There is an urgent need for novel antibacterial agents. Many species have become resistant to current antibiotics and there is the potential use of Select Agents and other bacteria in bioterrorist attacks. In particular, some isolates of Yersinia pestis, the causative agent of plague, are known to harbor multiple resistance determinants to commonly used antibiotics. Phage-encoded bacteriolytic enzymes are promising reagents to treat a wide range of bacterial infections because their mechanisms of action are different from those of antibiotics. Several recent reports have evaluated the potential of phage enzymes for both topical and systemic use, but only lysins active against Gram-positive bacteria have recently been successfully tested therapeutically. However, these enzymes are not active against Gram-negative bacteria, which are responsible for the majority of hospital infections. The outer membrane of Gram-negative bacteria is impermeable to macromolecules. We propose to develop novel antibacterial agents based on phage enzymes capable of lysing Gram-negative bacteria. Phages that grow on these bacteria have obviously developed mechanisms for penetrating the outer membrane. We will use Yersinia pestis as the initial model organism because it is rough and thus possesses a less complex outer membrane than most major Gram-negative pathogens. Phage ?XA1122, which is known to grow on and lyse virtually all Y. pestis strains, is closely related to coliphage T7, and T7 mutants that grow well on Y. pestis are available. We propose to purify the phage-encoded muralytic enzymes, and also to display them on phage virions in order to maintain high local concentrations of the enzyme during therapeutic treatment. Muralytic enzymes will be used with two proteins to promote access to the cell wall: T7 gp14 is ejected from infecting virions and makes a channel across the outer membrane, and the Bdellovibrio bacteriovorus surface protein CAE77837 is used for invasion of Gram-negative hosts. Both proteins will be purified, CAE77837 also as a ~100 residue protease domain that will be displayed on T7 virions. The proteins, or common outer membrane-destabilizing chemicals, will be tested for their ability to act synergistically with the muralytic enzymes. Our long-term goal is to develop novel antibacterial agents that can be used therapeutically for Gram-negative infections. We will optimize conditions for bacteriolytic and bacteriocidal activity of combinations of the reagents in vitro against Y. pestis, and then test the most effective formulations in vivo, in both prophylactic and therapeutic treatments, using a murine model of plague infection.
The work described in this proposal will provide a thorough assessment of the feasibility of using phage-encoded lytic enzymes, in conjunction with outer membrane permeants, as novel antibacterial agents directed against Y. pestis as a model Gram-negative bacterium. This class of enzymes has been demonstrated to be active against Gram-positive bacteria but the outer membrane of Gram-negatives prevents their direct application. In preliminary studies we have shown that phage-infected crude lysates are competent for killing and lysing Y. pestis. The choice of Y. pestis as a model organism is predicated mainly on its incomplete core LPS and minimal capsule. A priori, development of an antibacterial formulation based on phage enzymes is expected to be less problematic with Y. pestis than with bacteria containing a complete LPS and O-antigen. However, if we can demonstrate prophylactic and/or therapeutic activity against Y. pestis in a mouse model, future studies will extend the technology to other Gram-negative pathogens. Our approach to allowing the phage lysins access to the Gram-negative bacterial cell wall is to make a formulation that also contains either common membrane-destabilizing chemicals, the phage virion protein that forms a channel across the outer membrane at the initiation of infection, or the surface endopeptidase of B. bacteriovorus, which bores its way into the periplasm of a target Gram-negative cell. We are therefore asking that the """"""""accessory reagents"""""""" promote access of the phage lysins to the bacterial cell wall. We are also proposing to test Y. pestis phage ?XA1122, used by the CDC as a diagnostic reagent, for its ability to provide prophylactic and/or therapeutic benefits against Y. pestis infections. These in vivo studies will parallel those involving lysins using the mouse model of plague infection. ? ? ?
