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- Bacterial cell membrane (1)
- Bacterial cell wall (1)
- CD44 (1)
- Cell aggregation (1)
- Cell division defect (1)
- D61Y mutation (1)
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- Gedächtniszelle (1)
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Escherichia coli has been commonly used as a platform for recombinant protein production and accounts for approximately 30% of current biopharmaceuticals on the market. Nowadays, many recombinant proteins require post-translational modifications which E. coli normally cannot facilitate. Therefore, novel technological advancements are unceasingly being developed to improve the E. coli expression system. In this work, some of the most recently engineered platforms for the production of disulfide bond-containing proteins were used to study the E. coli proteome under heterologous protein production stress. The effects of protein secretion via the Sec and Tat translocation pathways were examined using a comparative LC-MS/MS analysis. The E. coli proteome responds to foreign protein production by activation of several overlapping stress responses with a high degree of interaction. In consequence, a number of important cellular processes such as cellular metabolism, protein transport, redox state of the cytoplasm and membrane structure are altered by the production stress. These changes lead to the reduction of cellular growth and recombinant product yields. Resolving the identified bottlenecks will increase the efficiency of recombinant protein expression processes in E. coli.
The proteasome is a major part of the ubiquitin-proteasome-system playing an important role in cell homeostasis due to its protein quality control function. Moreover, the proteasome is involved in cell cycle regulation and in the regulation of transcription factors. Upon induction of interferons, or treatment with lipopolysaccharides, an isoform of the standard-proteasome is composed, named immunoproteasome (i-proteasome). The i-proteasome is constitutively expressed in immune cells and deficiency of proteolytic subunits of this multiprotein complex has been associated with a poor outcome during infectious diseases. I-proteasome-deficiency has been shown to result in reduced MHC class I presentation. Using mice which are deficient for all three proteolytic active subunits LMP2, MECL-1 and LMP7, we could demonstrate that i-proteasome-deficiency lead to an altered recruitment of immune cells to the CNS when challenged with the intracellular parasite Toxoplasma gondii, resulting in increased frequencies of neutrophils and other cells of myeloid origin. The shift to reduced frequencies of CD45highCD11blow lymphocytes can be further explained by a decreased migratory capacity of i-proteasome-deficient CD8+ T cells. In contrast to previous studies using other pathogens, effector function of CD8+ as well as CD4+ T cells, measured by frequencies of IFNγ, TNF, IL-2 and granzyme B producing cells, were not impaired in these mice, whereas induction of CD4+ Tregs was strongly reduced. In addition, we found that parasite control was comparable to control mice and that i-proteasome deletion caused an overall pro-inflammatory cytokine milieu within the brain. Our results indicate that i-proteasome-deficiency lead to prolonged tissue inflammation during T. gondii infection which could be an explanation for the more severe course of disease observed in these mice.
The Src homology domain containing phosphatase 2 (SHP2) is a tyrosine phosphatase modulating several signaling pathways and therefore has an influence in cell cycle, differentiation, proliferation and cell activation. However, SHP2 is assumed to play a negative role during T-cell activation as the phosphatase has been shown to inhibit T-cell receptor-induced signaling cascades. Although, various gain-of-function mutations in the SH2 or PTP domain of this phosphatase, such as D61Y, have been associated with myeloproliferative diseases such as juvenile myelomonocytic leukemia (JMML), effects of such mutations on T cells have not been addressed in scientific literature so far. Therefore, in the second part of this thesis we could demonstrate that D61Y mutation in the SH2 domain of SHP2 did not cause JMML pathology when only introduced into T cells. Especially in aged mice, T cells of SHP2 mutant mice showed an increased expression of cell adhesion molecule CD44. In accordance with these findings, we observed increased influenza A virus-specific T cells in the bone marrow of SHP2 D61Y mutant mice, indicating a role of the phosphatase in memory formation or maintenance of CD8+ Tem. Although SHP2D61Y mice revealed a comparable viral clearance, IFNγ production of virus experienced CD4+ and CD8+ T cells was diminished compared to control mice, underlining a negative involvement of the phosphatase in the JAK/STAT1 signaling axis as suggested before by studies using mice with SHP2-/- T cells.
Reactive species play an essential role in orchestrating wound healing responses. They act as secondary messengers and drive redox-signaling pathways that are involved in the hemostatic, inflammatory, proliferative and remodeling phases of wound healing. Cold plasma produces a profusion of short- and long-lived redox species that promotes wound healing, however, until today, the knowledge of CAP mediated wound healing remained scarce. In this thesis, CAP mediated wound healing mechanism and their effect on extracellular matrix and adhesion molecules have been investigated. To this end, a keratinocyte cell line (HaCaT), skin fibroblast cell line (GM Fbs) and an in vitro coculture model including both HaCaT and GM Fbs at a 2:1 ratio, were employed to investigate the cross talk between these two skin cell types.
We examined the impact of CAP on extracellular matrix proteins and cell adhesion molecules in GM Fbs and observed a significant impact of cold plasma treatment on the expression level of collagen moieties, cell adhesion molecule like integrin, cadherin, versican, MMPs as well as extracellular matrix proteins.
Moreover, scratch assays with monocultures of HaCaT, GM Fbs and coculture of these two cell types were performed. We detected that, CAP accelerated the migratory capability of HaCaT cells cocultured with fibroblasts. In fact, compared to HaCaT monoculture, a significant acceleration on cell migration was observed in coculture upon CAP treatment. NAC, a potent antioxidant could abrogate this CAP-stimulated cell migration in coculture, further pointing towards the importance of well-orchestrated reactive species in wound healing. To better understand this CAP-mediated effect on cell migration, we examined the signaling pathways involved in tissue homeostasis and regeneration. We checked the HIPPO signaling pathway and observed an upregulation of several signaling molecules at transcriptional level in GM Fbs upon CAP treatment.
YAP is the central nuclear executer of HIPPO signaling pathway. YAP was upregulated in both HaCaT cells and GM Fbs. The major downstream effectors of the HIPPO signaling pathway (CTGF and Cyr61) were also upregulated in dermal fibroblasts at both transcriptional and protein level. However, administration of antioxidant NAC inhibited CAP-mediated wound healing and abrogated the gene expression of the HIPPO downstream effectors. These results confirm that the upregulation of YAP-CTGF-CYR61 axis is due to CAP-generated redox species. In HaCaT cells, both CTGF and Cyr61 was minimally transcribed. Even though CTGF was rarely detected in HaCaT cells on the protein level,Cyr61 remained undetected. This again shows the importance of the cross talk between fibroblasts and keratinocytes.
The coculture with the inclusion of fibroblasts showed an accelerated migration rate, compared to HaCaT monoculture which specifies a cross talk between these two cell types. Thus, monoculture of HaCaT cells were incubated with CAP-treated and untreated fibroblast conditioned medium. Interestingly, we observed that HaCaT cells exhibited an improved cell migration rate when incubated with CAP-treated fibroblast-conditioned media compared to that observed after incubation with untreated media. Upon investigation, an induction of CTGF and Cyr61 secretion was observed upon CAP treatment in the fibroblast-conditioned media. Furthermore, exposure to recombinant CTGF and Cyr61 could also significantly improve HaCaT cell migration which confirms that CAP mediated accelerated cell migration is due to activation of YAP-CTGF-Cyr61 axis.
In conclusion, this study revealed a completely new mechanical insight of CAP mediated wound healing. Along with several other ECM molecules, CAP activates a regenerative signaling pathway i.e., HIPPO signaling pathway in dermal fibroblasts at the onset of wound healing. Dermal fibroblasts drive a paracrine interaction by secreting CTGF and Cyr61 in close vicinity of wound, resulting in accelerated keratinocyte migration and wound healing in coculture.
Lipoproteins of Staphylococcus aureus represent a major class of surface proteins, which are anchored to the outer leaflet of the cell membrane. Although they play a key role in the immune response and virulence, the majority of lipoproteins in this organism is still of unknown function. The aim of our study was to investigate the function of so far poorly or uncharacterized lipoproteins in S. aureus strain Newman. To this end, an integrated bioinformatical approach was applied to define the pan-lipoproteome of 123 completely sequenced S. aureus strains. In total, this analysis predicted 192 different potential lipoproteins, with a core lipoproteome of 39 and a variable lipoproteome of 153 lipoproteins. Out of those 192 lipoproteins, 141 are so far functionally uncharacterized. Primarily focusing on members of the core-lipoproteome with unknown or poorly characterized function, 24 lipoproteins or co-encoded neighbor proteins were selected for further characterization. Of those 24 proteins, 20 S. aureus markerless deletion mutants were constructed (S. aureus delta l01 - delta l20) and screened for an altered growth behavior under various conditions. Here, three mutants showed a temperature-sensitive phenotype, two mutants formed aggregates in the TSB of the manufacturer Merck (TSBMerck), and four mutants showed reduced growth under osmotic stress with 8% NaCl. An altered aggregation behavior was observed for four mutants in the presence of Triton X-100 and for eleven mutants in the presence of SDS. Furthermore, ten mutants revealed an impaired biofilm formation capacity as well as reduced hemolytic activity. Interestingly, S. aureus deletion mutants delta l14 (delta NWMN_1435) and delta l16 (delta NWMN_0646) showed an altered phenotype under nearly all tested growth and stress conditions. Most strikingly, both deletion mutants demonstrated dramatic defects in cell morphology and cell division during the transient growth phase in TSBMerck and were therefore selected for further detailed characterization. Electron microscopy imaging of the two mutants revealed an irregular cell shape, increased cell size, multiple displaced division septa, and incomplete separation of daughter cells resulting in the formation of cell aggregates in TSBMerck. Complementarily, microarray-based transcriptome analysis and whole-genome sequencing of S. aureus delta l14 and delta l16 suppressor mutants strongly point to a functional association of both lipoproteins with cell envelope- or cell division-related processes. Specifically, multiple hints suggest a functional connection of both lipoproteins with lipo- or wall teichoic acids. Of note, the phenotypes of S. aureus delta l14 and delta l16 are conditional and appear under some, but not all growth conditions. Thus, it is conceivable that the function of L14 and L16 is modulated by metabolic processes, or that the proteins might be part of a “backup system” becoming important only under certain conditions. Collectively, we propose that L14 and L16 fulfill a basic role in cell envelope- or cell division-related processes under specific growth conditions. Particularly, the activity of L14 and L16 might be necessary for the function or localization of lipo- or wall teichoic acids, and thus, might be linked to the regulation of autolysins. In conclusion, this study reveals important insights into the function of two so far uncharacterized but highly conserved lipoproteins in S. aureus.
