CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage

The Ff family of filamentous bacteriophages infect gram-negative bacteria, but do not cause lysis of their host cell. Instead, new virions are extruded via the phage-encoded pIV protein, which has homology with bacterial secretins. Here, we determine the structure of pIV from the f1 filamentous bacteriophage at 2.7 Å resolution by cryo-electron microscopy, the first near-atomic structure of a phage secretin. Fifteen f1 pIV subunits assemble to form a gated channel in the bacterial outer membrane, with associated soluble domains projecting into the periplasm. We model channel opening and propose a mechanism for phage egress. By single-cell microfluidics experiments, we demonstrate the potential for secretins such as pIV to be used as adjuvants to increase the uptake and efficacy of antibiotics in bacteria. Finally, we compare the f1 pIV structure to its homologues to reveal similarities and differences between phage and bacterial secretins.

Filamentous Bacteriophage—A Powerful Carrier for Glioma Therapy

Glioma is a life-threatening malignant tumor. Resistance to traditional treatments and tumor recurrence present major challenges in treating and managing this disease, consequently, new therapeutic strategies must be developed. Crossing the blood-brain barrier (BBB) is another challenge for most drug vectors and therapy medications. Filamentous bacteriophage can enter the brain across the BBB. Compared to traditional drug vectors, phage-based drugs offer thermodynamic stability, biocompatibility, homogeneity, high carrying capacity, self-assembly, scalability, and low toxicity. Tumor-targeting peptides from phage library and phages displaying targeting peptides are ideal drug delivery agents. This review summarized recent studies on phage-based glioma therapy and shed light on the developing therapeutics phage in the personalized treatment of glioma.

CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage

The Ff family of filamentous bacteriophages infect gram-negative bacteria, but do not cause lysis of their host cell. Instead, new virions are extruded via the phage-encoded pIV protein, which has homology with bacterial secretins. Here, we have determined the structure of pIV from the f1 filamentous bacteriophage at 2.7 A resolution by cryo-electron microscopy, the first near-atomic structure of a phage secretin. Fifteen f1 pIV monomers assemble to form a gated channel in the bacterial outer membrane, with associated soluble domains projecting into the periplasm. We model channel opening and propose a mechanism for phage-mediated gate movement. By single-cell microfluidics experiments, we demonstrate the potential for secretins such as pIV to be used as adjuvants to increase the uptake and efficacy of antibiotics in bacteria. Finally, we compare the f1 pIV structure to its homologues to reveal similarities and differences between phage and bacterial secretins.

Analysis of the Consolidation Phase of Immunological Memory within the IgG Response to a B Cell Epitope Displayed on a Filamentous Bacteriophage

Immunological memory can be defined as the ability to mount a response of greater magnitude and with faster kinetics upon re-encounter of the same antigen. We have previously reported that a booster dose of a protein antigen given 15 days after the first dose interferes with the development of memory, i.e., with the ability to mount an epitope-specific IgG response of greater magnitude upon re-encounter of the same antigen. We named the time-window during which memory is vulnerable to disruption a “consolidation phase in immunological memory”, by analogy with the memory consolidation processes that occur in the nervous system to stabilize memory traces. In this study, we set out to establish if a similar memory consolidation phase occurs in the IgG response to a B cell epitope displayed on a filamentous bacteriophage. To this end, we have analyzed the time-course of anti-β-amyloid IgG titers in mice immunized with prototype Alzheimer’s Disease vaccine fdAD(2-6), which consists of a fd phage that displays the B epitope AEFRH of β -amyloid at the N-terminus of the Major Capsid Protein. A booster dose of phage fdAD(2-6) given 15 days after priming significantly reduced the ratio between the magnitude of the secondary and primary IgG response to β-amyloid. This analysis confirms, in a phage vaccine, a consolidation phase in immunological memory, occurring two weeks after priming.

Filamentous Bacteriophage Delay Healing of Pseudomonas-Infected Wounds

We have identified a novel role for filamentous bacteriophage in the delayed healing associated with chronic Pseudomonas aeruginosa (Pa) wound infections. In a mouse model of chronic Pa-infected wounds, Pf, a filamentous phage produced by Pa, impaired keratinocyte migration, prevented wound re-epithelialization, and delayed healing in both the absence and presence of live bacteria. Mechanistically, the immune response to Pf phage produces soluble factors that impair keratinocyte migration and delay wound re-epithelialization. In a prospective cohort study of 113 human patients, Pa was detected in 36 patients and 25 of these (69%) were positive for Pf phage. Pf(+) wounds were significantly older and more likely to increase in size over time than Pf(-) wounds. Together, these data implicate Pf in the delayed wound healing associated with Pa infection. We propose that Pf phage may have potential as a biomarker and therapeutic target for delayed wound healing.

Arming Filamentous Bacteriophage, a Nature-Made Nanoparticle, for New Vaccine and Immunotherapeutic Strategies

The pharmaceutical use of bacteriophages as safe and inexpensive therapeutic tools is collecting renewed interest. The use of lytic phages to fight antibiotic-resistant bacterial strains is pursued in academic and industrial projects and is the object of several clinical trials. On the other hand, filamentous bacteriophages used for the phage display technology can also have diagnostic and therapeutic applications. Filamentous bacteriophages are nature-made nanoparticles useful for their size, the capability to enter blood vessels, and the capacity of high-density antigen expression. In the last decades, our laboratory focused its efforts in the study of antigen delivery strategies based on the filamentous bacteriophage ‘fd’, able to trigger all arms of the immune response, with particular emphasis on the ability of the MHC class I restricted antigenic determinants displayed on phages to induce strong and protective cytotoxic responses. We showed that fd bacteriophages, engineered to target mouse dendritic cells (DCs), activate innate and adaptive responses without the need of exogenous adjuvants, and more recently, we described the display of immunologically active lipids. In this review, we will provide an overview of the reported applications of the bacteriophage carriers and describe the advantages of exploiting this technology for delivery strategies.

Cryo-electron microscopy structure of the filamentous bacteriophage IKe

The filamentous bacteriophage IKe infectsEscherichia colicells bearing IncN pili. We report the cryo-electron microscopy structure of the micrometer-long IKe viral particle at a resolution of 3.4 Å. The major coat protein [protein 8 (p8)] consists of 47 residues that fold into a ∼68-Å-long helix. An atomic model of the coat protein was built. Five p8 helices in a horizontal layer form a pentamer, and symmetrically neighboring p8 layers form a right-handed helical cylinder having a rise per pentamer of 16.77 Å and a twist of 38.52°. The inner surface of the capsid cylinder is positively charged and has direct interactions with the encapsulated circular single-stranded DNA genome, which has an electron density consistent with an unusual left-handed helix structure. Similar to capsid structures of other filamentous viruses, strong capsid packing in the IKe particle is maintained by hydrophobic residues. Despite having a different length and large sequence differences from other filamentous phages, π–π interactions were found between Tyr9 of one p8 and Trp29 of a neighboring p8 in IKe that are similar to interactions observed in phage M13, suggesting that, despite sequence divergence, overall structural features are maintained.