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Clostridioides difficile is an intestinal human pathogen that uses the opportunity of a depleted microbiota to cause an infection. It is known, that the composition of the intestinal bile acid cocktail has a great impact on the susceptibility toward a C. difficile infection. However, the specific response of growing C. difficile cells to diverse bile acids on the molecular level has not been described yet. In this study, we recorded proteome signatures of shock and long-term (LT) stress with the four main bile acids cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), and lithocholic acid (LCA). A general overlapping response to all tested bile acids could be determined particularly in shock experiments which appears plausible in the light of their common steroid structure. However, during LT stress several proteins showed an altered abundance in the presence of only a single or a few of the bile acids indicating the existence of specific adaptation mechanisms. Our results point at a differential induction of the groEL and dnaKJgrpE chaperone systems, both belonging to the class I heat shock genes. Additionally, central metabolic pathways involving butyrate fermentation and the reductive Stickland fermentation of leucine were effected, although CA caused a proteome signature different from the other three bile acids. Furthermore, quantitative proteomics revealed a loss of flagellar proteins in LT stress with LCA. The absence of flagella could be substantiated by electron microscopy which also indicated less flagellated cells in the presence of DCA and CDCA and no influence on flagella formation by CA. Our data break down the bile acid stress response of C. difficile into a general and a specific adaptation. The latter cannot simply be divided into a response to primary and secondary bile acids, but rather reflects a complex and variable adaptation process enabling C. difficile to survive and to cause an infection in the intestinal tract.
Die Maul- und Klauenseuche (MKS) stellt eine Tierseuche dar, die bei Ausbruch zu dramatischen wirtschaftlichen Verlusten durch Beeinträchtigung der anfälligen Tiere führt. Das verursachende Agens der Krankheit ist das Maul- und Klauenseuche Virus (MKSV), welches der Familie der Picornaviren und der Gattung der Aphthoviren angehört. Ausbrüche der MKS weltweit sind selten, jedoch sollte der Erreger weiterhin erforscht werden, um ablaufende Prozesse besser verstehen, sichere und effiziente Impfstoffe entwickeln und im Falle eines Ausbruches angemessene Gegenmaßnahmen ausüben zu können. Das aus in etwa 8500 Basenpaaren bestehende Genom des Virus codiert unter anderem für die Leader Protease, ein wichtiger Virulenzfaktor, welcher unter anderem die Expression der Wirtsproteine durch die Spaltung des eukaryotischen Initiationsfaktors 4G (eIF4G) beeinträchtigt und ebenfalls die Immunantwort des Wirtes beeinflusst. In dieser Arbeit sollten verschiedene Systeme zur Untersuchung und Differenzierung der Funktionen der Leader Protease analysiert werden. Unter Zuhilfenahme unterschiedlich komplexer Systeme (Infektionssystem, Replikonsystem und Einzelexpressionssystem) erfolgte die Untersuchung der Translationsinhibierung, vermittelt über die eIF4G-Spaltung und der damit einhergehende Einfluss auf die Analyse auf andere Funktionen der Leader Protease. Sowohl im Infektionssystem als auch im hier etablierten Replikonsystem konnte die MKSV-induzierte Spaltung des eukaryotischen Initiationsfaktors 4G verifiziert werden. Hingegen konnte im Einzelexpressionssystem die Spaltung erst nach Lyse der Zellen nachgewiesen werden. Diese bisher noch nicht beschriebene Problematik lässt die bisher veröffentlichten Resultate und Schlussfolgerungen aus Einzelexpressionssystemen in Frage stellen. Die mit Hilfe des Einzelexpressionssystems durchgeführten Studien dieser Arbeit weisen darauf hin, dass ein zusätzlicher translationsinhibierender Mechanismus der Leader Protease, unabhängig von der proteolytischen eIF4G-Spaltung, ausgeübt wird. Dementsprechend sind die Möglichkeiten der Untersuchung zur Leader Protease mittels Einzelexpressionssystem sehr eingeschränkt, da eine Analyse spezifischer Leader Protease-Effekte unabhängig von einer Translationsinhibierung, dem „host-shut-off“, experimentell kaum möglich ist. Dies betrifft insbesondere die durchgeführten InterferonReportergenversuche. Somit konnten unter Verwendung verschiedener Untersuchungssysteme experimentelle Möglichkeiten, aber auch entscheidende experimentelle Limitierungen für die proteinbiochemische Analyse der Leader Protease-vermittelten eIF4G-Spaltung aufgezeigt werden.
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.
Microbial cell factories have been largely exploited for the controlled production of recombinant proteins, including industrial enzymes and biopharmaceuticals. The advent of high-throughput ‘-omics’ techniques have boosted the design of these production systems due to their valuable contribution to the field of systems metabolic engineering, a discipline integrating metabolic engineering with systems and synthetic biology. In order to thrive, the field of systems metabolic engineering needs absolute proteomics data to be generated, as proteins are the central players in the complex metabolic and adaptational networks. Due to advent of mass spectrometry-based proteomics, a substantial amount of absolute proteomic data became available in the past decade. However, membrane proteins remained inaccessible to these efforts.
Nonetheless, comparative studies targeting the membrane proteome have been quite successful in characterizing physiological processes. Hence, label-free proteomics was used in a study (Quesada-Ganuza et al, 2019 – Article I) to identify and optimize PrsA in Bacillus subtilis, for improved yield of amylase. Amylase is one of the most relevant enzymes in the biotechnological sector. By employing a label-free mass spectrometry approach targeting the membrane proteome of this bacterium, relative changes in heterologous and native levels of PrsA could be quantified. The results of this study evidenced that each PrsA shows different relative abundancies, but with no relevant impact in the yield of amylase.
Even though relative protein quantification can already provide a good visualization of the physiological changes occurring between different conditions, they are not sufficient to understand how resources are allocated in the cell under certain physiological conditions. Therefore, a global method for absolute membrane protein quantification remains the biggest requirement for systems metabolic engineering.
Hence, with this work, we successfully developed a mass spectrometry-based approach enabling the absolute quantification of membrane proteins (Antelo-Varela et al, 2019 – Article II). This study was also performed in the Gram-positive model organism Bacillus subtilis, regarded as a prolific microbial cell factory. The method developed in this work combines the comprehensiveness of shotgun proteomics with the sensitivity and accuracy of targeted mass spectrometry. Fundamental to the method is that it relies on the application of a correction and an enrichment factor to calibrate absolute membrane protein abundances derived from shotgun mass spectrometry. This has permitted, for the first time reported, the calculation of absolute membrane protein abundances in a living organism.
The newly developed approach enabled to accurately quantify ~40% of the predicted proteome of this bacterium, offering a clear visualization of the physiological rearrangements occurring upon the onset of osmotic stress. In addition, this work also provides evidence for new membrane protein stoichiometries.
Overall, this study enabled the development of a straightforward methodology long-needed in the scientific and biotechnological community and, for the first time reported, providing absolute abundances of one of the most puzzling fractions of the cell – the membrane proteome.
The next step of the work summarized here was to implement the afore described method to a biotechnological relevant strain, as absolute membrane protein abundances are essential to understand the fundamental principles of protein secretion and production stress. Hence, this work was applied in a genome-reduced B. subtilis strain, ‘midiBacillus’, expressing the major staphylococcal antigen IsaA (Antelo-Varela et al, submitted – Article III). The employed absolute membrane protein quantification methodology enabled the analysis of physiological rearrangements occurring upon the induction of heterologous protein production. This work showed that, even though IsaA was successfully secreted into the growth medium, one of the main requirements for the biotechnological sector, it was still partly accumulated in the cell membrane of this bacterium. This led to an exacerbated physiological response where membrane proteins involved in the management of secretion stress were activated. In addition, this study also showed that a rearrangement of the cell’s translocation machinery occurs upon induction of production, where a ‘game’ of in- and decrease of transporters takes place.
Anticipating the impact of genetic and environmental insults, such as the ones caused by production stress, is essential for the field of systems metabolic engineering. Thus, the highly accurate and comprehensive dataset generated during this work can be implemented in predictive mathematical models, thereby contributing in the rational design of next-generation secretion systems.