Bacteriophage trigger antiviral immunity and prevent clearance of bacterial infection.

Bacteriophage are abundant at sites of bacterial infection, but their effects on mammalian hosts are unclear. We have identified pathogenic roles for filamentous Pf bacteriophage produced by Pseudomonas aeruginosa (Pa) in suppression of immunity against bacterial infection. Pf promote Pa wound infection in mice and are associated with chronic human Pa wound infections. Murine and human leukocytes endocytose Pf, and internalization of this single-stranded DNA virus results in phage RNA production. This triggers Toll-like receptor 3 (TLR3)- and TIR domain-containing adapter-inducing interferon-ß (TRIF)-dependent type I interferon production, inhibition of tumor necrosis factor (TNF), and the suppression of phagocytosis. Conversely, immunization of mice against Pf prevents Pa wound infection. Thus, Pf triggers maladaptive innate viral pattern-recognition responses, which impair bacterial clearance. Vaccination against phage virions represents a potential strategy to prevent bacterial infection.

In Vivo Studies on the Influence of Bacteriophage Preparations on the Autoimmune Inflammatory Process.

Phage preparations used for phage therapy may have not only direct antibacterial action but also immunomodulating effects mediated by phages themselves as well as by bacterial antigens. Therefore phage application in patients with immune disorders, and especially with autoimmune diseases, requires special attention. The aim of this study was to investigate the effect of phage lysates (staphylococcal phages A3/R, phi200, and MS-1 cocktail, enterococcal phage 15/P, Pseudomonas phage 119x, and E. coli T4 phage) as well as purified T4 phage on the course of murine collagen-induced arthritis (CIA), commonly used as an animal model of rheumatoid arthritis. Intraperitoneal application of phage lysates or purified T4 phage did not aggravate the course of autoimmune joint disease. Moreover, although endotoxins are known to potentiate CIA, the systemic administration of phage lysate of Pseudomonas aeruginosa, which contains debris of this Gram-negative bacillus, did not significantly influence CIA although the sonicate of the corresponding bacterial strain did. Interestingly, a purified T4 phage revealed some anti-inflammatory activity when applied under the therapeutic scheme. Our preliminary results do not suggest that phages may aggravate the symptoms of rheumatoid arthritis. In contrast T4 phage may even exert an immunosuppressive effect.

Immunogenicity and antimicrobial effectiveness of Pseudomonas aeruginosa specific bacteriophage in a human lung in vitro model.

The rise of antibiotic resistant bacteria is posing a serious threat to human health. For example, resistant strains of Pseudomonas aeruginosa have resulted in untreatable and potentially lethal infections in both cystic fibrosis and immunocompromised patients. Due to the growing need for alternative treatment options, bacteriophage, or phage, therapy is gaining considerable attention. While previous studies have demonstrated the effectiveness of phage in combating persistent bacterial infections, there is currently a lack of knowledge regarding the host immunological response following phage exposure. In the present study, the bioresponses of an enhanced in vitro model were characterized following exposure to either DMS3 or PEV2, P. aeruginosa targeting phages. Results demonstrated a PEV2-dependent increase in IL-6 and TNF-α production, but no changes associated with DMS3 exposure. Additionally, following the establishment of an in vitro infection model, DMS3 was found to successfully protect mammalian lung cells from P. aeruginosa. Taken together, the biocompatibility and antibacterial effectiveness distinguish DMS3 bacteriophage as a strong candidate for phage therapy. However, as DMS3 is pilin dependent and bacterial receptor expression varies significantly, this work highlights the necessity of generating phage cocktails.

Mammalian Host-Versus-Phage immune response determines phage fate in vivo.

Emerging bacterial antibiotic resistance draws attention to bacteriophages as a therapeutic alternative to treat bacterial infection. Examples of phage that combat bacteria abound. However, despite careful testing of antibacterial activity in vitro, failures nevertheless commonly occur. We investigated immunological response of phage antibacterial potency in vivo. Anti-phage activity of phagocytes, antibodies, and serum complement were identified by direct testing and by high-resolution fluorescent microscopy. We accommodated the experimental data into a mathematical model. We propose a universal schema of innate and adaptive immunity impact on phage pharmacokinetics, based on the results of our numerical simulations. We found that the mammalian-host response to infecting bacteria causes the concomitant removal of phage from the system. We propose the notion that this effect as an indirect pathway of phage inhibition by bacteria with significant relevance for the clinical outcome of phage therapy.

Unlocking the mystery of the hard-to-sequence phage genome: PaP1 methylome and bacterial immunity.

BACKGROUND: Whole-genome sequencing is an important method to understand the genetic information, gene function, biological characteristics and survival mechanisms of organisms. Sequencing large genomes is very simple at present. However, we encountered a hard-to-sequence genome of Pseudomonas aeruginosa phage PaP1. Shotgun sequencing method failed to complete the sequence of this genome. RESULTS: After persevering for 10 years and going over three generations of sequencing techniques, we successfully completed the sequence of the PaP1 genome with a length of 91,715 bp. Single-molecule real-time sequencing results revealed that this genome contains 51 N-6-methyladenines and 152 N-4-methylcytosines. Three significant modified sequence motifs were predicted, but not all of the sites found in the genome were methylated in these motifs. Further investigations revealed a novel immune mechanism of bacteria, in which host bacteria can recognise and repel modified bases containing inserts in a large scale. This mechanism could be accounted for the failure of the shotgun method in PaP1 genome sequencing. This problem was resolved using the nfi- mutant of Escherichia coli DH5α as a host bacterium to construct a shotgun library. CONCLUSIONS: This work provided insights into the hard-to-sequence phage PaP1 genome and discovered a new mechanism of bacterial immunity. The methylome of phage PaP1 is responsible for the failure of shotgun sequencing and for bacterial immunity mediated by enzyme Endo V activity; this methylome also provides a valuable resource for future studies on PaP1 genome replication and modification, as well as on gene regulation and host interaction.

Superinfection exclusion reveals heteroimmunity between Pseudomonas aeruginosa temperate phages.

Temperate siphophages (MP29, MP42, and MP48) were isolated from the culture supernatant of clinical Pseudomonas aeruginosa isolates. The complete nucleotide sequences and annotation of the phage genomes revealed the overall synteny to the known temperate P. aeruginosa phages such as MP22, D3112, and DMS3. Genome-level sequence analysis showed the conservation of both ends of the linear genome and the divergence at the previously identified dissimilarity regions (R1 to R9). Protein sequence alignment of the c repressor (ORF1) of each phage enabled us to divide the six phages into two groups: D3112 group (D3112, MP29, MP42, and MP48) and MP22 group (MP22 and DMS3). Superinfection exclusion was observed between the phages belonging to the same group, which was mediated by the specific interaction between the c repressor and the cognate operator. Based on these, we suggest that the temperate siphophages prevalent in the clinical strains of P. aeruginosa represent at least two distinct heteroimmunity groups.

Hepatitis C virus, human immunodeficiency virus and Pseudomonas phage PS5 triad share epitopes of immunogenic determinants.

A lytic phage for Pseudomonas aeruginosa belongs to the Myoviridea family was isolated from urine for use in therapeutics. Pair of hepatitis C virus (HCV) primers highlighted segments on the genome of this phage. The sequence of these PCR products as well as the possible serological cross reactivity/relationship between HCV and the phage were investigated. One hundred HCV positive human sera were analyzed by ELISA. Ninety six well plates were coated with multiple epitopes of HCV proteins (Kit), phage and Pseudomonas cells. Initially the positive and negative control sera supplied in the test kit were used to evaluate the cross reactivity between the phage and anti-HCV antibodies. The results suggested a value over than 0.105 for a HCV positive reaction. Of the 100 HCV positive sera tested, sixty five and thirty percent showed cross reaction with phage lysate and Pseudomonas aeruginosa, respectively. High HCV antibody titer correlated to high cut off value for phage cross reaction, whereas no such correlation existed between HCV antibody titer and Pseudomonas cross reaction. The PCR products were sequenced and aligned with the HCV genome of H77. Sequence homology was detected in the 5', 3' UTRs and NS3 regions. Further these products showed similarity with HIV-1 Env, Pol & 3'LTR regions as well.