Abstract
Lectin-like bacteriocins consist of tandem monocot mannose-binding domains and display a genus-specific killing activity. Here we show that pyocin L1, a novel member of this family from Pseudomonas aeruginosa, targets susceptible strains of this species through recognition of the common polysaccharide antigen (CPA) of P. aeruginosa lipopolysaccharide that is predominantly a homopolymer of d-rhamnose. Structural and biophysical analyses show that recognition of CPA occurs through the C-terminal carbohydrate-binding domain of pyocin L1 and that this interaction is a prerequisite for bactericidal activity. Further to this, we show that the previously described lectin-like bacteriocin putidacin L1 shows a similar carbohydrate-binding specificity, indicating that oligosaccharides containing d-rhamnose and not d-mannose, as was previously thought, are the physiologically relevant ligands for this group of bacteriocins. The widespread inclusion of d-rhamnose in the lipopolysaccharide of members of the genus Pseudomonas explains the unusual genus-specific activity of the lectin-like bacteriocins.
Highlights
The ability to target a subgroup of pathogenic bacteria in a complex bacterial community has potential applications in medicine and agriculture where the maintenance of a ‘normal’ microbiome is beneficial
To test the idea that the observed binding of D-rhamnose to sites C1 and C2 is reflective of common polysaccharide antigen (CPA) binding and that this binding is critical to pyocin L1 cytotoxicity, we created pyocin L1 variants in which the conserved aspartic acids of the QxDxNxVxY motifs of the C1 and C2 sugar binding sites were mutated to alanine and compared their cytotoxicity and ability to bind the CPA by isothermal titration calorimetry (ITC) with the wild-type protein
In this work we have shown that pyocin L1 targets susceptible cells through binding to the CPA component of LPS and that primary recognition of CPA occurs through binding of Drhamnose at the conserved QxDxNxVxY sugar binding motifs of the C-terminal lectin domain
Summary
The ability to target a subgroup of pathogenic bacteria in a complex bacterial community has potential applications in medicine and agriculture where the maintenance of a ‘normal’ microbiome is beneficial. The use of broad spectrum antibiotics to treat bacterial infections is known to cause a range of complications associated with collateral damage to the microbiome, including antibiotic associated diarrhea and Clostridium difficile infection [1,2]. In contrast to the broad spectrum antibiotics that are widely used in medicine and agriculture, protein antibiotics known as bacteriocins often target a specific bacterial species or a group of closely related bacterial species [10,11,12,13]. Well characterised bacteriocins include the S-type pyocins from P. aeruginosa and the closely related colicins of E. coli [12,13]. In the case of S-type pyocins it is thought that their activity is limited to strains of P. aeruginosa, whereas colicins show activity against E. coli and some strains of closely related bacteria such as Salmonella spp. In the case of the opportunistic human pathogen P. aeruginosa there is an urgent requirement for the development of novel therapeutic options since its ability to form drug-resistant biofilms in
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