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Identification of isp, a locus encoding an immunogenic secreted protein conserved among group A streptococci
K S McIver1, S Subbarao, E M Kellner
1Department of Microbiology and Immunology, Emory University, Atlanta, Georgia 30322, USA.
Abstract:
The protein Mga (mga), which is required for transcription of several virulence genes of group A streptococci (GAS), including the antiphagocytic M protein, was suggested to act as the response regulator element of a bacterial two-component pathway. To investigate whether a gene encoding a cognate sensor protein is located upstream of mga, 3.1 kb of DNA 5' of the mga translational start site was cloned from serotype M6 GAS strain JRS4. Sequence analysis of this region revealed two adjacent open reading frames, a previously described orf and a new locus, isp (immunogenic secreted protein), which could encode proteins of 9 and 59 kDa, respectively. Inactivation of either open reading frame had no significant effect on transcription of the gene encoding M protein (emm) under normal growth conditions, suggesting that neither isp nor orf is involved in the Mga regulatory circuit. A protein migrating at an apparent molecular weight of 65,000 was produced when isp was transcribed and translated in vitro. The predicted isp product (Isp) contains an amino-terminal signal sequence region homologous to that found in bacterial secreted proteins, and expression of isp in Escherichia coli resulted in the presence of Isp in the periplasmic fraction. Convalescent-phase serum from a patient with an active GAS infection recognized forms of Isp both from the periplasm of E. coli and the supernatant of a GAS strain. Both isp and orf are highly conserved among strains of GAS, as shown by hybridization analyses.
Insights
Group A Streptococcus virulence regulation was investigated. Researchers identified a new gene, isp, but found it and another gene, orf, are not involved in the Mga regulatory pathway controlling M protein.
Area of Science:
- Microbiology
- Bacterial Genetics
- Molecular Biology
Background:
- The Mga protein regulates virulence genes in Group A Streptococcus (GAS).
- Mga's role as a response regulator in a two-component system was hypothesized.
- Investigating upstream genes for a cognate sensor protein was necessary.
Purpose of the Study:
- To identify and characterize genes located upstream of the mga gene in GAS.
- To determine if these upstream genes are involved in the Mga regulatory pathway.
- To analyze the function and localization of the newly identified immunogenic secreted protein (Isp).
Main Methods:
- Cloning and sequencing of 3.1 kb DNA upstream of the mga gene in GAS serotype M6.
- Inactivation of identified open reading frames (orf and isp) to assess their effect on emm gene transcription.
- In vitro transcription and translation of isp.
- Expression of isp in Escherichia coli and analysis of Isp localization.
- Immunological detection of Isp using convalescent-phase GAS infection serum.
Main Results:
- Two adjacent open reading frames, orf and isp, were identified upstream of mga.
- Inactivation of isp or orf did not affect M protein (emm) gene transcription.
- The isp gene encodes a 59 kDa protein (Isp) with a signal sequence, localized to the periplasm in E. coli.
- Isp was recognized by human convalescent-phase serum, indicating immunogenicity.
- Both isp and orf are conserved among GAS strains.
Conclusions:
- The identified genes, isp and orf, are not part of the Mga regulatory circuit controlling GAS virulence.
- Isp is an immunogenic secreted protein in GAS, but its specific role requires further investigation.
- The study contributes to understanding GAS virulence factor regulation and potential vaccine targets.