Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Izghirean, N.
Effects of ribosomal protein S10 mutations on tetracycline and tigecycline susceptibility of Escherichia coli.
[ Diplomarbeit/Master Thesis (UNI) ] Universität Graz; 2022.
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- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Zarfel Gernot
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- Abstract:
- Every time an antibiotic is used, it can lead to resistance development. The sensitive bacteria get killed through/after antibiotic treatment, but resistant ones may be left. Those can grow, multiply and spread further to other people and cause infections that common antibiotics cannot cure anymore. Excessive use of antibiotics speeds up this whole process. This represents a recent problem and a challenge for the health system. As a reaction to increased resistance development and in order to soften its consequences, special treatments like last line antibiotics exist. For example, Tigecycline represents a last line antibiotic that is used against multidrug resistant bacteria. But resistances also develop against this antibiotic. Resistance to tigecycline can be caused by different mechanisms. One is mutations in ribosomal proteins. So far, isolates with such mutations for Enterobacteriaceae are only known from clinical isolates and from cultivation under tigecycline pressure. The exact influence of the mutations in the ribosomal proteins cannot be precisely determined for these strains, as they also show numerous other adaptations to an antibiotic-rich environment. One of these mutations occurred in the ribosomal protein S10, more accurate in the S10 flexible loop tip residue valine 57 (V57). S10 represents a ribosomal tertiary binding protein, which binds to the 16S rRNA of the 30S ribosomal subunit. Therefore, the aim of this thesis project was the generation of mutations in this ribosomal protein S10, in Escherichia coli. Further, the generated mutants were analyzed. The analyses contained growth experiments in order to identify the impact of the mutations on the general phenotype and antibiotic resistance testing, to gain information about the antibiotic susceptibility (to tigecycline/tetracycline and other antibiotics) of the mutant strains. Further ribosome biogenesis/translation analysis were done through polysome profiling/real time q-PCR, to gain information of ribosome assembly and translational processes of the S10 mutants. Additionally, mass spectrometry was performed as a means to obtain information about the composition of the 30S samples of the S10 mutants in comparison to wild type and to observe differences of present (ribosomal) proteins in the 30S peak. The generation of the S10 mutant strains was successful. The results revealed growth defects, thermosensitivity and altered antibiotic susceptibility to tigecycline and tetracycline for almost all of the S10 mutants. Moreover, severe ribosomal maturation defects, more specifically 30S accumulation and accumulations of 17S/pre23S rRNA of the generated S10 mutants, were observed. Furthermore, interestingly identified composition differences of ribosomal proteins such as RimM and RbfA of S10 mutants in comparison to wild type were identified. In conclusion, it is clear that additional mutations beside the S10 loop mutations are necessary to achieve high-level tigecycline resistance in E. coli. Furthermore, the S10 loop tip residue is critical for the correct functioning of S10. Due to the fact that both the S10 flexible loop and tigecycline are in contact with helix h31 of the 16S rRNA, we speculate that exchanges or deletion of V57 alter the positioning of h31 and therefore influence both tigecycline binding and S10 function.