Chemie  |  Biochemie  |  Medizin

 

Alea Ducret, 2007 | Muttenz, BL

 

The Type VI Secretion System (T6SS) is a bacterial nanoweapon key to the pathogenicity of Gram-negative bacteria and to interbacterial competition. Rapid contraction of the TssB-TssC sheath complex propels a central tube through cell membranes to deliver toxins into target cells. However, the precise functions of the sheath proteins TssB and TssC of T6SS remain unknown despite extensive research. This study identified potentially essential amino acid residues on TssB and TssC by generating Acinetobacter baylyi ADP1 mutants and a mutant library via selection with Pseudomonas aeruginosa. This library predominantly consists of T6SS-negative variants, as confirmed by live-cell fluorescent microscopy, CPRG assays and FACS analysis. Results revealed two highly frequent point mutations in the T6SS sheath: Thr24Ala in TssB, likely impairing proper sheath assembly, and Leu350Leu in TssC, potentially affecting cotranslational folding kinetics despite being synonymous. While these point mutations require independent verification, the findings of this study deepen our understanding of T6SS function, demonstrate a novel selection method, and highlight the impact of synonymous mutations.

Introduction

Bacteria use the dynamic T6SS to inject toxins into neighbouring cells, yet the precise function of the TssB-TssC sheath remains unclear. This study addressed this with a loss-of-function approach using the model organism A. baylyi ADP1, known for its high natural competence, non-pathogenicity and active T6SS. A mutant library was generated and then enriched using P. aeruginosa and subsequent analysis identified essential amino acid residues in TssB and TssC.

Methods

A loss-of-function approach generated a predominantly T6SS-inactive A. baylyi ADP1 mutant library. Knockouts were first prepared using a kanamycin-resistant, streptomycin-sensitive cassette produced via overlap extension PCR. Mutants were then obtained by transformation with error-prone PCR-generated TssB-TssC mutant cassettes, followed by selection on streptomycin. An innovative counterselection step exploiting P. aeruginosa’s counterattack mechanism eliminated most remaining T6SS-active cells. Phenotypes were assessed by live-cell fluorescent microscopy (GFP-tagged TssB for assembly, mCherry-tagged ClpV for disassembly), CPRG assays and FACS analysis. Finally, whole genome sequencing was performed on the mutant libraries before and after selection using Nanopore sequencing.

Results

All phenotypic assays were consistent and strongly supported the efficiency of the experimental procedures, with microscopy data showing a significant 51.8% decrease in T6SS+ cells after mutant library generation (t (2) = 32.6, p = 0.0009), and a 95.4% decrease after the P. aeruginosa selection with significant reductions after the first round (t (5) = 25.4, p < 0.0001) and the second (t (5) = 7.19, p = 0.0008). Moreover, Nanopore sequencing identified three highly frequent sheath mutations in (at least 88% frequency), including Thr24Ala in TssB as well as Leu350Leu and Gln443GlyfsTer9 in TssC.

Discussion

The significant difference in T6SS-active bacteria confirms successful mutagenesis and demonstrates the effectiveness of exploiting P. aeruginosa’s selective counterattack to select for T6SS- bacteria. Structural data from the closely related Vibrio cholerae sheath model suggest that Thr24Ala in TssB, placed at the TssB-TssC interface, weakens sheath interactions and impairs assembly. Furthermore, the synonymous Leu350Leu in TssC might alter cotranslational folding by slowing translation due to rare codon usage. This mutation is particularly noteworthy because, although synonymous mutations have been reported to affect cellular fitness, their effects remain poorly understood. Lastly, the Gln443GlyfsTer9 mutation in TssC produces a truncated protein, likely being selected from an early clonal dominance. However, its lack of consistent co-occurrence with the other mutations suggests that they act independently.

Conclusions

This study identified amino acid residues in TssB and TssC which are potentially essential for T6SS function. Results suggest that Thr24Ala in TssB weakens TssB-TssC sheath interactions, preventing assembly, while the synonymous Leu350Leu may alter cotranslational folding and impair function. Independent verification of these mutations remains necessary and could be pursued using site-directed mutagenesis. Overall, these findings deepen our understanding of T6SS mechanisms and provide a foundation for future research on bacterial secretion systems, with potential implications for microbial ecology and antibacterial strategies.

 

 

Würdigung durch den Experten

Dr. Enea Maffei

This project is notable for its scientific maturity and the Alea’s strong commitment to a complex biological question. She combined demanding experimental methods with care and evaluated the results thoughtfully and critically. Although the candidate mutations identified in this study still require independent verification, the work shows commendable self-criticism, a clear understanding of how scientific knowledge is generated and refined, and builds a strong foundation of hypotheses for follow-up studies.

Prädikat:

Gold

Sonderpreis «Taiwan International Science Fair (TISF)» gestiftet von den Odd Fellows, Helvetia Loge Nr. 1

 

 

 

Gymnasium Muttenz
Lehrerin: Cheryl Gysel