MOLECULAR PATHOGENESIS OF MYCOPLASMA HYOPNEUMONIAE-INDUCED DAMAGE IN AIRWAY EPITHELIA

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: NRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 0192791
• Grant No.: 2002-35204-12299
• Proposal No.: 2002-02737
• Project Start Date: Aug 1, 2002
• Project End Date: Jul 31, 2006
• Grant Year: 2002
• Program Code: [44.0]- (N/A)

Recipient Organization
IOWA STATE UNIVERSITY
S. AND 16TH ELWOOD
AMES,IA 50011

Performing Department
VETERINARY MEDICINE

Non Technical Summary
Mycoplasmal pneumonia causes significant economic losses to the swine industry. Surveys indicate that M. hyopneumoniae (Mhyo)-induced pneumonia lesions are present in over 80% of the swine slaughtered in the U.S. The most important mechanism by which Mhyo induces pneumonia is through damage to airway ciliated epithelia. Although it is known that binding of Mhyo to ciliated epithelium is the initial step of infection, the events this binding initiates in the epithelium are poorly understood. If our hypothesis is validated, a vaccine made from this protein and antibodies produced specifically against it may be used to control mycoplasmal pneumonia. Therefore, the results obtained from this research have a potential for generating both the academic and commercial interests for the development of strategies to control this major problem in the swine industry.

Animal Health Component

30%

Research Effort Categories

Basic

70%

Applied

30%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3510	1020	30%
311	3510	1090	50%
311	3510	1101	20%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3510 - Swine, live animal;

Field Of Science
1090 - Immunology; 1101 - Virology; 1020 - Physiology;

Keywords

swine

mycoplasma hyopneumoniae

polyclonal antibodies

pathogenesis

airways

epithelium

animal diseases

animal physiology

immunology

host pathogen relations

infection

binding

endoplasmic reticulum

receptors

phospholipase

calcium

pathways

membrane proteins

mechanisms

protein purification

amino acid sequence

recombinant proteins

biological activity

adhesion

protein characterization

antagonists

intervention

molecular biology

hplc (chromatography)

Goals / Objectives
Mycoplasma pneumonia causes significant economic losses to the swine industry. Surveys indicate that M. hyopneumoniae (Mhyo)-induced pneumonia lesions are present in over 80 percent of the swine slaughtered in the U.S. The most important mechanism by which Mhyo induces pneumonia is through damage to airway ciliated epithelia. Although it is known that binding of Mhyo to ciliated epithelium is the initial step of infection, the events this binding initiates in the epithelium are poorly understood. We found that inoculation of pathogenic Mhyo increased calcium release from the endoplasmic reticulum of ciliated epithelia, whereas nonpathogenic Mhyo did not. The Mhyo-induced calcium release is mediated by a G (i/o) protein-coupled receptor, via the phospholipase C-IP(3) pathway. Since intracellular calcium concentrations may induce cilia loss, we hypothesize that the Mhyo membrane protein-induced increase in calcium mediates this loss. Our long-range goal is to elucidate the cellular mechanisms that mediate the pathogenesis of the mycoplasmal pneumonia. The objectives of this project are to characterize the membrane protein that mediates M. hyopneumoniae-induced increases in calcium and to determine if this protein can cause cilia loss in tracheal cells. The central hypothesis of the research is that the membrane protein of M. hyopneumoniae binds to its receptors in the ciliated tracheal epithelia, thereby increasing calcium through a rise in calcium release from the endoplasmic reticulum. These changes in the calcium may evoke the epithelia to shed their cilia. Specific aims are: 1) Purify and sequence the solubilized Mhyo membrane protein, which increases calcium in tracheal cells; and 2) Express recombinant calcium-elevating protein and determine if it can cause cilia loss in tracheal cells; induce polyclonal antibodies against the purified membrane protein fragment and determine if it can block Mhyo-induced adhesion and cilia loss. Another long-term goal is to characterize this protein receptor in ciliated epithelium, the technology of which can be used to develop receptor antagonists for pharmacological intervention.

Project Methods
We ask two questions: 1) Can this protein be purified and characterized through protein chemistry and molecular biological techniques? and 2) Can this protein mimic intact pathogenic Mhyo soluble protein fragments, which increase calcium in ciliated epithelia at least ten times more potently than undigested membrane. We will purify the soluble tryptic fragments by HPLC and determine partial sequence from the active protein. This will allow us to identify the gene from the Mhyo genomic sequence. With the genomic sequence, we will produce a recombinant protein, which will be tested for the ability to increase calcium and cause cilia loss in tracheal epithelia. To confirm the Mhyo membrane protein-induced cilia loss, we will raise antibodies against this protein to determine if they will block the effect of the protein. This is a novel approach to help us understand the pathogenesis of mycoplasma infection.

Progress 08/01/02 to 07/31/06

Outputs
OUTPUTS: The protein that caused the increase in intracellular Ca2+ concentrations of porcine ciliated tracheal cells were found in pathogenic Mycoplasma hyopneumoniae was tentatively identified as a serine protease. These findings were shared with animal health industry in the U.S. We have received fundings from industry to continue the project. PARTICIPANTS: Dr. M. Ameri-Mahabadi, M. and Dr.E.-M. Zhou of the Veterinary Diagnostic and Production Medicine, Iowa State University Dr. S.C. Park, Laboratory of Veterinary Pharmacology and Toxicology, Kyungpook National University, Daegu, Republic of Korea. Dr. F. C. Minion, Department of Veterinary Microbiology and Preventive Medicine, Iowa State University Dr. G.X. Bai, Dr. N. Debevalya, Mr. N. Syed, and Dr. W.H. Hsu, Department of Biomedical Sciences. TARGET AUDIENCES: Veterinary scientists are the target audeiences, which include microbiologists in academia and industry. PROJECT MODIFICATIONS: The purification of the target protein using HPLC was the proposed approach. We changed the approach to utilization of molecular cloning, since it was very difficult to purify the protein using HPLC.

Impacts
This protein may be important for tahe pathogenesis of Mycoplasma hyopneumoniae, since it was not found in nonpathogenic strains of the microorganism. This protein could be further explored for production of vaccines against the microorganism. The USDA NRI grant was the principal source to generate these findings.

Publications

• 1. Ameri-Mahabadi, M., Zhou, E.-M., Hsu, W.H. Comparison of two swine Mycoplasma hyopneumoniae enzyme-linked immunorsorbent assays for detection of antibodies from vaccinated pigs and field serum samples. J. Vet. Diagn. Invest. 17:61-64, 2005.
• 2. Ameri-Mahabadi, M., Zhou, E.-M., and Hsu, W.H. A Western blot immunoassay as an ELISA confirmatory test for detecting swine antibodies against Mycoplasma hyopneumoniae. J. Vet. Diagn. Invest. 18:198-201, 2006.
• 3. Hwang, M.H., Lim, J.H., Yun, H.I., Kim, B.Y., Hsu, W.H., Park, S.C. The effect of polyclonal antibody on intracellular calcium increase induced by Mycoplasma hyopneumoniae in porcine tracheal cells. Vet. J. 172:556-560, 2006.

Progress 01/01/02 to 12/31/02

Outputs
New project: no progress report at this time.

Impacts
(N/A)

Publications

• No publications reported this period