.

Interferon alpha-armed nanoparticles trigger rapid and sustained STAT1-dependent anti-viral cellular responses.

Interferon-α (IFNα) has enormous potential for anti-proliferative and anti-viral treatments. However, clinical success is still hampered due to its limited bioavailability and thus, lack of sustained modulation of disease-relevant protective programs. Consequently, we here examined whether IFNα immobilized on nanoscale ferromagnetic R-Chitosan carriers is capable of inducing rapid and sustained activation of STAT1 signaling. We report the spontaneous formation of a stable nanoparticle-IFNα protein corona, which was exploited to generate IFNα-loaded spheres, obviating the need to specifically couple the cytokine to the nanoparticles (NPs). Notably, comprehensive experimental approaches ensure that formation of the IFNα NP-corona does not affect the biological activity of the cytokine, as STAT1 signaling was efficiently activated. Employing human prostate cancer and melanoma cell models, we found that the intensity and duration of STAT1 phosphorylation as well as the downstream activation of pathobiologically relevant genes were dose and particle dependent. In comparison with free IFNα, IFNα-loaded spheres resulted in a more sustained biologically relevant STAT1 activation, demonstrated also by conferring innate cellular immunity against vesicular stomatitis virus (VSV) infection. For one, our study demonstrates the advantages of biodegradable IFNα-coated R-Chitosan NPs for controlled cytokine release, and thereby improved therapy. Second, we reveal that the permanent presence of IFNα and not just the initial STAT1 phosphorylation ensures sustained IFNα-dependent signaling.

Effects of Salmonella enterica serovar Enteritidis on cellular recruitment and cytokine gene expression in caecum of vaccinated chickens.

Although vaccination of poultry is a suitable method to limit human food borne gastroenteritis caused by Salmonella (S.), the immune mechanisms responsible for a longer lasting protection against Salmonella infection in birds are not completely understood. To reveal unique protection-related immune parameters, day-old chicks were vaccinated with a commercial live S. Enteritidis vaccine and challenged with wild-type S. Enteritidis 147N at day 56 of life. The bacterial cell count was determined in gut and liver, while the immune cell composition and cytokine gene expression patterns were analysed by immunohistochemistry and quantitative real-time RT-PCR in caecum samples. The presented data suggest that the vaccine-elicited immune protection against the Salmonella wild-type infection was rather related to the bacterial count in gut mucosa and liver than to the colonisation in gut lumen. The higher number of Salmonella wild-type organisms found in caecal wall and liver of the non-immunised compared to immunised birds after challenge correlated with a more pronounced gene expression rate for IL-8, LITAF, iNOS, IL-12 and IFN-gamma. In contrast, immunised birds exhibited higher amounts of CD8(+) T cells as well as IgA than the non-immunised chickens after S. Enteritidis 147N infection in caecum. The results demonstrated a distinctive immune reaction pattern of previously vaccinated compared to non-vaccinated chickens upon S. Enteritidis wild-type challenge.

Circulating gamma delta T cells in response to Salmonella enterica serovar enteritidis exposure in chickens.

gammadelta T cells are considered crucial to the outcome of various infectious diseases. The present study was undertaken to characterize gammadelta (T-cell receptor 1(+) [TCR1(+)]) T cells phenotypically and functionally in avian immune response. Day-old chicks were orally immunized with Salmonella enterica serovar Enteritidis live vaccine or S. enterica serovar Enteritidis wild-type strain and infected using the S. enterica serovar Enteritidis wild-type strain on day 44 of life. Between days 3 and 71, peripheral blood was examined flow cytometrically for the occurrence of gammadelta T-cell subpopulations differentiated by the expression of T-cell antigens. Three different TCR1(+) cell populations were found to display considerable variation regarding CD8alpha antigen expression: (i) CD8alpha(+high) TCR1(+) cells, (ii) CD8alpha(+dim) TCR1(+) cells, and (iii) CD8alpha(-) TCR1(+) cells. While most of the CD8alpha(+high) TCR1(+) cells expressed the CD8alphabeta heterodimeric antigen, the majority of the CD8alpha(+dim) TCR1(+) cells were found to express the CD8alphaalpha homodimeric form. After immunization, a significant increase of CD8alphaalpha(+high) gammadelta T cells was observed within the CD8alpha(+high) TCR1(+) cell population. Quantitative reverse transcription-PCR revealed reduced interleukin-7 receptor alpha (IL-7Ralpha) and Bcl-x expression and elevated IL-2Ralpha mRNA expression of the CD8alphaalpha(+high) gammadelta T cells. Immunohistochemical analysis demonstrated a significant increase of CD8alpha(+) and TCR1(+) cells in the cecum and spleen and a decreased percentage of CD8beta(+) T cells in the spleen after Salmonella immunization. After infection of immunized animals, immune reactions were restricted to intestinal tissue. The study showed that Salmonella immunization of very young chicks is accompanied by an increase of CD8alphaalpha(+high) gammadelta T cells in peripheral blood, which are probably activated, and thus represent an important factor for the development of a protective immune response to Salmonella organisms in chickens.