Mutans Murder Machine: A Targeted Treatment for Dental Cavities

The oral microbiome comprises a variety of both commensal and detrimental microbes. Oral health requires a fine balance of these organisms, which can be upset by broad-spectrum antibiotics. Our experiments involved the use of the peptide based antibiotic actagardine, known to have activity against Streptococci. Homologs of actagardine were also incorporated into the designed gene cluster, in an effort to develop novel antimicrobial compounds. We sought to use synthetic biology tools to create a targeting system for an antibiotic to kill only Streptococcus mutans, the primary causative agent of dental cavities. A combinatorial approach applying phage display and heterologous expression of modified lantibiotics was applied to develop this targeted S. mutans killing machine.

Introduction

Antibiotics have helped to shape modern medicine and increase life expectancies considerably around the world. Penicillin, the first biologically-derived antibiotic, has saved millions of lives since its discovery in 1942 (1). Hundreds of antibiotics have been developed since then, which target a variety of pathogenic microorganisms. However, due to widespread overuse of antibiotics, antibiotic resistance has become a major obstacle in the treatment of infections with multi-drug resistant tuberculosis and MRSA(5). Combined with a decreasing rate of antibiotic discovery, antibiotic resistance in human pathogens has precipitated a significant crisis in modern medicine.

Due to the limited number of antibiotics remaining on the “front lines” of treatment, there is a need for a novel approach to antibiotic discovery. Lantibiotics are ribosomally-synthesized peptides that incorporate a number of characteristically modified amino acids. They are produced by a number of Gram-positive bacteria, and act to inhibit other microorganisms in competition with them (6). Thus, lantibiotics act as a unique platform for the identification of novel antimicrobial compounds. The mechanisms of action of lantibiotics are poorly characterized, although it is known that they target the bacterial cell wall (6). The peptide nature of lantibiotics renders them particularly amenable for modification by direct genetic manipulation and the potential for the creation of novel antibiotics from existing biological examples is high (6). (Emerson and Sathee: how lantibiotics are produced) (Team Phage Display: Why the oral microbiome? Future applications/foundational advance)

We heterologously expressed the lantibiotic actagardine, which was modified to directly target the Gram-positive pathogen Streptococcus mutan. S. mutans is found in the oral microbiome and is widely regarded as one of the major causative agents of tooth decay (7). Dental cavities affect a significant portion of the global population yearly, and resultant bacterial infections have the potential to spread systemically and cause serious life-threatening health problems. However, the application of common clinical antibiotics in the treatment of S. mutans-related dental cavities is currently limited by their broad-spectrum activity. Antibiotic treatment causes damage to the entire oral microbiome, eradicating commensal organisms important to maintaining good oral health. The application of a targeted antibiotic will be an important step forward in helping to manage the impact of dental cavities worldwide, and our results can be extended to other diseases caused by oral pathogens, including periodontal disease, halitosis and some forms of oral cancer (Dragana ajdic et al, 2002 PNAS)

Phage display – building a targeted antibiotic

In order to construct a targeted antibiotic, we utilized phage display to generate a targeting peptide that binds selectively to S. mutans. Phage display was first described in 1985 by George P. Smith, and has since become a powerful method for screening molecular libraries. It enables the selection of proteins from a pool of similar molecules, so that only those with a desired function propagate (8). This is accomplished by attaching the “library” of proteins to the coat of bacteriophage, and applying the modified phage to a target molecule. After incubating, any unbound phage are washed away, while those phage expressing favourably binding proteins are eluted. This process is repeated until only the most functional from the library remain. Phage display procedures typically make use of filamentous phage due to the flexibility of their genome, the retention of infectivity when attaching targeting peptides, and the fact that large titers of phage can easily be produced as their production does not kill the host cells (8).

Our experimental approach incorporates a slight modification in the traditional phage display procedure - panning completed sequentially against a panel of organisms, including our target, S. mutans, and three commensals, S. mitis, S. oralis, and S. salivarius. These commensals serve as negative selection in order to ensure that our targeting peptide does not recognize closely related species that are not pathogenic, thus eliminating the possibility of targeting and killing commensal Streptococcus species.

This specialized methodology also addresses the problem of antibiotic resistance. The possibility exists that should S. mutans be treated in a non-specific method, as in the use of broad spectrum antibiotics that, over time, would induce resistance in one or several microbes in oral microbiome. Combined with the natural competence displayed by several Streptococci (9), horizontal gene transfer of resistance elements between S. mutans and these commensal organisms would increase the likelihood of the development of antibiotic resistance to our novel antibiotic in S. mutans. Therefore, a highly selective treatment, as outlined in our project, offers a safer and more stable approach.

Novel lantibiotics through combinatorial biochemistry

Another aspect of this project was the construction of novel lantibiotics that can be targeted towards S. mutans. Traditional means of antibiotic discovery have yielded less than satisfactory returns for a number of years, and new methods of creating antibiotics are sorely needed. The peptide nature of lantibiotics makes them relatively simple to modify by means of genetic manipulation and we take advantage of this property to construct a number of novel lantibiotic-like biomolecules from a library of actagardine homologues in hopes of creating a molecule with novel bioactivity.

To accomplish this, homologues of garA, the gene encoding the actagardine pro-prepeptide, found in a number of Gram-positive species were cloned downstream of garM, which encodes an enzyme that cyclizes actagardine and functionalizes it. A number of the homologues are expected to be recognized by GarM due to their structural similarity to GarA and be similarly modified and functionalized. We expect this process to yield a number of novel biomolecules not found in nature that have the potential to act as antibiotics or have other forms of bioactivity.

In order to screen for bioactivity, our novel compounds will be used in a number of bioassays to test for their ability to inhibit the growth of a number of Gram-positive and negative bacteria, including S. mutans. Any compound that displays activity in our screens will be further screened for toxicity against eukaryotic cells and subsequently characterized by mass spectrometry.