Protein-mediated killing among bacteria: structure-function

relationship of an antibacterial Pseudomonas lectin

Maarten G. K. Ghequire1, Abel Garcia-Pino2,3, Remy Loris2,3 and René De Mot1

1 Centre of Microbial and Plant Genetics, University of Leuven, 3001 Heverlee-Leuven, Belgium

2 Molecular Recognition Unit, Flanders Interuniversity Institute for Biotechnology (VIB),

1050 Brussel, Belgium

3 Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussel, Belgium

Introduction – The LlpA protein from Pseudomonas putida BW11M1 represents the prototype of a novel family of antimicrobial proteins with bacteriocin-like properties. Functional members have been identified in several other Pseudomonas species, as well as in some Xanthomonas species [1,2]. In addition to these -proteobacterial producers, LlpA-like proteins are also found in strains of the β-proteobacterial genus Burkholderia [3]. LlpAs display strain-specific intra-genus killing, but in contrast to typical Gram-negative bacteriocins such as colicins or pyocins they do not need a cognate immunity protein.

Results – The 3D-structure of LlpA was elucidated, showing it is built from two tightly interacting β-prism-folded ‘monocot mannose-binding lectin’ (MMBL) domains stabilized by a β-hairpin [4]. MMBL domains are commonly found in eukaryotic lectins, being particularly widespread among monocot plants but recently also identified in several fish and fungal species. Such domain typically contains three (putative) mannose-binding motifs, that may exhibit different levels of sequence degeneracy and, hence, may differ in carbohydrate-binding capacity [1]. In LlpA only three of the six sites are potentially left intact. The binding with millimolar affinity of methyl-mannoside and several oligomannosides to only one of these sites, located in the carboxyterminal domain, was demonstrated. LlpA mutant proteins with a sterically occluded sugar-binding site displayed reduced bacteriocin activity, coupling antibacterial function with carbohydrate-binding capacity. Exchanging domains of P. putida LlpA with the equivalent domains from a P. fluorescens LlpA with a different target spectrum, still generated active proteins but conferred a spectrum shift. This indicates that the N-terminal domain determines host specificity. Compared to the sugar-binding C-terminal domain, the N-domain is structurally more divergent from the plant lectin domains [4].

Conclusion – Our results unequivocally identify LlpA as the first prokaryotic member of the MMBL lectin family. An intact mannoside-binding site in the C-terminal domain is necessary to obtain a fully active bacteriocin and the N-terminal domain represents the main determinant of target strain specificity. The occurrence of LlpA proteins in soil-dwelling and plant-associated bacteria suggests they evolved antibacterial activity following acquisition of MMBL modules from an eukaryotic source.

  1. Ghequire, M., Loris, R., De Mot, R. 2012. MMBL proteins: from lectin to bacteriocin. Biochemical Society Transactions, 40:1553-1559.
  2. Ghequire, M., Li, W., Proost, P., Loris, R., De Mot, R. 2012. Plant lectin-like antibacterial proteins from phytopathogens Pseudomonas syringae and Xanthomonas citri. Environmental Microbiology Reports, 4:373-380
  3. Ghequire, M., De Canck, E., Wattiau, P., Van Winge, I., Loris, R., Coenye, T., De Mot, R. 2013. Antibacterial activity of a lectin-like Burkholderia cenocepacia protein. MicrobiologyOpen. [accepted]
  4. Ghequire, M., Garcia-Pino, A., Lebbe, E., Spaepen, S., Loris, R., De Mot, R. 2013. Structural determinants for activity and specificity of the bacterial toxin LlpA. PLoS Pathogens, 9:e1003199.