High prevalence of lipopolysaccharide mutants and R2-pyocin susceptible variants in Pseudomonas aeruginosa populations sourced from cystic fibrosis lung infections.

IMPORTANCE: Cystic fibrosis (CF) patients often experience chronic, debilitating lung infections caused by antibiotic-resistant Pseudomonas aeruginosa, contributing to antimicrobial resistance (AMR). The genetic and phenotypic diversity of P. aeruginosa populations in CF lungs raises questions about their susceptibility to non-traditional antimicrobials, like bacteriocins. In this study, we focused on R-pyocins, a type of bacteriocin with high potency and a narrow killing spectrum. Our findings indicate that a large number of infectious CF variants are susceptible to R2-pyocins, even within diverse bacterial populations, supporting their potential use as therapeutic agents. The absence of a clear correlation between lipopolysaccharide (LPS) phenotypes and R-pyocin susceptibility suggests that LPS packing density may play a significant role in R-pyocin susceptibility among CF variants. Understanding the relationship between LPS phenotypes and R-pyocin susceptibility is crucial for developing effective treatments for these chronic infections.

Outer membrane translocation of pyocins via the copper regulated TonB-dependent transporter CrtA.

Pseudomonas aeruginosa is a common cause of serious hospital-acquired infections, the leading proven cause of mortality in people with cystic fibrosis and is associated with high levels of antimicrobial resistance. Pyocins are narrow-spectrum protein antibiotics produced by P. aeruginosa that kill strains of the same species and have the potential to be developed as therapeutics targeting multi-drug resistant isolates. We have identified two novel pyocins designated SX1 and SX2. Pyocin SX1 is a metal-dependent DNase while pyocin SX2 kills cells through inhibition of protein synthesis. Mapping the uptake pathways of SX1 and SX2 shows these pyocins utilize a combination of the common polysaccharide antigen (CPA) and a previously uncharacterized TonB-dependent transporter (TBDT) PA0434 to traverse the outer membrane. In addition, TonB1 and FtsH are required by both pyocins to energize their transport into cells and catalyze their translocation across the inner membrane, respectively. Expression of PA0434 was found to be specifically regulated by copper availability and we have designated PA0434 as Copper Responsive Transporter A, or CrtA. To our knowledge these are the first S-type pyocins described that utilize a TBDT that is not involved in iron uptake.

Remotely Controllable Engineered Bacteria for Targeted Therapy of Pseudomonas aeruginosa Infection.

Pseudomonas aeruginosa (P. aeruginosa) infection has become an intractable problem worldwide due to the decreasing efficacy of the mainstay therapy, antibiotic treatment. Hence, exploring new drugs and therapies to address this issue is crucial. Here, we construct a chimeric pyocin (ChPy) to specifically kill P. aeruginosa and engineer a near-infrared (NIR) light-responsive strain to produce and deliver this drug. Our engineered bacterial strain can continuously produce ChPy in the absence of light and release it to kill P. aeruginosa via remotely and precisely controlled bacterial lysis induced by NIR light. We demonstrate that our engineered bacterial strain is effective in P. aeruginosa-infected wound therapy in the mouse model, as it eradicated PAO1 in mouse wounds and shortened the wound healing time. Our work presents a potentially spatiotemporal and noninvasively controlled therapeutic strategy of engineered bacteria for the targeted treatment of P. aeruginosa infections.

F-Type Pyocins Are Diverse Noncontractile Phage Tail-Like Weapons for Killing Pseudomonas aeruginosa.

Most Pseudomonas aeruginosa strains produce bacteriocins derived from contractile or noncontractile phage tails known as R- and F-type pyocins, respectively. These bacteriocins possess strain-specific bactericidal activity against P. aeruginosa and likely increase evolutionary fitness through intraspecies competition. R-type pyocins have been studied extensively and show promise as alternatives to antibiotics. Although they have similar therapeutic potential, experimental studies on F-type pyocins are limited. Here, we provide a bioinformatic and experimental investigation of F-type pyocins. We introduce a systematic naming scheme for genes found in R- and F-type pyocin operons and identify 15 genes invariably found in strains producing F-type pyocins. Five proteins encoded at the 3' end of the F-type pyocin cluster are divergent in sequence and likely determine bactericidal specificity. We use sequence similarities among these proteins to define eleven distinct F-type pyocin groups, five of which had not been previously described. The five genes encoding the variable proteins associate in two modules that have clearly reassorted independently during the evolution of these operons. These proteins are considerably more diverse than the specificity-determining tail fibers of R-type pyocins, suggesting that F-type pyocins may have emerged earlier. Experimental studies on six F-type pyocin groups show that each displays a distinct spectrum of bactericidal activity. This activity is strongly influenced by the lipopolysaccharide O-antigen type, but other factors also play a role. F-type pyocins appear to kill as efficiently as R-type pyocins. These studies set the stage for the development of F-type pyocins as antibacterial therapeutics. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen that causes antibiotic-resistant infections with high mortality rates, particularly in immunocompromised individuals and cystic fibrosis patients. Due to the increasing frequency of multidrug-resistant P. aeruginosa infections, there is great need for the development of alternative therapeutics. In this study, we investigate one such potential therapeutic: F-type pyocins, which are bacteriocins naturally produced by P. aeruginosa that resemble noncontractile phage tails. We show that they are potent killers of P. aeruginosa and identify their probable bactericidal specificity determinants, which opens up the possibility of engineering them to precisely target strains of pathogenic bacteria. The resemblance of F-type pyocins to well-characterized phage tails will greatly facilitate their development into effective antibacterials.

[Functional synergism of pyoverdine and the S-type pyocins of Pseudomonas aeruginosa].

Pyocin S2 and S4 in Pseudomonas aeruginosa use the same uptake channels as the pyoverdine does in bacteria, indicating a possible connection between them. In this study, we characterized the single bacterial gene expression distribution of three S-type pyocins (Pys2, PA3866, and PyoS5) and examined the impact of pyocin S2 on bacterial uptake of pyoverdine. The findings demonstrated that the expression of the S-type pyocin genes was highly differentiated in bacterial population under DNAdamage stress. Moreover, exogenous addition of pyocin S2 reduces the bacterial uptake of pyoverdine so that the presence of pyocin S2 prevents the uptake of environmental pyoverdine by non-pyoverdine synthesizing 'cheaters', thereby reducing their resistance to oxidative stress. Furthermore, we discovered that overexpression of the SOS response regulator PrtN in bacteria significantly decreased the expression of genes involved in the synthesis of pyoverdine, significantly decreasing the overall synthesis and exocytosis of pyoverdine. These findings imply a connection between the function of the iron absorption system and the SOS stress response mechanism in bacteria.

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.

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.

DNA Damage-Inducible Pyocin Expression Is Independent of RecA in xerC-Deleted Pseudomonas aeruginosa.

Pyocins are interbacterial killing complexes made by Pseudomonas aeruginosa primarily to enact intraspecific competition. DNA damage and the ensuing activation of RecA initiate canonical pyocin expression. We recently discovered that deletion of xerC, which encodes a tyrosine recombinase involved in chromosome decatenation, markedly elevates basal pyocin production independently of RecA. Interestingly, the already-elevated basal pyocin expression in ΔxerC cells is substantially further increased by ciprofloxacin treatment. Here, we asked whether this further increase is due to DNA damage additionally activating the canonical RecA-dependent pyocin expression pathway. We also interrogated the relationship between XerC recombinase activity and pyocin expression. Surprisingly, we find that DNA damage-induced pyocin stimulation in ΔxerC cells is independent of RecA but dependent on PrtN, implying a RecA-independent means of DNA damage sensing that activates pyocin expression via PrtN. In sharp contrast to the RecA independence of pyocin expression in ΔxerC strains, specific mutational inactivation of XerC recombinase activity (XerCY272F) caused modestly elevated basal pyocin expression and was further stimulated by DNA-damaging drugs, but both effects were fully RecA dependent. To test whether pyocins could be induced by chemically inactivating XerC, we deployed a previously characterized bacterial tyrosine recombinase inhibitor. However, the inhibitor did not activate pyocin expression even at growth-inhibitory concentrations, suggesting that its principal inhibitory activity resembles neither XerC absence nor enzymatic inactivation. Collectively, our results imply a second function of XerC, separate from its recombinase activity, whose absence permits RecA-independent but DNA damage-inducible pyocin expression. IMPORTANCE The opportunistic pathogen Pseudomonas aeruginosa produces pyocins-intraspecific, interbacterial killing complexes. The canonical pathway for pyocin production involves DNA damage and RecA activation. Pyocins are released by cell lysis, making production costly. We previously showed that cells lacking the tyrosine recombinase XerC produce pyocins independently of RecA. Here, we show that DNA-damaging agents stimulate pyocin expression in ΔxerC strains without involving RecA. However, strains mutated for XerC recombinase activity display strictly RecA-dependent pyocin production, and a known bacterial tyrosine recombinase inhibitor does not elicit pyocin expression. Our results collectively suggest that the use of XerC inhibition as an antipseudomonal strategy will require targeting the second function of XerC in regulating noncanonical pyocin production rather than targeting its recombinase activity.

Bacterial Competition Systems Share a Domain Required for Inner Membrane Transport of the Bacteriocin Pyocin G from Pseudomonas aeruginosa.

Bacteria exploit a variety of attack strategies to gain dominance within ecological niches. Prominent among these are contact-dependent inhibition (CDI), type VI secretion (T6SS), and bacteriocins. The cytotoxic endpoint of these systems is often the delivery of a nuclease to the cytosol. How such nucleases translocate across the cytoplasmic membrane of Gram-negative bacteria is unknown. Here, we identify a small, conserved, 15-kDa domain, which we refer to as the inner membrane translocation (IMT) domain, that is common to T6SS and bacteriocins and linked to nuclease effector domains. Through fluorescence microscopy assays using intact and spheroplasted cells, we demonstrate that the IMT domain of the Pseudomonas aeruginosa-specific bacteriocin pyocin G (PyoG) is required for import of the toxin nuclease domain to the cytoplasm. We also show that translocation of PyoG into the cytosol is dependent on inner membrane proteins FtsH, a AAA+ATPase/protease, and TonB1, the latter more typically associated with transport of bacteriocins across the outer membrane. Our study reveals that the IMT domain directs the cytotoxic nuclease of PyoG to cross the cytoplasmic membrane and, more broadly, has been adapted for the transport of other toxic nucleases delivered into Gram-negative bacteria by both contact-dependent and contact-independent means. IMPORTANCE Nuclease bacteriocins are potential antimicrobials for the treatment of antibiotic-resistant bacterial infections. While the mechanism of outer membrane translocation is beginning to be understood, the mechanism of inner membrane transport is not known. This study uses PyoG as a model nuclease bacteriocin and defines a conserved domain that is essential for inner membrane translocation and is widespread in other bacterial competition systems. Additionally, the presented data link two membrane proteins, FtsH and TonB1, with inner membrane translocation of PyoG. These findings point to the general importance of this domain to the cellular uptake mechanisms of nucleases delivered by otherwise diverse and distinct bacterial competition systems. The work is also of importance for the design of new protein antibiotics.

Pyocin-mediated antagonistic interactions in Pseudomonas spp. isolated in James Ross Island, Antarctica.

Interactions within bacterial communities are frequently mediated by the production of antimicrobial agents. Despite the increasing interest in research of new antimicrobials, studies describing antagonistic interactions among cold-adapted microorganisms are still rare. Our study assessed the antimicrobial interactions of 36 Antarctic Pseudomonas spp. and described the genetic background of these interactions in selected strains. The overall bacteriocinogeny was greater compared to mesophilic Pseudomonas non-aeruginosa species. R-type tailocins were detected on transmission electron micrographs in 16 strains (44.4%); phylogenetic analysis of the corresponding gene clusters revealed that the P. prosekii CCM 8878 tailocin was related to the Rp3 group, whereas the tailocin in Pseudomonas sp. CCM 8880 to the Rp4 group. Soluble antimicrobials were produced by eight strains (22.-2%); gene mining found pyocin L homologues in the genomes of P. prosekii CCM 8881 and CCM 8879 and pyocin S9-like homologues in P. prosekii CCM 8881 and Pseudomonas sp. CCM 8880. Analysis of secretomes confirmed the production of all S- and L-type pyocin genes. Our results suggest that bacteriocin-based inhibition plays an important role in interactions among Antarctic soil bacteria, and these native, cold-adapted microorganisms could be a promising source of new antimicrobials.

SOS-Independent Pyocin Production in P. aeruginosa Is Induced by XerC Recombinase Deficiency.

Pyocins are phage tail-like protein complexes that can be used by Pseudomonas aeruginosa to enact intraspecies competition by killing competing strains. The pyocin gene cluster also encodes holin and lysin enzymes that lyse producer cells to release the pyocins. The best-known inducers of pyocin production under laboratory conditions are DNA-damaging agents, including fluoroquinolone antibiotics, that activate the SOS response. Here, we report the discovery of an alternate, RecA-independent pathway of strong pyocin induction that is active in cells deficient for the tyrosine recombinase XerC. When ΔxerC cells were examined at the single-cell level, only a fraction of the cell population strongly expressed pyocins before explosively lysing, suggesting a that a built-in heterogenous response system protects the cell population from widespread lysis. Disabling the holin and lysin enzymes or deleting the entire pyocin gene cluster blocked explosive lysis and delayed but did not prevent the death of pyocin-producing cells, suggesting that ΔxerC cells activate other lysis pathways. Mutating XerC to abolish its recombinase activity induced pyocin expression to a lesser extent than the full deletion, suggesting that XerC has multiple functions with respect to pyocin activation. Our studies uncover a new pathway for pyocin production and highlight its response across a genetically identical population. Moreover, our finding that ΔxerC populations are hypersensitive to fluoroquinolones raises the intriguing possibility that XerC inhibition may potentiate the activity of these antibiotics against P. aeruginosa infections. IMPORTANCE Pseudomonas aeruginosa is a versatile and ubiquitous bacterium that frequently infects humans as an opportunistic pathogen. P. aeruginosa competes with other strains within the species by producing killing complexes termed pyocins, which are only known to be induced by cells experiencing DNA damage and the subsequent SOS response. Here, we discovered that strains lacking a recombinase enzyme called XerC strongly produce pyocins independently of the SOS response. We also show that these strains are hypersensitive to commonly used fluoroquinolone antibiotic treatment and that fluoroquinolones further stimulate pyocin production. Thus, XerC is an attractive target for future therapies that simultaneously sensitize P. aeruginosa to antibiotics and stimulate the production of bactericidal pyocins.

Recombinant R2-pyocin cream is effective in treating Pseudomonas aeruginosa-infected wounds.

Pseudomonas aeruginosa, a gram-negative opportunistic pathogen, is one of the major species isolated from infected chronic wounds. The multidrug resistance exhibited by P. aeruginosa and its ability to form biofilms that are difficult to eradicate, along with the rising cost of producing new antibiotics, has necessitated the search for alternatives to standard antibiotics. Pyocins are antimicrobial compounds produced by P. aeruginosa that protect themselves from their competitors. We synthesized and purified recombinant P. aeruginosa R2 pyocin and used it in an aqueous solution (rR2P) or formulated in polyethylene glycol (rR2PC) to treat P. aeruginosa-infected wounds. Clinical strains of P. aeruginosa were found to be sensitive (completely), partially sensitive, or resistant to rR2P. In the in vitro biofilm model, rR2P inhibited biofilm development by rR2P-sensitive isolates, while rR2PC eliminated partial biofilms formed by these strains in an in vitro wound biofilm model. In the murine model of excision wounds, and at 24 h post-infection, rR2PC application significantly reduced the bioburden of the clinical isolate BPI86. Application of rR2PC containing two glycoside hydrolase antibiofilm agents eliminated BPI86 from infected wounds. These results suggest that the topical application of rR2PC is an effective therapy for treating wounds infected with R2P-senstive P. aeruginosa strains.

Spatial transcriptomics of planktonic and sessile bacterial populations at single-cell resolution.

Capturing the heterogeneous phenotypes of microbial populations at relevant spatiotemporal scales is highly challenging. Here, we present par-seqFISH (parallel sequential fluorescence in situ hybridization), a transcriptome-imaging approach that records gene expression and spatial context within microscale assemblies at a single-cell and molecule resolution. We applied this approach to the opportunistic pathogen Pseudomonas aeruginosa, analyzing about 600,000 individuals across dozens of conditions in planktonic and biofilm cultures. We identified numerous metabolic- and virulence-related transcriptional states that emerged dynamically during planktonic growth, as well as highly spatially resolved metabolic heterogeneity in sessile populations. Our data reveal that distinct physiological states can coexist within the same biofilm just several micrometers away, underscoring the importance of the microenvironment. Our results illustrate the complex dynamics of microbial populations and present a new way of studying them at high resolution.

Pyocin efficacy in a murine model of Pseudomonas aeruginosa sepsis.

BACKGROUND: Bloodstream infections with antibiotic-resistant Pseudomonas aeruginosa are common and increasingly difficult to treat. Pyocins are naturally occurring protein antibiotics produced by P. aeruginosa that have potential for human use. OBJECTIVES: To determine if pyocin treatment is effective in a murine model of sepsis with P. aeruginosa. METHODS: Recombinant pyocins S5 and AP41 were purified and tested for efficacy in a Galleria mellonella infection model and a murine model of P. aeruginosa sepsis. RESULTS: Both pyocins produced no adverse effects when injected alone into mice and showed good in vitro antipseudomonal activity. In an invertebrate model of sepsis using G. mellonella, both pyocins significantly prolonged survival from 1/10 (10%) survival in controls to 80%-100% survival among groups of 10 pyocin-treated larvae. Following injection into mice, both showed extensive distribution into different organs. When administered 5 h after infection, pyocin S5 significantly increased survival from 33% (2/6) to 83% (5/6) in a murine model of sepsis (difference significant by log-rank test, P < 0.05). CONCLUSIONS: Pyocins S5 and AP41 show in vivo biological activity and can improve survival in two models of P. aeruginosa infection. They hold promise as novel antimicrobial agents for treatment of MDR infections with this microbe.

Heterogenous Susceptibility to R-Pyocins in Populations of Pseudomonas aeruginosa Sourced from Cystic Fibrosis Lungs.

Bacteriocins are proteinaceous antimicrobials produced by bacteria that are active against other strains of the same species. R-type pyocins are phage tail-like bacteriocins produced by Pseudomonas aeruginosa Due to their antipseudomonal 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 organisms 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 to 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. aeruginosa strains 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) a large number of strains lack R-pyocin genes, and (iii) isolates collected from the same patient have the same R-type. We then assessed the impact of intrastrain 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.IMPORTANCE R-pyocins have potential as alternative therapeutics against Pseudomonas aeruginosa in chronic infection; however, little is known about the efficacy of R-pyocins in heterogeneous bacterial populations. P. aeruginosa is known to become resistant to multiple antibiotics and to evolve phenotypic and genotypic diversity over time; thus, it is particularly difficult to eradicate in chronic cystic fibrosis (CF) lung infections. In this study, we found that P. aeruginosa populations from CF lungs maintain the same R-pyocin genotype but exhibit heterogeneity in susceptibility to R-pyocins from other strains. Our findings suggest there is heterogeneity in response to other types of LPS-binding antimicrobials, such as phage, highlighting the necessity of further studying the potential of LPS-binding antimicrobial particles as alternative therapies in chronic infections.

Characterization of R-pyocin activity against Gram-positive pathogens for the first time with special focus on Staphylococcus aureus.

AIM: This study is aimed at characterization of both antimicrobial and anti-biofilm activity of R-pyocin from clinical Pseudomonas aeruginosa against Gram-positive pathogens including Staphylococcus aureus. METHODS AND RESULTS: Pyocinogenic P. aeruginosa was detected using reverse-side method, and pyocinogeny typing was confirmed using revised-spotting method. Transmission electron microscopy (TEM) was used for morphological characterization of R-pyocin and for detection of changes in membrane of R-pyocin-treated S. aureus. SDS-PAGE analysis was used for detection of the molecular weight of R-pyocin protein-subunits and Poisson-killing-distribution assay for burst-size calculation. Lipotechoic-acid (LTA) adsorption-assay was used to confirm whether LTA in Gram-positive bacteria served as R-pyocin receptor. Moreover, R-pyocin production at 10-60°C was assessed herein. Host-range of activity of R-pyocin was tested against antimicrobial resistant (AMR) pathogens. The anti-biofilm activity of R-pyocin was detected against sensitive bacterial strains. Chemical, enzymatic, pH and thermo-stability of R-pyocin were evaluated. TEM micrographs revealed a typical morphology of myotailocins indicating the production of R-pyocin designated as RPU15. TEM revealed pores formation in S. aureus membrane, and bacteriophage-like plaques were obvious on plates of R-pyocin-treated S. aureus. R-pyocin activity was neutralized by LTA of S. aureus and Listeria monocytogenes. Pseudomonas aeruginosa PU15 produced ˜428 non-inducible R-pyocin particles. RPU15 sheath and tube protein-subunits exhibited a molecular weight of 38 and 23 kDa, respectively. RPU15 possessed activity against S. aureus, L. monocytogenes, Bacillus cereus and Candida albicans and reduced biofilm-biomasses of tested AMR strains. CONCLUSION: Our results show the potential therapeutic use of R-pyocin due to its effectiveness on tested bacterial biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that investigates antimicrobial and anti-biofilm activity of R-pyocin activity against S. aureus. R-pyocin shows new phenomenon of bacteriophage-like plaques. Our findings represent a future therapeutic agent targeting both methicillin-resistant and vancomycin-resistant S. aureus.

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.

Bacteriocins: New Potential Therapeutic Candidates in Cancer Therapy.

Cancer is one of the most important disorders which is associated with high mortality and high costs of treatment for patients. Despite several efforts, finding, designing and developing, new therapeutic platforms in the treatment of cancer patients are still required. Utilization of microorganisms, particularly bacteria has emerged as new therapeutic approaches in the treatment of various cancers. Increasing data indicated that bacteria could be used in the production of a wide range of anti-cancer agents, including bacteriocins, antibiotics, peptides, enzymes, and toxins. Among these anti-cancer agents, bacteriocins have attractive properties, which make them powerful anti-cancer drugs. Multiple lines evidence indicated that several bacteriocins (i.e., colcins, nisins, pediocins, pyocins, and bovocins) via activation/inhibition different cellular and molecular signaling pathways are able to suppress tumor growth in various stages. Hence, identification and using various bacteriocins could lead to improve and introduce them to clinical practices. Here, we summarized various bacteriocins which could be employed as anti-cancer agents in the treatment of many cancers.

The Pyocin Regulator PrtR Regulates Virulence Expression of Pseudomonas aeruginosa by Modulation of Gac/Rsm System and c-di-GMP Signaling Pathway.

In Pseudomonas aeruginosa, the second messenger cyclic-di-GMP and Gac/Rsm signaling pathways are associated with the transition from acute to chronic infection. Therefore, identification of the molecular mechanisms that govern lifestyle choice in bacteria is very important. Here, we identified a novel cyclic-di-GMP modulator, PrtR, which was shown to repress pyocin production by inhibition of PrtN and activate the type III secretion system (T3SS) through PtrB. Compared to a wild-type strain or a prtN mutant, the prtR prtN double mutant exhibited a wrinkly colony and hyperbiofilm phenotype, as well as an increase in intracellular c-di-GMP levels. Interestingly, a diguanylate cyclase (DGC) gene, siaD, was repressed by PrtR. Further experiments revealed that PrtR directly interacts with SiaD and facilitates the accumulation of c-di-GMP in cells. We also demonstrated that PrtR regulates the activity of the Gac/Rsm system, thus affecting expression of the T3SS and type VI secretion system (T6SS) and the formation of biofilm. Taken together, the present findings indicate that PrtR, as a c-di-GMP modulator, plays key roles in the adaptation to opportunistic infection of P. aeruginosa Additionally, this study revealed a novel mechanism for PrtR-mediated regulation of the lifestyle transition via the Gac/Rsm and c-di-GMP signaling networks.

Spatial structure maintains diversity of pyocin inhibition in household Pseudomonas aeruginosa.

Nearly all bacteria produce narrow-spectrum antibiotics called bacteriocins. Studies have shown that bacteriocins can mediate microbial interactions, but the mechanisms underlying patterns of inhibition are less well understood. We assembled a spatially structured collection of isolates of Pseudomonas aeruginosa from bathroom and kitchen sink drains in nine households. Growth inhibition of these P. aeruginosa by bacteriocins, known as pyocins in this species, was measured using pairwise inhibition assays. Carbon source usage of these isolates was measured, and genetic distance was estimated using multilocus sequencing. We found that as the distance between sites of isolation increased, there was a significantly higher probability of inhibition, and that pyocin inhibition and susceptibility vary greatly among isolates collected from different houses. We also detected support for other mechanisms influencing diversity: inhibition outcomes were influenced by the type of drain from which isolates were collected, and while we found no indication that carbon source utilization influences inhibition, inhibition was favoured at an intermediate genetic distance. Overall, these results suggest that the combined effects of dispersal limitation among sites and competitive exclusion within them maintain diversity in pyocin inhibition and susceptibility phenotypes, and that additional processes such as local adaptation and effects of phylogenetic distance could further contribute to spatial variability.