Michael C. Haymore and Dr. Noel Owen, Chemistry and Biochemistry
Introduction
The discovery of penicillin by Alexander Flemming in the early twentieth century is seen as one of the most significant medical advances in the past 100 years. Millions of people have had their lives preserved or prolonged due to the use of effective antibiotics since their discovery and wide-spread use. However, with the incredibly diverse and constantly evolving infectious diseases older antibiotics such as penicillin are becoming obsolete in the fight against infection. Thus, there remains a constant need to discover antibiotics effective against today’s diseases. Plants are a rich source of antibiotic compounds. Because plants live and compete in environments with infectious disease just as humans do, they have developed effective defense mechanisms against potentially harmful diseases. One method of defense used by plants is that of a symbiotic relationship developed between the plants themselves and “good” microorganisms. Some microbes, such as fungi or bacteria, excrete compounds that kill any nearby competing microbes. These microbes live inside the tissue of plants and find a constant source of nutrients. In return, the microbe offers the plant protection from other more harmful bacteria or fungi. Experimental
Our research group collected plant samples from the Peruvian rain forest and from more arid regions of southern Utah. The microorganisms growing inside these plants were grown and screened for antibiotic activity. Our hypothesis was that plants competing in areas of greater natural resources, such as a rain forest, have developed more extensive defense mechanisms than those in areas with fewer resources, and thus contain microbes with greater antibiotic activity.
Screening
Each microbe was grown on solid and liquid media. The liquid media containing the chemical compounds produced by the microbes underwent a filtering process at which point it was screened for antibacterial, antifungal and cytotoxic (anticancer) activity. The media extracts were tested against two strains of gram-negative bacteria, Salmonella salamae and Pseudomonas aeruginosa, and one gram-positive bacterium, Staphylococcus aureus. The media extracts were also tested against Geotrichum and Pythium fungi and a HeLa cancer cell line.
Results
The Peruvian plants yielded approximately 275 fungal and bacterial cultures. Of the 275 microbes screened approximately 7 showed significant antifungal activity, 5 significant antibacterial activity, and none showed significant cytotoxic activity. Two bacteria, identified as similar or identical strains of the bacterium Paenibacillus polymyxa, showed significant antifungal and antibacterial activity against gram-positive bacteria. Active portions of the media extract from P. polymyxa cultures were semi-purified using butanol extractions and a silica gel column. The semi-purified extract was run on Electro-Spray Ionization Mass Spectrometry in order to determine the molecular weight of major extract components. Molecular weights of 892 and 894 kD resulted. Research conducted by a group at the University of Alberta showed similar results to ours.1 In their research of P. polymyxa they found antifungal and antibacterial compounds with molecular weights similar to those indicated above. Through structural experiments this research group found that these compounds were derivatives of a class of antifungal and antibacterial cyclic depsipeptides called fusaricidins. Fusaricidins are circular proteins with varying side chains. A picture of one type of fusaricidin is found below.
With this information we were able to determine with strong assurance that the active compounds isolated were also fusaricidin derivatives. Further mass-spectrometry experiments determined that the two compounds detected were in fact fusaricidin A and C, or close derivatives.
Current & Future Research
The southern Utah plants, taken from an area known as San Rafael Swell, yielded approximately 250 fungi and bacteria. Of those, 12 showed significant antifungal activity, two showed significant antibacterial activity and 10 showed significant cytotoxic activity. A rose colored bacterium particularly active against the HeLa cell line has recently become the focus of my research. Interestingly but not surprisingly, recent cytotoxic assays have shown that media extracts from this bacterium show greater activity when the bacterium is grown in the presence of a fungus, indicating that the bacteria can be induced to produce its defensive cytotoxic compounds by having a competing microbe. While a past researcher attained some structural data on the active compound produced by this bacteria, more of the pure material must be isolated before a complete structure can be determined. This is current focus of my research. In addition to this goal, future studies will aim at determining what chemical compound produced by the competing fungus induces the bacterium to “turn on” the gene for the cytotoxic compound. This information combined with the complete structure of the cytotoxic compound will make for an interesting story that will hopefully see publication.
It is also interesting to note the difference in antibiotic activity between the Peruvian and southern Utah plants. Not only did the microbes isolated from the Utah plants show as much or more antibiotic activity than those from Peru, there were far more microbes active against cancer cells.
Notes
Paenibacillus polymyxa produces fusaricidin-type antifungal antibiotics active against Leptosphaeria maculans, the causative agent of blackleg disease of canola. Beatty, Perrin H.; Jensen, Susan E. Can. J. Microbiol. 2002. 48, 2: 159–169.