Structure determination of the CAMP factor of streptococcus agalactiae with the aid of an MBP tag and insights into membrane-surface attachment

Authors: LI, YAJUAN - University Of Science And Technology Of China; ZENG, WEIHONG - University Of Science And Technology Of China; LI, YUELONG - University Of Science And Technology Of China; FAN, WEIRONG - Shanghai Jiaotong University; MA, HUAN - University Of Science And Technology Of China; FAN, XIAOJIAO - University Of Science And Technology Of China; JIANG, JIANSHENG - National Institutes Of Health (NIH); BREFO-MENSAH, ERIC - University Of Waterloo; Zhang, Yuzhu; YANG, MEIXIANG - Jinan University; DONG, ZHONGJUN - Tsinghua University; PALMER, MICHAEL - University Of Waterloo; JIN, TENGCHUAN - University Of Science And Technology Of China

Submitted to: Acta Crystallographica, Section D: Biological Crystallography
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/25/2019
Publication Date: 7/31/2019
Citation: Li, Y., Zeng, W., Li, Y., Fan, W., Ma, H., Fan, X., Jiang, J., Brefo-Mensah, E., Zhang, Y., Yang, M., Dong, Z., Palmer, M., Jin, T. 2019. Structure determination of the CAMP factor of streptococcus agalactiae with the aid of an MBP tag and insights into membrane-surface attachment. Acta Crystallographica, Section D: Biological Crystallography. 75:772-781. https://doi.org/10.1107/S205979831901057X.
DOI: https://doi.org/10.1107/S205979831901057X

Interpretive Summary: Streptococcus agalactiae (group B streptococcus; GBS) was first discovered as common bacteria causing mastitis in cattle. Now, it is known as a significant pathogen that causes severe invasive diseases in human and farm animals, particularly in farmed fishes. It represents a zoonotic hazard that also compromises food safety. Thus, GBS has been studied in the context of agriculture, veterinary medicine, in addition to human health. The CAMP factor was discovered in the human pathogen and is expressed in all GBS. It the infection process, CAMP attaches to the cell membrane and forms circular pores. Thus, studying the structural basis of the mechanism of the activity of this toxin is highly desired. This study reported a protocol that overcomes the obstacles in the structure determination of CAMP and the insights into the mechanisms of CAMP-host cell membrane interactions. The methodology and results may will facilitate solving difficult structures in general and help the development of efficient treatment and prevention GBS infection of farm animals to increase food safety in particular.

Technical Abstract: CAMP factor is a unique a-helical bacterial toxin that is known for its co-hemolytic activity in combination with staphylococcal sphingomyelinase. It was first discovered in the human pathogen Streptococcus agalactiae (also known as group B streptococcus), but homologous genes have been found in many other Gram-positive pathogens. In this study, the efforts that led to the determination of the first structure of a CAMP-family toxin are reported. Initially, it was possible to produce crystals of the native protein which diffracted to near 2.45 Å resolution. However, a series of technical obstacles were encountered on the way to structure determination. Over a period of more than five years, many methods, including selenomethionine labeling, mutations, crystallization chaperones and heavy-atom soaking, were attempted, but these attempts resulted in limited progress. The structure was finally solved using a combination of iodine soaking and molecular replacement using the crystallization chaperone maltose-binding protein (MBP) as a search model. Analysis of native and MBP-tagged CAMP-factor structures identified a conserved interaction interface in the C-terminal domain (CTD). The positively charged surface may be critical for binding to acidic ligands. Furthermore, mutations on the interaction interface at the CTD completely abolished its co-hemolytic activities. This study provides novel insights into the mechanism of the membrane-permeabilizing activity of CAMP factor.