|Alejandro P. Heuck
Assistant Professor Biology, University of Massachusetts
Ph.D. : University of Buenos Aires, Argentina
Pore-forming toxins and translocation of virulence factors in bacterial pathogenesis.
Perfringolysin O Recognition of Cholesterol in the Target Membrane
Some bacterial protein toxins function by binding to the surface of mammalian cells, inserting into the bilayer, and creating holes in the membrane that lead to cell death. Perfringolysin O (PFO) is secreted by Clostridium perfringens , the pathogenic bacteria that cause gas gangrene. PFO binds to cholesterol-containing membranes and oligomerizes to form huge pores with diameters of ~300 Å. The C-terminus of PFO (domain 4) mediates its initial binding to the membrane, and this binding triggers the structural rearrangements required to initiate the oligomeriztion of PFO monomers.
Using multiple independent fluorescence techniques we are investigating the role of cholesterol in pore formation and the nature of the PFO-cholesterol interaction. For example, does PFO bind to a single cholesterol molecule or to a surface feature of a cholesterol-rich domain in the membrane? Is cholesterol required only for membrane binding, or it also plays an additional role in toxin oligomerization and/or membrane insertion?
Injection of Virulence Factors Through the Cell Membrane
Several pathogenic bacteria including Yersina ssp., Salmonella ssp., enterophatogenic E. coli , Pseudomonas aeruginosa , Shigella flexneri , etc., inject proteins directly into the eukaryotic cell cytoplasm to interfere with and to alter host processes. These proteins are presumably injected through the eukaryotic cell membrane via a proteinaceous transmembrane channel known as translocon, which is of bacterial origin. The translocons are thought to be transmembrane protein complexes consisting of several components. Our goal is to understand, at a molecular level, how the translocon is assembled into the target membrane and functions to translocate virulence factors into the cell cytoplasm.
We employ a variety of biophysical, biochemical, and molecular biological approaches to study protein structure, protein-membrane and protein-protein interactions.
Romano F.B., Rossi K.C., Sava C.G., Holzenburg A., Clerico E.M., Heuck A.P. (2011) Efficient isolation of Pseudomonas aeruginosa type III secrertion translocators and assembly of heteromeric transmembrane pores in model membranes. Biochemistry, 50, 7117-7131.
Luthra A., Zhu G., Desrosiers D.C., Eggers C.H., Mulay V., Romano F.R., Caimano M.J., Heuck A.P., Malkowski M.G., Radolf J.D. (2011) The transition from closed to open conformation of Treponema pallidum outer membrane-associated lipoprotein TP0453 involves membrane sensing and integration by two amphipathic helices. 2011 Journal of Biological Chemistry. 286, 41656-41668.
Raghava S., Giorda K.M., Romano F.B., Heuck A.P., Hebert, D. (2011) The SV40 late protein VP4 is a viroporin that forms pores to disrupt membranes for viral release. PLoS Pathogens, 7, e1002116.
Moe P.C., Heuck A.P. (2010) Phospholipid Hydrolysis Caused by Clostridium perfringens a-toxin Facilitates the Targeting of Perfringolysin O to Membrane Bilayers. Biochemistry, 49, 9498-9507.
Heuck A.P., Moe, P.C., Johnson B.B. (2010) "The cholester-dependent cytolysins family of Gram-positive bacterial toxins" in Cholesterol-binding and Cholesterol Transport Proteins: Stucture and Function in Health and Disease. Subcellular Biochemistry Series, Vol. 51, Chapter 20, 551-577. Edited by Harris R. Springer.