Assessment of bacteriocin production by clinical Pseudomonas aeruginosa isolates and their potential as therapeutic agents.

INTRODUCTION: Bacterial infections and the rising antimicrobial resistance pose a significant threat to public health. Pseudomonas aeruginosa produces bacteriocins like pyocins, especially S-type pyocins, which are promising for biological applications. This research focuses on clinical P. aeruginosa isolates to assess their bacteriocin production, inhibitory spectrum, chemical structure, antibacterial agents, and preservative potential. METHODS: The identification of P. aeruginosa was conducted through both phenotypic and molecular approaches. The inhibitory spectrum and antibacterial potential of the isolates were assessed. The kinetics of antibacterial peptide production were investigated, and the activity of bacteriocin was quantified in arbitrary units (AU ml-1). Physico-chemical characterization of the antibacterial peptides was performed. Molecular weight estimation was carried out using SDS-PAGE. qRT-PCR analysis was employed to validate the expression of the selected candidate gene. RESULT: The antibacterial activity of P. aeruginosa was attributed to the secretion of bacteriocin compounds, which belong to the S-type pyocin family. The use of mitomycin C led to a significant 65.74% increase in pyocin production by these isolates. These S-type pyocins exhibited the ability to inhibit the growth of both Gram-negative (P. mirabilis and P. vulgaris) and Gram-positive (S. aureus, S. epidermidis, E. hirae, S. pyogenes, and S. mutans) bacteria. The molecular weight of S-type pyocin was 66 kDa, and its gene expression was confirmed through qRT-PCR. CONCLUSION: These findings suggest that S-type pyocin hold significant potential as therapeutic agents against pathogenic strains. The Physico-chemical resistance of S-type pyocin underscores its potential for broad applications in the pharmaceutical, hygiene, and food industries.

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.

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.

Fis Contributes to Resistance of Pseudomonas aeruginosa to Ciprofloxacin by Regulating Pyocin Synthesis.

Factor for inversion stimulation (Fis) is a versatile DNA binding protein that plays an important role in coordinating bacterial global gene expression in response to growth phases and environmental stresses. Previously, we demonstrated that Fis regulates the type III secretion system (T3SS) in Pseudomonas aeruginosa In this study, we explored the role of Fis in the antibiotic resistance of P. aeruginosa and found that mutation of the fis gene increases the bacterial susceptibility to ciprofloxacin. We further demonstrated that genes related to pyocin biosynthesis are upregulated in the fis mutant. The pyocins are produced in response to genotoxic agents, including ciprofloxacin, and the release of pyocins results in lysis of the producer cell. Thus, pyocin biosynthesis genes sensitize P. aeruginosa to ciprofloxacin. We found that PrtN, the positive regulator of the pyocin biosynthesis genes, is upregulated in the fis mutant. Genetic experiments and electrophoretic mobility shift assays revealed that Fis directly binds to the promoter region of prtN and represses its expression. Therefore, our results revealed novel Fis-mediated regulation on pyocin production and bacterial resistance to ciprofloxacin in P. aeruginosaIMPORTANCEPseudomonas aeruginosa is an important opportunistic pathogenic bacterium that causes various acute and chronic infections in human, especially in patients with compromised immunity, cystic fibrosis (CF), and/or severe burn wounds. About 60% of cystic fibrosis patients have a chronic respiratory infection caused by P. aeruginosa The bacterium is intrinsically highly resistant to antibiotics, which greatly increases difficulties in clinical treatment. Therefore, it is critical to understand the mechanisms and the regulatory pathways that are involved in antibiotic resistance. In this study, we elucidated a novel regulatory pathway that controls the bacterial resistance to fluoroquinolone antibiotics, which enhances our understanding of how P. aeruginosa responds to ciprofloxacin.

Pseudomonas aeruginosa Oligoribonuclease Contributes to Tolerance to Ciprofloxacin by Regulating Pyocin Biosynthesis.

Bacterial oligoribonuclease (Orn) is a conserved 3'-to-5' exonuclease. In Pseudomonas aeruginosa, it has been demonstrated that Orn plays a major role in the hydrolysis of pGpG, which is required for cyclic-di-GMP homeostasis. Meanwhile, Orn is involved in the degradation of nanoRNAs, which can alter global gene expression by serving as transcription initiation primers. Previously, we found that Orn is required for the type III secretion system and pathogenesis of P. aeruginosa, indicating a role of Orn in the bacterial response to environmental stimuli. Here we report that Orn is required for the tolerance of P. aeruginosa to ciprofloxacin. Transcriptome analysis of an orn mutant revealed the upregulation of pyocin biosynthesis genes. Mutation of genes involved in pyocin biosynthesis in the background of an orn mutant restored bacterial tolerance to ciprofloxacin. We further demonstrate that the upregulation of pyocin biosynthesis genes is due to RecA-mediated autoproteolysis of PrtR, which is the major negative regulator of pyocin biosynthesis genes. In addition, the SOS response genes were upregulated in the orn mutant, indicating a DNA damage stress. Therefore, our results revealed a novel role of Orn in bacterial tolerance to ciprofloxacin.

Biological cost of pyocin production during the SOS response in Pseudomonas aeruginosa.

LexA and two structurally related regulators, PrtR and PA0906, coordinate the Pseudomonas aeruginosa SOS response. RecA-mediated autocleavage of LexA induces the expression of a protective set of genes that increase DNA damage repair and tolerance. In contrast, RecA-mediated autocleavage of PrtR induces antimicrobial pyocin production and a program that lyses cells to release the newly synthesized pyocin. Recently, PrtR-regulated genes were shown to sensitize P. aeruginosa to quinolones, antibiotics that elicit a strong SOS response. Here, we investigated the mechanisms by which PrtR-regulated genes determine antimicrobial resistance and genotoxic stress survival. We found that induction of PrtR-regulated genes lowers resistance to clinically important antibiotics and impairs the survival of bacteria exposed to one of several genotoxic agents. Two distinct mechanisms mediated these effects. Cell lysis genes that are induced following PrtR autocleavage reduced resistance to bactericidal levels of ciprofloxacin, and production of extracellular R2 pyocin was lethal to cells that initially survived UV light treatment. Although typically resistant to R2 pyocin, P. aeruginosa becomes transiently sensitive to R2 pyocin following UV light treatment, likely because of the strong downregulation of lipopolysaccharide synthesis genes that are required for resistance to R2 pyocin. Our results demonstrate that pyocin production during the P. aeruginosa SOS response carries both expected and unexpected costs.

Membrane vesicle formation is associated with pyocin production under denitrifying conditions in Pseudomonas aeruginosa†PAO1.

Many Gram-negative bacteria produce membrane vesicles (MVs) that serve as vehicles to mediate intraspecies and interspecies interactions. Despite their ubiquity in Gram-negative bacteria and their biological importance, how MV formation is regulated is poorly understood. Pseudomonas aeruginosa is a ubiquitous bacterium that is one of the most extensively studied model organism in MVs. Recent studies highlight the importance of a quorum-sensing signal, Pseudomonas quinolone signal (PQS), in the formation of MVs; however, PQS synthesis requires oxygen and is not produced under anoxic conditions. This situation leads to the question of MV production under anoxic conditions. Here, we examined whether MVs are produced under denitrifying conditions and what kind of factors are involved in the MV production under such condition. Under denitrifying condition, P. aeruginosa PAO1 produced a considerable amount of MVs. Interestingly, pyocin components were found to be accumulated in the isolated MVs. Pyocin-related protein mutants produced less MVs compared with the wild type. We further indicate that pyocin production is activated by nitric oxide, in which the SOS response is involved. This study presents a regulatory mechanism where pyocin is associated with MV production, and further implies how the environment impacts MV production in P. aeruginosa.

The role of 'soaking' in spiteful toxin production in Pseudomonas aeruginosa.

The ubiquitous production of antibacterial toxins, such as bacteriocins, is an ecologically significant class of interbacterial interactions that have primarily evolved through their indirect fitness benefits to the producer. Bacteria release bacteriocins into the environment at a cost to individual cell, but individual bacteriocin-producing cells are unlikely to gain any direct benefit from their own toxin; indeed, cell lysis is required in many species. There is a growing body of research describing the ecological conditions that can favour the evolution of bacteriocin production. However, an important aspect of many bacteriocins has yet to be investigated: the ability of bacteriocin-producing cells to neutralize toxin ('soaking') produced by other clonemates. By competing Pseudomonas aeruginosa bacteriocin-producing wild-type and 'non-soaking' strains against a bacteriocin-susceptible strain, we find that soaking markedly reduces the fitness of a bacteriocin-producing strain at both high and low frequencies.

Spite versus cheats: competition among social strategies shapes virulence in Pseudomonas aeruginosa.

Social interactions have been shown to play an important role in bacterial evolution and virulence. The majority of empirical studies conducted have only considered social traits in isolation, yet numerous social traits, such as the production of spiteful bacteriocins (anticompetitor toxins) and iron-scavenging siderophores (a public good) by the opportunistic pathogen Pseudomonas aeruginosa, are frequently expressed simultaneously. Crucially, both bacteriocin production and siderophore cheating can be favored under the same competitive conditions, and we develop theory and carry out experiments to determine how the success of a bacteriocin-producing genotype is influenced by social cheating of susceptible competitors and the resultant impact on disease severity (virulence). Consistent with our theoretical predictions, we find that the spiteful genotype is favored at higher local frequencies when competing against public good cheats. Furthermore, the relationship between spite frequency and virulence is significantly altered when the spiteful genotype is competed against cheats compared with cooperators. These results confirm the ecological and evolutionary importance of considering multiple social traits simultaneously. Moreover, our results are consistent with recent theory regarding the invasion conditions for strong reciprocity (helping cooperators and harming noncooperators).

[Screening of producers of bacteriocin-like substances active against Pseudomonas aeruginosa].

The strains (n = 94) of 16 Pseudomonas species have been screened for producers of substances active against Pseudomonas aeruginosa. Investigated cultures were divided into two groups. The majority of Pseudomonas species have been included in the first group. These species were able to produce substances with low and medium activity spectrum. In the first group P. mendocina, P. fragi and P. taetrolens lysates were the most active and influenced 30-50% of indicator cultures. Only P. aeruginosa strains belong to the second group. The microorganisms of this group were able to produce substances with considerably higher activity spectrum. Among all investigated pseudomonades four P. aeruginosa strain lysates possessed the highest activity and were active against more than 75% of used cultures. It was shown that the main active killer components of these lysates belonged to low-weight pyocins.

Engineering microbes to sense and eradicate Pseudomonas aeruginosa, a human pathogen.

Synthetic biology aims to systematically design and construct novel biological systems that address energy, environment, and health issues. Herein, we describe the development of a synthetic genetic system, which comprises quorum sensing, killing, and lysing devices, that enables Escherichia coli to sense and kill a pathogenic Pseudomonas aeruginosa strain through the production and release of pyocin. The sensing, killing, and lysing devices were characterized to elucidate their detection, antimicrobial and pyocin release functionalities, which subsequently aided in the construction of the final system and the verification of its designed behavior. We demonstrated that our engineered E. coli sensed and killed planktonic P. aeruginosa, evidenced by 99% reduction in the viable cells. Moreover, we showed that our engineered E. coli inhibited the formation of P. aeruginosa biofilm by close to 90%, leading to much sparser and thinner biofilm matrices. These results suggest that E. coli carrying our synthetic genetic system may provide a novel synthetic biology-driven antimicrobial strategy that could potentially be applied to fighting P. aeruginosa and other infectious pathogens.

MexT modulates virulence determinants in Pseudomonas aeruginosa independent of the MexEF-OprN efflux pump.

In the human pathogen Pseudomonas aeruginosa, the LysR-family regulator MexT modulates the induction of the tripartite MexEF-OprN resistance nodulation-division multi-drug efflux system resulting in increased resistance to diverse antibiotics. The MexEF-OprN system is normally quiescent in wild-type cells, but is highly induced in nfxC-type phenotypic mutants in a MexT dependent manner. In addition to antibiotic resistance, induction of mexEF-oprN in nfxC-type mutants has been linked to reduced levels of homoserine lactone-dependent virulence traits, including pyocyanin, elastase, rhamnolipids and PQS and to reduced expression of type three secretion effector proteins. In this study, MexT is overexpressed in wild-type PAO1 and an isogenic mexEF deletion mutant to determine if MexT regulates diverse virulence phenotypes dependent or independent of MexEF-OprN. It is shown that MexT regulates type three secretion, pyocyanin production and early surface attachment independent of MexEF-OprN. In contrast, MexT modulation of the expression of the virulence genes rhlA, lasB and hcnB is dependent on MexEF-OprN, which apparently mediates these effects via efflux of cell-signaling intermediates. The data presented demonstrates that MexT may play a more global role in modulating P. aeruginosa virulence than previously reported and suggests that MexT regulates diverse targets that mediate phenotypic alterations independent of MexEF-OprN.

Pseudomonas aeruginosa PAO1 pyocin production affects population dynamics within mixed-culture biofilms.

Transcriptomic and phenotypic studies showed that pyocins are produced in Pseudomonas aeruginosa PAO1 aerobic and anaerobic biofilms. Pyocin activity was found to be high in slow-growing anaerobic biofilms but transient in aerobic biofilms. Biofilm coculture of strain PAO1 and a pyocin-sensitive isolate showed that pyocin production had a significant impact on bacterial population dynamics, particularly under anaerobic conditions.

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.

PtrB of Pseudomonas aeruginosa suppresses the type III secretion system under the stress of DNA damage.

In a search for regulatory genes of the type III secretion system (TTSS) in Pseudomonas aeruginosa, transposon (Tn5) insertional mutants of the prtR gene were found defective in the TTSS. PrtR is an inhibitor of prtN, which encodes a transcriptional activator for pyocin synthesis genes. In P. aeruginosa, pyocin synthesis is activated when PrtR is degraded during the SOS response. Treatment of a wild-type P. aeruginosa strain with mitomycin C, a DNA-damaging agent, resulted in the inhibition of TTSS activation. A prtR/prtN double mutant had the same TTSS defect as the prtR mutant, and complementation by a prtR gene but not by a prtN gene restored the TTSS function. Also, overexpression of the prtN gene in wild-type PAK had no effect on the TTSS; thus, PrtN is not involved in the repression of the TTSS. To identify the PrtR-regulated TTSS repressor, another round of Tn mutagenesis was carried out in the background of a prtR/prtN double mutant. Insertion in a small gene, designated ptrB, restored the normal TTSS activity. Expression of ptrB is specifically repressed by PrtR, and mitomycin C-mediated suppression of the TTSS is also abolished in a ptrB mutant strain. Therefore, PtrB is a new TTSS repressor that coordinates TTSS repression and pyocin synthesis under the stress of DNA damage.

Cytotoxic effects of pyocin S2 produced by Pseudomonas aeruginosa on the growth of three human cell lines.

Pyocin typing of 82 Pseudomonas aeruginosa strains, collected from different Iranian clinical sources, revealed that one isolate, P. aeruginosa 42A, produced pyocin S2, a protease-sensitive bacteriocin. Pyocin S2 production was induced by mitomycin C (2 micro g/mL) in the pyocin S2 producer P. aeruginosa 42A. Pyocin S2 was purified using ion exchange chromatography with CM-Sepharose CL-6B and sodium phosphate buffer (pH 8) from an 80% ammonium sulfate precipitate of whole-cell lysates. Pyocin activity of the fractions was detected using the Govan spot testing method. The purity of the active fraction was confirmed by SDS-PAGE, where a single band with a molecular mass of 74 kDa was detected. Cytotoxic effects of purified pyocin S2 and partially purified pyocin from P. aeruginosa 42A on the human tumor cell lines HepG2 and Im9 and the normal human cell line HFFF (Human Foetal Foreskin Fibroblast) were studied by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The results demonstrated that partially purified pyocin and pyocin S2 exhibited substantial inhibitory effects on the growth of the tumor cell lines HepG2 and Im9, while no inhibitory effects were observed on the normal cell line HFFF. Pure lipopolysaccharide was used as a control and was found to have no inhibitory effect on any of the cell lines tested.

The pyocins of Pseudomonas aeruginosa.

Pyocins are produced by more than 90% of Pseudomonas aeruginosa strains and each strain may synthesise several pyocins. The pyocin genes are located on the P. aeruginosa chromosome and their activities are inducible by mutagenic agents such as mitomycin C. Three types of pyocins are described. (i). R-type pyocins resemble non-flexible and contractile tails of bacteriophages. They provoke a depolarisation of the cytoplasmic membrane in relation with pore formation. (ii). F-type pyocins also resemble phage tails, but with a flexible and non-contractile rod-like structure. (iii). S-type pyocins are colicin-like, protease-sensitive proteins. They are constituted of two components. The large component carries the killing activity (DNase activity for pyocins S1, S2, S3, AP41; tRNase for pyocin S4; channel-forming activity for pyocin S5). It interacts with the small component (immunity protein). The synthesis of pyocins starts when a mutagen increases the expression of the recA gene and activates the RecA protein, which cleaves the repressor PrtR, liberating the expression of the protein activator gene prtN. R and F-pyocins are derived from an ancestral gene, with similarities to the P2 phage family and the lambda phage family, respectively. The killing domains of S1, S2, AP41 pyocins show a close evolutionary relationship with E2 group colicins, S4 pyocin with colicin E5, and S5 pyocin with colicins Ia, and Ib.

Characteristics of sheep-rumen isolates of Pseudomonas aeruginosa inhibitory to the growth of Escherichia coli O157.

Screening facultative sheep-rumen bacteria which inhibit growth of Escherichia coli produced 11 strains of Pseudomonas aeruginosa. The isolates showed three different pulsed-field gel electrophoresis patterns and strains from different sheep produced pyocins that varied in strain specificity. Representative strains were resistant to ampicillin, methicillin, erythromycin, fusidic acid and augmentin, but not to tetracycline or nalidixic acid. Tested strains attached in large numbers to cultured rumen epithelial cells, potentially providing a means of survival in this ecosystem.

Antibiotic stress induces a large amount of outer membrane protein in Pseudomonas aeruginosa.

To investigate the bacterial response to antibiotic stress, we analyzed the outer membrane proteins of Pseudomonas aeruginosa grown in the presence of a sub-minimum inhibitory concentration of antibiotics. Among the antibiotics tested, fluoroquinolones and streptonigrin induced a large amount of outer membrane protein with a molecular mass of 43 kDa. This protein is most likely the stress-responsive protein, since the quinolone-resistant mutants with a higher minimum inhibitory concentration of antibiotic than the wild-type strain produced a large amount of 43-kDa protein only in the presence of sub-minimum inhibitory concentration of the mutants itself, but not that of the antibiotic-susceptible wild-type strain. The sequence of N-terminal 15 amino acids of the 43-kDa protein was identical to that of pyocin R1. However, purified pyocin R1 failed to accumulate in the outer membrane. Thus, we concluded that the 43-kDa protein (pyocin R1) is the antibiotic-stress-induced outer membrane protein.

Cloning, complementation, and characterization of an rfaE homolog from Neisseria gonorrhoeae.

Neisseria gonorrhoeae WS1 is a spontaneous pyocin (a bacteriocin produced by Pseudomonas aeruginosa)-resistant mutant of N. gonorrhoeae FA19 that produces a truncated lipooligosaccharide (LOS) and is non-transformable. The LOS-specific mutation in WS1 was moved into a transformable background by transforming FA19 with chromosomal DNA from WS1 (generating strain JWS-1). A clone (pJCL2) capable of restoring JWS-1 to wild-type LOS expression, as detected by its acquisition of reactivity with monoclonal antibodies and by its complemented sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile, was isolated. Sequential unidirectional deletion and DNA sequence analysis of pJCL2 identified an open reading frame, designated lsi-7, that could complement the defect in JWS-1. Homology searches against various databases indicated that lsi-7 bad homology with several Escherichia coli genes involved in the phosphorylation of sugars. lsi-7 is adjacent to the lsi-6 gene, another gene involved in LOS biosynthesis. Complementation studies using Salmonella typhimurium lipopolysaccharide mutants showed lsi-6 and lsi-7 to be gonococcal homologs of S. typhimurium rfaD and rfaE, respectively. Reverse transcriptase PCR analysis demonstrated that lsi-6 and lsi-7 are part of the same transcriptional unit.