Progress 10/01/08 to 09/30/14
Outputs
Target Audience: This project may be relevant for the dairy indsudtry as well as the National Antimicrobial Resistance Monitoring System (NARMS) Changes/Problems: Water Buffalo becauseis widely recognized to bemore resilient to infection of the mammary gland than domestic cattle, and this was the basis of our working hypotheisi.Therefore we proposed that it is highly likely that natural antibiotics isolated and synthesizedfrom these species are more potent than the natural antibiotics that cow produced. To date we have isolated and characterized a verypromising bioactive moleculefrom Water Buffalo. This ,molecule is a small protein (cathelicidin peptide) which possess antimicrobial activity against relevant pathogens indairy cows. The economical impact of this discovery may be of great importance considering that the economical burden of mastitis world-wide is substantial, and a new natural antibiotic to combat mastitis would be ground-breaking for the dairy cattle industry worldwide. However, one of the main problems we have encountered is that we need to test the molecule in an in vivo system which is an expensiveendeavor. We are currently submitting grant proposals to Animal Health insudtries that may be interestedincontinuing with the research and development of theses natural antibiotics. What opportunities for training and professional development has the project provided? The project has provided my research group not only with basic molecular biology training, but also with the exposure to Corporate Sponsors. How have the results been disseminated to communities of interest? Some of the data has been reportedat presentationson KSU capus seminars as well as to Animal Heatlh companies (Centaur Animal Health). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The main goal of using HDPs as food packing material shifted early on during the 5-year peoject However,the original goal of looking at the discovery of novel natural antibiotics from animal origin remained.Consequently, the researchhasbeen focusing on the study these molecules as templates for the development, and eventual clinical use of natural antibiotics for animal health.
Publications
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience:
Nothing Reported
Changes/Problems: Problems arre inherent to research but none so far that has prevented the progress of the original plans What opportunities for training and professional development has the project provided? Training my doctoral student Kate Osei-Boadion currentmolecular, clelular and microbiological techniques. How have the results been disseminated to communities of interest? In Laboratory meetings only due to the IP protection of the novel molecules What do you plan to do during the next reporting period to accomplish the goals? To continue with the developpment and testing of the novel antimicrobialas to be eventually used as part of the food packing material
Impacts
What was accomplished under these goals?
We have identified several natural molecules from animal origen. Some of they have been already protected with a US patent (US patent # 7,985,832). The research and development of novel antimicrobials is ongoing in my laboratory.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
ISSN:2155-9899 JCCI, an open access journal
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: This project continues with the original goal of looking at the discovery of novel natural antibiotics from animal origin. As such, the research is focusing on the study these molecules as templates for the development, and eventual clinical use of natural antibiotics for animal health. Cathelicidins are multifunctional antimicrobial peptides expressed in multiple tissue in mammalian systems, making them readily available as part of the innate immune defense system (Selsted 1993). We are highly interested in the study of buffalo because it is widely recognized to be more resilient to infection than domestic cattle (Joshi 2006), and therefore, it is likely that antibiotics of these species are more potent than the natural antibiotics that cow produced cows. Our studies are now fine tuning the evaluation of the activity of synthetic Water Buffalo cathelicidin peptidomimetics which possess antimicrobial activity against relevant pathogens in farm animals, specifically dairy cows. The output for the project was then focused on a single area: A) The structure-activity relationship of the peptidomimetics using computational methods as outlined below PARTICIPANTS: Annika Linde, DVM, PhD Kate Osei-Boadi MS Lushington Gerald, PhD - University of Kansas faculty collaborator TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A) Small peptide scaffolds provide a rich basis for the formulation of antimicrobial agents since such species numerous naturally occurring examples exist that are utilized by innate immune responses, they are chemically very amenable to synthesis, and they are composed of a small orderly series of building blocks whose key composite properties are easy to computationally characterize. To this end, we will apply techniques such as Quantitative Structure-Activity Relationship (QSAR) modeling to guide the optimization of wide spectrum antimicrobial formulations for the treatment of mastitis. To date we have aided in the development of promising bioactive molecules based on empirical assumption that bioactivity requires peptides capable of forming stable transmembrane spanning helices with strong amphiphilicity. Although we have a hypothesis regarding the cellular mechanism of action, we are only now accruing the informational basis that will be required for detailed understanding of the structure-activity relationships from which it will be possible to develop detailed predictive models that will support the systematic development of new peptides with optimal target potency and selectivity. B) Among the four main mechanisms for cellular peptidic antimicrobial action, two (the barrel-stave and toroidal pore) models require stable amphiphilic helical peptides, and a third (in-plane diffusion) requires significant intramolecular stabilization as may be achieved by secondary (helices or sheets) or tertiary (disulphide bonds or salt bridges) structural elements that can guarantee preservation of peptidic amphiphilicity. For peptide families such as ours that are known to possess antimicrobial activity and has structural aspects indicative of helix formation, it is reasonable to assume that the bioactive conformation of the peptide is helical, and that the mechanism of action is one of the three listed above. The success of our preliminary studies in suggesting novel, antimicrobially-potent peptide formulations based primarily on improving helical propensity and amphiphilicity profiles affords us confidence that the observed activity arises from one of the above two mechanisms, however our data is not yet sufficiently comprehensive as to differentiate between the two. To refine our knowledge, we are considering further studies that may help to differentiate between the two mechanisms of action paradigms described in the previous section.
Publications
- No publications reported this period
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: The original project is still aimed at the discovery of novel antimicrobial molecules from animal origin and in its current form it is directed to study HDPs as templates for the development of natural antibiotics for animal health. Cathelicidins are multifunctional, but structurally conserved HDPs and typically expressed by bone marrow and circulatory cells, while beta-defensins are classical epithelial HDPs - with the exception of the bovine, which is unique in that beta-defensins are produced by neutrophils (BNBDs) (Selsted 1993). The evolution of multiple HDPs in different species is most likely in part a result of different microbial loads in diverse ecological niches, which has allowed varying degrees of resistance to infections in distinct animal species. The buffalo is generally considered more resistant to infection than domestic cattle (Joshi 2006), and it is thus possible that the HDPs of this species are more potent than the congeners isolated from cows. Our lab has assessed the activity of two synthetic WB-HDPs (cathelicidin) and found that one of the two cathelicidin peptides (WBCAT-14) has significant antimicrobial activity against clinical isolates of common mastitis pathogens (table 1) The outputs for the first part of the project were the following: A) In vitro assessment of the Water Buffalo {Bubalus bubalis) Cathelicidin (WBCATH-14) antimicrobial activity against mastitis-producing bacteria in dairy cattle. B) Computational design of WBCATH-14 peptide analogs to enhance antimicrobial activity and physicochemical stability of the resulting peptidomimetics PARTICIPANTS: Jessie Kiotzer Annika Linde, DVM, PhD Kate Osei-Boadi MS Lushington Gerald, PhD - University of Kansas faculty collaborator TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The first part of the outcome for the first part of the project was the discovery of synthetic antibiotic agents from the Water Buffalo. One of the Water Buffalo cathelicidins, WBCATH-14, displayed potent antimicrobial activity against pathogenic bacteria in naturally occurring mastitis.. The economical impact of this discovery may be of great importance considering that the economical burden of mastitis world-wide is substantial, and a new natural antibiotic to combat mastitis would be ground-breaking for the dairy cattle industry worldwide. - Table 1- Antimicrobial activity of WBCATH-14 peptides (minimum inhibitory concentration, MIC) Cow ID # MIC (uM) Bacterial strain 335 B-5 12.5 Streptococcus dysgalactiae 335B-15 25.0 Klebsiella pneumoniae 404C-18 12.5 Corynebacterium species 416B-10 25.0 Staphylococcus aureus 331 C-10 25.0 Staphylococcus aureus 422C-10 25.0 Staphylococcus aureus The second output of the project is an ongoing study. The computationally-derived WBCATH-14 analogues will be grouped into two functional series as follows: 1. Mutants to improve helicity of the molecule. The helicity score (3.64) of the wild-type peptide (WBCATH-14) suggests that it is helical at some concentration under certain physiological conditions, but that the helix is likely to exist in equilibrium with a non- helical conformer. Consequently, we will be designed computational analogues with helicity scores from approx. from 3.2 to 23.00. 2. Analogues with enhanced activity against Gram-positive bacteria. Indolicidin (GenBank NP 777252) is a cathelicidin HDP with activity against Gram-positive bacteria. Indolicidin possesses an amino acid sequence that is similar to the WBCATH-14. It will consequently be utilized to design additional WBCATH-14 analogues with different charge localizations and helicity gradients ranging from marginal (3.64) to excellent (>20).
Publications
- Jessie Kiotzer, Kate Osei-Boadi, Tonatiuh Melgarejo. 2011. Water Buffalo Host Defense Peptides as a Novel Natural Therapy for the Treatment of Mastitis in Dairy Cattle. Poster presented at the National Conference for the Basic Research Immersion Training Experience (BRITE) sponsored by the National Institutes of Health (NIH) held at Orlando, FL on August, 2011.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: This project is aimed at the discovery of novel antimicrobial molecules from animal originand explore not only their potential use as novel natural food packaging preservatives but also as temples for the development of natural antibiotics for human and animalhealth. Drug development is known to be a time consuming and lengthy scientific process and therefore the original output of this project has remained essentially unmodified. The results derived from this Action Plan are in direct accordance with Kansas State University's commitment to research in food safety and security. The outputs for the first part of the project were the following: A) Molecular characterization, chemical synthesis and structure-function NMR studies of Water Buffalo (Bubalus bubalis) Host Defense Peptides B) Partial Characterization of a novel HDP from the Domestic Cat - Felis catus PARTICIPANTS: Madeline Miller, student Annika Linde, DVM, PhD Kate Osei-Boadi MS Lushington Gerald, PhD - University of Kansas faculty collaborator Susan Brown PhD - Biology faculty collaborator Tony Grace PhD - Director Bioinformatics center KSU TARGET AUDIENCES: Research and development groups from pharmaceutical companies PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The first part of the outcome for the first part of the project was the discovery of synthetic antibiotic agents from the Water Buffalo. These peptides were biologically characterized in our laboratory, work that resulted in filing a temporary patent at the United States Patent and Trademark Office early fall 2010. The economical/medical impact of this discovery may be reflected within the next few years when the in vivo studies are final completed. The second output of the project is an ongoing project with very good success. Analysis of a Cathelicidin gene (CatCATH) in the domestic cat is been further evaluated To the best of our knowledge, this is the first study that clearly demonstrates the expression of a natural putative antibiotic peptide in cat tissues. Whereas some animal species (e.g. cows, guinea pigs) possess multiple cathelicidin (>5), other species such as monkeys, dogs and rabbits are considered monocathelicidin animals. In our study, the cat appeared to belong to the latter. This may suggest that the feline cathelicidin displays greater potency and broader antimicrobial spectrum in order to allow a single antimicrobial peptide to defend the host. Ongoing studies evaluating the biological activity of the CatCATH (using a chemically synthesized analog) will determine the feasibility of using CatCATH as a template for the development of natural antibiotics of animal origin. Following the original objectives of our research project, we are currently evaluating the antimicrobial activity of felineCAtCATH. The objective # 1 of this proposal encompassed five different experiments to achieve full biological/structural characterization of the HDP isolated from the mammalian species studied in this project. So far, experiments 1, 2 and 3 have been completed with regard of the Bubalus bubalis and data collected from these experiments are the backbone of our US patent. These results will pave the way for the development of the best natural antibiotic that can be used as our leading molecule.
Publications
- 1) U.S. Provisional Patent Application No. 61/377,365 A Novel Class of Natural Antibiotics for the Treatment of Clinically Relevant Infectious Diseases in Cattle, Docket No. 42270-PRO (2010). Linde A, Lushington G & Melgarejo T
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: This project involves the discovery, full biological characterization of novel Host Defense Peptides from animal origin as well as their potential use as novel natural food packaging preservatives. The discovery and characterization of novel antimicrobial molecules is a time consuming and laborious scientific process and as such the original output listed for this project has remained basically unmodified. The results derived from this Action Plan are in direct accordance with Kansas State University's commitment to research in food safety and security. The outputs for the first part of the project were the following: A) Molecular characterization, chemical synthesis and structure-function NMR studies of Water Buffalo (Bubalus bubalis) Host Defense Peptides B) Partial Characterization of a novel HDP from hepatic tissue of the Domestic Cat Felis catus. PARTICIPANTS: Lea Dib, MS; Annika Linde, DVM, PhD; Kate Osei-Boadi, BS; Amy Hanson, DVM student. TARGET AUDIENCES: Researchers working with novel synthetic antibiotic agents from animal origin. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The major outcome for the first part of the project is the discovery of two novel synthetic antibiotic agents from animal origin (from the Bovidae family). These HDPs were identified, synthesized and partially characterized in our laboratory during 2008 and 2009. Furthermore, one feline HDP - Hepcidin - has been isolated, sequenced,synthesized and functionally evaluated. Feline hepcidin functions in other species in maintaining iron homeostasis in the body by internalizing the erroportin protein that transports iron from the intestine to the bloodstream thus reducing the amount of iron taken into the cells. It is a highly conserved gene found in a variety of species and now my lab has identified and characterized this peptide in the feline species. RNA from a cat liver was extracted and the gene encoding the 25-amino acid peptide was sequenced using RT-PCR. The sequenced gene had 83% homology with the previously characterized human hepcidin gene. The sequence was used to produce a synthetic feline hepcidin peptide and its function was compared to a synthetic human hepcidin peptide. Caco-2 intestinal cells were cultured on Transwell membrane inserts and exposed to three different concentrations of the feline hepcidin peptide and human hepcidin. Fe-59 was added to the upper chamber of the membrane and the amount of Fe-59 crossing the cells into the lower chamber was measured with scintillation counting at 60 minutes and 120 minutes. The feline hepcidin functioned similarly to the human hepcidin in that iron transport from the upper chamber to the lower chamber was decreased when compared to control cells containing Fe-59, but no hepcidin from either species. Following the original objectives of our research project, we are currently evaluating the antimicrobial activity of feline Hepcidin. The objective # 1 of this proposal encompassed five different experiments to achieve full biological/structural characterization of the HDP isolated from the mammalian species studied in this project. So far, experiments 1, 2 and 3 have been completed with regard of the Bubalus bubalis as well as the Felis catus peptide. These are pivotal steps forward towards the identification of the best natural antibiotic that can be used as our leading molecule. Contrary to classical antibiotics - which are made in a sequential fashion, involving different enzymatic steps - HDPs are all gene encoded peptides originating from an RNA template and their antimicrobial activity is immediately displayed when the bacteria become in contact with them. Their consistency in efficacy throughout eons would furthermore speak against common belief that microorganisms inevitably will develop resistance against any imaginable antimicrobial compound over time. Our research team will keep investigating the expression of HDPs in other mammalian species during the next two periods of the project.
Publications
- No publications reported this period
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: The original proposed Action Plan covered the following dual rationale: Firstly, the experimental studies will establish the physicochemical characteristics, including antimicrobial activity, of host defense peptides from different species (including scavengers), and assess their application as novel natural food packaging preservatives. Secondly, the results derived from this Action Plan are in direct accordance with Kansas State University's commitment to research in food safety and security. The outputs for the first part of the project were the following: A) Identification and partial characterization of 2 novel Host Defense Peptides (HDPs) from the bone marrow of the Water Buffalo - Bubalus bubalis B) Identification of one novel HDP from hepatic tissue of the Domestic Cat - Felis catus PARTICIPANTS: Lea Dib, MS Annika Linde, DVM, PhD Kate Osei-Boadi BS TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The major outcome for the first part of the project is the discovery of two novel synthetic antibiotic agents from animal origin (from the Bovidae family). These HDPs have been identified, synthesized and partially characterized in our laboratory. The objective # 1 of this proposal encompassed five different experiments to achieve full biological/structural characterization of the HDP isolated from the mammalian species studied in this project. So far, experiments 1, 2 and 3 have been completed with regard of the Bubalus bubalis. These are pivotal steps forward towards the identification of the best natural antibiotic that can be used as our leading molecule. Contrary to classical antibiotics - which are made in a sequential fashion, involving different enzymatic steps - HDPs are all gene encoded peptides originating from an RNA template and their antimicrobial activity is immediately displayed when the bacteria become in contact with them. Their consistency in efficacy throughout eons would furthermore speak against common belief that microorganisms inevitably will develop resistance against any imaginable antimicrobial compound over time. Our research team will keep investigating the expression of HDPs in other mammalian species during the next two periods of the project.
Publications
- No publications reported this period
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