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Title: Structural Determinants for Activity and Specificity of the Bacterial Toxin LlpA}
Article:
Abstract:
Lectin-like bacteriotoxic proteins, identified in several plant-associated bacteria, are able to selectively kill closely related species, including several phytopathogens, such as Pseudomonas syringae and Xanthomonas species, but so far their mode of action remains unrevealed. The crystal structure of LlpABW, the prototype lectin-like bacteriocin from Pseudomonas putida, reveals an architecture of two monocot mannose-binding lectin( MMBL) domains and a C-terminal β-hairpin extension. The C-terminal MMBL domain( C-domain) adopts a fold very similar to MMBL domains from plant lectins and contains a binding site for mannose and oligomannosides. Mutational analysis indicates that an intact sugar-binding pocket in this domain is crucial for bactericidal activity. The N-terminal MMBL domain( N-domain) adopts the same fold but is structurally more divergent and lacks a functional mannose-binding site. Differential activity of engineered N/C-domain chimers derived from two LlpA homologues with different killing spectra, disclosed that the N-domain determines target specificity. Apparently this bacteriocin is assembled from two structurally similar domains that evolved separately towards dedicated functions in target recognition and bacteriotoxicity.
Introduction:
In most natural settings, complex interactions occur among microorganisms, ranging from nutritional co-operation to warfare among competitors. Examples of such interplay have been reported not only between unrelated microorganisms( e. g. fungi and bacteria[1],[2]), but also between distant relatives( e. g. members of different bacterial genera[3]), and even between close relatives( e. g. at inter- and intra-species levels[4],[5]). A major strategy in niche colonization is the production of growth inhibitors or toxins directed at microbial competitors[6]. While a huge variety of secondary metabolites is used to target phylogenetically-distant competitors, ribosome-synthesized peptides or proteins are typically active against close relatives. These protein toxins are collectively referred to as bacteriocins, and may either be released into the environment or transferred to the host via specialized contact-dependent delivery systems[7]–[9]. Bacteriocins are structurally and mechanistically very diverse. This is reflected in the bacteriocinogenic potential of the genus Pseudomonas[10]. Their R- and F-type pyocins are multi-subunit protein complexes evolutionarily related to contractile tails of bacteriophages[11]–[13]. R-pyocins attach to specific lipopolysaccharide moieties at the cell surface of susceptible cells and insert their core structure through the cell envelope, causing depolarization of the cytoplasmic membrane[14]. The S-type pyocins of Pseudomonas aeruginosa share structural and functional features with Escherichia coli colicins[15]. Following docking onto surface-exposed targets such as siderophore receptors[16],[17], S-pyocins kill cells by nucleic acid degradation[10],[17], cytoplasmic membrane damage[18], or inhibition of peptidoglycan synthesis[19],[20]. Putidacin A( or LlpABW), first identified in Pseudomonas putida BW11M1[21], represents a class of Pseudomonas-specific antibacterial proteins not related to any known bacteriocin. Additional llpA-like genes encoding functional bacteriocins were identified by genome mining in the biocontrol strain Pseudomonas fluorescens Pf-5[22] and in the phytopathogen Pseudomonas syringae pv. syringae 642[23]. Identification of this type of protein in two Xanthomonas pathovars extended its occurrence as a genus-specific killer protein[23]. The Xanthomonas LlpA precursor is proteolytically processed by removal of a characteristic Type II secretion signal peptide, whereas such N-terminal sequence is lacking in Pseudomonas homologues, indicating that secretory routes may differ among LlpA producers. The amino acid sequence of LlpA suggests the presence of two related domains belonging to the ‘monocot mannose-binding lectin’( MMBL) family[24]. The MMBL domain consists of a β-prism fold containing three potential carbohydrate-binding pockets, each generated by a QxDxNxVxY sequence( with x, any amino acid), but some sites may be inactive due to degeneracy of the signature motif[25]. This domain( Pfam domain: B_lectin- PF01453) was initially identified in lectins of monocot plants[26],[27], but a more widespread occurrence of MMBL lectins has become evident and includes representatives in fungi[28],[29], slime molds[30], sponges[31], and fishes[32]–[34]. The LlpA branch occupies a unique position among MMBL-domain proteins, harboring non-eukaryotic representatives and being equipped with the capacity to kill bacterial cells with bacteriocin-like specificity, a property not yet demonstrated for other family members[25]. Next to proteins with the LlpA-type tandem-MMBL organization, many other predicted MMBL proteins are encoded by bacterial genomes. Often the MMBL module is embedded in a larger protein. For one such protein, bacteriocin-like activity among Ruminococcus species, Gram-positive bacteria colonizing the rumen, was demonstrated[35]. Here we report on the crystal structure of LlpABW as the prototype of a novel family of antibacterial proteins and explore how domain architecture and specific structural elements contribute to its activity and specificity.
Discussion:
Structure elucidation of LlpABW from P. putida BW11M1 unequivocally assigns this bacteriocin to the MMBL lectin family, in which it constitutes the first prokaryotic member, representative for a group of bacterial proteins composed of two MMBL domains[21]–[23]. Systematic inactivation of the three potential carbohydrate-binding sites present in the N-domain( IIIN) and in the C-domain( IIIC and IIC) of LlpABW, revealed that a non-occluded IIIC pocket is required to obtain a fully active LlpABW molecule. A negative co-operative effect on activity resulted when the IIC site was additionally modified. Although mannose-containing carbohydrates can bind to the IIIC pocket of LlpABW, it remains unclear if these are part of or mimic the natural ligand required for biological activity since bacteriocin activity is not impaired in the presence of excess mannose. A mutated IIIN site did not provoke a negative effect on antibacterial activity. However, the N-domain appears to play a major role in target selection. This was demonstrated by assessing the differential activity of domain chimers against two target strains, diagnostic for the LlpABW- and LlpA1-specific killing. The β-hairpin does not appear to be a specificity determinant, although it constitutes the most variable region among LlpA-like bacteriocins. Possibly, it is required for thermodynamic stability since it needs to be intact in LlpABW. An equivalent C-terminal stretch is absent from the Xanthomonas citri LlpA-like bacteriocin[23]. From our results relying on heterologous expression in E. coli and a bacteriocin assay with permeabilized cells, it cannot be excluded that this structural element may play a role in the way an LlpA protein is exported by its native producer cells. In general, a defensive role has been proposed for the( oligo) mannose-binding MMBL lectins based on insecticidal, nematicidal, antifungal, or even antiviral activities demonstrated for several of these proteins that are abundantly found in monocot plants[40]–[46]. Some of these plant lectins trigger apoptosis in cancer cells[47]. Also their identification in fish mucus and epithelial cells is in line with a general protective( antimicrobial) function for MMBL domains[32]. LlpABW as a bactericidal protein fits within this picture of MMBL domains being involved in general defense mechanisms. Since no antibacterial activity has been assigned to the eukaryotic MMBL proteins, it is challenging to identify structural features that confer the intragenus-specific bacteriocin activity of LlpA, as shown for proteins from P. putida[21], P. fluorescens[22], P. syringae[23], and Xanthomonas citri[23]. Their target spectra are narrower than reported for the mammalian antibacterial C-type lectins of the RegIII family, such as mouse RegIIIγ and its human homolog HIP/PAP that bind to the surface-exposed peptidoglycan layer of Gram-positive bacteria[48], and RegIIIβ that also binds to the lipid A moiety of lipopolysaccharides on the cell envelope of Gram-negative bacteria[49]. The absence of any known secretory signal sequence in LlpABW and its homologues in other Pseudomonas species is intriguing in view of their extracellular location[21]. The translocation of the outer membrane-associated mannose/fucose-specific lectin LecB of P. aeruginosa, that also lacks such signal sequence[50], is dependent on its glycosylation[51]. Contrary to LlpA that is exported to the culture supernatant to exert its antagonistic activity, LecB remains associated with the cell envelope through interaction with the major outer membrane protein OprF[52], in line with its role in biofilm formation.
In their natural environments , microorganisms compete for space and nutrients , and a major strategy to assist in niche colonization is the deployment of antagonistic compounds directed at competitors , such as secondary metabolites ( antibiotics ) and antibacterial peptides or proteins ( bacteriocins ) . The latter selectively kill closely related bacteria , which is also the case for members of the LlpA family . Here , we investigate the structure-function relationship for the prototype LlpABW from a saprophytic plant-associated Pseudomonas whose genus-specific target spectrum includes several phytopathogenic pseudomonads . By determining the 3D structure of this protein , we could assign LlpA to the so-called monocot mannose-binding lectin ( MMBL ) family , representing its first prokaryotic member , and also add a new type of protective function , as the eukaryotic MMBL members have been linked with antiviral , antifungal , nematicidal or insecticidal activities . For the protein containing two similarly folded domains , we constructed site-specific mutants affected in carbohydrate binding and domain chimers from LlpA homologues to show that mannose-specific sugar binding mediated by one domain is required for activity and that the other domain determines target strain specificity . The strategy that evolved for these bacteriocins is reminiscent of the one used by mammalian bactericidal proteins of the RegIII family that recruited a C-type lectin fold to kill bacteria .