Antibacterial potency of type VI amidase effector toxins is dependent on substrate topology and cellular context

Abstract

Members of the bacterial T6SS amidase effector (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 (NMR) 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.

Lipopolysaccharide integrity primes bacterial sensitivity to a cell wall-degrading intermicrobial toxin

“…To investigate, we used an orthogonal in vivo assay to directly test the effect of Tae1 activity in msbA-KD cells in the absence of Pae and other co-delivered H1-T6SS toxins. We measured lysis for rfp-KD and msbA-KD upon induction of exogenous wild-type Tae1 (Tae1 WT ) expression in the cell wall-containing periplasm 8,46 and found that msbA-KD had increased survival against Tae1 WT relative to rfp-KD ( Fig. 4a ).…”

“…Plasmids for periplasmic Tae1 overexpression in Eco were constructed using splice-overlap extension cloning of tae1 WT and tae1 C30A coding sequences derived from P . aeruginosa (PAO1) into pBAD24 46,72 . A pelB leader sequence was fused to tae1 for localization to the periplasm.…”

Pseudomonas fluorescensMFE01 delivers a putative type VI secretion amidase that confers biocontrol against the soft‐rot pathogen Pectobacterium atrosepticum

“…Based on the usual genomic environment of T6SS effector genes, we screened the MFE01 genome and identified a putative amidase of the Tae3 family, encoded downstream of an hcp gene in an hcp‐vgrG island, and located upstream of a gene of unknown function. Peptidoglycan‐targeting enzymes have been shown to be common T6SS effectors in pseudomonads, including the opportunistic pathogen P. aeruginosa (Chou et al, 2012; Radkov et al, 2022; Russell et al, 2011, 2012; T. Wang et al, 2020; Whitney et al, 2013). While MFE01 T6SS antibacterial activity and T6SS‐mediated potato protection are not abolished in a strain lacking Tae3, our results showed that Tae3 Pf contributes significantly to Pca and E. coli rounding and lysis.…”

Lipopolysaccharide transport regulates bacterial sensitivity to a cell wall-degrading intermicrobial toxin

“…To investigate, we used an orthogonal in vivo assay to directly test the effect of Tae1 activity in msbA-KD cells in the absence of Pae and other co-delivered H1-T6SS toxins. We measured lysis for rfp-KD and msbA-KD upon induction of exogenous wild-type Tae1 (Tae1 WT ) expression in the cell wall-containing periplasm [8,48] and found that msbA-KD had increased survival against Tae1 WT Given the multigenic knockdown in msbA-lpxK-ycaQ in msbA-KD, these data suggest that msbA is a partial determinant of Tae1 susceptibility in the strain.…”

“…Plasmids for periplasmic Tae1 overexpression in Eco were constructed using splice-overlap extension cloning of tae1 WT and tae1 C30A coding sequences derived from P. aeruginosa (PAO1) into pBAD24 [48,81]. A pelB leader sequence was fused to tae1 for localization to the periplasm.…”