Structural and Biochemical analysis of a β-carbonic anhydrase from Streptococcus sanguinis, an opportunistic pathogen involved in subacute infective endocarditis

Emily Bedea, and Vijayakumar Somalinga, Department of Biological Sciences, Southwestern Oklahoma State University, 100 Campus Drive, Weatherford, OK

Streptococcus sanguinis is a pathobiont associated with healthy oral biofilms in humans. S. sanguinis is one of the leading causes of infective endocarditis (IE) and its pathogenesis is linked to its ability to survive in blood and colonize endocardial surfaces. The mechanism by which S. sanguinis survives blood remains unclear. Survival in blood requires nutrients which are scarce in vivo. Pathogens overcome this scarcity by upregulating metabolic pathways required for the biosynthesis of nutrients. Several enzymes involved in biosynthesis of these nutrients utilize bicarbonate which is supplied by hydration of CO2. This process is carried out by carbonic anhydrase (CA), which catalyzes the hydration of CO2 to bicarbonate. Several studies have also shown that inactivating CA’s negatively impacts virulence in pathogens. Given the importance of CA’s in metabolic pathways and in pathogenesis, the goal of this study is to purify CA from S. sanguinis for biochemical and structural studies. We identified an 18.2 kDa protein in S. sanguinis using BLAST analysis with homology to well-studied β-CA’s. Homology modeling and structure analysis revealed that SsaCanB has a α/β fold typical of β-CA’s. Active site analysis of SsaCanB shows conserved residues similar to other well-studied β-CA’s. Furthermore, the catalytic dyad (Asp/Arg) required for water molecule activation is also conserved in SsaCanB. In order to over-express SsaCanB, the gene coding for SsaCanB was cloned into the pET28a using traditional cloning strategies. The construct was then transformed into E. coli BL21(DE3) and SsaCanB over-expression was induced with IPTG at 20°C. SDS-PAGE analysis revealed 19.8 kDa band indicating successful over-expression of SsaCanB and the presence of 19.8 kDa protein band in soluble fraction indicated that SsaCanB is soluble under the conditions tested. Furthermore, overexpressed SsaCanB was purified using immobilized metal affinity chromatography. Purified SsaCanB was found to have CA activity using an in-gel protonography assay.

Additional Abstract Information

Presenter: Emily Bedea

Institution: Southwestern Oklahoma State University

Type: Poster

Subject: Biology

Status: Approved

Time and Location

Session: Poster 3
Date/Time: Mon 4:30pm-5:30pm
Session Number: 3054