Background and Training:
PhD: University of Buenos Aires, Argentina
Postdoctoral training: Texas A&M University, College of Medicine
Engineering bacterial toxin to measure cholesterol levels in cell membranes
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 large pores with diameters of ~300 Å. The C-terminus of PFO (domain 4) mediates its initial binding to the membrane, and this binding trigger the structural rearrangements required to initiate the oligomerization of PFO monomers.
Our goal is to develop molecular probes that will ultimately allow the quantification and imaging of distinct cholesterol levels in cell membranes.
Assembly of the Type III secretion translocon in membranes
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, the assembly mechanism of the Type III secretion translocon into the target cell membrane.
We employ a variety of biophysical, biochemical, and molecular biological approaches to study protein structure, protein-membrane and protein-protein interactions.
Tang Y., Romano F.B., Brena M., and Heuck A.P. The Pseudomonas aeruginosa type III secretion translocator PopB assists the insertion of the PopD translocator into host cell membranes J. Biol. Chem. 2018 293, 8982-8993
Savinov S.N. and Heuck A.P. Interaction of cholesterol with Perfringolysin O: what have we learned from functional analysis? Toxins 2017, 9 (12), 381
Johnson, B. B., Breña, M., Anguita, J. & Heuck, A. P. Mechanistic Insights into the Cholesterol-dependent Binding of Perfringolysin O-based Probes and Cell Membranes. Scientific Reports 2017 7, 13793.
Vermeulen A.J., Tang Y., & Heuck A.P. Translocation of Toxins by Gram Negative Pathogens Using the Type III Secretion System in Gopalakrishnakone, P., Alape-Girón, A., Stiles, B., Dubreuil, J.D., Mandal, M. (Eds.) Microbial Toxins, Toxinology. 2016.
Romano F.B., Tang Y., Rossi K.C., Monopoli K.R., Ross J.L., Heuck A.P. Type 3 Secretion translocators spontaneously assemble a hexadecameric transmembrane complex. J. Biol. Chem. 291, 6304-6315.
Johnson B.B. and Heuck A.P. Perfringolysin O structure and mechanism of pore formation as a paradigm for cholesterol-dependent cytolysins. Subcell. Biochem. 2014 80: pp 63-81.
Divakaruni AS, Wiley SE, Rogers GW, Andreyev AY, Petrosyan S, Loviscach M, Wall EA, Yadava N, Heuck AP, Ferrick DA, Henry RR, McDonald WG, Colca JR, Simon MI, Ciaraldi TP, Murphy AN.* Thiazolidinediones are acute, specific inhibitors of the mitochondrial pyruvate carrier. Proc Natl Acad Sci U S A. 2013 110, pp 5422-5427.
Johnson B.B., Moe P.C., Wang Y.D., Rossi K., Trigatti B.L., and Heuck A.P. Modifications in Perfringolysin O domain 4 alter the threshold of cholesterol concentration required for binding. Biochemistry 2012, 51, pp 3373-3382
Romano F. B.,; Rossi, K.; Sava, C.G.; Holzenburg, A.; Clerico, E.M.; Heuck A. P. Efficient isolation of Pseudomonas aeruginosa type III secretion translocators and assembly of heteromeric transmembrane pores in model membranes. Biochemistry 2011 50, pp 7117–7131