Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: The project was designed to characterize the Streptococcus equi homologue of Group A Streptococcus antiphagocytosis-required protein ARP. We have the following outputs: (1) We found that this protein is a secreted esterase (SeE) and characterized its enzymatic activity. SeE hydrolyzes ethyl acetate, acetylsalicylic acid, and tributyrin but not ethyl butyrate. This substrate specificity pattern does not match those of the three conventional types of non-specific carboxylic ester hydrolases (carboxylesterases, arylesterases, and acetylesterases). To determine whether SeE has lipase activity, a number of triglycerides and vinyl esters were tested in SeE-catalyzed hydrolysis. SeE does not hydrolyze triglycerides and vinyl esters of long chain carboxylic acids nor display interfacial activation, indicating that SeE is not a lipase. Like the conventional carboxylesterases, SeE is inhibited by diisopropylfluorophosphate. These findings indicate that SeE is a novel carboxylesterae with optimal activity against acetyl ester. (2) Intranasal immunization with SeE protects mice in a model of intranasal S. equi infection. (3) We obtained putative S. equi mutants defective in the see gene. We need to confirm that the SeE gene was knocked out and to compare this mutant with the wild-type strain in virulence using the mouse model of intranasal S. equi infection, growth ex vivo, and anti-phagocytosis ability. PARTICIPANTS: Dr. Xie Gang (Postdoctoral fellow), Dr. Hui Zhu (Postdoctoral fellow), Mengyao Liu, Michael McClure, JoAnne Schurman, and Yanchao Ran (Graduate Student) TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Extracellular carboxylic ester hydrolases are produced by many bacterial pathogens and have been shown recently to be important for virulence of some pathogens. However, these hydrolases are poorly characterized in enzymatic activity. Our characterization of SeE indicates that SeE is a novel esterase with optimal activity against acetyl esterase, which suggest that SeE may SeE may modify the mucosa by hydrolyzing the O-acetyl group of mucin. The protective effect of SeE we observed in the mouse immunization and challenge experiment will be very significant if we can observe the protective effect of SeE in horse immunization and challenge. If so, SeE can be included in a subunit vaccine formulation against equine strangles.
Publications
- Xie, G., Liu, M., Zhu, H., Lei, B. 2008. Esterase SeE of Stretpcoccus equi ssp. equi is a novel non-specific carboxylic ester hydrolase. FEMS Microbiol. Lett. 289:181-186.
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Progress 10/01/06 to 09/30/08
Outputs
OUTPUTS: The overall goals of our research on Streptococcus equi are to understand the basis of S. equi pathogenesis and develop an effective and safe subunit or attenuated live vaccine against infection in horses. The specific objective of this project is to test whether the secreted protein of S. equi, ARP (now renamed as SeE for S. equi esterase), is involved in evasion of host innate immunity and is important for virulence. To achieve our objective, we have the following oupputs: 1) Defining the enzymatic activity of See; 2) performing SeE immunization and S. equi challenge using a mouse model of intranasal S. equi challenge; 3) generating a S. equi mutant defective in the see gene; 4) testing the see knockout mutant in virulence using the mouse model; 5) examining the effect of the see inactivation on phagocytosis of S. equi by horse neutrophils; and 6) training 2 postdoctoral fellows, a graduate student, and 9 undergraduate students in research. PARTICIPANTS: Dr. Gang Xie, Mengyao Liu, Dr. Zhu Hui, Michael McClure, JoAnne Schurman, and Yanchao Ran. The project provided the opportunities for the following undergraduate students to gain research experience: Jarred Page, Wesley Tanaka, Cameron Marlow, Kelly Lye, Kien S. Lim, Shyam S. Shanmugam, Paramjit S. Karam Singh, Timoney Lin, Jennifer Martin, and Beth Zhou. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We found that SeE can hydrolyze a number of esters, including ethyl acetate, acetylsalicylic acid, and tributyrin. The substrate specificity pattern does not match those of the three conventional types of non-specific carboxylic ester hydrolases (carboxylesterases, arylesterases, and acetylesterases). To determine whether SeE has lipase activity, a number of triglycerides and vinyl esters were tested in SeE-catalyzed hydrolysis. SeE does not hydrolyze triglycerides and vinyl esters of long chanin carboxylic acids nor display interfacial activation, indicating that SeE is not a lipase. Like the conventional carboxylesterases, SeE is inhibited by diisopropylfluorophosphate. These findings indicate that SeE is a novel carboxylesterase with optimal activity against acetyl ester. These results are published in FEMS Microbiology Letters. Immunization of mice with SeE protected mice against intranasal S. equi infection. However, knockout of the see gene did not attenuate the virulence of S. equi in the mouse model of intranasal infection. The see-deficient mutant also retains the capacity of S. equi to resist phagocytosis. These results suggest that the antibodies of SeE may cross-react with a protective antigen. Although the see gene is not critical for virulence of S. equi as tested in mice, the characterization of the SeE enzymatic activity suggest that SeE may modify the mucosa by hydrolyzing the O-acetyl group of mucin. The protection conferred by SeE immunization suggests that there is a protective antigen that can be recognized by anti-SeE antibodies.
Publications
- Xie, G., Liu, M., Zhu, H., Lei, B. 2008. Esterase SeE of Streptococcus equi ssp. equi is a novel non-specific carboxylic ester hydrolase. FEMS Microbiol. Lett. 289:181-186.
- Zhu, H., Liu, M., Suby, P., Lei, B. 2009. The secreted esterase of group a Streptococcus is important for invasive skin infection and dissemination in mice. Infect. Immun. 77:5225-5235.
- Liu, M, Lei, B. 2010. IgG Endopeptidase SeMac does not Inhibit Opsonophagocytosis of Streptococcus equi Subspecies equi by Horse Polymorphonuclear Leukocytes. Open Microbiol. J. In press.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: We have made progress on the function of the Streptococcus equi homologue of Group A stretpococcus antiphagocytosis-required protein ARP. We found that the protein has an enzymatic activity to catalyze hydrolysis of esters, which suggests that the protein may be involved in modifying the host protein(s)and hydrolyzing lipid molecules to promote bacterial adhesion to mucus and process nutrients for bacterial growth.
PARTICIPANTS: The following individuals parcipated in the project: Dr. Jinlian Zhang, Dr. Hui Zhu (postdoctoral fellow), Mengyao Liu. The project also provided the opportunities for the following undergraduate students to gain research experience: Jarred Page, Wesley Tanaka, Cameron Marlow, Kelly Lye, Kien S. Lim, Shyam S. Shanmugam, and Paramjit S Karam Singh.
Impacts
The ARP is homologous to carboxyl esterases. We used a pH-stat to potentiometrically determine whether ARP catalyzes the hydrolysis of various ester compounds. We found that the protein catalyzes the hydrolysis of actylserine, tripropionin, tributyrin, tripropionine, and vinyl propionate but not vinyl laurate and trioctanoin. Thus, the protein is an esterase but not a lipase. The mucin of the mucus in the repiratory tract is acetylated. The protein may be involved in the modification of the mucin and thus modulation of bacteria-host interaction and nutrient processing. Our findings have led the directions clues to figure out the basis on why ARP is a protective antigen.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
The formula fund in 2007 supports the study on Streptococcus equi homologue of Group A Streptococcus antiphagocytosis-required protein ARP with the objectives to generate a S. equi mutant defective in arp and characterize the mutant. We have constructed the plasmid for the generation of the mutant, but we have not obtained the mutant yet. As an additional effort, we have prepared recombinant S. equi ARP protein and found that it is an antigen. More importantly, immunization of mice with the recombinant ARP protected mice against lethal intranasal infection of S. equi.
Impacts
S. equi is a new protective antigen, which may be used to develop a multivalent S. equi subunit vaccine.
Publications
- No publications reported this period
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