Phage Tail-like Bacteriocins Research Articles

Specialized Listeria monocytogenes produce tailocins to provide a population-level competitive growth advantage.

Tailocins are phage tail-like bacteriocins produced by various bacterial species to kill kin competitors. Given that tailocin release is dependent upon cell lysis, regulation of tailocin production at the single-cell and population level remains unclear. Here we used flow cytometry, competition assays and structural characterization of tailocin production in a human bacterial pathogen, Listeria monocytogenes. We revealed that a specialized subpopulation, constituting less than 1% of the total bacterial population, differentiates to produce, assemble and store thousands of tailocin particles. Tailocins are packed in a highly ordered manner, clustered in a liquid crystalline phase that occupies a substantial volume of the cell. Tailocin production confers a competitive growth advantage for the rest of the population. This study provides molecular insights into tailocin production as a form of altruism, showing how cell specialization within bacterial populations can confer competitive advantages at the population level.

The long and sinuous road to phage-based therapy of Clostridioides difficile infections.

With the antibiotic crisis and the rise in antimicrobial resistance worldwide, new therapeutic alternatives are urgently needed. Phage therapy represents one of the most promising alternatives but for some pathogens, such as Clostridioides difficile, important challenges are being faced. The perspective of phage therapy to treat C. difficile infections is complicated by the fact that no strictly lytic phages have been identified so far, and current temperate phages generally have a narrow host range. C. difficile also harbors multiple antiphage mechanisms, and the bacterial genome is often a host of one or multiple prophages that can interfere with lytic phage infection. Nevertheless, due to recent advances in phage host receptor recognition and improvements in genetic tools to manipulate phage genomes, it is now conceivable to genetically engineer C. difficile phages to make them suitable for phage therapy. Other phage-based alternatives such as phage endolysins and phage tail-like bacteriocins (avidocins) are also being investigated but these approaches also have their own limitations and challenges. Last but not least, C. difficile produces spores that are resistant to phage attacks and all current antibiotics, and this complicates therapeutic interventions. This mini-review gives a brief historical overview of phage work that has been carried out in C. difficile, presents recent advances in the field, and addresses the most important challenges that are being faced, with potential solutions.

Wild-type Lactococcus lactis producing bacteriocin-like prophage lysins.

Lactococcus is a genus of lactic acid bacteria used in the dairy industry as a starter. Lactococci have been found to produce altogether more than 40 different bacteriocins, ribosomally synthesized antimicrobial proteins. All known Lactococcus spp. bacteriocins belong to classes I and II, which are mainly heat-resistant peptides. No class III bacteriocins, bigger heat-sensitive proteins, including phage tail-like bacteriocins, have been found from the Lactococcus spp. Unlike phage tail-like bacteriocins, prophage lysins have not been regarded as bacteriocins, possibly because phage lysins contribute to autolysis, degrading the host's own cell wall. Wild-type Lactococcus lactis strain LAC460, isolated from spontaneously fermented idli batter, was examined for its antimicrobial activity. We sequenced the genome, searched phage lysins from the culture supernatant, and created knock-out mutants to find out the source of the antimicrobial activity. The strain LAC460 was shown to kill other Lactococcus strains with protease- and heat-sensitive lytic activity. Three phage lysins were identified in the culture supernatant. The genes encoding the three lysins were localized in different prophage regions in the chromosome. By knock-out mutants, two of the lysins, namely LysL and LysP, were demonstrated to be responsible for the antimicrobial activity. The strain LAC460 was found to be resistant to the lytic action of its own culture supernatant, and as a consequence, the phage lysins could behave like bacteriocins targeting and killing other closely related bacteria. Hence, similar to phage tail-like bacteriocins, phage lysin-like bacteriocins could be regarded as a novel type of class III bacteriocins.

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.

R-Type Fonticins Produced by Pragia fontium Form Large Pores with High Conductance.

Fonticins are phage tail-like bacteriocins produced by the Gram-negative bacterium Pragia fontium from the family Budviciaceae. This bacterium produces contractile-type particles that adsorb on the surface of sensitive bacteria and penetrate the cell wall, probably during contraction, in a way similar to the type VI secretion system. We characterized the pore-forming activity of fonticins using both living cells and in vitro model membranes. Using a potassium leakage assay, we show that fonticins are able to permeabilize sensitive cells. On black lipid membranes, single-pore conductance is about 0.78 nS in 1 M NaCl and appears to be linearly dependent on the increasing molar strength of NaCl solution, which is a property of considerably large pores. In agreement with these findings, fonticins are not ion selective for Na+, K+, and Cl-. Polyethylene glycol 3350 (PEG 3350) molecules of about 3.5 nm in diameter can enter the fonticin pore lumen, whereas the larger molecules cannot pass the pore. The size of fonticin pores was confirmed by transmission electron microscopy. The terminal membrane-piercing complex of the fonticin tube probably creates a selective barrier restricting passage of macromolecules. IMPORTANCE Phage tail-like bacteriocins are now the subject of research as potent antibacterial agents due to their narrow host specificity and single-hit mode of action. In this work, we focused on the structure and mode of action of fonticins. According to some theories, related particles were initially adapted for passage of double-stranded DNA (dsDNA) molecules, but fonticins changed their function during the evolution; they are able to form large pores through the bacterial envelope of Gram-negative bacteria. As various pore-forming proteins are extensively used for nanopore sequencing and stochastic sensing, we decided to investigate the pore-forming properties of fonticin protein complexes on artificial lipid membranes. Our research revealed remarkable structural properties of these particles that may have a potential application as a nanodevice.

Heterogenous susceptibility to R-pyocins in populations of Pseudomonas aeruginosa sourced from cystic fibrosis lungs

Bacteriocins are proteinaceous antimicrobials produced by bacteria which are active against other strains of the same species. R-type pyocins are phage tail-like bacteriocins produced by Pseudomonas aeruginosa. Due to their anti-pseudomonal activity, R-pyocins have potential as therapeutics in infection. P. aeruginosa is a Gram-negative opportunistic pathogen and is particularly problematic for individuals with cystic fibrosis (CF). P. aeruginosa from CF lung infections develop increasing resistance to antibiotics, making new treatment approaches essential. P. aeruginosa populations become phenotypically and genotypically diverse during infection, however, little is known of the efficacy of R-pyocins against heterogeneous populations. R-pyocins vary by subtype (R1-R5), distinguished by binding to different residues on the lipopolysaccharide (LPS). Each type varies in killing spectrum, and each strain produces only one R-type. To evaluate the prevalence of different R-types, we screened P. aeruginosastrains from the International Pseudomonas Consortium Database (IPCD) and from our biobank of CF strains. We found that (i) R1-types were the most prevalent R-type among strains from respiratory sources; (ii) there are a large number of strains lacking R-pyocin genes, and (iii) isolates collected from the same patient have the same R-type. We then assessed the impact of diversity on R-pyocin susceptibility and found a heterogenous response to R-pyocins within populations, likely due to differences in the LPS core. Our work reveals that heterogeneous populations of microbes exhibit variable susceptibility to R-pyocins and highlights that there is likely heterogeneity in response to other types of LPS-binding antimicrobials, including phage.

Predicting bacteriophage hosts based on sequences of annotated receptor-binding proteins

Nowadays, bacteriophages are increasingly considered as an alternative treatment for a variety of bacterial infections in cases where classical antibiotics have become ineffective. However, characterizing the host specificity of phages remains a labor- and time-intensive process. In order to alleviate this burden, we have developed a new machine-learning-based pipeline to predict bacteriophage hosts based on annotated receptor-binding protein (RBP) sequence data. We focus on predicting bacterial hosts from the ESKAPE group, Escherichia coli, Salmonella enterica and Clostridium difficile. We compare the performance of our predictive model with that of the widely used Basic Local Alignment Search Tool (BLAST). Our best-performing predictive model reaches Precision-Recall Area Under the Curve (PR-AUC) scores between 73.6 and 93.8% for different levels of sequence similarity in the collected data. Our model reaches a performance comparable to that of BLASTp when sequence similarity in the data is high and starts outperforming BLASTp when sequence similarity drops below 75%. Therefore, our machine learning methods can be especially useful in settings in which sequence similarity to other known sequences is low. Predicting the hosts of novel metagenomic RBP sequences could extend our toolbox to tune the host spectrum of phages or phage tail-like bacteriocins by swapping RBPs.

Bacteriocins of Listeria monocytogenes and Their Potential as a Virulence Factor.

Intestinal microbiota exerts protective effects against the infection of various bacterial pathogens, including Listeria monocytogenes, a major foodborne pathogen whose infection can lead to a disease (listeriosis) with a high fatality rate. As a strategy to mitigate the action of the intestinal microbiota, pathogens often produce antimicrobial proteinaceous compounds such as bacteriocins. In this review, we summarize the information currently available for the well-characterized L. monocytogenes bacteriocin listeriolysin S, with the emphasis on its intriguing mode of action as a virulence factor, which promotes the infection of L. monocytogenes by changing the composition of the intestinal microbiota. We then discuss another intriguing L. monocytogenes bacteriocin Lmo2776 that specifically inhibits the inflammogenic species, Prevotella copri, in the intestinal microbiota, reducing superfluous inflammation while weakening virulence. In addition, we describe relatively less studied phage tail-like Listeria bacteriocins (monocins) and elaborate on the possibility that these monocins could be involved in enhancing pathogenicity. In spite of the burgeoning interest in the roles played by the intestinal microbiota against the L. monocytogenes infection, our understanding on the virulence factors affecting the intestinal microbiota is still lacking, calling for further studies on bacteriocins that could function as novel virulence factors.

Coordination of cohabiting phage elements supports bacteria\u2013phage cooperation

Bacterial pathogens often carry multiple prophages and other phage-derived elements within their genome, some of which can produce viral particles in response to stress. Listeria monocytogenes 10403S harbors two phage elements in its chromosome, both of which can trigger bacterial lysis under stress: an active prophage (ϕ10403S) that promotes the virulence of its host and can produce infective virions, and a locus encoding phage tail-like bacteriocins. Here, we show that the two phage elements are co-regulated, with the bacteriocin locus controlling the induction of the prophage and thus its activity as a virulence-associated molecular switch. More specifically, a metalloprotease encoded in the bacteriocin locus is upregulated in response to stress and acts as an anti-repressor for CI-like repressors encoded in each phage element. Our results provide molecular insight into the phenomenon of polylysogeny and its intricate adaptation to complex environments.

Engineering of receptor-binding proteins in bacteriophages and phage tail-like bacteriocins.

Bacteriophages and phage tail-like bacteriocins (PTLBs) rely on receptor-binding proteins (RBPs) located in tail fibers or spikes for an initial and specific interaction with susceptible bacteria. Bacteriophages kill bacteria through a lytic, replicative cycle, whereas PTLBs kill the target through membrane depolarization in a single hit mechanism. Extensive efforts in the engineering of RBPs of both phages and PTLBs have been undertaken to obtain a greater understanding of the structural organization of RBPs. In addition, a major goal of engineering RBPs of phages and PTLBs is the production of antibacterials with a customized spectrum. Swapping of the RBP of phages and PTLBs results in a shift in activity spectrum in accordance with the spectrum of the new RBP. The engineering of strictly virulent phages with new RBPs required significant technical advances in the past decades, whereas the engineering of RBPs of PTLBs relied on the traditional molecular techniques used for the manipulation of bacteria and was thus relatively straightforward. While phages and PTLBs share their potential for specificity tuning, specific features of phages such as their lytic killing mechanism, their self-replicative nature and thus different pharmacokinetics and their potential to co-evolve are clear differentiators compared with PTLBs in terms of their antibacterial use.

Crystal Structures of R-Type Bacteriocin Sheath and Tube Proteins CD1363 and CD1364 From Clostridium difficile in the Pre-assembled State.

Diffocins are high-molecular-weight phage tail-like bacteriocins (PTLBs) that some Clostridium difficile strains produce in response to SOS induction. Similar to the related R-type pyocins from Pseudomonas aeruginosa, R-type diffocins act as molecular puncture devices that specifically penetrate the cell envelope of other C. difficile strains to dissipate the membrane potential and kill the attacked bacterium. Thus, R-type diffocins constitute potential therapeutic agents to counter C. difficile-associated infections. PTLBs consist of rigid and contractile protein complexes. They are composed of a baseplate, receptor-binding tail fibers and an inner needle-like tube surrounded by a contractile sheath. In the mature particle, the sheath and tube structure form a complex network comprising up to 200 copies of a sheath and a tube protein each. Here, we report the crystal structures together with small angle X-ray scattering data of the sheath and tube proteins CD1363 (39 kDa) and CD1364 (16 kDa) from C. difficile strain CD630 in a monomeric pre-assembly form at 1.9 and 1.5 Å resolution, respectively. The tube protein CD1364 displays a compact fold and shares highest structural similarity with a tube protein from Bacillus subtilis but is remarkably different from that of the R-type pyocin from P. aeruginosa. The structure of the R-type diffocin sheath protein, on the other hand, is highly conserved. It contains two domains, whereas related members such as bacteriophage tail sheath proteins comprise up to four, indicating that R-type PTLBs may represent the minimal protein required for formation of a complete sheath structure. Comparison of CD1363 and CD1364 with structures of PTLBs and related assemblies suggests that several conformational changes are required to form complete assemblies. In the sheath, rearrangement of the flexible N- and C-terminus enables extensive interactions between the other subunits, whereas for the tube, such contacts are primarily established by mobile α-helices. Together, our results combined with information from structures of homologous assemblies allow constructing a preliminary model of the sheath and tube assembly from R-type diffocin.

Phage Tail-Like Bacteriocins.

Many dsDNA bacterial viruses (bacteriophages/phages) have long tail structures that serve as organelles for DNA delivery to host targets. These structures, particularly those of Myoviridae and Siphoviridae phages, have an evolutionary relationship with other cellular biological entities that share the common function of penetrating the bacterial envelope. Among these are type VI secretion systems, insecticidal protein complexes, and bacteriocins. Phage tail-like bacteriocins (PTLBs) are widespread in bacteria, comprising different types that likely evolved independently. They can be divided into two major classes: the R-type PTLBs, which are related to contractile Myoviridae phage tails, and the F-type PTLBs, which are related to noncontractile Siphoviridae phage tails. This review provides an overview of the history, biology, and diversity of these entities and also covers recent efforts to utilize these potent bactericidal agents as human therapeutics against bacterial disease.

Effect of a Pseudomonas fluorescens tailocin against phytopathogenic Xanthomonas observed by atomic force microscopy

Phage tail-like bacteriocins, called tailocins, represent a class of protein complexes produced by a multitude of bacteria. Pseudomonas fluorescens SF4c, a strain isolated from wheat rhizosphere, produces a bacteriocin similar to phage tail-like pyocins of Pseudomonas aeruginosa. This tailocin has antimicrobial activity against several phytopathogenic strains of the genus Xanthomonas and Pseudomonas. In this work, the effect of the SF4c tailocin on the phytopathogenic strain X. axonopodis pv vesicatoria Xcv Bv5–4a was analyzed through Atomic Force Microscopy (AFM). We demonstrated that tailocins adhere and cause damage to the cell envelope of strain Xcv Bv5-4a. This results in a rapid leakage of intracellular materials, with the subsequent decrease of cell volume. Finally, lysis of sensitive bacteria occurs. This study provides, to our knowledge, the first evidence about the effect of a tailocin analyzed by AFM. Further studies are in progress to evaluate the use of SF4c tailocin in the biocontrol of bacterial spot on tomato.

F-Type Bacteriocins of Listeria monocytogenes: a New Class of Phage Tail-Like Structures Reveals Broad Parallel Coevolution between Tailed Bacteriophages and High-Molecular-Weight Bacteriocins.

Listeria monocytogenes is a significant foodborne human pathogen that can cause severe disease in certain high-risk individuals. L. monocytogenes is known to produce high-molecular-weight, phage tail-like bacteriocins, or "monocins," upon induction of the SOS system. In this work, we purified and characterized monocins and found them to be a new class of F-type bacteriocins. The L. monocytogenes monocin genetic locus was cloned and expressed in Bacillus subtilis, producing specifically targeted bactericidal particles. The receptor binding protein, which determines target cell specificity, was identified and engineered to change the bactericidal spectrum. Unlike the F-type pyocins of Pseudomonas aeruginosa, which are related to lambda-like phage tails, monocins are more closely related to TP901-1-like phage tails, structures not previously known to function as bacteriocins. Monocins therefore represent a new class of phage tail-like bacteriocins. It appears that multiple classes of phage tails and their related bacteriocins have coevolved separately in parallel. Phage tail-like bacteriocins (PTLBs) are structures widespread among the members of the bacterial kingdom that are evolutionarily related to the DNA delivery organelles of phages (tails). We identified and characterized "monocins" of Listeria monocytogenes and showed that they are related to the tail structures of TP901-1-like phages, structures not previously known to function as bacteriocins. Our results show that multiple types of envelope-penetrating machines have coevolved in parallel to function either for DNA delivery (phages) or as membrane-disrupting bacteriocins. While it has commonly been assumed that these structures were coopted from phages, we cannot rule out the opposite possibility, that ancient phages coopted complex bacteriocins from the cell, which then underwent adaptations to become efficient at translocating DNA.

Occurrence of Strains Producing Specific Antibacterial Inhibitory Agents in Five Genera of Enterobacteriaceae

Striking differences in the production of specific inhibitory agents affecting other strains of the same (or of related) species were found between genera of the family Enterobacteriaceae. We tested 50-163 strains each of the potentially pathogenic genera: Escherichia, Citrobacter, Enterobacter, Kluyvera, and Leclercia for their ability to produce bacteriophages, high-molecular-weight (HMW) and low-molecular-weight (LMW) bacteriocins and siderophores against the same sets of strains, using the cross-test method. The genus Escherichia differs substantially from all other Enterobacteriaceae, harboring a notable proportion of lysogenic (36.6%) and colicinogenic (13.9%) strains. Only 18.2% of the Citrobacter strains are lysogenic and only rarely are they colicinogenic, although in 7.3%, they produce phage tail-like bacteriocins. On the other hand, Kluyvera strains were only in 1.8% lysogenic, no colicinogenic strains were found, but in 7.3%, they produced siderophores causing zones of growth inhibition in agar cultures of strains of the same genus. In Leclercia, 10.0% of the strains were lysogenic, 2.0% produced HMW bacteriocins, no colicinogenic strains were found and 2.0% produced siderophores. Enterobacter has shown 23.1% of strains producing siderophores, whereas merely 7.7% were lysogenic, 1.9% colicinogenic and 3.8% formed phage tail-like bacteriocins. HMW bacteriocins of Enterobacter strains disposed of an unusually wide spectrum of activity. The siderophore activity spectrum was rather wide in any genus, but the siderophores were usually not produced by strains producing phages or colicins.

Phage Tail-Like (High-Molecular-Weight) Bacteriocins of Budvicia aquatica and Pragia fontium ( Enterobacteriaceae )

Electron microscopic analysis of contractile phage tail-like bacteriocins of three Pragia fontium strains and one Budvicia aquatica strain was performed. Fonticin and aquaticin are remarkably heat sensitive but trypsin resistant. Simultaneous production of contractile and flexible phage tail-like bacteriocins in the P. fontium 64613 strain is shown for the first time.

Purification and characterization of xenorhabdicin, a phage tail-like bacteriocin, from the lysogenic strain F1 of Xenorhabdus nematophilus

Xenorhabdicin, the phage tail-like bacteriocins of Xenorhabdus nematophilus, and phage head particles, elements produced together after mitomycin induction in X. nematophilus lysogenic strain F1 cultures, were separated by DEAE chromatography, examined by transmission electron microscopy, and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electrophoresis of xenorhabdicin showed two major subunits of 43 and 20 kDa corresponding to the sheath and the inner core, respectively. At least five other minor subunits of 67, 54, 35, 28, and 16 kDa were also characterized. Electrophoresis of the phage head capsids showed a major 40-kDa subunit and two minor 50- and 34-kDa subunits. Bactericidal activity recorded against closely related bacterial species and spontaneously produced by X. nematophilus resides in the xenorhabdicin particles and is another antimicrobial barrier to save the symbiotic association.

Genetic comparison of bacteriophage PS17 and Pseudomonas aeruginosa R-type pyocin

PS17 is a bacteriophage of Pseudomonas aeruginosa that is serologically cross-reactive with phage tail-like bacteriocins called R-type pyocins. In addition to having immunological cross-reactivity, certain genes are functionally complementable between PS17 and R-type pyocins. To compare the genetic structures of PS17 and R-type pyocins, a physical map of PS17 genes was constructed by cloning phage DNA fragments on RSF1010-derived vector plasmids. The head and tail gene clusters were tandemly arrayed and together occupied about half of the 41-kilobase-pair PS17 chromosome. With use of these phage clones, the following results were obtained with respect to the genetic relationship between PS17 and R-type pyocins: (i) serological cross-reaction between PS17 and pyocin occurred for the major sheath protein and two components of the fiber, (ii) a certain pyocin mutation was complemented by cloned phage fragments, and (iii) the phage DNA fragment carrying sheath and core tube genes was shown to hybridize to the DNA fragment carrying the pyocin R2 genes.