Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Feb;104(2):424-32.
doi: 10.1002/jps.24047. Epub 2014 Jun 10.

Biophysical characterization of the type III secretion tip proteins and the tip proteins attached to bacterium-like particles

Shyamal P Choudhari  1 , Xiaotong Chen, Jae Hyun Kim, Maarten L Van Roosmalen, Jamie C Greenwood 2nd, Sangeeta B Joshi, William D Picking, Kees Leenhouts, C Russell Middaugh, Wendy L Picking
Affiliations

Biophysical characterization of the type III secretion tip proteins and the tip proteins attached to bacterium-like particles

Shyamal P Choudhari et al. J Pharm Sci. 2015 Feb.

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.

Keywords: circular dichroism; fluorescence spectroscopy; light scattering (static); pH; physical characterization; physical stability.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Three-index empirical phase diagrams (EPDs) for IpaD (A), SipD (B) and LcrV (C), representing the conformational stability of the tip proteins as a function of pH and temperature. The red, green and blue panels at the right define individual component indices for secondary structure (CD), tertiary structure (fluorescence peak position) and aggregation behavior (LS), respectively.
Figure 2
Figure 2
Three-index empirical phase diagrams (EPDs) for IpaD-PA (A), SipD-PA (B) and LcrV-PA (C), representing the conformational stability of the proteins as a function of pH and temperature. The red, green and blue panels at the right define individual component indices for secondary structure (CD), tertiary structure (fluorescence peak position) and aggregation behavior (LS), respectively.
Figure 3
Figure 3
Three-index empirical phase diagrams (EPDs) for IpaD-BLPs (A), SipD-BLPs (B) and LcrV-BLPs (C), representing the conformational stability of the tip proteins attached to the BLPs as a function of pH and temperature. The red, green and blue panels at the right define individual component indices for secondary structure (CD), tertiary structure (fluorescence peak position) and aggregation behavior (LS), respectively.
See this image and copyright information in PMC

References

    1. Leenhouts K. Mimopath-Based Vaccine Delivery. In: Singh M, editor. Novel Immune Potentiators and Delivery Technologies for Next Generation Vaccines. New York: Springer; 2013. pp. 245–265.
    1. van Roosmalen ML, Kanninga R, El Khattabi M, Neef J, Audouy S, Bosma T, Kuipers A, Post E, Steen A, Kok J, Buist G, Kuipers OP, Robillard G, Leenhouts K. Mucosal vaccine delivery of antigens tightly bound to an adjuvant particle made from food-grade bacteria. Methods. 2006;38(2):144–149. - PubMed
    1. Ramirez K, Ditamo Y, Rodriguez L, Picking WL, van Roosmalen ML, Leenhouts K, Pasetti MF. Neonatal mucosal immunization with a non-living, non-genetically modified Lactococcus lactis vaccine carrier induces systemic and local Th1-type immunity and protects against lethal bacterial infection. Mucosal immunology. 2010;3(2):159–171. - PMC - PubMed
    1. Keijzer CMT, Voorn P, de Haan A, Haijema BJ, Leenhouts K, van Roosmalen ML, van Eden W, Broere F. Inactivated influenza vaccine adjuvanted with bacterium-like particles induce systemic and mucosal influenza A virus specific T-cell and B-cell responses after nasal administration in a TLR2 dependent fashion. Vaccine. 2013 in press. - PubMed
    1. Van Braeckel-Budimir N, Haijema BJ, Leenhouts K. Bacterium-like particles for efficient immune stimulation of existing vaccines and new subunit vaccines in mucosal applications. Frontiers in immunology. 2013;4:282. - PMC - PubMed

Publication types

MeSH terms