M protein is an important virulence factor found on the surface of virtually all clinical isolates of group A streptococci, and over 80 distinct serological types of M protein have been identified. Our recent studies indicate that streptococcal M proteins fall into two basic structural classes, and that nearly all serotypes associated with rheumatic fever (RF) are Class I. Several other biological properties (egs., opacity factor production, Fc-receptor activity) appear to be closely associated with streptococcal class. Class I streptococci share a surface-exposed antigenic domain within the M protein molecule that is absent from Class II M proteins. Our concepts of the structure of M proteins are based almost exclusively on Class I M protein types. Since Class I and II M proteins appear to be substantially different in molecular and immunological structure, the major objective of this proposal is to bring our knowledge of Class II M protein structure up to the current level of understanding for Class I M proteins. This will be achieved by cloning and sequencing the DNA of two Class II M protein genes of different serotypes. Predicted Class II amino acid sequences will be compared to those reported for Class I M proteins in order to identify homologous and nonhomologous regions. The purported Class II-specific antigenic site and the immunodeterminants shared among Class I and II M proteins will be identified by using antipeptide and monoclonal antibodies directed to defined epitopes; whether these epitopes are buried or surface-exposed will also be established. Understanding the similarities and differences between Class I and II M proteins should lead to a deeper insight into the basic biology of group A streptococci and their role in specific diseases. In addition, identification of the Class II-specific antigenic domain will aid in the development of a vaccine against Class II streptococci. GRANT-R43AI29260 Genetic engineering techniques will be used to enable yeast cells to produce functional monoclonal antibody fragments. The long-term applications are: 1) Reliable production of kilogram quantities of monoclonal antibodies (needed in cancer therapy and diagnostic imaging) from yeast fermentations rather than from more difficult hybridoma cultures; and 2) Rapid engineering and testing of improved antibody-related molecules through in vitro recombination and mutagenesis. For example, vectors containing the hypervariable coding regions from rodent genes can be recombined with human constant or framework coding regions or other interesting protein coding regions to produce new molecules. Also, the variable coding regions can be randomly mutagenized in vitro and rapidly expressed in yeast cells. Millions of yeast colonies, each producing a different singly mutated molecule, can be screened for improved antibody binding specificity, enzymatic activity, etc. on petri dishes or filters. The key to the success of this project is the recently improved technology for secretion of foreign proteins from yeast. The specific experiments proposed for Phase I involve: 1) Creating yeast expression vectors for the light and heavy chain portions of the Fab fragment of a well-characterized monoclonal antibody. 2) Selecting a yeast host stain with the genetic mutations rendering it capable of secreting functional monoclonal antibody fragments. 3) Demonstrating fermentation conditions under which functional Fab molecules are made.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI028944-01A1
Application #
3143622
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1990-05-01
Project End
1993-04-30
Budget Start
1990-05-01
Budget End
1991-04-30
Support Year
1
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Rockefeller University
Department
Type
Other Domestic Higher Education
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
Kalia, Awdhesh; Bessen, Debra E (2004) Natural selection and evolution of streptococcal virulence genes involved in tissue-specific adaptations. J Bacteriol 186:110-21
Kalia, Awdhesh; Bessen, Debra E (2003) Presence of streptococcal pyrogenic exotoxin A and C genes in human isolates of group G streptococci. FEMS Microbiol Lett 219:291-5
Svensson, Mikael D; Sjobring, Ulf; Luo, Feng et al. (2002) Roles of the plasminogen activator streptokinase and the plasminogen-associated M protein in an experimental model for streptococcal impetigo. Microbiology 148:3933-45
Kalia, Awdhesh; Spratt, Brian G; Enright, Mark C et al. (2002) Influence of recombination and niche separation on the population genetic structure of the pathogen Streptococcus pyogenes. Infect Immun 70:1971-83
Bessen, Debra E; Kalia, Awdhesh (2002) Genomic localization of a T serotype locus to a recombinatorial zone encoding extracellular matrix-binding proteins in Streptococcus pyogenes. Infect Immun 70:1159-67
Kalia, A; Enright, M C; Spratt, B G et al. (2001) Directional gene movement from human-pathogenic to commensal-like streptococci. Infect Immun 69:4858-69
Enright, M C; Spratt, B G; Kalia, A et al. (2001) Multilocus sequence typing of Streptococcus pyogenes and the relationships between emm type and clone. Infect Immun 69:2416-27
Scaramuzzino, D A; McNiff, J M; Bessen, D E (2000) Humanized in vivo model for streptococcal impetigo. Infect Immun 68:2880-7
Bessen, D E; Carapetis, J R; Beall, B et al. (2000) Contrasting molecular epidemiology of group A streptococci causing tropical and nontropical infections of the skin and throat. J Infect Dis 182:1109-16
Svensson, M D; Scaramuzzino, D A; Sjobring, U et al. (2000) Role for a secreted cysteine proteinase in the establishment of host tissue tropism by group A streptococci. Mol Microbiol 38:242-53

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