title: Biophysical characterization of the type III secretion tip proteins and the tip proteins attached to bacterium-like particles.
abstract: Bacterium-like particles (BLPs), derived from Lactococcus lactis, offer a self-adjuvanting delivery vehicle for subunit protein vaccines. Proteins can be specifically loaded onto the BLPs via a peptidoglycan anchoring (PA) domain. In this study, the tip proteins IpaD, SipD, and LcrV belonging to type III secretion systems of Shigella flexneri, Salmonella enterica, and Yersinia enterocolitica, respectively, were fused to the PA and loaded onto the BLPs. Herein, we biophysically characterized these nine samples and condensed the spectroscopic results into three-index empirical phase diagrams (EPDs). The EPDs show distinctions between the IpaD/SipD and LcrV subfamilies of tip proteins, based on their physical stability, even upon addition of the PA. Upon attachment to the BLPs, the BLPs become defining moiety in the spectroscopic measurements, leaving the tip proteins to have a subtle yet modulating effect on the structural integrity of the tip proteins-BLPs binding. In summary, this work provides a comprehensive view of physical stability of the tip proteins and tip protein-BLPs and serves as a baseline for screening of excipients to increase the stability of the tip protein-BLPs for future vaccine formulation.
authors.affiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, Oklahoma.
authors.forename: Shyamal P
authors.initials: SP
authors.lastname: Choudhari
authors.forename: Xiaotong
authors.initials: X
authors.lastname: Chen
authors.forename: Jae Hyun
authors.initials: JH
authors.lastname: Kim
authors.forename: Maarten L
authors.initials: ML
authors.lastname: Van Roosmalen
authors.forename: Jamie C
authors.initials: JC
authors.lastname: Greenwood
authors.forename: Sangeeta B
authors.initials: SB
authors.lastname: Joshi
authors.forename: William D
authors.initials: WD
authors.lastname: Picking
authors.forename: Kees
authors.initials: K
authors.lastname: Leenhouts
authors.forename: C Russell
authors.initials: CR
authors.lastname: Middaugh
authors.forename: Wendy L
authors.initials: WL
authors.lastname: Picking
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D058947:Bacterial Secretion Systems; D055592:Biophysical Phenomena; D013294:Lactococcus lactis / Q000737:chemistry*; D010316:Particle Size; D022223:Vaccines, Subunit / Q000008:administration & dosage* / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D058947:Bacterial Secretion Systems; D022223:Vaccines, Subunit
keywords: circular dichroism; fluorescence spectroscopy; light scattering (static); pH; physical characterization; physical stability