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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 oligomerization 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 does it also play an additional role in toxin oligomerization and/or membrane insertion?

Support: American Heart Association.

Injection of Virulence Factors Through the Cell Membrane

Bacterial pathogens of animals and plants have evolved sophisticated strategies to infect their eukaryotic hosts. Many proteins that play a role in the virulence of Gram-negative pathogenic bacteria are not only secreted, but are also translocated directly across the eukaryotic cell membrane into the host cell cytoplasm. Once inside the cytosol they can interfere with cellular processes and suppress host defenses. The long-term goal of this project is to determine the molecular mechanism of protein translocation across the host cell cytoplasmic membrane.
Type III Gram-negative bacteria use a specialized secretion apparatus known as the type III secretion system (TTSS) to inject proteins directly into the eukaryotic cell cytoplasm. Bacterial proteins that are delivered by a TTSS are presumably injected through the eukaryotic cell membrane via a proteinaceous transmembrane channel known as the type III translocon, which is of bacterial origin. The type III translocons are thought to be transmembrane protein complexes consisting of several components. For example, in Yersinia spp., erythrocyte lysis depends on the putative transmembrane translocon proteins YopB, YopD, and LcrV, which have been shown to be essential for type III-mediated translocation of proteins. Similar proteins have been found in several other pathogens, including Salmonella enterica, Shigela flexineri, enteropathogenic E. coli, and Pseudomonas aeruginosa.
In the well-studied Yersinia TTSS, the pathogenicity arsenal is encoded by a large virulence plasmid (which codes for >20 proteins) and the proposed members of the translocation machinery are encoded by the lcrGVsycDyopBD operon. Notably, YopB and YopD are two proteins suggested as forming the translocon itself, and possess functional counterparts in other pathogenic bacteria, such as enteropathogenic Escherichia coli (EspB/EspD), Pseudomonas aeruginosa (PopB/PopD), Salmonella enterica (SipB/SipC), and Shigella spp. (IpaB/IpaC).
We are using site directed fluorescence labeling, multiple independent fluorescence techniques, and many other biochemical and biophysical techniques to characterize the structure and function of the type III translocon complex.

Support: American Lung Association and the Massachusetts Thoracic Society, NIH


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Last modified February 2008