Intramuscular Immunization of Streptococcus pyogenes SF370 protein extract and identification of multiple virulence factors through proteomic profiling in RHD induced Balb/c mice

Abstract

Group A streptococcus (GAS) and autoimmunity are associated with heart related mitral valve damage, in adults. In this study Balb/c mice were intramuscularly immunized with S. pyogenes SF370 for 4 weeks. Prior to euthanization, physiological parameters like body weight and electrical signalling of the heart were recorded. After euthanization, the heart tissue homogenate was prepared and proteomic alterations were studied using SDS-PAGE and 2D electrophoresis. The expression levels of inflammatory genes like TNFα, IFNγ and TGF-β were quantified using real time PCR. Insilico analysis was performed to identify the functions of hypothetical proteins and virulence factors involved in the induction of rheumatic carditis. The results showed a reduction in body weight, ulceration, inflammation, cardiac lesions and prolonged PR interval in mice immunized with S. pyogenes SF370, as a result of RHD. The heart related proteins like α-actinin, fatty acid binding protein-heart, myosin light chain 3, hemoglobin subunit alpha, myoglobin regulatory light chain 2, (ventricular/cardiac muscle isoform), myosin-6, troponin-1 were found to be up-regulated when compared with the control. The functional annotation of S. pyogenes (SF370) was carried out by retrieving 1696 identified proteins and 653 hypothetical protein sequences in NCBI genome database. The conserved domain was identified for 505 proteins. The pfam database documented that the super families of 279 sequences and 40 signal peptides enabled the classification of proteins in different categories like biological (20%), cellular (22%) and molecular functions (36%). Putative transcription repair coupling factor and putative lysine aminopeptidase N terminal are the two virulence factors identified by VICMPRED in S. pyogenes SF370. The two identified virulence factors are involved in altering the mice heart proteome and thereby controlling the streptococcus pyogenes infection. Thus, the results of the present study reveals the role of immunogenic proteins in induction of rheumatic carditis and to elucidate the molecular mechanisms leading to autoimmune reactions in Balb/c mice.

Keywords: Immunogenic proteins; Rheumatic heart disease; S.pyogenes; Vaccine; Virulence factors.