Zuzanna Kaźmierczak

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.

Oral Application of T4 Phage Induces Weak Antibody Production in the Gut and in the Blood

A specific humoral response to bacteriophages may follow phage application for medical purposes, and it may further determine the success or failure of the approach itself. We present a long-term study of antibody induction in mice by T4 phage applied per os: 100 days of phage treatment followed by 112 days without the phage, and subsequent second application of phage up to day 240. Serum and gut antibodies (IgM, IgG, secretory IgA) were analyzed in relation to microbiological status of the animals. T4 phage applied orally induced anti-phage antibodies when the exposure was long enough (IgG day 36, IgA day 79); the effect was related to high dosage. Termination of phage treatment resulted in a decrease of IgA again to insignificant levels. Second administration of phage induces secretory IgA sooner than that induced by the first administrations. Increased IgA level antagonized gut transit of active phage. Phage resistant E. coli dominated gut flora very late, on day 92. Thus, the immunological response emerges as a major factor determining phage survival in the gut. Phage proteins Hoc and gp12 were identified as highly immunogenic. A low response to exemplary foreign antigens (from Ebola virus) presented on Hoc was observed, which suggests that phage platforms can be used in oral vaccine design.

Facing Antibiotic Resistance: Staphylococcus aureus Phages as a Medical Tool

Staphylococcus aureus is a common and often virulent pathogen in humans. This bacterium is widespread, being present on the skin and in the nose of healthy people. Staphylococcus aureus can cause infections with severe outcomes ranging from pustules to sepsis and death. The introduction of antibiotics led to a general belief that the problem of bacterial infections would be solved. Nonetheless, pathogens including staphylococci have evolved mechanisms of drug resistance. Among current attempts to address this problem, phage therapy offers a promising alternative to combat staphylococcal infections. Here, we present an overview of current knowledge on staphylococcal infections and bacteriophages able to kill Staphylococcus, including experimental studies and available data on their clinical use.

Spectroscopic, structural and in vitro cytotoxicity evaluation of luminescent, lanthanide doped core@shell nanomaterials GdVO4:Eu3+5%@SiO2@NH2

The luminescent GdVO4:Eu(3+)5%@SiO2@NH2 core@shell nanomaterials were obtained via co-precipitation method, followed by hydrolysis and co-condensation of silane derivatives: tetraethyl orthosilicate and 3-aminopropyltriethoxysilane. Their effect on human erythrocytes sedimentation and on proliferation of human lung microvascular endothelial cells was examined and discussed. The luminescent nanoparticles were synthesized in the presence of polyacrylic acid or glycerin in order to minimalize the agglomeration and excessive growth of nanostructures. Surface coating with amine functionalized silica shell improved their biocompatibility, facilitated further organic conjugation and protected the internal core. Magnetic measurements revealed an enhanced T1-relaxivity for the synthesized GdVO4:Eu(3+)5% nanostructures. Structure, morphology and average grain size of the obtained nanomaterials were determined by X-ray diffraction, transmission electron microscopy and dynamic light scattering analysis. The qualitative elemental composition of the nanomaterials was established using energy-dispersive X-ray spectroscopy. The spectroscopic characteristic of red emitting core@shell nanophosphors was completed by measuring luminescence spectra and decays. The emission spectra revealed characteristic bands of Eu(3+) ions related to the transitions (5)D0-(7)F0,1,2,3,4 and (5)D1-(7)F1. The luminescence lifetimes consisted of two components, associated with the presence of Eu(3+) ions located at the surface of the crystallites and in the bulk.

T4 Phage Tail Adhesin Gp12 Counteracts LPS-Induced Inflammation In Vivo

Bacteriophages that infect Gram-negative bacteria often bind to the bacterial surface by interaction of specific proteins with lipopolysaccharide (LPS). Short tail fiber proteins (tail adhesin, gp12) mediate adsorption of T4-like bacteriophages to Escherichia coli, binding surface proteins or LPS. Produced as a recombinant protein, gp12 retains its ability to bind LPS. Since LPS is able to exert a major impact on the immune response in animals and in humans, we have tested LPS-binding phage protein gp12 as a potential modulator of the LPS-induced immune response. We have produced tail adhesin gp12 in a bacterial expression system and confirmed its ability to form trimers and to bind LPS in vitro by dynamic light scattering. This product had no negative effect on mammalian cell proliferation in vitro. Further, no harmful effects of this protein were observed in mice. Thus, gp12 was used in combination with LPS in a murine model, and it decreased the inflammatory response to LPS in vivo, as assessed by serum levels of cytokines IL-1 alpha and IL-6 and by histopathological analysis of spleen, liver, kidney and lungs. Thus, in future studies gp12 may be considered as a potential tool for modulating and specifically for counteracting LPS-related physiological effects in vivo.

Molecular imaging of T4 phage in mammalian tissues and cells

Advances in phage therapy encourage scientific interest in interactions of phages with human and animal organisms. This has created a need for developing tools that facilitate studies of phage circulation and deposition in tissues and cells. Here we propose a new green fluorescent protein (GFP)-based method for T4 phage molecular imaging in living systems. The method employs decoration of a phage capsid with GFP fused to the N-terminus of Hoc protein by in vivo phage display. Fluorescent phages were positively assessed as regards their applicability for detection inside living mammalian cells (by phagocytosis) and tissues (filtering and retention by lymph nodes and spleen). Molecular imaging provides innovative techniques that have brought substantial progress in life sciences. We propose it as a useful tool for studies of phage biology.

Bacteriophages displaying anticancer peptides in combined antibacterial and anticancer treatment

AIMS Novel anticancer strategies have employed bacteriophages as drug carriers and display platforms for anticancer agents; however, bacteriophage-based platforms maintain their natural antibacterial activity. This study provides the assessment of combined anticancer (engineered) and antibacterial (natural) phage activity in therapies. MATERIALS & METHODS An in vivo BALB/c mouse model of 4T1 tumor growth accompanied by surgical wound infection was applied. The wounds were located in the areas of tumors. Bacteriophages (T4) were modified with anticancer Tyr-Ile-Gly-Ser-Arg (YIGSR) peptides by phage display and injected intraperitoneally. RESULTS & CONCLUSION Tumor growth was decreased in mice treated with YIGSR-displaying phages. The acuteness of wounds, bacterial load and inflammatory markers in phages-treated mice were markedly decreased. Thus, engineered bacteriophages combine antibacterial and anticancer activity.

Addendum: Kaźmierczak, Z.; Górski, A.; Dąbrowska, K. Facing Antibiotic Resistance: Staphylococcus aureus Phages as a Medical Tool. Viruses 2014, 6, 2551-2570.

The authors would like to add the following Acknowledgements section: “Acknowledgements This work was funded by the National Science Centre in Poland grant UMO-2012/05/E/NZ6/03314.” [...]

Interaction of Bacteriophages with Mammalian Cells

Natural bacteriophages (present in the microbiome) and those applied as therapeutic agents may interact with mammalian cells and tissues. Adhesion interactions may define bacteriophage pharmacokinetics and resulting efficiency of bacteriophage agents in therapeutic applications by shaping bacteriophage homing to tissues and organs. Here we propose protocols for testing direct adhesion of bacteriophages or bacteriophage proteins to mammalian cells (in vitro). We further propose an animal model for investigation of accumulation/homing of bacteriophages in tissues (in vivo).

Real-Time qPCR as a Method for Detection of Antibody-Neutralized Phage Particles

The most common method for phage quantitation is the plaque assay, which relies on phage ability to infect bacteria. However, non-infective phage particles may preserve other biological properties; specifically, they may enter interactions with the immune system of animals and humans. Here, we demonstrate real-time quantitative polymerase chain reaction (qPCR) detection of bacteriophages as an alternative to the plaque assay. The closely related staphylococcal bacteriophages A3R and 676Z and the coliphage T4 were used as model phages. They were tested in vivo in mice, ex vivo in human sera, and on plastic surfaces designed for ELISAs. T4 phage was injected intravenously into pre-immunized mice. The phage was completely neutralized by specific antibodies within 5 h (0 pfu/ml of serum, as determined by the plaque assay), but it was still detected by qPCR in the amount of approximately 107 pfu/ml of serum. This demonstrates a substantial timelapse between “microbiological disappearance” and true clearance of phage particles from the circulation. In human sera ex vivo, qPCR was also able to detect neutralized phage particles that were not detected by the standard plaque assay. The investigated bacteriophages differed considerably in their ability to immobilize on plastic surfaces: this difference was greater than one order of magnitude, as shown by qPCR of phage recovered from plastic plates. The ELISA did not detect differences in phage binding to plates. Major limitations of qPCR are possible inhibitors of the PCR reaction or free phage DNA, which need to be considered in procedures of phage sample preparation for qPCR testing. We propose that phage pharmacokinetic and pharmacodynamic studies should not rely merely on detection of antibacterial activity of a phage. Real-time qPCR can be an alternative for phage detection, especially in immunological studies of bacteriophages. It can also be useful for studies of phage-based drug nanocarriers or biosensors.