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Purification, crystallization and halide phasing of a Streptococcus agalactiae backbone pilin GBS80 fragment
Krishnan Vengadesan1, Xin Ma, Prabhat Dwivedi
1Center for Biophysical Sciences and Engineering, School of Optometry, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Insights
Structural studies of Gram-positive pili are emerging. This research reports the purification, crystallization, and initial phasing of the major pilin GBS80 from Streptococcus agalactiae, advancing our understanding of GBS pilus structure.
Area of Science:
- Microbiology
- Structural Biology
- Biochemistry
Background:
- Streptococcus agalactiae (GBS) is a leading cause of neonatal infections worldwide.
- GBS utilizes PI-1 and PI-2 pili for adherence to host cells.
- The PI-1 pilus is composed of major pilin GBS80 and minor pilins GBS104/GBS52.
Purpose of the Study:
- To investigate the structural characteristics of the GBS PI-1 pilus backbone.
- To facilitate future structural studies of Gram-positive pili.
Main Methods:
- Purification of the 35 kDa major fragment of GBS80.
- Crystallization of GBS80 in two space groups (P2(1) and C2).
- Single-wavelength anomalous dispersion (SAD) phasing using an iodide derivative.
Main Results:
- Successful purification and crystallization of GBS80 fragment.
- Obtained initial phases and interpretable electron-density maps via SAD.
- Identified two distinct crystal forms (P2(1) and C2).
Conclusions:
- This study provides a foundation for determining the high-resolution structure of GBS80.
- The structural insights will aid in understanding GBS pathogenesis and developing novel therapeutics.
- Advances the emerging field of Gram-positive pilus structural biology.
Abstract:
The Gram-positive pathogen Streptococcus agalactiae or group B streptococcus (GBS) is the leading cause of bacterial septicemia, pneumonia and meningitis among neonates around the world. The pathogen assembles two types of pili on its surface, named PI-1 and PI-2, that mediate bacterial adherence to host cells. The GBS PI-1 pilus is formed by the major pilin GBS80, which forms the pilus shaft, and two minor pilins GBS104 and GBS52, which are incorporated into the pilus structure. While considerable structural information exists on Gram-negative pili, the structural study of Gram-positive pili is an emerging area of research. Here, the purification, crystallization and initial phasing of the 35 kDa major fragment of the backbone pilin GBS80 are reported. Crystals were obtained in two different space groups: P2(1) and C2. SAD data collected from an iodide-derivative crystal at the home source were used to obtain initial phases and interpretable electron-density maps.
