Antibacterial potency of type VI amidase effector toxins is dependent on substrate topology and cellular context.
Atanas RadkovAnne L SapiroSebastian FloresCorey HendersonHayden SaundersJungyun KimSteven MassaSamuel ThompsonChase MateusiakJacob BiboyZiyi ZhaoLea M StaritaWilliam L HatlebergWaldemar VollmerAlistair B RussellJean-Pierre SimorreSpencer Anthony-CahillPeter BrzovicBeth M HayesSeemay ChouPublished in: eLife (2022)
Members of the bacterial T 6SS a midase e ffector (Tae) superfamily of toxins are delivered between competing bacteria to degrade cell wall peptidoglycan. Although Taes share a common substrate, they exhibit distinct antimicrobial potency across different competitor species. To investigate the molecular basis governing these differences, we quantitatively defined the functional determinants of Tae1 from Pseudomonas aeruginosa PAO1 using a combination of nuclear magnetic resonance and a high-throughput in vivo genetic approach called deep mutational scanning (DMS). As expected, combined analyses confirmed the role of critical residues near the Tae1 catalytic center. Unexpectedly, DMS revealed substantial contributions to enzymatic activity from a much larger, ring-like functional hot spot extending around the entire circumference of the enzyme. Comparative DMS across distinct growth conditions highlighted how functional contribution of different surfaces is highly context-dependent, varying alongside composition of targeted cell walls. These observations suggest that Tae1 engages with the intact cell wall network through a more distributed three-dimensional interaction interface than previously appreciated, providing an explanation for observed differences in antimicrobial potency across divergent Gram-negative competitors. Further binding studies of several Tae1 variants with their cognate immunity protein demonstrate that requirements to maintain protection from Tae activity may be a significant constraint on the mutational landscape of tae1 toxicity in the wild. In total, our work reveals that Tae diversification has likely been shaped by multiple independent pressures to maintain interactions with binding partners that vary across bacterial species and conditions.
Keyphrases
- cell wall
- magnetic resonance
- gram negative
- high throughput
- single cell
- pseudomonas aeruginosa
- multidrug resistant
- staphylococcus aureus
- body mass index
- high resolution
- cystic fibrosis
- biofilm formation
- copy number
- stem cells
- dendritic cells
- acinetobacter baumannii
- small molecule
- escherichia coli
- genome wide
- immune response
- computed tomography
- genetic diversity
- bone marrow
- anti inflammatory
- protein protein
- electron microscopy