Search Results (11)

Pseudomonas aeruginosa is a common opportunistic pathogen associated with nosocomial infections. The primary treatment for infections typically involves antibiotics, which can lead to the emergence of multidrug-resistant strains. Therefore, there is a pressing need for safe and effective alternative methods. Phage therapy stands out as a promising approach. However, filamentous prophages (Pfs) commonly found in P. aeruginosa encode genes with phage defense activity, thereby reducing the efficacy of phage therapy. Through a genomic analysis of the Pf4 prophage, we identified a 102 bp gene co-transcribed with the upstream gene responsible for phage release (zot gene), giving rise to a 33-amino-acid polypeptide that we have named Pf4-encoded toxic polypeptide (PftP4). The overexpression of PftP4 demonstrated cellular toxicity in P. aeruginosa, with subcellular localization indicating its presence in the cell membrane and a subsequent increase in membrane permeability. Notably, PftP4 homologues are found in multiple Pf phages and exhibit specificity in their toxicity towards P. aeruginosa among the tested bacterial strains. Our study reveals that the novel Pf-encoded polypeptide PftP4 has the potential to selectively target and eradicate P. aeruginosa, offering valuable insights for combating P. aeruginosa infections. Full article

Pseudomonas are significant spoilage bacteria in raw milk and dairy products, primarily due to their ability to form biofilms and resist disinfection. This study explored the effects of the UFJF_PfSW6 phage combined with sodium hypochlorite in reducing Pseudomonas fluorescens biofilms on stainless steel at various temperatures and ages. Biofilms were formed using P. fluorescens UFV 041 in UHT milk, incubated at 4 °C and 30 °C for 2 and 7 days. Two lytic phages were compared, with UFJF_PfSW6 showing superior activity, reducing cell counts by 0.8 to 2.0 logs CFU/cm² depending on conditions. Increasing the contact time of the UFJF_PfSW6 phage from 4 to 8 h did not significantly affect the reduction in mature biofilms. The individual treatments of the phage and sodium hypochlorite (100 mg/L) reduced bacterial counts by 0.9 and 0.6 log CFU/cm² at 30 °C, and 1.3 and 1.2 log CFU/cm² at 4 °C, respectively. However, their sequential application achieved greater reductions, reaching 1.3 and 1.8 log CFU/cm² for biofilms formed at 30 °C and 4 °C, respectively. These findings suggest a promising strategy for controlling P. fluorescens in the food industry. Our findings suggest that the UFJF_PfSW6 phage combined with chlorine improves the removal of P. fluorescens biofilms. Full article

Background/Objectives: This study explores the genome sequencing data from the infection of Pseudomonas fluorescens UFV 041 by the bacteriophage Pijolavirus UFJF_PfSW6, aiming to identify and characterize prophages induced in the host bacterium during the infection. Methods: Scaffolds from sequencing data were analyzed, and reads were mapped to identify potential prophages using phage-to-host coverage metrics. The putative prophage scaffold was annotated, taxonomically classified, and its integration in the host bacterium was verified by PCR amplification of two target genes. We also tested whether mitomycin treatment could induce the prophage to enter the lytic cycle. Results: The prophage UFJF_PfPro was identified with a high phage-to-host coverage ratio. Its genome is 32,700 bp in length, containing 42 genes, 3 terminators, and 11 promoters, with 98.84% completeness. PCR confirmed its integration into P. fluorescens UFV 041, but mitomycin treatment did not induce the lytic cycle. The UFJF_PfPro genome shares 38.60% similarity with the closest lytic phages in the Phitrevirus genus, below genus and species assignment thresholds. A viral proteomic tree clustered UFJF_PfPro with Phitrevirus in a clade representing the Peduoviridae family. Conclusions: The UFJF_PfPro is a prophage integrated into the P. fluorescens UFV 041 genome, but we were unable to induce it to enter the lytic cycle using mitomycin treatment. The genome of UFJF_PfPro encodes all structural proteins typical of the Caudoviricetes class and shares low genomic similarity with species of the genus Phitrevirus, suggesting that UFJF_PfPro represents a new genus and species within the Peduoviridae family. Full article

Pseudomonas aeruginosa (Pa), a WHO priority 1 pathogen, resulted in approximately 559,000 deaths globally in 2019. Pa has a multitude of host-immune evasion strategies that enhance Pa virulence. Most clinical isolates of Pa are infected by a phage called Pf that has the ability to misdirect the host-immune response and provide structural integrity to biofilms. Previous studies demonstrate that vaccination against the coat protein (CoaB) of Pf4 virions can assist in the clearance of Pa from the dorsal wound model in mice. Here, a consensus peptide was derived from CoaB and conjugated to cross-reacting material 197 (CRM197). This conjugate was adjuvanted with a novel synthetic Toll-like receptor agonist (TLR) 4 agonist, INI-2002, and used to vaccinate mice. Mice vaccinated with CoaB-CRM conjugate and INI-2002 developed high anti-CoaB peptide-specific IgG antibody titers. Direct binding of the peptide-specific antibodies to whole-phage virus particles was demonstrated by ELISA. Furthermore, a functional assay demonstrated that antibodies generated from vaccinated mice disrupted the replicative cycle of Pf phages. The use of an adjuvanted phage vaccine targeting Pa is an innovative vaccine strategy with the potential to become a new tool targeting multi-drug-resistant Pa infections in high-risk populations. Full article

Pseudomonas aeruginosa is an opportunistic pathogen that can cause infections in humans, especially in hospital patients with compromised host defence mechanisms, including patients with cystic fibrosis. Filamentous bacteriophages represent a group of single-stranded DNA viruses infecting different bacteria, including P. aeruginosa and other human and animal pathogens; many of them can replicate when integrated into the bacterial chromosome. Filamentous bacteriophages can contribute to the virulence of P. aeruginosa and influence the course of the disease. There are just a few isolated and officially classified filamentous bacteriophages infecting P. aeruginosa, but genomic studies indicated the frequent occurrence of integrated prophages in many P. aeruginosa genomes. An analysis of sequenced genomes of P. aeruginosa isolated from upper respiratory tract (throat and nasal swabs) and sputum specimens collected from Russian patients with cystic fibrosis indicated a higher diversity of filamentous bacteriophages than first thought. A detailed analysis of predicted bacterial proteins revealed prophage regions representing the filamentous phages known to be quite distantly related to known phages. Genomic comparisons and phylogenetic studies enabled the proposal of several new taxonomic groups of filamentous bacteriophages. Full article

The phage T7 RNA polymerase (RNAP) and lysozyme form the basis of the widely used pET expression system for recombinant expression in the biotechnology field and as a tool in microbial synthetic biology. Attempts to transfer this genetic circuitry from Escherichia coli to non-model bacterial organisms with high potential have been restricted by the cytotoxicity of the T7 RNAP in the receiving hosts. We here explore the diversity of T7-like RNAPs mined directly from Pseudomonas phages for implementation in Pseudomonas species, thus relying on the co-evolution and natural adaptation of the system towards its host. By screening and characterizing different viral transcription machinery using a vector-based system in P. putida., we identified a set of four non-toxic phage RNAPs from phages phi15, PPPL-1, Pf-10, and 67PfluR64PP, showing a broad activity range and orthogonality to each other and the T7 RNAP. In addition, we confirmed the transcription start sites of their predicted promoters and improved the stringency of the phage RNAP expression systems by introducing and optimizing phage lysozymes for RNAP inhibition. This set of viral RNAPs expands the adaption of T7-inspired circuitry towards Pseudomonas species and highlights the potential of mining tailored genetic parts and tools from phages for their non-model host. Full article

More than 20% of all Pseudomonas aeruginosa are infected with Pf4-related filamentous phage and although their role in virulence of P. aeruginosa strain PAO1 is well documented, its properties related to therapy are not elucidated in detail. The aim of this study was to determine how phage and antibiotic therapy induce Pf4, whether the released virions can infect other strains and how the phage influences the phenotype of new hosts. The subinhibitory concentrations of ciprofloxacin and mitomycin C increased Pf4 production for more than 50% during the first and sixth hour of exposure, respectively, while mutants appearing after infection with obligatory lytic phage at low MOI produced Pf4 more than four times after 12–24 h of treatment. This indicates that production of Pf4 is enhanced during therapy with these agents. The released virions can infect new P. aeruginosa strains, as confirmed for models UCBPP-PA14 (PA14) and LESB58, existing both episomally and in a form of a prophage, as confirmed by PCR, RFLP, and sequencing. The differences in properties of Pf4-infected, and uninfected PA14 and LESB58 strains were obvious, as infection with Pf4 significantly decreased cell autoaggregation, pyoverdine, and pyocyanin production, while significantly increased swimming motility and biofilm production in both strains. In addition, in strain PA14, Pf4 increased cell surface hydrophobicity and small colony variants’ appearance, but also decreased twitching and swarming motility. This indicates that released Pf4 during therapy can infect new strains and cause lysogenic conversion. The infection with Pf4 increased LESB58 sensitivity to ciprofloxacin, gentamicin, ceftazidime, tetracycline, and streptomycin, and PA14 to ciprofloxacin and ceftazidime. Moreover, the Pf4-infected LESB58 was re-sensitized to ceftazidime and tetracycline, with changes from resistant to intermediate resistant and sensitive, respectively. The obtained results open a new field in phage therapy—treatment with selected filamentous phages in order to re-sensitize pathogenic bacteria to certain antibiotics. However, this approach should be considered with precautions, taking into account potential lysogenic conversion. Full article

In this study, we have presented the genomic characterisation of UFJF_PfDIW6, a novel lytic Pseudomonas fluorescens-phage with potential for biocontrol in the dairy industry. This phage showed a short linear double-stranded DNA genome (~42 kb) with a GC content of 58.3% and more than 50% of the genes encoding proteins with unknown functions. Nevertheless, UFJF_PfDIW6’s genome was organised into five functional modules: DNA packaging, structural proteins, DNA metabolism, lysogenic, and host lysis. Comparative genome analysis revealed that the UFJF_PfDIW6’s genome is distinct from other viral genomes available at NCBI databases, displaying maximum coverages of 5% among all alignments. Curiously, this phage showed higher sequence coverages (38–49%) when aligned with uncharacterised prophages integrated into Pseudomonas genomes. Phages compared in this study share conserved locally collinear blocks comprising genes of the modules’ DNA packing and structural proteins but were primarily differentiated by the composition of the DNA metabolism and lysogeny modules. Strategies for taxonomy assignment showed that UFJF_PfDIW6 was clustered into an unclassified genus in the Podoviridae clade. Therefore, our findings indicate that this phage could represent a novel genus belonging to the Podoviridae family. Full article

Phytopathogenic pseudomonads are widespread in the world and cause a wide range of plant diseases. In this work, we describe the Pseudomonas phage Pf-10, which is a part of the biopesticide “Multiphage” used for bacterial diseases of agricultural crops caused by Pseudomonas syringae. The Pf-10 chromosome is a dsDNA molecule with two direct terminal repeats (DTRs). The phage genomic DNA is 39,424 bp long with a GC-content of 56.5%. The Pf-10 phage uses a packaging mechanism based on T7-like short DTRs, and the length of each terminal repeat is 257 bp. Electron microscopic analysis has shown that phage Pf-10 has the podovirus morphotype. Phage Pf-10 is highly stable at pH values from 5 to 10 and temperatures from 4 to 60 °C and has a lytic activity against Pseudomonas strains. Phage Pf-10 is characterized by fast adsorption rate (80% of virions attach to the host cells in 10 min), but has a relatively small number of progeny (37 ± 8.5 phage particles per infected cell). According to the phylogenetic analysis, phage Pf-10 can be classified as a new phage species belonging to the genus Pifdecavirus, subfamily Studiervirinae, family Autographiviridae, order Caudovirales. Full article

It has been shown that the filamentous phage, Pf4, plays an important role in biofilm development, stress tolerance, genetic variant formation and virulence in Pseudomonas aeruginosa PAO1. These behaviours are linked to the appearance of superinfective phage variants. Here, we have investigated the molecular mechanism of superinfection as well as how the Pf4 phage can control host gene expression to modulate host behaviours. Pf4 exists as a prophage in PAO1 and encodes a homologue of the P2 phage repressor C and was recently named Pf4r. Through a combination of molecular techniques, ChIPseq and transcriptomic analyses, we show a critical site in repressor C (Pf4r) where a mutation in the site, 788799A>G (Ser4Pro), causes Pf4r to lose its function as the immunity factor against reinfection by Pf4. X-ray crystal structure analysis shows that Pf4r forms symmetric homo-dimers homologous to the E.coli bacteriophage P2 RepC protein. A mutation, Pf4r*, associated with the superinfective Pf4r variant, found at the dimer interface, suggests dimer formation may be disrupted, which derepresses phage replication. This is supported by multi-angle light scattering (MALS) analysis, where the Pf4r* protein only forms monomers. The loss of dimerisation also explains the loss of Pf4r’s immunity function. Phenotypic assays showed that Pf4r increased LasB activity and was also associated with a slight increase in the percentage of morphotypic variants. ChIPseq and transcriptomic analyses suggest that Pf4r also likely functions as a transcriptional regulator for other host genes. Collectively, these data suggest the mechanism by which filamentous phages play such an important role in P. aeruginosa biofilm development. Full article

Pseudomonas aeruginosa PAO1 has an integrated Pf4 prophage in its genome, encoding a relatively well-characterized filamentous phage, which contributes to the bacterial biofilm organization and maturation. Pf4 variants are considered as superinfectives when they can re-infect and kill the prophage-carrying host. Herein, the response of P. aeruginosa H103 to Pf4 variant infection was investigated. This phage variant caused partial lysis of the bacterial population and modulated H103 physiology. We show by confocal laser scanning microscopy that a Pf4 variant-infection altered P. aeruginosa H103 biofilm architecture either in static or dynamic conditions. Interestingly, in the latter condition, numerous cells displayed a filamentous morphology, suggesting a link between this phenotype and flow-related forces. In addition, Pf4 variant-infection resulted in cell envelope stress response, mostly mediated by the AlgU and SigX extracytoplasmic function sigma factors (ECFσ). AlgU and SigX involvement may account, at least partly, for the enhanced expression level of genes involved in the biosynthesis pathways of two matrix exopolysaccharides (Pel and alginates) and bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) metabolism. Full article