ISOLATION,ACTIVITY AND CHARACTERIZATION
OF A NOVEL ANTIMICROBIAL PEPTIDE
FROM HYDRA LITTORALIS
Lagang, Marie Khatrina C., Lappay, Jeffrey I., Lasiste, Jade Marie Edenvirg F.,
Leomo, Victoria Nastassa M., Licaros, Andro Reginald L., Lopez, Sheryl Mae J.,
Mallari, Ardynne Martin C., Manalo, Minette Krisel A., Marcelo, Wendel T.,
Martin, Aireen Paula V., Masiddo, Dan Jared T., Medrano, Jose Miguel M.,
Mendoza, John Michael T., Mendoza, Maria Christel M.,
Mercene, Roberto Martin C. and Miranda, Ronald Jason P.
A research proposalsubmitted to the
Department of Pharmacology
College of Medicine
University of the Philippines Manila
In partial fulfillment of the requirements for
Therapeutics 201
30 October 2009
ABSTRACT
The perennial need for new antimicrobials, against a background of increasing resistance to antibiotics, has led to the discovery and characterization of small molecules and antimicrobial peptides (AMPs) from eukaryotic organisms that, studies have revealed, form the stronghold of innate immunity. In 2008, the antimicrobial peptide hydramacin-1, novel in structure and tested to have a broad spectrum of activity, was isolated from the freshwater polyp Hydra magnipapillata. Noted also was the observation that putative antimicrobial peptides have yet to be identified from hydra polyp crude protein extracts. It is on this principle that this study aims to isolate, characterize and determine the spectrum of activity of a novel antimicrobial protein from the basal metazoan Hydralittoralis, a closely related species of H. magnipapillata.
This research employs a qualitative descriptive study design that will be run for two (2) weeks. The H.littoralis obtained will be starved and immunologically challenged via exposure to Psuedomonas aeruginosa cultured filtrate. Protein will be extracted from ten (10) grams of immune-challenged H.littoralis and will be subjected to fast-protein liquid chromatography (FPLC) using a heparin-sepharose column, after which it will be subjected to repeated runs of C2-C18 reverse phase high performance liquid chromatography (RP-HPLC) against increasing concentrations of acetonitrile. Fractions collected from the each run of RP-HPLC purification will be tested for antimicrobial activity against Klebsiella pneumoniae, Staphylococcus aureus and P. aeruginosa using the broth microdilution assay. Purity of the isolated fraction will be assessed through non-denaturing sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Once purity has been confirmed, the isolated protein will be sent for further characterization and analysis via mass spectrometry. The antimicrobial activity and the minimum inhibitory concentrations (MICs) of the antimicrobial peptide for the test microorganisms implicated in nosocomial infections—P. aeruginosa, Acinetobacter baumannii, K. pneumoniae, Staphylococcus epidermidis, Escherichia coli, S. aureus, Pseudomonas putida, Enterobacter cloacae, Enterococcus faecium and Klebsiella ozanae—will also be determined via the broth microdilution assay, as tested on clinical cultures. No statistical tests are necessary for interpretation of the data obtained from this study.
TABLE OF CONTENTS
Title Page / iAbstract / iii
List of Tables / iv
List of Figures / v
Introduction / 1
Research Objectives / 4
Review of Related Literature
Antibiotic Resistance
Antimicrobial Peptides
Hydra / 5
9
14
Materials and Methods
Study Design / 19
Test Subjects / 19
Study Maneuver
Immune Challenge / 19
Protein Extraction / 20
Protein Purification / 20
Screening for Antimicrobial Activity / 21
Further Purification of the Protein Extract / 21
Outcome Measures / 22
Data Management and Analysis / 23
Literature Cited / 25
Appendix
A: Formulations of Culture Media / 28
B: Sample Data Tables / 29
C: Working Timetable / 31
D: Budget Proposal / 32
LIST OF TABLES
1. Setups in screening for antimicrobial susceptibility. / 21
2. Test organisms for determination of MICs. / 22
3.Setups in Determining the Activity Spectrum and MICs. / 23
A1. Components of Hydra medium. / 28
A2. Components of Mueller Hinton Broth. / 28
B1. Screening for antimicrobial activity of fractionated protein extracts. / 29
B2. Determination of the minimum inhibitory concentrations (MICs) of the / 29
purified protein and the antibiotic control for the test organisms.
B3. Summary of the minimum inhibitory concentrations of the purified antimicrobial protein and antibiotic solutions against the test organisms. / 30
C. Working Timetable. / 31
D. Estimated cost of the proposed study. / 32
LIST OF FIGURES
1.Timeline of antibiotic deployment and resistance. / 82.Worldwide distribution of multi-drug resistant bacteria. / 9
1
INTRODUCTION
Since Alexander Fleming’s serendipitous discovery of the bacteria-killing property of penicillin in 1928, medical history has changed dramatically. Fatalities from diseases thought to be incurable were diminished with only a few pills. Treatment of prevalent bacterial diseases such as tuberculosis, syphilis, strep throat and pneumonia was improved remarkably. These incited the search and subsequent discovery of more antibacterial drugs from molds, plants, animal products or other bacteria. Pharmaceutical companies began to create artificial antibiotics or combine natural and synthetic components to form semi-synthetic antibiotics.
As the development of more antimicrobial drugs progresses, the spectrum of its benefits and uses continue to expand. Aside from its therapeutic value, these drugshave also proven useful as prophylaxis for infections such as endometritis (Dumaset al., 2008) as well as in pre-surgical operations.
However, the beneficial effects of these drugs come with some detrimental consequences. Streptomycin, for example, discovered by Selman Waksman in 1943 for treating tuberculosis, lists ototoxicity and disturbance of vestibular function among its adverse effects. Penicillin, though relatively safe for most people, can cause allergy and hypersensitivity reactions in certain individuals. Interestingly, antibiotics may bring about immunosuppresion, which may paradoxically induce susceptibility to infection in patients. In addition, because antimicrobials are among the medications most popularly prescribed, more bacterial pathogens are mutating into strains that are resistant to the drug. Improper compliance with regards to antibacterial drugs, as well as the lengthened period of usage, may also predispose the development of resistant strains of bacteria (Hildrethet al.,2009). Mechanisms of mutations may differ among species, but the ultimate goal remains the same—adaptation for survival.
The difficulty of treating infections with antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus and Clostridium difficile,is that it poses serious threats inboth in-patient and outpatient care (Matlow and Morris, 2009). Nosocomial infections continue to be highly prevalent, especially in hospitals of the developing countries. Aside from the danger it imposes to the patient's health, it also implies a longer, morecostly and moretoxic treatment necessary during extended hospitalstays. As of late, even other bacteria such as Streptococcusare also developingresistance to antibiotics (Hildrethet al., 2009).Dug-resistant organisms also have significant implications in infection control (Siegelet al., 2006). These are but few of the reasons why further studies on novel sources and molecules of antibiotics, especially those with sufficient potency and efficacy to act on resistant strains, is a continuous need. Indeed, there will never be a lack of necessity to embark on the search for new drugs.
From the soil to the sky, from prokaryotes to eukaryotes and from nature to the laboratory—pharmacologic research continues to blaze new trails in its quest for antibiotics, and just in the last decade, another shift in existing trends has emerged. Methods of isolating compounds with antimicrobial property has focused on small molecules until recently, when studies on the innate immune system revealed the potential array of biomolecules, specifically proteins, capable of defending the organism from the invasion of pathogens.
Referred to as antimicrobial peptides (AMPs), these defensive biomolecules are found in great diversity throughout the animal and plant kingdoms and are theorized to have played a significant role throughout evolution (Augustinet al., 2009). AMPs have been isolated from moths (Mak, 2001), pigs (Kyoungsooet al., 2002), frogs, bacteria (Schadich and Cole, 2009) and even humans. The pursuit of AMPs is compounded by the current inclination to explore the rich yet untapped resources in aquatic environments, both marine and freshwater, as exemplified by the isolation of such molecules from the sea squirt and the shrimp.
Only last year, an antimicrobial protein was isolated from the freshwater polyp Hydra magnipapillata, a soft bodied Cnidarian lacking mobile phagocytes, hemolymph and impermeable barriers, making them seem more vulnerable to pathogens (Boschet al., 2008). Named hydramacin-1, its protein sequence reveals homology only with three other proteins that have previously been isolated from the leech and scorpion, thus prompting the creation of a new class of antibiotics (Jung et al., 2009). Studies suggest that antimicrobial activity among Hydra species is directed against different microbes (Augustin et al., 2009), while Bosch et al. (2008) also noted the presence of other putative antimicrobial peptides in crude extracts, yet to be identified. Notwithstanding these is the fact that Hydra has a simple nervous system and that its action appears to correlate inversely with its antimicrobial activity (Kasahara and Bosch, 2003). Such collective observations then warrant further investigations on Hydra, especially on its other biomolecules with therapeutic potential that have yet to harness.
The purpose of this study is to isolate a novel antimicrobial peptide from Hydra littoralis, a closely related species of H. magnipapillata(Mari-Beffa and Knight, 2005), using protein purification protocols adapted from literature. The antibacterial spectrum of activity of the isolated AMP will be determined using strains of bacterial species labeled to be responsible for majority of the nosocomial infections at the Philippine General Hospital. The structural and chemical properties of the AMP will also be characterized via mass spectrometry.
Most significant in the study of antimicrobial peptides is that it sheds light on the mechanisms with which innate immunity works to defend the organism from attacks of a variety of pathogens. Increased understanding of the defenses living organisms possess will, in the long run, be pivotal in medicine, and it starts with the discovery of a new AMP. For through this begins the journey of developing an antimicrobial drug that brings with it prospects of improved pharmacotherapy—and with these new molecules, the possibilities are endless. Elucidation of the structure of the AMP can be the basis for the synthesis of novel drugs, and modifications to its elemental configuration can be made to improve suitability of the drug for a variety of purposes. With techniques of recombinant DNA expression employed in full swing, mass production of these biomolecules is not too difficult to imagine.
RESEARCH OBJECTIVES
Thegeneral objective of this research is to isolate, characterize and determine the spectrum of activity of a novel antimicrobial protein from the basal metazoan Hydra littoralis. Specifically, this research aims to:
- Obtain a crude protein extract from Hydra littoralis polyps challenged immunologically with the culture supernatant of Pseudomonas aeruginosa;
- Isolate an antimicrobial peptide from the crude extract using chromatographic methods based on affinity, size, andhydrophobicity;
- Evaluate the fractions for activity against a variety of microorganismscausing the majority of the nosocomial infections in the Philippine General Hospital, including Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus aureus;
- Determine the molecular weight and structure of the purified antimicrobial peptide using mass spectrometry.
REVIEW OF RELATED LITERATURE
ANTIBIOTIC RESISTANCE
Definition.The never-ending search for a panacea to all of the diseases plaguing humanity has yielded the discovery of hundreds of herbal medication, thousands of remedies and treatments, and perhaps the most famous a set “wonder-drugs” used and abused so often that nature is learning to fight back. Indeed, anti-microbial agents and antibiotics have since decreased the morbidity and mortality attributed to infectious diseases resulting in a dramatic rise of the average life expectancy of a human being in the Twentieth Century (Todar, 2008).
However, wanton abuse and irrational drug prescription have transformed our once thought panacea into nothing more than a catalyst for the evolution of the very bacteria we seek to kill with each generation seemingly deadlier than the last. Worst of all this, we are now becoming acutely aware that our stockpile of wonder drugs is desperately low, without additional ammunition in sight.
In essence antibiotic resistance is evolution; for the bacteria that is. They adapt to our treatments, growing more complex as our treatments do, striving to thrive even in the most anti-bacterial of environments, changing non-stop so that the status quo of the pre-antibiotic era is fully restored. And the losses on our side of the conflict are severe. Infections caused by resistant microbes fail to respond to treatment, resulting in prolonged illness and greater risk of death. Compounding this situation, treatment failures also lead to longer periods of infectivity, which increase the numbers of infected people moving in the community and thus expose the general population to the risk of contracting a resistant strain of infection (WHO, 2002).
Prevalence & Importance of Antibiotic Resistance in the Current Hospital Setting.Given all the characteristics of antibiotic resistant organisms, it is important to recognize several factors that allow resistance to spread. According to the WHO Fact Sheet on Antibiotic Resistance(2002):
“Hospitals are a critical component of the antimicrobial resistance, problem worldwide. The combination of highly susceptible patients, intensive and prolonged antimicrobial use, and cross-infection, has resulted in nosocomial infections with highly resistant bacterial pathogens. Resistant hospital-acquired infections are expensive to control and extremely difficult to eradicate. Failure to implement simple infection control practices, such as handwashing and changing gloves before and after contact with patients, is a common cause of infection spread in hospitals throughout the world. Hospitals are also the eventual site of treatment for many patients with severe infections due to resistant pathogens acquired in the community. In the wake of the AIDS epidemic, the prevalence of such infections can be expected to increase.”
In a recent US consensus statement on strategies to prevent and control the emergence and spread of antibiotic-resistant microorganisms in hospitals, one major point was “to develop a system to recognize and promptly report significant changes and trends in antimicrobial resistance” (Sorberg, 2003). From an international perspective, the prevalence of infections is usually higher in ICUs than in other hospital wards, and nosocomial outbreaks are also more frequent in ICUs. Antibiotic consumption is consequently relatively high in ICUs. Because of these factors, selection of resistant strains is expected, and hence many antibiotic resistance surveillance studies have been carried out in ICUs only (Sorberg, 2003).
Yet, why must we focus so much on hospitals? According to the director of the Pan American Health Organization (PAHO), globally, first-line antibiotics are no longer effective in treating resistant strains of several of the world's most threatening infectious diseases, including tuberculosis, pneumonia, cholera and many sexually transmitted infections. Everywhere, the consequences of growing antibiotic resistance are no longer limited to hospital settings and immunocompromised patients, but are showing up in community-acquired infections as well, essentially as a manifestation of indiscriminate use.
Intrinsic Antibiotic Resistance. Klebsiella spp. and Enterobacter spp. are widespread throughout the environment and also carried by humans. Both genera are well-recognized community and nosocomial pathogens and cause significant infections. They are a common cause of respiratory and nonrespiratory infections. Klebsiellaspp. is responsible for 1% to 5% of all cases of community-acquired pneumonia and between 0% to 23% of those acquired in the hospital, and its frequency is greater in alcoholic patients. The majority of cases are unilateral in the posterior segment of the right upper lobe. Lung abscess can occur after a pneumonic process or secondarily to Klebsiella spp. infections and have high rates of morbidity and mortality. K. pneumoniae is one of the most common microorganisms responsible for empyema. Klebsiella spp. and Enterobacter spp. rank fourth and third, respectively, as causes of hospital-acquired pneumonia mainly in patients during the early period of mechanical ventilation. Klebsiella spp. are intrinsically resistant to penicillins and can acquire resistance to third- and fourth-generation cephalosporins owing to the production of plasmid-mediated extended-spectrum beta-lactamases (ESBLs). These plasmids frequently carry aminoglycoside-modifying enzymes. Enterobacter spp. are intrinsically resistant to ampicillin, amoxicillin, amoxicillin-clavulanate, first-generation cephalosporins, and cefoxitin owing to the production of constitutive AmpC beta-lactamase. The derepression of this enzyme is increasingly frequent among clinical isolates and confers resistance to third-generation cephalosporins, and ureido- and carboxypenicillins; fourth-generation cephalosporins retain reasonable activity against depressed strains. Most isolates of Klebsiella spp. and Enterobacter spp. are susceptible to fluoroquinolones, trimethoprimsulfamethoxazole, aminoglycosides, and carbapenems. In some instances, treatment of severe infections caused by these microorganisms may benefit from the combination of beta-lactams (or fluoroquinolones) with aminoglycosides. Because of the high risk for developing resistance during treatment, all severe infections should be carefully watched during therapy (Bouza, 2002).
Extrinsically Developed Resistance.The Enterococci are a group of gram-positive cocci that are part of the normal resident flora of both humans and some animals. Generally not considered virulent, their intrinsic resistance to many antibiotics (including cephalosporins, penicillin, and aminoglycosides) has made them important opportunistic pathogens and one of the most common causes of nosocomial infections. Mortality rates in enterococcal bacteremia can reach 70%. The traditional method of treatment has been a combination of an aminoglycoside and ampicillin or a glycopeptide. By the 1970's, only ampicillin and vancomycin (a glycopeptide) were effective treatment options in most cases. As of 2000, high level resistance to ampicillin and aminoglycosides was common, leaving vancomycin as the treatment of last resort. Yet in one study in New York City, (Frieden et al.), it was found that 98% of vancomycin resistant enterococci (VRE) infections were acquired nosocomially and 19% of these were resistant to all antibiotics (Gangle, 2005).
Antibiotic resistance in Staphylococci is also very common and was observed very early in the antibiotic era. When general use of penicillin began, nearly all staphylococcal isolates were susceptible when tested in the laboratory, but resistance to penicillin and other N-lactam antibiotics, including ampicillin, in hospitals began to appear almost immediately, so that by 1948, 59% of S. aureus strains