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Creation Date
4-24-2025
Description
Identification and Characterization of a DNA-binding Protein from Starved (Dps) Cells Homolog from Streptococcus sanguinis, an opportunistic pathogen involved in subacute infective endocarditis
Dps proteins are ubiquitous metalloproteins that play an important role in bacterial oxidative stress tolerance. Although Dps proteins contain iron-bound active sites, other metals such as manganese (Mn) have also been shown in the active sites of some Dps proteins and have been implicated in Mn homeostasis. Streptococcus sanguinis is an opportunistic pathogen capable of causing subacute infective endocarditis (SIE) in humans. Hydrogen peroxide (H2O2) production and Mn utilization is critical for its survival and pathogenesis. A previous study demonstrated that S. sanguinis dps mutants impaired in H2O2 mediated oxidative stress tolerance. The role of Dps in oxidative stress and the importance of Mn in virulence prompted us to investigate its role in S. sanguinis. We recently identified a 19.2 kDa Dps homolog (SSA0644) in S. sanguinis with 58% identity to a Dpr protein from S. mutans. Sequence analysis of SSA0644 revealed the presence of conserved iron entry and active site iron binding residues. In addition, preliminary homology structure analysis showed that the monomer of SSA0644 is composed of a four-helix bundle typical of Dps family of proteins. The ssa0644 gene has already been cloned into pET28a expression vector and transformed in E. coli BL21(DE3) cells. Protein overexpression and purification will be done use standard methods. Preliminary crystallization screens will be set up using vapor-diffusion method and based on the preliminary crystallization screens, optimization screens will be set up. Iron oxidation and DNA binding activity of SSA0644 will be evaluated using ferroxidase and electrophoretic mobility shift assays respectively.
Keywords
SWOSU Research Fair, Research and Scholarly Activity Fair, Scholarly Activity, Research, Student Research, Research Fair