Action of a minimal contractile bactericidal nanomachine.

R-type bacteriocins are minimal contractile nanomachines that hold promise as precision antibiotics1-4. Each bactericidal complex uses a collar to bridge a hollow tube with a contractile sheath loaded in a metastable state by a baseplate scaffold1,2. Fine-tuning of such nucleic acid-free protein machines for precision medicine calls for an atomic description of the entire complex and contraction mechanism, which is not available from baseplate structures of the (DNA-containing) T4 bacteriophage5. Here we report the atomic model of the complete R2 pyocin in its pre-contraction and post-contraction states, each containing 384 subunits of 11 unique atomic models of 10 gene products. Comparison of these structures suggests the following sequence of events during pyocin contraction: tail fibres trigger lateral dissociation of baseplate triplexes; the dissociation then initiates a cascade of events leading to sheath contraction; and this contraction converts chemical energy into mechanical force to drive the iron-tipped tube across the bacterial cell surface, killing the bacterium.

Molecular Structure and Functional Analysis of Pyocin S8 from Pseudomonas aeruginosa Reveals the Essential Requirement of a Glutamate Residue in the H-N-H Motif for DNase Activity.

Multidrug resistance (MDR) is a serious threat to public health, making the development of new antimicrobials an urgent necessity. Pyocins are protein antibiotics produced by Pseudomonas aeruginosa strains to kill closely related cells during intraspecific competition. Here, we report an in-depth biochemical, microbicidal, and structural characterization of a new S-type pyocin, named S8. Initially, we described the domain organization and secondary structure of S8. Subsequently, we observed that a recombinant S8 composed of the killing subunit in complex with the immunity (ImS8) protein killed the strain PAO1. Furthermore, mutation of a highly conserved glutamic acid to alanine (Glu100Ala) completely inhibited this antimicrobial activity. The integrity of the H-N-H motif is probably essential in the killing activity of S8, as Glu100 is a highly conserved residue of this motif. Next, we observed that S8 is a metal-dependent endonuclease, as EDTA treatment abolished its ability to cleave supercoiled pUC18 plasmid. Supplementation of apo S8 with Ni2+ strongly induced this DNase activity, whereas Mn2+ and Mg2+ exhibited moderate effects and Zn2+ was inhibitory. Additionally, S8 bound Zn2+ with a higher affinity than Ni2+ and the Glu100Ala mutation decreased the affinity of S8 for these metals, as shown by isothermal titration calorimetry (ITC). Finally, we describe the crystal structure of the Glu100Ala S8 DNase-ImS8 complex at 1.38 Å, which gave us new insights into the endonuclease activity of S8. Our results reinforce the possibility of using pyocin S8 as an alternative therapy for infections caused by MDR strains, while leaving commensal human microbiota intact.IMPORTANCE Pyocins are proteins produced by Pseudomonas aeruginosa strains that participate in intraspecific competition and host-pathogen interactions. They were first described in the 1950s and since then have gained attention as possible new antibiotics. However, there is still only scarce information about the molecular mechanisms by which these molecules induce cell death. Here, we show that the metal-dependent endonuclease activity of pyocin S8 is involved with its antimicrobial action against strain PAO1. We also describe that this killing activity is dependent on a conserved Glu residue within the H-N-H motif. The potency and selectivity of pyocin S8 toward a narrow spectrum of P. aeruginosa strains make this protein an attractive antimicrobial alternative for combatting MDR strains, while leaving commensal human microbiota intact.

Contractile injection systems of bacteriophages and related systems.

Contractile tail bacteriophages, or myobacteriophages, use a sophisticated biomolecular structure to inject their genome into the bacterial host cell. This structure consists of a contractile sheath enveloping a rigid tube that is sharpened by a spike-shaped protein complex at its tip. The spike complex forms the centerpiece of a baseplate complex that terminates the sheath and the tube. The baseplate anchors the tail to the target cell membrane with the help of fibrous proteins emanating from it and triggers contraction of the sheath. The contracting sheath drives the tube with its spiky tip through the target cell membrane. Subsequently, the bacteriophage genome is injected through the tube. The structural transformation of the bacteriophage T4 baseplate upon binding to the host cell has been recently described in near-atomic detail. In this review we discuss structural elements and features of this mechanism that are likely to be conserved in all contractile injection systems (systems evolutionary and structurally related to contractile bacteriophage tails). These include the type VI secretion system (T6SS), which is used by bacteria to transfer effectors into other bacteria and into eukaryotic cells, and tailocins, a large family of contractile bacteriophage tail-like compounds that includes the P. aeruginosa R-type pyocins.

A Colicin M-Type Bacteriocin from Pseudomonas aeruginosa Targeting the HxuC Heme Receptor Requires a Novel Immunity Partner.

Pyocins are bacteriocins secreted by Pseudomonas aeruginosa, and they assist in the colonization of different niches. A major subset of these antibacterial proteins adopt a modular organization characteristic of polymorphic toxins. They include a receptor-binding domain, a segment enabling membrane passage, and a toxin module at the carboxy terminus, which eventually kills the target cells. To protect themselves from their own products, bacteriocin-producing strains express an immunity gene concomitantly with the bacteriocin. We show here that a pyocin equipped with a phylogenetically distinct ColM toxin domain, PaeM4, mediates antagonism against a large set of P. aeruginosa isolates. Immunity to PaeM4 is provided by the inner membrane protein PmiC, which is equipped with a transmembrane topology not previously described for the ColM family. Given that strains lacking a pmiC gene are killed by PaeM4, the presence of such an immunity partner likely is a key criterion for escaping cellular death mediated by PaeM4. The presence of a TonB box in PaeM4 and enhanced bacteriocin activity under iron-poor conditions strongly suggested the targeting of a TonB-dependent receptor. Evaluation of PaeM4 activities against TonB-dependent receptor knockout mutants in P. aeruginosa PAO1 revealed that the heme receptor HxuC (PA1302) serves as a PaeM4 target at the cellular surface. Because other ColM-type pyocins may target the ferrichrome receptor FiuA, our results illustrate the versatility in target recognition conferred by the polymorphic nature of ColM-type bacteriocins.IMPORTANCE The antimicrobial armamentarium of a bacterium is a major asset for colonizing competitive environments. Bacteriocins comprise a subset of these compounds. Pyocins are an example of such antibacterial proteins produced by Pseudomonas aeruginosa, killing other P. aeruginosa strains. A large group of these molecules show a modular protein architecture that includes a receptor-binding domain for initial target cell attachment and a killer domain. In this study, we have shown that a novel modular pyocin (PaeM4) that kills target bacteria via interference with peptidoglycan assembly takes advantage of the HxuC heme receptor. Cells can protect themselves from killing by the presence of a dedicated immunity partner, an integral inner membrane protein that adopts a transmembrane topology distinct from that of proteins currently known to provide immunity against such toxin activity. Understanding the receptors with which pyocins interact and how immunity to pyocins is achieved is a pivotal step toward the rational design of bacteriocin cocktails for the treatment of P. aeruginosa infections.

Bacteriocins: perspective for the development of novel anticancer drugs.

Antimicrobial peptides (AMPs) from prokaryotic source also known as bacteriocins are ribosomally synthesized by bacteria belonging to different eubacterial taxonomic branches. Most of these AMPs are low molecular weight cationic membrane active peptides that disrupt membrane by forming pores in target cell membranes resulting in cell death. While these peptides known to exhibit broad-spectrum antimicrobial activity, including antibacterial and antifungal, they displayed minimal cytotoxicity to the host cells. Their antimicrobial efficacy has been demonstrated in vivo using diverse animal infection models. Therefore, we have discussed some of the promising peptides for their ability towards potential therapeutic applications. Further, some of these bacteriocins have also been reported to exhibit significant biological activity against various types of cancer cells in different experimental studies. In fact, differential cytotoxicity towards cancer cells as compared to normal cells by certain bacteriocins directs for a much focused research to utilize these compounds as novel therapeutic agents. In this review, bacteriocins that demonstrated antitumor activity against diverse cancer cell lines have been discussed emphasizing their biochemical features, selectivity against extra targets and molecular mechanisms of action.

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.

Study of 32 new phage tail-like bacteriocins (pyocins) from a clinical collection of Pseudomonas aeruginosa and of their potential use as typing markers and antimicrobial agents.

Phage tail-like bacteriocins (PTLBs) are large proteomic structures similar to the tail phages. These structures function in bacterial competition by making pores in the membrane of their competitors. The PTLBs identified in Pseudomonas aeruginosa are known as R-type and F-type pyocins, which have a narrow spectrum of action. Their specificity is determined by the tail fiber and is closely related to the lipopolysaccharide type of the target competitor strain. In this study, the genome sequences of 32 clinical of P. aeruginosa clinical isolates were analysed to investigate the presence of R-type and F-type pyocins, and one was detected in all strains tested. The pyocins were classified into 4 groups on the basis of the tail fiber and also the homology, phylogeny and structure of the cluster components. A relationship was established between these groups and the sequence type and serotype of the strain of origin and finally the killing spectrum of the representative pyocins was determined showing a variable range of activity between 0 and 37.5%. The findings showed that these pyocins could potentially be used for typing of P. aeruginosa clinical isolates, on the basis of their genomic sequence and cluster structure, and also as antimicrobial agents.

Rapid Single-Shot Synthesis of the 214 Amino Acid-Long N-Terminal Domain of Pyocin S2.

The impermeable outer membrane of Pseudomonas aeruginosa is bypassed by antibacterial proteins known as S-type pyocins. Because of their properties, pyocins are investigated as a potential new class of antimicrobials against Pseudomonas infections. Their production and modification, however, remain challenging. To address this limitation, we employed automated fast-flow peptide synthesis for the rapid production of a pyocin S2 import domain. The N-terminal domain sequence (PyS2NTD) was synthesized in under 10 h and purified to yield milligram quantities of the desired product. To our knowledge, the 214 amino acid sequence of PyS2NTD is among the longest peptides produced from a "single-shot" synthesis, i.e., made in a single stepwise route without the use of ligation techniques. Biophysical characterization of the PyS2NTD with circular dichroism was consistent with the literature reports. Fluorescently labeled PyS2NTD binds to P. aeruginosa expressing the cognate ferripyoverdine receptor and is taken up into the periplasm. This selective uptake was validated with confocal and super resolution microscopy, flow cytometry, and fluorescence recovery after photobleaching. These modified, synthetic S-type pyocin domains can be used to probe import mechanisms of P. aeruginosa and leveraged to develop selective antimicrobial agents that bypass the outer membrane.

Characterization of a phage-like pyocin from the plant growth-promoting rhizobacterium Pseudomonas fluorescens SF4c.

R-type and F-type pyocins are high-molecular-mass bacteriocins produced by Pseudomonas aeruginosa that resemble bacteriophage tails. They contain no head structures and no DNA, and are used as defence systems. In this report, we show that Pseudomonas fluorescens SF4c, a strain isolated from the wheat rhizosphere, produces a high-molecular-mass bacteriocin which inhibits the growth of closely related bacteria. A mutant deficient in production of this antimicrobial compound was obtained by transposon mutagenesis. Sequence analysis revealed that the transposon had disrupted a gene that we have named ptm, since it is homologous to that encoding phage tape-measure protein in P. fluorescens Pf0-1, a gene belonging to a prophage similar to phage-like pyocin from P. aeruginosa PAO1. In addition, we have identified genes from the SF4c pyocin cluster that encode a lytic system and regulatory genes. We constructed a non-polar ptm mutant of P. fluorescens SF4c. Heterologous complementation of this mutation restored the production of bacteriocin. Real-time PCR was used to analyse the expression of pyocin under different stress conditions. Bacteriocin was upregulated by mitomycin C, UV light and hydrogen peroxide, and was downregulated by saline stress. This report constitutes, to our knowledge, the first genetic characterization of a phage tail-like bacteriocin in a rhizosphere Pseudomonas strain.

Structural design principles for specific ultra-high affinity interactions between colicins/pyocins and immunity proteins.

The interactions of the antibiotic proteins colicins/pyocins with immunity proteins is a seminal model system for studying protein-protein interactions and specificity. Yet, a precise and quantitative determination of which structural elements and residues determine their binding affinity and specificity is still lacking. Here, we used comparative structure-based energy calculations to map residues that substantially contribute to interactions across native and engineered complexes of colicins/pyocins and immunity proteins. We show that the immunity protein α1-α2 motif is a unique structurally-dissimilar element that restricts interaction specificity towards all colicins/pyocins, in both engineered and native complexes. This motif combines with a diverse and extensive array of electrostatic/polar interactions that enable the exquisite specificity that characterizes these interactions while achieving ultra-high affinity. Surprisingly, the divergence of these contributing colicin residues is reciprocal to residue conservation in immunity proteins. The structurally-dissimilar immunity protein α1-α2 motif is recognized by divergent colicins similarly, while the conserved immunity protein α3 helix interacts with diverse colicin residues. Electrostatics thus plays a key role in setting interaction specificity across all colicins and immunity proteins. Our analysis and resulting residue-level maps illuminate the molecular basis for these protein-protein interactions, with implications for drug development and rational engineering of these interfaces.

Targeted Killing of Pseudomonas aeruginosa by Pyocin G Occurs via the Hemin Transporter Hur.

Pseudomonas aeruginosa is a priority pathogen for the development of new antibiotics, particularly because multi-drug-resistant strains of this bacterium cause serious nosocomial infections and are the leading cause of death in cystic fibrosis patients. Pyocins, bacteriocins of P. aeruginosa, are potent and diverse protein antibiotics that are deployed during bacterial competition. Pyocins are produced by more than 90% of P. aeruginosa strains and may have utility as last resort antibiotics against this bacterium. In this study, we explore the antimicrobial activity of a newly discovered pyocin called pyocin G (PyoG). We demonstrate that PyoG has broad killing activity against a collection of clinical P. aeruginosa isolates and is active in a Galleria mellonella infection model. We go on to identify cell envelope proteins that are necessary for the import of PyoG and its killing activity. PyoG recognizes bacterial cells by binding to Hur, an outer-membrane TonB-dependent transporter. Both pyocin and Hur interact with TonB1, which in complex with ExbB-ExbD links the proton motive force generated across the inner membrane with energy-dependent pyocin translocation across the outer membrane. Inner-membrane translocation of PyoG is dependent on the conserved inner-membrane AAA+ ATPase/protease, FtsH. We also report a functional exploration of the PyoG receptor. We demonstrate that Hur can bind to hemin in vitro and that this interaction is blocked by PyoG, confirming the role of Hur in hemin acquisition.

R pyocin tail fiber structure reveals a receptor-binding domain with a lectin fold.

R pyocins are ɸCTX-like myophage tailocins of Pseudomonas sp. Adsorption of R pyocins to target strains occurs by the interaction of tail fiber proteins with core lipopolysaccharide (LPS). Here, we demonstrate that N-terminally truncated R pyocin tail fibers corresponding to a region of variation between R-subtypes are sufficient to bind target strains according to R-subtype. We also report the crystal structures of these tail fiber proteins and show that they form an elongated helical trimer composed of three domains arranged linearly from N- to C-terminus: a baseplate proximal head, medial shaft, and distal foot. The head and shaft domains contain novel structural motifs. The foot domain, however, is composed of a conserved jellyroll fold and shares high structural similarity to the tail fiber of myophage AP22, podophage tailspike C-terminal domains (LKA-1 and ɸ297), and several eukaryotic adhesins (discoidin I/II, agglutinin, and octocoral lectin). Many of these proteins bind polysaccharides by means of their distal loop network, a series of highly variable loops at one end of the conserved jellyroll fold backbone. Our structures reveal that the majority of R-subtype specific polymorphisms cluster in patches covering a cleft formed at the oligomeric interface of the head domain and in a large patch covering much of the foot domain, including the distal loop network. Based on the structural variation in distal loops within the foot region, we propose that the foot is the primary sugar-binding domain of R pyocins and R-subtype specific structural differences in the foot domain distal loop network are responsible for binding target strains in an R-subtype dependent manner.

Structure and Analysis of R1 and R2 Pyocin Receptor-Binding Fibers.

The R-type pyocins are high-molecular weight bacteriocins produced by some strains of Pseudomonas aeruginosa to specifically kill other strains of the same species. Structurally, the R-type pyocins are similar to "simple" contractile tails, such as those of phage P2 and Mu. The pyocin recognizes and binds to its target with the help of fibers that emanate from the baseplate structure at one end of the particle. Subsequently, the pyocin contracts its sheath and drives the rigid tube through the host cell envelope. This causes depolarization of the cytoplasmic membrane and cell death. The host cell surface-binding fiber is ~340 Å-long and is attached to the baseplate with its N-terminal domain. Here, we report the crystal structures of C-terminal fragments of the R1 and R2 pyocin fibers that comprise the distal, receptor-binding part of the protein. Both proteins are ~240 Å-long homotrimers in which slender rod-like domains are interspersed with more globular domains-two tandem knob domains in the N-terminal part of the fragment and a lectin-like domain at its C-terminus. The putative substrate binding sites are separated by about 100 Å, suggesting that binding of the fiber to the cell surface causes the fiber to adopt a certain orientation relative to the baseplate and this then triggers sheath contraction.

Commensurability between protein nanotubes in contractile ejection nanomachines.

Contractile ejection nanomachines being sheath-tube assemblies create an opening in the cell membrane to translocate molecules or ions across it. Here, on the most structurally investigated examples of the bacteriophage T4 tail and pyocin R2, we show that the rearrangement of the sheath structure resulting in its contraction and twist occurs in such a way that the contracted sheath becomes commensurate with the inner tube. This fact dictates the previously unknown simple geometrical relationship between the nanotube symmetries. Using the Frank and van der Merwe classical theory of commensurability, we study an interaction between two protein nanotubes forming such nanomachines and obtain an expression for the corresponding energy, which depends on the tube structures and their mutual arrangement. The appearance of commensurability between the contracted sheath and the inner tube decreases both the interaction energy and the total energy of the system. It improves the nanomachine efficiency, since the energy gain obtained increases the torque of the inner tube piercing the cell membrane.

Identification and functional analysis of a bacteriocin, pyocin S6, with ribonuclease activity from a Pseudomonas aeruginosa cystic fibrosis clinical isolate.

S-type pyocins are bacteriocins produced by Pseudomonas aeruginosa isolates to antagonize or kill other strains of the same species. They have a modular organization comprising a receptor-binding domain recognizing a surface constituent of the target bacterium, a domain for translocation through the periplasm, and a killing or toxic domain with DNase, tRNase, or pore-forming activity. Pyocins S2, S3, S4, and S5 recognize TonB-dependent ferri-siderophore receptors in the outer membrane. We here describe a new nuclease bacteriocin, pyocin S6, encoded in the genome of a P. aeruginosa cystic fibrosis (CF) clinical isolate, CF_PA39. Similarly to pyocins S1 and S2, the S6 toxin-immunity gene tandem was recruited to the genomic region encoding exotoxin A. The pyocin S6 receptor-binding and translocation domains are identical to those of pyocin S1, whereas the killing domain is similar to the 16S ribonuclease domain of Escherichia coli colicin E3. The cytotoxic activity was abolished in pyocin S6 forms with a mutation in the colicin E3-equivalent catalytic motif. The CF_PA39 S6 immunity gene displays a higher expression level than the gene encoding the killing protein, the latter being only detected when bacteria are grown under iron-limiting conditions. In the S1-pyocinogenic strain P. aeruginosa ATCC 25324 and pyocin S2 producer P. aeruginosa PAO1, a remnant of the pyocin S6 killing domain and an intact S6-type immunity gene are located downstream of their respective pyocin operons. Strain PAO1 is insensitive for pyocin S6, and its S6-type immunity gene provides protection against pyocin S6 activity. Purified pyocin S6 inhibits one-fifth of 110 P. aeruginosa CF clinical isolates tested, showing clearer inhibition zones when the target cells are grown under iron limitation. In this panel, about half of the CF clinical isolates were found to host the S6 genes. The pyocin S6 locus is also present in the genome of some non-CF clinical isolates.

Microarray analysis of Pseudomonas aeruginosa reveals induction of pyocin genes in response to hydrogen peroxide.

BACKGROUND: Pseudomonas aeruginosa, a pathogen infecting those with cystic fibrosis, encounters toxicity from phagocyte-derived reactive oxidants including hydrogen peroxide during active infection. P. aeruginosa responds with adaptive and protective strategies against these toxic species to effectively infect humans. Despite advances in our understanding of the responses to oxidative stress in many specific cases, the connectivity between targeted protective genes and the rest of cell metabolism remains obscure. RESULTS: Herein, we performed a genome-wide transcriptome analysis of the cellular responses to hydrogen peroxide in order to determine a more complete picture of how oxidative stress-induced genes are related and regulated. Our data reinforce the previous conclusion that DNA repair proteins and catalases may be among the most vital antioxidant defense systems of P. aeruginosa. Our results also suggest that sublethal oxidative damage reduces active and/or facilitated transport and that intracellular iron might be a key factor for a relationship between oxidative stress and iron regulation. Perhaps most intriguingly, we revealed that the transcription of all F-, R-, and S-type pyocins was upregulated by oxidative stress and at the same time, a cell immunity protein (pyocin S2 immunity protein) was downregulated, possibly leading to self-killing activity. CONCLUSION: This finding proposes that pyocin production might be another novel defensive scheme against oxidative attack by host cells.

Biophysicochemical characterization of Pyocin SA189 produced by Pseudomonas aeruginosa SA189.

Pseudomonas aeruginosa, in spite of being a ubiquitous organism (as it is found in soil, water, and humans), is also an opportunistic pathogen. In order to maintain its diversity in the community, it produces various toxic proteins, known as, bacteriocins. In the present study, pyocin SA189, which is a bacteriocin produced by P. aeruginosa SA189 (isolated from a clinical sample) was characterized. P. aeruginosa SA189, as identified by the conventional and 16S rRNA gene amplification, produced pyocin SA189 of molecular weight of 66 k Da. The pyocin showed antimicrobial activity against several clinically relevant Gram-positive and Gram-negative bacteria and was substantially stable for wide ranges of temperature and pH. Furthermore, the pyocin also retained its biological activity upon treatment with metal ions, organic solvents, and various proteolytic and lipolytic enzymes. The data from the growth kinetics indicated that the maximum bacteriocin production occurred in the late log phase. Overall, our results signify the potential of pyocin SA189 as a bio-control agent.

Atomic structures of a bactericidal contractile nanotube in its pre- and postcontraction states.

R-type pyocins are representatives of contractile ejection systems, a class of biological nanomachines that includes, among others, the bacterial type VI secretion system (T6SS) and contractile bacteriophage tails. We report atomic models of the Pseudomonas aeruginosa precontraction pyocin sheath and tube, and the postcontraction sheath, obtained by cryo-EM at 3.5-Å and 3.9-Å resolutions, respectively. The central channel of the tube is negatively charged, in contrast to the neutral and positive counterparts in T6SSs and phage tails. The sheath is interwoven by long N- and C-terminal extension arms emanating from each subunit, which create an extensive two-dimensional mesh that has the same connectivity in the extended and contracted state of the sheath. We propose that the contraction process draws energy from electrostatic and shape complementarities to insert the inner tube through bacterial cell membranes to eventually kill the bacteria.