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Mass spectrometry-based Proteome analysis of porcine cells infected with African swine fever virus
(2023)
ASFV, a highly contagious, pathogenic and lethal pathogen of swine, poses a major threat to domestic and wild suids worldwide as neither vaccines nor treatments are available. Compared to other well-characterized similarly complex viruses like herpesviruses or adenoviruses, the understanding of ASFV biology is poor.
To improve the understanding of ASFV biology, following the establishment of a robust protocol for the isolation of primary monocyte-derived porcine macrophages (moMΦ) and their infection with ASFV for mass spectrometry (MS)-based proteome analysis was performed.
Under both conditions, naïve and infected, the isolated cells showed cell type-specific characteristics like phagocytosis and antigen presentation and protein expression patterns, including the expression of swine leucocyte antigens and CD markers. Furthermore, moMΦ could be reproducibly infected with ASFV isolates of different genotypes and pathogenicity.
The ASFV protein expression patterns in moMΦ correlate well with those observed in established cell lines at transcript and protein level. The expression of 27 ASFV proteins was confirmed at the protein level. Among them, 9 members of multi-gene families (MGF) and 12 novel open reading frames (nORFs) were recently predicted based on transcription start site mapping.
The direct comparison of closely related ASFV genotype II isolates revealed no virulence-associated protein expression patterns beyond those expected based on the genome sequences of the isolates.
Using different MS quantification strategies, it was shown that ASFV affects both static protein expression levels and protein synthesis. These changes in protein expression impact proteins and pathways known to be targeted by ASFV, including CD-markers, ER-stress and cell death pathways, and cellular antiviral responses. Beyond these observations that further validated the moMΦ infection model, novel effects of the ASFV infection on the cellular proteome were noticed.
These effects include the decreased expression levels of cathepsins, especially cathepsins D (CTSD), H (CTSH) and L (CTSL) as well as the transient activation of MAPK14/p38 prior to its strong downregulation. In addition to MAPK14/p38 further members of the MAPK14/p38 signaling pathway, like MAPKAPK2, were affected by ASFV infection.
As these modulations of the cellular proteome would in general result in decreased pro-inflammatory responses, it did stand out that the synthesis of interferon-response related genes including MX1 and ISG15 evaded the ASFV-induced global reduction of protein synthesis. In contrast, the synthesis of genes involved in RNA processing and splicing was significantly impaired. In total, the regulations of individual host proteins assessed in the context of the whole cellular proteome integrate well with each other and other cellular responses to ASFV infection and may help to improve the understanding of host-virus interactions.
Overall, this thesis provides novel insights into the expression of ASFV-encoded ORFs of different isolates and the host response to ASFV infection. It points out that the current knowledge of the ASFV coding capacity, temporal protein expression patterns, protein functionality, post-translational modifications and host interactions is still sketchy as many aspects of ASFV replication have yet to be understood. The established moMΦ-model to study ASFV infections in vitro provides a powerful tool for future applications to increase the understanding of ASFV biology.
The highly oncogenic alphaherpesvirus Marek’s disease virus (MDV) causes immense economic losses in the poultry industry. The main targets of in vivo MDV infection are primary B and T lymphocytes. The cytolytic infection of B cells leads to depletion of lymphoid cells results in severe immunosuppression. Infected B cells recruit and activate T cells. The close interaction between B cells and T cells enables efficient intercellular transfer of MDV. During infection of T cells, the virus enters a latent state. Infection of T cells can lead to transformation of these cells and formation of lymphoma, which manifest in various visceral organs. This study aimed at the characterization of the proteomes of MDV-infected lymphocytes during the lytic and latent phases of infection.
Previous in vitro studies concerning the MDV pathogenesis and host-virus interactions have been mainly conducted with primary fibroblasts or kidney cells, due to the short lifespan of primary lymphocytes in cell culture. Recently, a cultivation system has been established that extents the lifespan of primary lymphocytes through the addition of cytokines to the growth medium. This allowed the infection of B cells in vitro and to conduct quantitative proteomic analysis of primary lymphocytes. Infection with GFP labelled virus recombinants allowed the isolation of infected cells by FACS for the proteome analysis of MDV infected B lymphocytes. An efficient quantitative proteomic workflow was developed, which consisted of a filter-aided (FASP) digest of the extracted proteins, followed by differential dimethyl chemical labeling of the peptides for quantitative evaluation prior to LC-MALDI TOF/TOF mass spectrometry. Only few alterations of the protein and transcript expression profiles were observed after infection of primary B cells with the very virulent RB-1B and the live-attenuated vaccine strain CVI988/Rispens. Relevant changes in relative protein levels were found for only twelve and six interesting host proteins after RB1B and CVI988 infection, respectively. However, the regulations were confirmed by inspection of the spectra from all experiments. The identified candidates play a role in immune response, translation and inflammatory response.
To confirm the potential infection markers, RNA-seq analysis of three biological replicates of each RB-1B -, CVI988- and mock-infected B cells was performed. Eighty expressed MDV transcripts could be identified, which were associated with lytic infection. The same MDV proteins were identified after infection with RB-1B or CVI988. However, transcriptome and proteome analysis of MDV-infected primary B cells showed only poor correlation. This indicates that the changes in protein expression profiles are mostly due to posttranscriptional events. Infection marker candidates were identified by the RNA-seq analysis, for which the gene expression was altered by MDV infection. Although almost 12,000 transcripts were identified, only few transcript levels changed markedly after MDV infection. The biological processes immune response, apoptotic process, signal transduction, cell migration and response to virus were enriched after MDV infection. The RNA-seq results confirm the observation that alterations of protein levels early after MDV infection are rare.
Most notably, MDV induces transformation of lymphocytes leading to malignant T-cell lymphomas in visceral organs with mortalities of up to 100 %. While several factors involved in MDV tumorigenesis have been identified, the transformation process is not fully understood. Therefore, we set out to fill this knowledge gap using proteome analysis of transformed T-cells ex vivo. In addition, the role of the viral telomerase RNA during transformation was assessed by comparison of tumors that had formed after infection with WT-virus or a telomerase RNA negative mutant. A major obstacle for tumor proteome analyses is the preparation of sufficient amounts of homogenous tumor tissue, as tumors appear with a dispersed morphology in the affected organs. The quantitation of cell types within the tumors indicated varying portions of hepatocytes, connective tissue, and CD3+ lymphocytes even with the same virus strain in different animals. However, the ∆vTR-induced tumors contained lower levels of hepatocytes and higher levels of CD3+ lymphocytes compared to WT tumors in all tested tumor samples. Thus, ∆vTR tumors were chosen for determination of differences in protein expression profiles of tumors and naïve T cells for their lower content of liver cells. We developed a workflow for the proteome analysis of T cell tumors from livers of MDV-infected chickens. Samples included laser capture micro-dissected tissue cuts from tumors and surrounding healthy liver tissue as well as naïve T-cells prepared from thymus. To enable quantitative proteome analysis, samples were digested using the FASP protocol and peptides were isotope-coded by differential dimethyl labeling. To improve proteome analysis peptides were fractionated by preparative isoelectric focusing prior to nano-HPLC MALDI/TOF-TOF mass- spectrometric analysis.
Proteomic analyses of LCM dissected ΔvTR tumor compared to naïve T cells, the main targets of transformation, identified nineteen potential transformation markers but again only minor changes in relative levels were observed. Several of the identified markers could also be verified by RT-qPCR on transcript level. The identified transformation candidates were associated with nucleosome assembly, regulation of transcription, inflammatory response, immune response and oxidation-reduction process.
However, further functional analyses are necessary to fully elucidate the role of the identified markers during MDV infection and transformation.
Humanity is plagued by many diseases. Beside environmental influences, many --- if not all --- diseases are also subject to genetic predisposition and then display molecular alterations such as proteomic or metabolic aberrations. The elucidation of the molecular principles underlying human diseases is one of the prime goals of biomedical research. To this end, there has been an advent of large-scale omics profiling studies. While the field of molecular biology has experienced tremendous development, data analysis remains a bottleneck. In the context of this thesis, we developed a number of analysis strategies for different types of omics data resulting from different experimental settings. These include approaches for associations studies for plasma miRNAs and time-resolved plasma omics data. Furthermore, we devised analyses of different RNA-Seq transcriptome profiling studies coping with problems such as lack of replicates or multifactorial experimental design. We also designed machine learning frameworks for the identification of discriminatory biomolecular signatures analysing case-control or time-to-event data. All of the strategies mentioned above were developed and applied in the contexts of multi-disciplinary endeavours. They aided in the identification of plasma miRNAs associated with age, sex, and BMI as well as plasma miRNAs bearing potential as diagnostic biomarkers for non-alcoholic fatty liver disease (NAFLD). This thesis significantly contributed to a study demonstrating the utility of plasma miRNAs as prognostic biomarkers for major cardiovascular events such as ST-elevation myocardial infarction. Our approaches for analysing RNA-Seq data aided in the characterisation of murine models for Alzheimers disease and the transcriptional response of human gingiva fibroblasts to ionizing radiation exposure. Furthermore, the developed approaches were applied for studying a human model for thyrotoxicosis and for the successful identification of a multi-omics plasma biomarker signature of thyroid status. We are only beginning to understand the molecular principles underlying human diseases. The approaches and results presented in this thesis will contribute to improved understanding of biomolecular processes involved in common diseases such as Alzheimers disease, NAFLD, and cardiovascular diseases.
Microbial infections can be either caused by a single species or complex multi-species consortia. One of the most prominent opportunistic human pathogens leading to mono- or mixed-species infections is the Gram-negative bacterium Pseudomonas aeruginosa. Understanding the molecular basis of its adaptation to infection-related stresses is an essential prerequisite for the prevention and treatment of P. aeruginosa infections. We therefore employed state-of-the-art proteomics approaches to elucidate the molecular adaptation mechanisms of P. aeruginosa to infection-related conditions. Moreover, structure, function and interaction of complex microbial consortia containing P. aeruginosa and causing catheter-associated urinary tract infections were investigated by metaproteomics analyses. Our investigations revealed that the adaptation of P. aeruginosa during infection is either based on gene expression changes caused by environmental signal integration or by gene mutations leading to a selective advantage in a particular host environment. In study I, investigating the proteome response of P. aeruginosa biofilms to the clinical relevant antibiotic ciprofloxacin, global changes in the protein profile were observed. Ciprofloxacin induced the expression of proteins involved in the Lex-induced SOS-response, drug efflux pumps and gene products of the ciprofloxacin-responsive prophage cluster and repressed the expression of porins and DNA-binding proteins. In study II the transcriptome and proteome of two clonal P. aeruginosa lineages during long-term colonization of cystic fibrosis (CF) patient’s lungs were analyzed. Point mutations in global regulator genes, i.e. retS, gacS, and gacA, were identified by genomic sequencing. Inactivation of RetS, found two years after the initial colonization, induced the expression of genes involved in chronic infections and coding for the type 6-secretion system (T6SS). Additional mutations in the GacS/GacA two-component regulatory system (TCS) were found to repress the expression of T6SS proteins and to induce the expression of proteins belonging to the type 3-secretion system (T3SS). In study III we elucidated the niche-specific adaptation of P. aeruginosa isolates from different infection sites by investigating their protein expression patterns and glucose metabolic fluxes. We could show that isolates from the urinary tract express a higher amount of proteins involved in the acquisition of micronutrients (i.e. iron) and carbohydrates compared to isolates from the CF lung. In study IV 16S rDNA sequencing and metaproteomics were employed to demonstrate that the investigated CAUTI-related biofilms consisted of two to five different species with one or two species dominating the mixed community. Following this line of research, we investigated in study V structure and function of a biofilm of a long-term catheterized patient, which was predominantly composed of P. aeruginosa and Morganella morganii, but also contained a minor proportion of the obligate anaerobe Bacteroides sp.. The comparison of in vivo and in vitro protein expression profiles of P. aeruginosa and M. morganii indicated that iron and carbohydrates are the major growth-limiting factors in the bladder. These results indicate different nutritional strategies of the two pathogens in the bladder environment. A comparison of urinary protein profiles of healthy persons and catheterized patients suggested that the human innate immune system is induced by CAUTIs. Moreover, numerous proteins involved in nutritional immunity, e.g. iron-, calcium- and magnesium-binding proteins, were found to be more abundant in the urine of catheterized patients. A follow-up (meta)proteomics study (study VI) aiming at the elucidation of interspecies interactions during multi-species infections indicated that the urease-positive uropathogen Proteus mirabilis induces the precipitation of metal ions by urine alkalization and thereby limits the availability of these important micronutrients for other co-infecting bacteria. This limitation seems to be sensed by the P. aeruginosa PhoP-PhoQ two-component system (TCS) leading to an increased resistance to antimicrobial peptides and biofilm-forming capacity of the pathogen. Also during co-cultivation of P. aeruginosa with Staphylococcus aureus a slight increase in the expression of the PhoP-PhoQ TCS and the alkaline protease could be observed (study VII). In study VIII a combined metagenomics and metaproteomics approach was employed to investigate structure and function of the lichen Lobaria pulmonaria, a complex consortium consisting of a fungus, an algal partner, cyanobacteria, and a highly diverse bacterial microbiome. The results presented in this work contribute to a better understanding of the manifold and complex bacterial adaptation mechanisms to infection-related and environmental stress and thereby foster the development of novel treatment and prevention strategies.
This thesis will discuss the different fields of application of the two soft ionization techniques ESI and MALDI in microbial proteomics and their importance for a better understanding of bacteria physiology. The general development in the past 25 years coming from 2D-gel analysis and protein identification by peptide mass fingerprint analysis via MALDI-TOF to genome wide quantitative LC-ESI-MS experiments with fast and sensitive ESI instruments is exemplary shown for the Gram-positive bacterium Bacillus subtilis in article I. Even though 2D-PAGE in conjunction with MALDI-MS is still an important tool in proteomic research, the more recently established global quantitative LC-ESI-MS workflows gain more and more relevance as they overcome 2D-PAGE based protein restrictions and enable the acquisition of higher accurate protein quantities. In article II such a workflow was used to analyze the physiological adaptation of Staphylococcus aureus to vancomycin treatment on a global-scale. Also post-translational modifications of proteins, that are important for regulation of their activity and allow rapid adaption to changed environmental conditions, could be analyzed by LC-ESI-MS workflows using special enrichment strategies (article III and IV). Despite the mentioned discrimination and less accurate quantification of proteins, 2D-PAGE analyses are still advantageous when analyzing large-scale time series experiments. To gain highly time resolved data but also very accurate relative quantities on a global-scale, 2D-PAGE-MALDI-MS and LC-ESI-MS techniques have been combined to investigate dynamic proteome adaptations of B. subtilis during nutrition shift as part of a global systems biology approach (article V). Also absolute quantities of proteins are of high interest for systems biology, but are still challenging to obtain on large-scale as well as with sufficient accuracy. In article VI a method that again combined 2D-PAGE-MALDI-MS and LC-ESI-MS was introduced to gain absolute protein quantities on global-scale. Utilizing the complementarity of 2D-PAGE and LC-ESI-MS this new workflow enabled fast and cost efficient data acquisition on absolute scale. In article VII we described for the first time a global quantitative LC-MALDI-MS workflow. Cross validation with an LTQ Orbitrap proofed that LC-MALDI-MS is able to process complex samples and obtain highly reliable quantities. The comparative analysis of data gained with both instrument types revealed biases for certain biochemical properties of MALDI as well as ESI instruments, resulting in a general complementarity of both ionization techniques. Article I Becher, D., Büttner, K., Moche, M., Hessling, B., Hecker, M., 2011. From the genome sequence to the protein inventory of Bacillus subtilis. Proteomics 11, 2971–2980. Article II Hessling,B., Bonn,F., Herbst,F.-A., Rappen,G.-M., Bernhardt,J., Hecker,M. and Becher,D. Global proteome analysis of vancomycin stress in Staphylococcus aureus. Submitted to Mol. Cell Proteomics. Article III Elsholz, A.K.W., Turgay, K., Michalik, S., Hessling, B., Gronau, K., Oertel, D., Mäder, U., Bernhardt, J., Becher, D., Hecker, M., Gerth, U., 2012. Global impact of protein arginine phosphorylation on the physiology of Bacillus subtilis. Proc. Natl. Acad. Sci. U.S.A. 109, 7451–7456. Article IV Chi, B.K., Gronau, K., Mäder, U., Hessling, B., Becher, D., Antelmann, H., 2011. S-bacillithiolation protects against hypochlorite stress in Bacillus subtilis as revealed by transcriptomics and redox proteomics. Mol. Cell Proteomics 10, M111.009506. Article V Buescher,J.M., Liebermeister,W., Jules,M., Uhr,M., Muntel,J., Botella,E., Hessling,B., Kleijn,R.J., Le Chat,L., Lecointe,F., et al. (2012) Global network reorganization during dynamic adaptations of Bacillus subtilis metabolism. Science, 335, 1099–1103. Article VI Maass, S., Sievers, S., Zühlke, D., Kuzinski, J., Sappa, P.K., Muntel, J., Hessling, B., Bernhardt, J., Sietmann, R., Völker, U., Hecker, M., Becher, D., 2011. Efficient, global-scale quantification of absolute protein amounts by integration of targeted mass spectrometry and two-dimensional gel-based proteomics. Anal. Chem. 83, 2677–2684. Article VII Hessling,B., Büttner,K., Hecker,M. and Becher,D. Global relative quantification with LC-MALDI – cross-validation with LTQ-Orbitrap proves reliability and reveals complementary ionization preferences. Submitted to Mol. Cell Proteomics.
Dilated Cardiomyopathy is a chronic myocardial disease characterized by progressive depression of contractile function and ventricular dilatation. It is the leading cause of heart failure and the most common reason for heart transplantation. Besides genetic causes, viral infection and autoimmune response are considered to play a major role in the etiology of the disease. Among different viruses that cause the disease, Coxsackievirus B3 (CVB3) is predominantly associated with the development and progression of the disease. Moreover, Coxsackievirus induced myocarditis in the mouse mimics human myocarditis and dilated cardiomyopathy. In the murine model, the disease progresses over a period of 90 days from acute myocarditis to chronic myocarditis and further develops into dilated cardiomyopathy and congestive heart failure. Though much is known about the progression of the disease, the molecular events occurring after infection with CVB3 are not completely understood. In the current study, comparative proteomic analysis of A.BY/SnJ mouse hearts 84 days post infection (84 d p.i.) with CVB3 and age-matched non-infected mouse hearts was performed. 2D-DIGE and gel-free LC-MS/MS were used to characterize the changes occurring at the molecular level and Western Blot analysis as well as immunohistochemical staining was carried out for validation of results. A total of 101 distinct proteins were identified as displaying dilated cardiomyopathy-associated changes in A.BY/SnJ mouse hearts 84 d p.i. compared to age matched controls. Comprehensive analysis by both DIGE and gel-free proteomics revealed proteins related to lipid metabolism (18%), carbohydrate metabolism (14%), cell morphogenesis (14%) and respiratory electron transport chain (9%) to display significantly altered levels in diseased mouse hearts. The significant increase in extracellular matrix proteins observed in mouse hearts 84 d p.i. indicated extensive fibrosis. On the other hand, proteins related to energy metabolism were identified at lower levels in infected mouse hearts than in controls. These proteomics data and the decrease in activities measured for complexes I-IV of the respiratory electron transport chain in A.BY/SnJ mouse hearts 84 d p.i compared to age matched controls, indicate a diminished energy supply in the dilated hearts of CVB3 infected mice. Furthermore, proteins associated with muscle contraction were identified at lower levels in mouse hearts 84 d p.i. compared to age matched controls indicating compromised myocardial contractility due to virus induced dilated cardiomyopathy. While extracellular matrix proteins and contractile proteins were identified in the DIGE analysis, proteins of lipid metabolism which are mostly mitochondrial in origin and have a pI > 7 were identified by gel-free proteomics indicating the advantages of both methods. Gel based analysis also aided in the identification of protein isoforms/ species which allows conclusions on post translational modifications and protein processing. Thus, the current study also identified infection related changes in the phosphorylation of selected proteins. Phosphospecific staining of the gels demonstrated increased phosphorylation of myosin regulatory light chain - ventricular isoform, actin - aortic smooth muscle isoform, heat shock protein 90B, and heat shock protein beta-1 in infected mouse hearts. Extensive degradation of proteins was not observed in the dilated heart. As described earlier, virus induced dilated cardiomyopathy develops over a period of 90 days in the murine model during which the mice also grow and undergo aging. Since aging is one of the factors influencing the susceptibility of animals to disease, age dependent changes in the proteome of mouse hearts were also studied by comparing 4 months old (84d) A.BY/SnJ mice with 1 month old mice as controls. Complementary analyses by 2D-DIGE and gel-free LC-MS/MS analysis revealed 96 distinct proteins displaying age associated differences in intensity. These proteins are related to lipid metabolism (19%), protein transport (17%) and electron transport chain (12%). Mitochondrial proteins such as carnitine-o-palmitoyltranferase 1, carnitine-o-palmitoyltranferase 2, and carnitine-O-acetyltransferase involved in lipid metabolism and transport were identified at significantly higher levels indicating higher energy demand in 4 months old mice compared to controls. This conclusion is complemented by observation of decreases in the levels of respiratory electron transport chain proteins especially of subunits of ATP synthase as a member of complex V. Furthermore, an increase in intracellular transport proteins was also observed in 4 months old mouse hearts compared to one month old controls. An increase in the level of vesicular transport proteins likely constitutes a secondary effect leading to endoplasmic reticulum associated protein degradation. In the two studies described above, altered mitochondrial functioning and thereby decreased energy/ATP production was very prominent indicating the role of mitochondria in health and disease. The exchange of ADP/ATP across the mitochondrial membrane is carried out by the carrier protein adenine nucleotide translocase1 (ANT1). To improve understanding of the influence of ANT1 in the heart, comparative proteomic analysis using gel-free LC-MS/MS was performed with hearts of 3 months old rats over-expressing ANT1 using hearts from age-matched wild type animals as controls. A total of four hundred and thirty three proteins were identified with at least two peptides, of which eighty seven proteins displayed small but significant (p<0.05) changes in intensity. Proteins related to integrin linked kinase signalling and myocardial contraction displayed increased levels whereas proteins of the mitochondrial respiratory electron transport chain displayed decreased levels in ANT1 overexpressing hearts compared to wild type animals. Oxyblot analysis performed to study changes in the protein oxidation did not reveal any significant difference in the oxidative state of the proteins between the wild type and transgenic animals. To understand the influence of ANT1 overexpression in virus induced dilated cardiomyopathy, comparative proteomic analyses was performed for the mitochondrial fractions from the hearts of 8 months old rats of the wild type and ANT1 transgenic animals infected with CVB3. Of a total of 370 identified proteins, 83 proteins displayed altered levels in ANT1 overexpressing animals compared to controls. Proteins related to mitochondrial electron transport chain, fatty acid metabolism, contractility and cell structure displayed decreased levels in the infected transgenic animals compared to controls indicating decreased energy metabolism and myocardial contractility besides compromised cell structure. Besides viral causes of dilated cardiomyopathy, autoimmunity also plays a major role in the development of myocarditis and dilated cardiomyopathy. Therefore proteomic analyses of experimental models of autoimmune myocarditis generated by active immunization of rats with peptides of FcγIIa receptor -CEPPWIQVLKEDTVTL (peptide 1) designated as FcR animals and CRCRMEETGISEPI (peptide 2) designated as FcR2 animals- was performed. Of the 303 proteins identified with at least two peptides by gel-free LC-MS/MS analysis. 43 proteins displayed intensities greater than 1.2 fold in FcR rat hearts and 49 proteins displayed intensities greater than 1.2 fold in FcR2 rat hearts compared to animals injected with KLH adjuvant treated as controls. The majority of the alterations (>70%) were observed in both autoimmune models. Thus, immunization leading to an induction of the acute phase response signalling was observed in both experimental setups. Furthermore, the increased amount of proteins such as lumican or procollagen alpha 1, type 1 indicated the presence of fibrosis after immunization independent of the peptide used. In summary, using proteomics the current thesis addresses the changes in protein profiles of two models of dilated cardiomyopathy, namely, virus induced dilated cardiomyopathy and autoimmunity induced dilated cardiomyopathy in mouse and rat models of disease. 2D-DIGE and gel-free LC-MS/MS analysis are complementary techniques which provided a comprehensive view of the changes in the protein profile of hearts of the different animal models. Altered mitochondrial function resulting in decreased energy metabolism and compromised myocardial contractility were prominent in viral models of cardiomyopathy whereas intense acute phase response signalling was observed as a characteristic feature of autoimmune dilated cardiomyopathy. Altered mitochondrial function was also prominent in age associated changes in the heart of A.BY/SnJ mice indicating the role and influence of mitochondria in health and in disease.
Non-thermal atmospheric pressure plasma has recently been shown to have broad application potential for medical as well as industrial purposes. Improved wound healing and tissue decontamination have been described as consequences of non- thermal plasma treatment. However, thus far the underlying molecular mechanisms in human tissues have only been partially characterized. In this work a two-dimensional difference in-gel electrophoresis (2D-DIGE) approach was used and an analysis-workflow to study the response of human cells to atmospheric pressure non-thermal plasma was established. Human S9 bronchial epithelial cells were used as a model for airway epithelial cells. They were treated with atmospheric pressure plasma jet (APPJ) for different periods of time. Subsequently, time-resolved comparative proteome analysis was used to study the complex cellular adaptation reactions after a 120 sec plasma treatment, which accelerated wound healing in a clinically relevant model. The results indicate, that intracellular oxidative stress due to the non-thermal plasma treatment either leads to cell death or to proliferation. The oxidative stress response, mediated by Nrf2, appears to play a pivotal role in molecular signalling and might be a key pathway determining the fate of stressed cells. This thesis demonstrates changes in Nrf2-expression after non-thermal plasma treatment. Furthermore, potential protein biomarker candidates for evaluation of oxidative stress after non-thermal plasma treatment were identified. Finally, it is shown, that the cytosolic concentrations of IL-1beta and IL-33 were decreased following non-thermal plasma treatment. Thus, modulation of innate immune response by non-thermal plasma treatment of epithelial cells (ENTplas treatment) is concluded.
Staphylococcus aureus is a pathogenic bacterium infecting the human host. It’s multifaced adaptation to various environmental conditions is mediated by a tight regulation of the virulence factors influencing the host’s immune system. In this thesis two regulators of gene expression were analysed: (i) the global influence of the two-component system SaePQRS and (ii) the regulation of superantigen gene expression by the alternative sigma factor σB. At the outset of this thesis, single target genes induced by SaeRS were known (hla, hlb, cap5, fnbA, coa). In order to get a general idea of the Sae-regulon, the influence of SaePQRS on gene-expression was analysed in two strain backgrounds by proteomics and transcriptomics aproaches. Recapitulatory, expression of at least 18 secreted and two covalently cell-wall bound proteins was decreased following inactivation of the Sae-system. Sae-dependently expressed were, amongst others, well decribed virulence factors like the y-hemolysins HlgA, HlgB, HlgC, LukM and LukF, the innate immune system modulating proteins Efb, CHIPS and SCIN-B as well as the enterotoxin SEB. SaeR acts as an activator of its target genes. Some proteins were detected in increased amounts in the extracellular proteome of the Sae-deficient strain. However, these changes did not occur at the transcriptional level. The expression of virulence factors is determined by other global regulators. No influence of SaePQRS on the transcription of five substancial regulators, namely the Agr-system and its effector molecule RNAIII, the alternative sigma factor σB, the two-component system ArlRS and the DNA-binding protein SarA, could be shown. In the second part of this thesis the issue was broached to the regulation of gene-expression of a subgroup of virulence factors, the superantigens (SAgs) of S. aureus by SaePQRS and σB. In contrast to their well described molecule structure and function, the regulation of their gene expression was largely unknown. Six different S. aureus strains (two laboratory strains and four clinical isolates) encoding one to seven SAg-genes each, were used for analysis of a total of twelve SAgs regarding their transcription and mitogenic activity. The transcriptional units were characterized using Northern-Blotting. The expression of SAgs could be correlated to the respective growth phase. While egc-SAgs were expressed mainly at low optical densities, seb was induced during late growth phase. In contrast, the transcription of sea, seh, sek, tst and sep remained constant and growth-phase independent. The transcriptional dataset was verified using T-cell proliferation assays. The expression of seh, tst and the egc-operon was dependent on σB. A potential σB-dependent promotor could be identified preceeding seo, the first gene of the egc-operon. In contrast, the expression of seb was increased in sigB-deficient background. This might be due to indirect effects. Expression of seb required SaePQRS. Transcriptional datasets were verified by Immuno-Blotting and T-cell-proliferation assays. In conclusion, the same mutation in sigB but in different strain backgrounds could result in opposite phenotypes with respect to their mitogenic activity. Besides well characterized virulence factors, some secreted proteins with so far unknown function belong to the Sae-regulon. Given that the influence of SaePQRS was restricted to virulence factors and induced especially modulators of the innate immune system, it can be assumed, that these proteins potentially play a role in virulence of S. aureus. In the third part of this thesis, one of these potential new virulence factors, namely SACOL0908, was analysed in detail. In cooperation with the group of Prof. Stehle, Tübingen, the crystal structure was solved. The protein folding of SACOL0908 is new with only minor similarities to described protein structures. Recombinantly expressed SACOL0908 binds to granulocytes. These cells belong to the innate immune system, incorporate bacteria by phagocytosis and kill them. The receptor for SACOL0908 on the surface of granulocytes could not be identified using immunoprecipitation, antibody-blocking assays and functional assays in cooperation with the group of Prof. Peschel, Tübingen. The gene encoding SACOL0908 was deleted in two S. aureus strain backgrounds (COL and Newman). These mutants are currently in use to characterize their phenotype in mouse-infection studies.
Macrophages are cells of immune system and distributed throughout the body. They provide the first line of defense against microbial pathogen infections. Using bone marrow macrophages (BMMs) which derived from mice of strain BALB/c and strain C57BL/6, this study aimed to identify the changes in proteome of the macrophages due to IFN gamma stimulation and S. aureus infection. Two quantitative proteomic techniques, two-dimensional difference gel electrophoresis (2D-DIGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were applied in this study. The analysis results indicated that many proteins which play important roles in immunological functions of macrophages were changed due to IFN gamma stimulation and S. aureus infection. This study also identified the differences in proteome of macrophages derived from mice of strain BALB/c in comparing to macrophages of strain C57BL/6.
In the post genomic era, novel “Omics” technologies like genomics and proteomics can be used in powerful screening approaches to provide unbiased lists of candidate genes and proteins and thus facilitate a comprehensive analysis of complex diseases such as cancer, which would not have been possible applying traditional genetic and biochemical approaches alone. During my PhD tenure I applied functional genomics screening technologies including proteomics in combination with traditional biochemical and cell biology approaches in two disease oriented projects: 1. Characterization of the role of BCL11b in Human T cell lymphomas (and) 2. Elucidation of the mechanism of pathophysiology of Johanson Blizzard Syndrome using UBR1 knockout mice and JBS patients’ lymphoblasts cell lines.
1.Characterization of the role of BCL11b in Human T cell lymphomas
: The Bcl11b protein belongs to the C2H2-family of Krueppel-like zinc finger proteins and thus is a member of the largest family of transcription factors in eukaryotes. It was shown to be important for a variety of functions such as T cell differentiation, normal development of central nervous system and DNA damage response. Malignant T cells undergo apoptotic cell death upon BCL11B down-regulation. However, the detailed mechanism of this cell death is not fully understood. Two dimensional difference in-gel electrophoresis (2D-DIGE), mass spectrometry and cell biological experiments were employed to investigate the functional impact of knock down of BCL11B in malignant T cell lines such as Jurkat and huT78. To further confirm the findings of these experiments, changes in protein patterns were also recorded after down-regulation of BCL11B expression in Jurkat cells over expressing BcL-xL and in Jurkat cells over expressing BCL11B. These experiments provide evidence for the involvement of the mitochondrial apoptotic pathway and increased levels of cleavage fragments of known caspase targets such as myosin, spectrin and vimentin were observed after BCL11B knockdown. The findings suggest an involvement of ERM proteins, which were up-regulated and phosphorylated upon BCL11B down-regulation. Besides ERM proteins, PDCD5, a key regulator of apoptosis, was also found at increased levels upon down regulation of BCL11B. Moreover, the levels of several proteins implicated in cell cycle entry, including DUT-N, UCK2, MAT1, CDK6, MCM4 and MCM6 were elevated, which might lead to uncontrolled cell cycle progression, uracil misincorporation and cell death. Interestingly, an inverse regulation pattern, i.e. decreased levels of ERM proteins, DUT-N, UCK2 and PDCD5 was seen upon over expression of BCL11B in Jurkat cells. In summary, proteome analyses revealed several previously unidentified mechanisms which could significantly contribute to the cell death following BCL11B knockdown.
2.Elucidation of the mechanism of pathophysiology of Johanson Blizzard Syndrome using UBR1 knockout mice and JBS patients’ lymphoblasts cell lines
: Johanson-Blizzard syndrome (JBS; OMIM 243,800), which was first described in 1971, is a rare autosomal recessively inherited genetic disorder with a unique combination of congenital abnormalities. The most constant clinical feature of JBS is the loss of exocrine pancreatic function due to progressive destruction of pancreatic acini. Genome wide linkage analysis identified the disease associated locus in the 15q14-q21 chromosome region and high-throughput sequencing of this region revealed several truncated and some missense mutations in the UBR1 gene. UBR1 gene contains 47 exons and spans over 161 kilobases. The UBR1 protein belongs to the E3 ubiquitin ligase family and is an important component of the N-end rule pathway of ubiquitous protein degradation. It was hypothesized that stabilization of direct and unique substrates of UBR1 could be the main cause of the JBS pathophysiology. So far sequencing of the UBR1 gene is the only available diagnostic procedure. However, sequencing might not always allow precise prediction of residual UBR1 activity. Hence, this study was started to develop a protein based diagnostic assay for the detection of subclinical cases of JBS and to identify signalling pathways contributing to the pathophysiology of this complex disorder using a murine UBR1 knockout model. 2D-DIGE proteome analysis was carried out for a comparative evaluation of lymphoblast samples of 14 patients and 11 controls. Principal component Analysis (PCA) clearly discriminated JBS patients from controls. However, 4 JBS patients differed from the rest and resembled controls more closely. Western-blot analysis revealed residual UBR1 levels in these patients, which were linked to a milder phenotype. Hierarchical clustering of the three groups (controls, patients with residual UBR1 levels and patients without UBR1) showed group-specific characteristic differences in the abundance of differentially regulated proteins. Quantification of a panel of five selected protein spots encompassing Interferon-induced GTP binding protein, HLA class II histocompatibility antigen, Annexin A6, FK506-binding protein 4 and GRP78 permitted discrimination of controls and JBS patients with mild phenotypes. Of note, the molecular chaperones GRP78 (BiP) and FK506BP were consistently altered in level in JBS patients and probably constitute UBR1 dependent substrates. This suggested JBS as an ER-stress related disease also indicating a possible way of therapeutic intervention. Comparative proteome analysis of UBR1 knockout and wild type animals after caerulein treatment revealed a significant accumulation of pancreatic proteases such as chymotrypsin B, anionic trypsin and pancreatic elastase in animals lacking UBR1. Furthermore, an up-regulation of ER-stress proteins and inflammation related proteins was observed. Phenotypic characterisation revealed in UBR1 knockout animals significantly increased lipase levels, a significantly increased histological score and significantly increased elastase activity 8h after the onset of pancreatitis. In isolated pancreatic acini of UBR1 knockout animals we found a significant increase in intracellular elastase activation upon supramaximal CCK stimulation, which was associated with a significant rise in the rate of necrosis explaining the more severe phenotype in the UBR1 knock-out animals. A TUNEL assay showed that there was more apoptosis in wild type compared to UBR1 knockout mice. Another set of experiments was designed to identify physiologically important substrates of UBR1. Inhibition of such substrates might then in turn allow reversion or prevention of the severe form of pancreatitis in UBR1 knockout mice. However, using the trypsin specific and reversible inhibitor S-124 it was shown that impaired trypsin degradation and thereby prolonged activation of this protease did not critically influence the phenotype. Calcium analysis after physiological stimulation revealed an increase of pathological Ca2+ signalling events, i.e. significant decrease of spike number and significant increase of spike duration. Of the candidates potentially influencing Ca2+ signalling RGS4 turned out to be of particular importance. Pre-incubation of pancreatic acini of UBR1 knockout animals with a specific RGS4 inhibitor (CCG-4986, 10 µM) normalized Ca2+ patterns, did not affect trypsin activity itself but prevented Ca2+-triggered premature trypsin activation and thus acinar disintegration. In summary, using lymphoblasts samples of JBS patients we were able to deduce a protein panel which could be developed as a possible diagnostic tool for confirmation of JBS syndrome. Furthermore, using UBR1 knockout mice in an experimental model we were able to elucidate the vital function of UBR1 and its direct substrate RGS4 in the defense against pathologic pancreatic damage thereby manifesting JBS as an inflammatory disorder due to an inadequate UBR1 mediated defense.