Progress 10/01/03 to 09/30/06
Outputs
The formula fund in 2006 partially supported the study on the cell surface proteins of Streptococcus equi with the following aims: 1) To continue to characterize the cell surface proteins of S. equi and 2) to examine the roles of the cell surface proteins of S. equi in pathogenesis. The cell surface protein SeShp and extracellular lipoprotein SeHtsA were found to bind heme, and SeShp transfers its heme to apoSeHtsA. The study identified the heme acquisition system in S. equi. The genes encoding cell wall-linked proteins 2 and 5 (CWP2 and CWP5) were inactivated by gene replacement. The effects of the gene inactivation on virulence of S. equi were evaluated using a mouse model of an intranasal S. equi infection. The inactivation of cwp5, but not cwp2, significantly protected mice against lethal S. equi challenge. Consistent with this result, immunization of mice with CWP5, but not CWP2, protected mice against S. equi infection. The results indicate that CWP5 is a
virulence factor and protective antigen.
Impacts
The new finding identified the heme acquisition system in S. equi, which will advance our understanding on how S. equi acquires essential iron for its growth and survival. The identification of a new virulence factor and protective antigen advances our understanding on the pathogenesis of S. equi and may accelerate the development of an effective vaccine to prevent strangles in horses.
Publications
- Nygaard, T. K., M. Liu, M. J. McClure, and B. Lei. 2006. Identification and characterization of the heme-binding proteins SeShp and SeHtsA of Streptococcus equi subspecies equi. BMC Microbiol. 6:82.
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Progress 01/01/05 to 12/31/05
Outputs
The formula fund in 2005 mainly covered the expenses of buying two horses and per-diem charge for the horses and part of the expense for the purchase of a Surface Plasmom Refractometer (SPR). These expenditures made the experiments possible to pursue the following specific aims: 1) To determine whether seven dominant cell-surface protein antigens of Streptococcus equi elicit protective antibodies and 2) to identify host proteins which interact with S. equi cell-surface proteins. Phagocytosis and bactericidal activity assays have been set up. The bactericidal activity assay monitors the growth of S. equi in rabbit and horse blood with or without antibodies specific for the cell-surface protein antigens, while the phagocytosis assay examines whether the specific antibodies enhance the phagocytosis of S. equi by horse and rabbit neutrophils (PMNs). Antibodies specific for two of 6 proteins tested appears to enhance the phagocytosis of S. equi by PMNs. Further tests are
being conducted to determine whether these two proteins really elicit protective antibodies. SPR measurement was used to identify horse protein in serum which interacts with one of the seven antigenic cell surface proteins. A dominant serum protein was found to bind to one of the two proteins which could enhance S. equi phagocytosis by PMNs. We are determining the identity of the serum protein. These studies will confirm whether the two proteins are protective antigens and provide clues about the function of the protein that can interact with the horse serum protein.
Impacts
Two new protective antigens of S. equi, if confirmed, would accelerate the development of effective vaccines to prevent strangles in horses.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
The formula funds provided partial support of the work with focus on the first of the following objectives of a proposal which was later funded by a NRI competitive grant (MONB00409): 1. Characterize seven dominant antigenic cell wall-linked proteins (CWPs) of S. equi; 2.identify antigenic CWPs which can elicit opsonic and bactericidal antibodies; 3. determine whether immunization with antigenic CWPs protects mice against S. equi infection. Four (RSEQ291, 702, 1407, and 2168) of seven antigenic CWPs have been purified. However, there were some difficulties to get homogenous form of the other tree proteins (RSEQ200, 2110 and 2341). These proteins had 6xHis tag at their amino-terminus to facilitate purification using affinity column NTA-Ni. However, these three proteins could not bind to NTA-Ni well, suggesting that the amino-terminus may not be on protein surface. To solve this problem, subcloning procedure has been performed to engineer the His tag at the
carboxy-terminus. The newly modified recombinant proteins bound NTA-Ni better. Purification of these proteins is in process. To prepare specific mouse antisera and horse antibodies for objectives 2 and 3, mice and horse were immunized with purified RSEQ291, 702, 1407, and 2168 and sera were obtained. Specific horse antibodies have been purified using affinity columns with immobilized RSEQ291, 702, 1407, or 2168. ELISA analysis was then used to investigate whether the target proteins were produced in host during infection, the antibodies titers in convalescent sera from horses with strangles were determined to be more than 2000 for RSEQ291, 702, 1407, and 2168, indicating that these proteins were produced in vivo during infection. To determine whether the protein is produced in vitro and localized on cell surface, the binding of specific antibody to S. equi cell surface was analyzed by flow cytometry. Antibodies specific for RSEQ291, 702, 1407, or 2168 bound to S. equi cell surface,
indicating that these proteins were produced in vitro and localized on the cell surface.
Impacts
Successful identification of Streptococcus equi vaccine candidates would accelerate the development of effective vaccines to prevent the most common infectious disease, strangles, in horses.
Publications
- No publications reported this period
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