Lectin-Like Bacteriocins

Bacteria produce a diverse array of antagonistic compounds to restrict growth of microbial rivals. Contributing to this warfare are bacteriocins: secreted antibacterial peptides, proteins and multi-protein complexes. These compounds typically eliminate competitors closely related to the producer. Lectin-like bacteriocins (LlpAs) constitute a distinct class of such proteins, produced by Pseudomonas as well as some other proteobacterial genera. LlpAs share a common architecture consisting of two B-lectin domains, followed by a short carboxy-terminal extension. Two surface-exposed moieties on susceptible Pseudomonas cells are targeted by the respective lectin modules. The carboxy-terminal domain binds D-rhamnose residues present in the lipopolysaccharide layer, whereas the amino-terminal domain interacts with a polymorphic external loop of the outer-membrane protein insertase BamA, hence determining selectivity. The absence of a toxin-immunity module as found in modular bacteriocins and other polymorphic toxin systems, hints toward a novel mode of killing initiated at the cellular surface, not requiring bacteriocin import. Despite significant progress in understanding the function of LlpAs, outstanding questions include the secretion machinery recruited by lectin-like bacteriocins for their release, as well as a better understanding of the environmental signals initiating their expression

A Natural Chimeric Pseudomonas Bacteriocin with Novel Pore-Forming Activity Parasitizes the Ferrichrome Transporter

Modular bacteriocins represent a major group of secreted protein toxins with a narrow spectrum of activity, involved in interference competition between Gram-negative bacteria. These antibacterial proteins include a domain for binding to the target cell and a toxin module at the carboxy terminus. Self-inhibition of producers is provided by coexpression of linked immunity genes that transiently inhibit the toxin''s activity through formation of bacteriocin-immunity complexes or by insertion in the inner membrane, depending on the type of toxin module. We demonstrate strain-specific inhibitory activity for PmnH, a Pseudomonas bacteriocin with an unprecedented dual-toxin architecture, hosting both a colicin M domain, potentially interfering with peptidoglycan synthesis, and a novel colicin N-type domain, a pore-forming module distinct from the colicin Ia-type domain in Pseudomonas aeruginosa pyocin S5. A downstream-linked gene product confers PmnH immunity upon susceptible strains. This protein, ImnH, has a transmembrane topology similar to that of Pseudomonas colicin M-like and pore-forming immunity proteins, although homology with either of these is essentially absent. The enhanced killing activity of PmnH under iron-limited growth conditions reflects parasitism of the ferrichrome-type transporter for entry into target cells, a strategy shown here to be used as well by monodomain colicin M-like bacteriocins from pseudomonads. The integration of a second type of toxin module in a bacteriocin gene could offer a competitive advantage against bacteria displaying immunity against only one of both toxic activities

Novel conopeptides of largely unexplored Indo Pacific <i>Conus</i> sp.

Cone snails are predatory creatures using venom as a weapon for prey capture and defense. Since this venom is neurotoxic, the venom gland is considered as an enormous collection of pharmacologically interesting compounds having a broad spectrum of targets. As such, cone snail peptides represent an interesting treasure for drug development. Here, we report five novel peptides isolated from the venom of Conus longurionis, Conus asiaticus and Conus australis. Lo6/7a and Lo6/7b were retrieved from C. longurionis and have a cysteine framework VI/VII. Lo6/7b has an exceptional amino acid sequence because no similar conopeptide has been described to date (similarity percentage C. asiaticus, has a typical framework III Cys arrangement, classifying the peptide in the M-superfamily. Asi14a, another peptide of C. asiaticus, belongs to framework XIV peptides and has a unique amino acid sequence. Finally, AusB is a novel conopeptide from C. australis. The peptide has only one disulfide bond, but is structurally very different as compared to other disulfide-poor peptides. The peptides were screened on nAChRs, NaV and KV channels depending on their cysteine framework and proposed classification. No targets could be attributed to the peptides, pointing to novel functionalities. Moreover, in the quest of identifying novel pharmacological targets, the peptides were tested for antagonistic activity against a broad panel of Gram-negative and Gram-positive bacteria, as well as two yeast strains

Lectin-like bacteriocins from pseudomonas spp. utilise D-rhamnose containing lipopolysaccharide as a cellular receptor

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

The antimicrobial compound xantholysin defines a new group of Pseudomonas cyclic lipopeptides

The rhizosphere isolate Pseudomonas putida BW11M1 produces a mixture of cyclic lipopeptide congeners, designated xantholysins. Properties of the major compound xantholysin A, shared with several other Pseudomonas lipopeptides, include antifungal activity and toxicity to Gram-positive bacteria, a supportive role in biofilm formation, and facilitation of surface colonization through swarming. Atypical is the lipopeptide’s capacity to inhibit some Gram-negative bacteria, including several xanthomonads. The lipotetradecadepsipeptides are assembled by XtlA, XtlB and XtlC, three co-linearly operating non-ribosomal peptide synthetases (NRPSs) displaying similarity in modular architecture with the entolysin-producing enzymes of the entomopathogenic Pseudomonas entomophila L48. A shifted serine-incorporating unit in the eight-module enzyme XtlB elongating the central peptide moiety not only generates an amino acid sequence differing at several equivalent positions from entolysin, but also directs xantholysin’s macrocyclization into an octacyclic structure, distinct from the pentacyclic closure in entolysin. Relaxed fatty acid specificity during lipoinitiation by XtlA (acylation with 3-hydroxydodec-5-enoate instead of 3-hydroxydecanoate) and for incorporation of the ultimate amino acid by XtlC (valine instead of isoleucine) account for the production of the minor structural variants xantholysin C and B, respectively. Remarkably, the genetic backbones of the xantholysin and entolysin NRPS systems also bear pronounced phylogenetic similarity to those of the P. putida strains PCL1445 and RW10S2, albeit generating the seemingly structurally unrelated cyclic lipopeptides putisolvin (undecapeptide containing a cyclotetrapeptide) and WLIP (nonapeptide containing a cycloheptapeptide), respectively. This similarity includes the linked genes encoding the cognate LuxR-family regulator and tripartite export system components in addition to individual modules of the NRPS enzymes, and probably reflects a common evolutionary origin. Phylogenetic scrutiny of the modules used for selective amino acid activation by these synthetases indicates that bacteria such as pseudomonads recruit and reshuffle individual biosynthetic units and blocks thereof to engineer reorganized or novel NRPS assembly lines for diversified synthesis of lipopeptides

Live cell dynamics of production, explosive release and killing activity of phage tail-like weapons for Pseudomonas kin exclusion.

Interference competition among bacteria requires a highly specialized, narrow-spectrum weaponry when targeting closely-related competitors while sparing individuals from the same clonal population. Here we investigated mechanisms by which environmentally important Pseudomonas bacteria with plant-beneficial activity perform kin interference competition. We show that killing between phylogenetically closely-related strains involves contractile phage tail-like devices called R-tailocins that puncture target cell membranes. Using live-cell imaging, we evidence that R-tailocins are produced at the cell center, transported to the cell poles and ejected by explosive cell lysis. This enables their dispersal over several tens of micrometers to reach targeted cells. We visualize R-tailocin-mediated competition dynamics between closely-related Pseudomonas strains at the single-cell level, both in non-induced condition and upon artificial induction. We document the fatal impact of cellular self-sacrifice coupled to deployment of phage tail-like weaponry in the microenvironment of kin bacterial competitors, emphasizing the necessity for microscale assessment of microbial competitions

Discovery, characterization and in vivo activity of pyocin SD2, a protein antibiotic from Pseudomonas aeruginosa

Increasing rates of antibiotic resistance among Gram-negative pathogens such as Pseudomonas aeruginosa means alternative approaches to antibiotic development are urgently required. Pyocins, produced by P. aeruginosa for intraspecies competition, are highly potent protein antibiotics known to actively translocate across the outer membrane of P. aeruginosa. Understanding and exploiting the mechanisms by which pyocins target, penetrate and kill P. aeruginosa is a promising approach to antibiotic development. In this work we show the therapeutic potential of a newly identified tRNase pyocin, pyocin SD2, by demonstrating its activity in vivo in a murine model of P. aeruginosa lung infection. In addition, we propose a mechanism of cell targeting and translocation for pyocin SD2 across the P. aeruginosa outer membrane. Pyocin SD2 is concentrated at the cell surface, via binding to the common polysaccharide antigen (CPA) of P. aeruginosa lipopolysaccharide (LPS), from where it can efficiently locate its outer membrane receptor FpvAI. This strategy of utilizing both the CPA and a protein receptor for cell targeting is common among pyocins as we show that pyocins S2, S5 and SD3 also bind to the CPA. Additional data indicate a key role for an unstructured N-terminal region of pyocin SD2 in the subsequent translocation of the pyocin into the cell. These results greatly improve our understanding of how pyocins target and translocate across the outer membrane of P. aeruginosa. This knowledge could be useful for the development of novel anti-pseudomonal therapeutics and will also support the development of pyocin SD2 as a therapeutic in its own right

Maintaining Integrity Under Stress:Envelope Stress Response Regulation of Pathogenesis in Gram-Negative Bacteria

The Gram-negative bacterial envelope is an essential interface between the intracellular and harsh extracellular environment. Envelope stress responses (ESRs) are crucial to the maintenance of this barrier and function to detect and respond to perturbations in the envelope, caused by environmental stresses. Pathogenic bacteria are exposed to an array of challenging and stressful conditions during their lifecycle and, in particular, during infection of a host. As such, maintenance of envelope homeostasis is essential to their ability to successfully cause infection. This review will discuss our current understanding of the σE- and Cpx-regulated ESRs, with a specific focus on their role in the virulence of a number of model pathogens

Community-led comparative genomic and phenotypic analysis of the aquaculture pathogen Pseudomonas baetica a390T sequenced by Ion semiconductor and Nanopore technologies

Pseudomonas baetica strain a390T is the type strain of this recently described species and here we present its high-contiguity draft genome. To celebrate the 16th International Conference on Pseudomonas, the genome of P. baetica strain a390T was sequenced using a unique combination of Ion Torrent semiconductor and Oxford Nanopore methods as part of a collaborative community-led project. The use of high-quality Ion Torrent sequences with long Nanopore reads gave rapid, high-contiguity and -quality, 16-contig genome sequence. Whole genome phylogenetic analysis places P. baetica within the P. koreensis Glade of the P. fluorescens group. Comparison of the main genomic features of P. baetica with a variety of other Pseudomonas spp. suggests that it is a highly adaptable organism, typical of the genus. This strain was originally isolated from the liver of a diseased wedge sole fish, and genotypic and phenotypic analyses show that it is tolerant to osmotic stress and to oxytetracycline.Microbial Biotechnolog

Protein-mediated killing among bacteria: structure and function of prokaryotic MMBL lectins

Production of bacteriocins represents a highly diversified defense mechanism, identified in virtually all lineages of bacteria. These proteinaceous toxins are specifically directed against related bacteria and their production confers to a eco-evolutionary advantage for the producing organism. In this work, the objective was to further investigate the LlpA family of lectin-like bacteriocins, originally identified in the banana rhizosphere isolate P. putida BW11M1.The solved crystal structure of LlpA, the prototype lectin-like bacteriocin, unequivocally assigned this protein to the class of the so-called monocot mannose-binding lectin (MMBL) proteins, also designated B-lectins. LlpA is built from two tightly interacting ß-prism-folded MMBL modules, stabilized by a C-terminal ß-hairpin extension. The MMBL modules of LlpA display pseudo-threefold symmetry, domain stabilization by a central tryptophan triad, and a ß-strand swap interconnecting these two domains, hallmarks of B-lectins. Each MMBL domain contains three putative mannose-binding motifs exhibiting different levels of sequence degeneracy. Of these six sites, only three retain their potential carbohydrate-binding capacity. Binding of methyl-D-alfa-mannopyranoside and oligomannosides could only be demonstrated for one of these sites, located in the C-terminal MMBL-domain. LlpA mutant proteins with a sterically occluded mannose-binding site displayed a reduced antibacterial function, clearly coupling the ability to bind carbohydrates with bacteriotoxicity. Differential activity of engineered domain chimers derived from two LlpA homologues with different killing spectra, revealed that the N-domain is the main determinant of target strain recognition. Plant MMBL proteins display a diverse range of antagonistic activities, including antifungal, insecticidal, nematicidal or antiviral function, but antibacterial activity could not be demonstrated however. Hence, assigning a bacteriocin to the MMBL family further expands its range of antagonistic functions.The MMBL domain is commonly found in eukaryotic lectins, being particularly widespread among plants but also recently identified in several fish and fungal species. In the proteomes of prokaryotes, this MMBL module may appear as a single unit, in tandem organization, or as a hybrid fused to at least one other domain. The former domain architectures are typically found in Gram-negative bacteria, particularly abundant among Pseudomonas and Burkholderia, while the latter are more often found in Gram-positive bacteria. So far, only one such hybrid protein, composed of a MMBL module fused to a putative peptidase domain, has been characterized. This protein, albusin B, equally acts as a bacteriocin. Phylogenetic analysis of the different MMBL domains from LlpAs demonstrated that N- and C-modules evolved separately: N-domains tend to cluster together, distant from equally clustered C-domains. This observation is in line with the functional segregation described previously. The antibacterial potentialof tandem-MMBL proteins retrieved from proteomes of different bacterial species from Pseudomonas, Xanthomonas and Burkholderia was explored. Using recombinantly-purified proteins, we were able to demonstrate narrow-spectrum, genus-specific activity for all these LlpAs. As different human and phytopathogenic strains are susceptible to these LlpAs, these results may provide interesting perspectives for future antibacterial strategies.An atypical tandem-MMBL protein was identified in an Arthrobacter strain. Having two highly homologous B-lectin modules, six perfectly conserved mannose-binding motifs, a virtually absent ß-hairpin and an oligomeric organization, it represents the sole LlpA-like protein identified in a Gram-positive bacterium so far. We were not able to identify an antibacterial function for this protein, but instead demonstrated anti-retroviral activity. Hence, this protein was called arthrohivin.In several pseudomonad genomes, a single-MMBL-domain protein, termed LlpB, is encoded. This protein displays antibacterial activity as well, which stands in contrast with the task division between domains in LlpA. LlpBs display pronounced similarity with the N-domains of LlpAs, which enabled to construct a model of LlpB based on the crystal structure of the LlpA from P. putida. Based on this structural prediction, LlpB can be assigned to the class of the MMBL proteins as well. This novel subtype lectin-like bacteriocin may represent a kind of minimal LlpA, with target strain recognition and carbohydrate-binding function, if present, condensed into only a single domain. Conceivably, domain duplication within an ancestral LlpB protein, may have given rise to the tandem architecture of LlpA proteins, as a way to diversify target recognition, uncoupled from actual killing activity.SUMMARY I SAMENVATTING III LIST OF ABBREVIATIONS V CHAPTER 1 ANTIBACTERIAL PEPTIDES AND PROTEINS IN GRAM-NEGATIVE BACTERIA 1 1.1 MICROCINS 2 1.2 COLICINS 4 1.2.1 Colicinogeny 4 1.2.2 Biological activities of colicins 4 1.2.3 Modular structure of colicins and self-protection 5 1.2.4 Colicin import 7 1.2.5 Regulation of colicin production 7 1.2.6 Colicins as promoters of microbial diversity? 9 1.2.7 Colicin-like bacteriocins from other γ-proteobacteria 10 1.3 S-TYPE PYOCINS 11 1.3.1 Modular architecture 11 1.3.2 S-pyocin uptake and immunity 12 1.3.3 Pyocin regulation 12 1.3.4 Pyocinogeny 12 1.4 PHAGE TAIL-LIKE BACTERIOCINS 13 1.4.1 R-type pyocins from Pseudomonas 13 1.4.2 F-type pyocins from Pseudomonas 14 1.4.3 Phage ancestry of R- and F-pyocins? 15 1.4.4 Phage tail-like bacteriocins in plant-associated bacteria 15 1.5 CONTACT-DEPENDENT GROWTH INHIBITION 16 1.5.1 Two-partner secretion system CDI 16 1.5.1.1 Structural organization of CdiA 16 1.5.1.2 Functionality of CdiI as a protector from auto-inhibition 17 1.5.1.3 Interaction with sensitive cells 17 1.5.1.4 CDI in other bacteria 18 1.5.1.5 Rhs elements as mediators of intercellular competition 19 1.6 INHIBITION MEDIATED BY TYPE VI SECRETION 19 1.7 MORE HIDDEN BACTERIOCIN DIVERSITY? 21 1.7.1 Ferredoxin-containing bacteriocins from Pectobacterium 21 1.7.2 Glycinecin A from Xanthomonas 21 1.7.3 Triple-subunit bacteriocin from Pseudomonas syringae 22 1.8 LECTIN-LIKE BACTERIOCINS: MEMBERS OF THE MMBL FAMILY 22 1.8.1 MMBL (monocot mannose-binding lectin)-type lectins: what is in a name? 22 1.8.2 Eukaryotic MMBLs revisited 24 1.8.3 Prokaryotic MMBLs: in search of a function 24 1.8.4 Bacterial killer MMBLs 26 1.8.5 Bacterial chimeric MMBLs: toxic proteins as well? 30 1.8.6 Bacteriocins with a novel mode of action? 32 1.9 SUPPORTING INFORMATION 33 1.10 SCOPE AND OUTLINE OF THE THESIS 34 1.11 BIBLIOGRAPHY 36 CHAPTER 2 ANTAGONISTIC ACTIVITIES OF PROKARTYOIC TANDEM-MMBL PROTEINS 45 ABSTRACT 45 2.1 PLANT LECTIN-LIKE ANTIBACTERIAL PROTEINS FROM PHYTOPATHOGENS PSEUDOMONAS SYRINGAE AND XANTHOMONAS CITRI 47 2.1.1 Introduction 47 2.1.2 Results 47 2.1.2.1 New candidate lectin-like bacteriocins 47 2.1.2.2 Expression of recombinant tandem MMBL lectins 48 2.1.2.3 Purification of recombinant proteins 50 2.1.2.4 Bacteriocin activity of novel LlpAs 50 2.1.3 Discussion 51 2.2 ANTIBACTERIAL ACTIVITY OF A LECTIN-LIKE BURKHOLDERIA CENOCEPACIA PROTEIN 54 2.2.1 Introduction 54 2.2.2 Results 55 2.2.2.1 Genes encoding LlpA-like proteins in Burkholderia genomes 55 2.2.2.2 Bacteriocin activity of B. cenocepacia LlpA6 56 2.2.2.3 Anti-biofilm activity of B. cenocepacia LlpA6 57 2.2.3 Discussion 59 2.3 ARTHROHIVIN, A TANDEM MONOCOT MANNOSE-BINDING LECTIN FROM AN ARTHROBACTER SOIL ISOLATE, INHIBITS HIV-1 INFECTION 60 2.3.1 Introduction 60 2.3.2 Results 60 2.3.2.1 Identification, recombinant production and purification of LlpA5 60 2.3.2.2 LlpA5 is an atypical tetrameric LlpA, lacking antibacterial activity 61 2.3.2.3 LlpA5 displays antiretroviral activity 61 2.3.3 Discussion 63 2.4 MATERIALS AND METHODS 64 2.4.1 Strains and culture conditions 64 2.4.2 Recombinant DNA methods 65 2.4.3 Overexpression and purification of recombinant LlpAs 65 2.4.4 Glycan array 66 2.4.5 Bacteriocin assay 66 2.4.6 Antifungal assays 67 2.4.7 Antiviral assay 67 2.4.8 Co-cultivation assay 68 2.4.9 Surface plasmon resonance (SPR) analysis 68 2.4.10 Determination of the MIC of LlpA6 68 2.4.11 Determination of the inhibition and eradication of Burkholderia biofilms 69 2.5 SUPPORTING INFORMATION 70 2.6 BIBLIOGRAPHY 70 CHAPTER 3 STRUCTURAL DETERMINANTS FOR ACTIVITY AND SPECIFICITY OF THE BACTERIAL TOXIN LLPA 75 ABSTRACT 75 3.1 INTRODUCTION 76 3.2 RESULTS 77 3.2.1 LlpA forms a rigid MMBL tandem 77 3.2.2 Domains of LlpABW are shaped by differential evolutionary pressure 79 3.2.3 LlpA is capable of binding mannose-containing carbohydrates 82 3.2.4 Carbohydrate-binding capacity is required for LlpA toxicity 86 3.2.5 All domains are necessary for LlpABW functionality 90 3.2.6 Target specificity of LlpA is hosted by the N-domain 91 3.3 DISCUSSION 93 3.4 MATERIALS AND METHODS 94 3.4.1 Strains and culture conditions 94 3.4.2 Recombinant DNA methods 94 3.4.3 Recombinant protein expression and purification 95 3.4.4 Antibacterial assays 96 3.4.5 Glycan array 96 3.4.6 Circular dichroism 97 3.4.7 Isothermal titration calorimetry 97 3.4.8 X-ray data collection and structure determination 97 3.4.9 Carbohydrate soaks 98 3.4.10 Flow cytometry 98 3.5 SUPPORTING INFORMATION 100 3.6 BIBLIOGRAPHY 100 CHAPTER 4 THE MONO-MMBL-DOMAIN PROTEIN LLPB FROM PSEUDOMONAS DEFINES A NEW CLASS OF LECTIN-LIKE BACTERIOCINS 103 ABSTRACT 103 4.1 INTRODUCTION 104 4.2 RESULTS 104 4.2.1 Identification of mono-MMBL-domain proteins in Pseudomonas 104 4.2.2 LlpBs display antibacterial but no antifungal activity 106 4.2.3 Heterologous expression of LlpB in Pseudomonas spp. 108 4.2.4 Monomeric nature of LlpB 108 4.2.5 LlpB as a member of the MMBL family 109 4.2.5.1 Structural similarities between LlpB and LlpA 109 4.2.5.2 Potential carbohydrate-binding capacity of LlpB 111 4.3 DISCUSSION 113 4.4 MATERIALS AND METHODS 114 4.4.1 Strains and culture conditions 114 4.4.2 DNA and cellular manipulations 115 4.4.3 Recombinant expression and purification of LlpBs 115 4.4.4 Point mutation analysis of LlpB and construction of chimeric tandem lectins 115 4.4.5 Antagonistic assays 116 4.4.6 Phylogenetic analysis 116 4.4.7 LlpB modeling 116 4.5 SUPPORTING INFORMATION 116 4.6 BIBLIOGRAPHY 116 CHAPTER 5 GENERAL DISCUSSION, CONCLUSIONS AND PERSPECTIVES 119 5.1 LLPA: A NATURAL CHILD OF THE MMBL FAMILY DISCLOSED 119 5.2 THE Β-HAIRPIN TAIL: IN CAUDA VENENUM? 121 5.3 LECTIN-BASED KILLING: TOWARDS A NEW MODE OF ACTION? 121 5.4 BEYOND MMBL BACTERIOCINS: ARTHROHIVIN AND MORE 123 5.5 BIBLIOGRAPHY 124 APPENDIX 125 LIST OF PUBLICATIONS 127nrpages: 127status: publishe