Chemie | Biochemie | Medizin
William Blättler, 2005 | Basel, BS
The bacterial Type VI secretion system (T6SS) enables bacteria to attack prey cells by translocating effector proteins. While its genetic components are well understood in some bacteria, the role of certain T6SS-related genes in Acinetobacter baylyi remained unclear. This study investigated these genes and their impact on bacterial competition. Using Pseudomonas aeruginosa as selective pressure, mutant A. baylyi strains were obtained and analysed via X-gal and CPRG assays, revealing insights into lysis and inhibition caused by the T6SS. Live-cell fluorescence microscopy as well as whole genome sequencing showed that mutations in VipB led to a short T6SS structure but did not reduce lysis or inhibition against prey cells. Deleting ClpV was also shown to halt disassembly of the T6SS, mirroring phenotypes previously observed in Vibrio cholerae. The study also confirmed that a truncated TslA protein completely stops T6SS activity. These findings enhanced the understanding of the T6SS and have potential implications for studying T6SS-related pathogens as well as providing the field with a foundation for further research.
Introduction
Bacteria and other pathogens use the T6SS mechanism to eliminate competitors. Although the roles of the T6SS’s essential components are well-defined, many remain unclear. A. baylyi is a well-studied bacterium with an active T6SS, making it an ideal model for studying its function. This study used competition with P. aeruginosa as a selective pressure to select for spontaneous T6SS mutants. The mutants were then identified and characterized to examine how the selected mutations influence T6SS structure and function, as well as their impact on the bacterium’s competitiveness.
Methods
Mutant A. baylyi strains were selected using a competition-based approach with P. aeruginosa, which counterattacks and kills T6SS-active cells, thereby allowing cells with T6SS defects to survive. The T6SS phenotypes of the selected strains were analysed using X-gal and CPRG assays to assess inhibition and lysis of prey E. coli cells. T6SS dynamics were visualised to quantify the activity of the T6SS, using live-cell fluorescence microscopy, where sheath structures were marked by GFP-tagged VipA and disassembly was indicated by RFP-tagged ClpV. To find genetic mutations in the T6SS gene cluster, several strains exhibiting interesting phenotypes were sequenced.
Results
Live-cell fluorescent microscopy showed that the proportion of cells with inactive T6SS rose to 57% (95% CI [±1.85%]) after the 4th round, indicating that P. aeruginosa is selecting T6SS mutants. X-Gal and CPRG assays revealed that the selected strains have a wide range of lysis and inhibition, with some similar to wild-type. Additionally, both live-cell fluorescent microscopy and full genome sequencing of the strains identified mutations and their corresponding phenotypes. A mutation in the TslA gene was found to halt assembly. It was also shown that a single base deletion in VipB shortens the sheath of the T6SS, while a mutation causing a deletion of ClpV was found to stop disassembly.
Discussion
The selection process successfully increased the recovery of A. baylyi mutants with altered T6SS activity, confirming that P. aeruginosa serves as an effective selective pressure. X-gal and CPRG assays detected a variety of T6SS activity, ranging from strains with partial or total loss of function to those with typical inhibition and lysis. A mutation in VipB was detected in several strains with a shortened T6SS structure, which most likely interfered with the VipA-VipB interaction which is critical during assembly. Interestingly, many of these mutants maintained their normal antibacterial activity. Similar to findings in V. cholerae, ClpV was found to strictly be used for disassembly. A truncated version of the TslA protein, that was previously described to be required for contact dependent assembly, was also discovered to negatively affect T6SS assembly.
Conclusions
This study gives insights into the role of genes in the A. baylyi T6SS. Findings showed that the T6SS sheath was shortened by a mutation in the VipB gene, demonstrating the significance of the VipA-VipB interaction in assembly. While a shortened TslA protein hindered T6SS function, ClpV was shown to be crucial for disassembly and performed similarly to its function in V. cholerae. Gene complementation should be performed to rule out mutations outside of the T6SS cluster contributing to the phenotypes. These findings enhance our understanding of how the T6SS works in A. baylyi, providing the field with a foundation to conduct further research.
Würdigung durch den Experten
Dr. Enea Maffei
William’s work demonstrates dedication and insight in exploring a scientific question. His approach advances understanding of A. baylyi’s T6SS and raises some new hypotheses, which are thoughtfully examined in the discussion. The text is clearly written, well-structured, and the findings are effectively presented, making his work a valuable scientific contribution.
Prädikat:
hervorragend
Sonderpreis «European Union Contest for Young Scientists (EUCYS)» gestiftet von der Stiftung Aldo e Cele Daccò
Freies Gymnasium Basel
Lehrer: M.Sc. Dominik Sutter