1. Field of the Invention
This invention relates generally to an antimicrobial agent and to methods of preventing microbial growth. In particular, the present invention involves compositions comprising an antimicrobial peptide and methods for its use.
2. Description of Related Art
The first antibiotics were used clinically in the 1940s and 1950s, and their use has been increasing significantly since this period. Although an invaluable advance, antibiotic and antimicrobial therapy suffers from several problems, particularly when strains of various bacteria appear that are resistant to antibiotics. Interestingly, bacteria resistant to streptomycin were isolated about a year after this antibiotic was introduced.
The development of antibiotic resistance is a serious and life-threatening event of worldwide importance. For example, strains of Staphylococcus are known that are immune to all antibiotics except one (Travis, 1994). Such bacteria often cause fatal hospital infections. Among other drug resistant organisms are: pneumococci that cause pneumonia and meningitis; Cryptosporidium and E. coli that cause diarrhea; and enterococci that cause blood-stream, surgical wound and urinary tract infections (Berkelman et. al., 1994). The danger is further compounded by the fact that antibiotic and antimicrobial resistance may be spread vertically and horizontally by plasmids and transposons.
Davies (1986) described seven basic biochemical mechanisms for naturally-occurring antibiotic resistance: (1) alteration (inactivation) of the antibiotic; (2) alteration of the target site; (3) blockage in the transport of the antibiotic; (4) by-pass of the antibiotic sensitive-step (replacement); (5) increase in the level of the inhibited enzyme (titration of drug); (6) sparing the antibiotic-sensitive step by endogenous or exogenous product; and (7) production of a metabolite that antagonizes action of inhibitor.
Antimicrobial peptides have been isolated from plants, insects, fish, amphibia, birds, and mammals (Gallo, 1998; Ganz and Lehrer, 1998). Vertebrate skin, trachea and tongue epithelia are rich sources of these peptides, which may explain the unexpected resistance of these tissues to infection (Russell et al. 1996). Although previously considered an evolutionarily primitive system of immune protection with little relevance beyond minimal antimicrobial activity, it has subsequently been determined that antimicrobial peptides are a primary component of an innate immune response and are expressed by mammalian cells during inflammatory events such as wound repair, contact dermatitis and psoriasis (Nilsson, 1999). The efficacy of antimicrobial peptides is based upon their ability to create pores in the cytoplasmic membrane of microorganisms (Oren et al., 1998). They also have been shown to stimulate syndecan expression, chemotaxis, and chloride secretion (Gallo, 1998).
The present invention seeks to employ antimicrobial compounds to overcome the deficiencies inherent in the prior art by providing new compositions, combined compositions, methods and kits, for treating infections and reducing resistance to antimicrobials and antibiotics.
The instant inventions seeks to overcome the noted deficiencies in the art by disclosing the previously unidentified antimicrobial peptide human beta-defensin-3 (HBD-3). The instant application provides the peptide and nucleic acid sequences for HBD-3 as well as methods of use for each.
As noted, the instant invention relates the peptide sequence of HD-3. A preferred embodiment of the instant invention is therefore an isolated antimicrobial peptide comprising the amino acid sequence: MRIHYLLFALLFLFLVPVPGHGGIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK (SEQ ID NO: 2). In an alternate embodiment of the invention, the HBD-3 peptide may be utilized in its functional state, i.e., absent the signaling sequence, and thus comprising the amino acid sequence: TLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK (SEQ ID NO: 3). It is contemplated that the HBD-3 peptide, as set forth may be administered to a host. For this or related purposes, the peptide may be dispersed within a pharmaceutically acceptable composition which in some aspects of the invention includes a pharmaceutically acceptable carrier. Alternate embodiments of the invention contemplate that the HBD-3 peptide, dispersed in a pharmaceutical composition may be used for, for example, topical administration, oral administration, or parenteral administration. Where HBD-3 is administered parenterally, the administration may be, for example, by injection or by inhalation.
Another aspect of the instant invention relates a beta-defensin encoding nucleic acid molecule isolated from other coding sequences where the nucleic acid molecule encodes a peptide of the amino acid sequence: MRIHYLLFALLFLFLVPVPGHGGIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK (SEQ ID NO: 2). In a preferred embodiment of the instant invention the nucleic acid molecule encoding HBD-3 is incorporated into a vector. Where a vector is utilized, it is particularly contemplated that the vector will be an expression vector.
Additional aspects of the invention contemplate that the expressed or mature peptide of the instant invention may be used to inhibit or prevent the growth of bacteria or other microbes. A preferred embodiment of the instant invention is therefore, a method of inhibiting the growth of a microbe by introducing into an environment an antimicrobial peptide comprising the amino acid sequence of the HBD-3 peptide. Where the peptide is introduced into an environment to prevent microbial growth, it is contemplated that the peptide will be dispersed in a composition capable of sustaining the antimicrobial properties of the peptide in the environment. A preferred embodiment is thus the introduction of the peptide into an environment, for example a host organism, with the peptide dispersed in a pharmaceutical composition.
The antimicrobial peptide of the instant invention is of particular interest because it does not appear that microbial species have selectively developed resistance to its effects. It is therefore contemplated that the HBD-3 peptide will be useful in combination with other antimicrobial agents, particularly those agents to which certain microbial strains may be developing resistance. Therefore, a preferred embodiment of the instant invention is a method of introducing the HBD-3 peptide into an environment in combination with an additional antimicrobial agent. The agents may be introduced concurrently, or the HBD-3 peptide may be introduced before or after the second antimicrobial agent.
Alternate embodiments of the invention contemplate the use of a variety of second antimicrobial agents in the context of the invention. Agents of particular relevance are, for example, antimicrobial agents that are protein synthesis inhibitors, cell wall growth inhibitors, cell membrane synthesis inhibitors, nucleic acid synthesis inhibitors, or competitive inhibitors.
While the HBD-3 peptide may be dispersed into formulations for delivery into a variety of environments, it is specifically contemplated that the peptide will be useful in the prevention of microbial growth in or on a host, particularly the growth of strains that exhibit some form of drug resistant phenotype. A preferred embodiment of the instant invention is therefore a method of inhibiting growth of a microbe in a host, comprising administering to the host the HBD-3 antimicrobial peptide. Alternate embodiments specifically contemplate that the HBD-3 peptide will be combined with a second antimicrobial agent, which may be administered as previously described. The second agent may comprise an antimicrobial agent exhibiting, for example, one of the following properties: protein synthesis inhibition, cell wall growth inhibition, cell membrane synthesis inhibition, nucleic acid synthesis inhibition, or competitive inhibition.
In another aspect of the invention, the HBD-3 peptide may be contained within a kit. In a preferred embodiment of the invention, the kit may further comprise an additional antimicrobial agent, for example an antimicrobial agent as previously described.