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Streptococcus pneumoniae is one of the leading human pathogen causing morbidity and mortality worldwide. The pneumococcus can cause a variety of different diseases ranging from mild illnesses like otitis media and sinusitis to life-threatening diseases such as pneumonia, meningitis and sepsis. Mostly affected are infants, elderly and immune-suppressed patients. Although, there are vaccines against pneumococci available, still hundreds of thousands of people got infected each year. These vaccines are targeting the pneumococcal polysaccharide capsule. Because of the high number of different serotypes, it is not possible to generate a vaccine against all present serotypes. In the last years a shift to non-vaccine serotypes was noticed. This strengthens the need for the development of vaccines which do not target polysaccharides. Thus, proteins came into focus as potential new vaccine candidates or targets for drug treatment, because several proteins are highly conserved among different strains or even genera. Proteome analyses can give insights into the protein composition in a certain state of a bacterium. So, targets can be identified, which are especially expressed under infection-relevant conditions. Iron limitation is one of these conditions and the knowledge on iron acquisition in pneumococci is still limited. Iron is an essential trace element and as redox-active catalyst or as cofactor involved in various key metabolic pathway in nearly all living organisms and thus also in bacteria. For instance, iron is necessary during biosynthesis of amino acids and in electron transport as well as in DNA replication. Within the human host iron is extremely limited due to its high insolubility under physiological conditions, which is part of the nutritional immunity of its human host. Hence, bacteria had to evolve mechanism to overcome iron starvation. In this thesis the adaptation process triggered by iron limitation in the S. pneumoniae serotype 2 strain D39 was investigated in a global mass spectrometry-based proteome analysis.
In preceding growth experiments the pneumococcal growth was adapted to the needs of proteomic workflows. In order to investigate the pneumococcal response to iron limitation, the organic iron-chelating agent 2,2’-bipyridine (BIP) was applied. For the quantification of changes in protein abundances comparing stress to control conditions the very reliable and robust metabolic labeling technique Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) was used. This method requires the bacterial cultivation in a chemically defined medium, for which reason modified RPMI 1640 medium was chosen. A pooled protein extract with heavy labeled amino acids was applied as an internal standard, which included proteins expressed under control and stress condition, to control, BIP and BIP-iron-complex (BIP control experiment) samples. Samples were analyzed by liquid chromatography coupled directly to a tandem mass spectrometer. It is described that under iron-restricted conditions proteins associated to pathogenesis are higher abundant in pathogenic bacteria like Staphylococcus aureus. Hence, similar observations were expected also for the proteomic adaptation of S. pneumoniae, but the first results showed a reduction in protein abundance of virulence factors. In order to explain these results inductively-coupled-plasma mass spectrometry was executed to determine the iron concentration of chemically defined medium (CDM) used in this experiment. The analysis revealed a relatively low iron concentration of approximately 190 µg l-1. Therefore, the iron concentration of the complex medium THY, in which pneumococci are usually grown, was investigated. THY contains four-fold (740 µg l-1) more iron than the CDM. Subsequently, an additional iron limitation approach was carried out in THY. As SILAC is not applicable in complex media like THY, MaxLFQ was applied as quantification method in this case. Because two different media were used, an additional comparative proteome analysis with regard to the two investigated media was executed.
Comparing the protein composition in both cultivation media it became clear that pneumococci exhibit a totally different proteome depending on the medium. Major differences were found in metabolisms of amino acids, vitamins and cofactors as well as in pathogenesis-associated proteins. These differences have to be taken into account during the analyses of both iron limitation approaches. Overall, more proteins were identified and quantified in CDM samples. The pneumococcal adaptation to iron limitation in both media was different; especially, the alterations in protein abundances of virulence factors. In contrast to the iron limitation in CDM, proteins involved in pathogenesis were higher abundant under iron limitation in THY, which was the expected result. Because of proteomic changes of cell division and lipid metabolism involved proteins in iron-limited pneumococci in CDM, electron microscopic pictures were taken in order to proof cell morphology. The pictures showed an impaired cell division in iron-limited CDM, but not in THY medium. However, both datasets have similarities as well. Thus, the iron uptake protein PiuA is strongly increased in iron-restricted conditions and the abundance of the iron storage protein Dpr is significantly decreased in both datasets. Notably, PiuA and Dpr seem to have important roles during the pneumococcal adaptation to iron-restricted environments.
One the basis of these results, it could be shown that the proteomic response of pneumococci to iron limitation is strongly dependent to the initial iron concentration of the environment. Hence, pneumococci will adapt differently to varying niches and thus potential vaccine candidates should be expressed independently of the localization within the human host.
The anaerobic pathogen Clostridioides difficile is perfectly equipped to survive and persist inside the mammalian intestine. When facing unfavorable conditions C. difficile is able to form highly resistant endospores. Likewise, biofilms are currently discussed as form of persistence. Here a comprehensive proteomics approach was applied to investigate the molecular processes of C. difficile strain 630Δerm underlying biofilm formation. The comparison of the proteome from two different forms of biofilm-like growth, namely aggregate biofilms and colonies on agar plates, revealed major differences in the formation of cell surface proteins, as well as enzymes of its energy and stress metabolism. For instance, while the obtained data suggest that aggregate biofilm cells express both flagella, type IV pili and enzymes required for biosynthesis of cell-surface polysaccharides, the S-layer protein SlpA and most cell wall proteins (CWPs) encoded adjacent to SlpA were detected in significantly lower amounts in aggregate biofilm cells than in colony biofilms. Moreover, the obtained data suggested that aggregate biofilm cells are rather actively growing cells while colony biofilm cells most likely severely suffer from a lack of reductive equivalents what requires induction of the Wood-Ljungdahl pathway and C. difficile’s V-type ATPase to maintain cell homeostasis. In agreement with this, aggregate biofilm cells, in contrast to colony biofilm cells, neither induced toxin nor spore production. Finally, the data revealed that the sigma factor SigL/RpoN and its dependent regulators are noticeably induced in aggregate biofilms suggesting an important role of SigL/RpoN in aggregate biofilm formation.