Host cell invasion by Toxoplasma gondii is temporally regulated by the host microtubule cytoskeleton.

Toxoplasma gondii is an obligate intracellular protozoan parasite that invades and replicates within most nucleated cells of warm-blooded animals. The basis for this wide host cell tropism is unknown but could be because parasites invade host cells using distinct pathways and/or repertoires of host factors. Using synchronized parasite invasion assays, we found that host microtubule disruption significantly reduces parasite invasion into host cells early after stimulating parasite invasion but not at later time points. Host microtubules are specifically associated with the moving junction, which is the site of contact between the host cell and the invading parasite. Host microtubules are specifically associated with the moving junction of those parasites invading early after stimulating invasion but not with those invading later. Disruption of host microtubules has no effect on parasite contact, attachment, motility, or rate of penetration. Rather, host microtubules hasten the time before parasites commence invasion. This effect on parasite invasion is distinct from the role that host microtubules play in bacterial and viral infections, where they function to traffic the pathogen or pathogen-derived material from the host cell's periphery to its interior. These data indicate that the host microtubule cytoskeleton is a structure used by Toxoplasma to rapidly infect its host cell and highlight a novel function for host microtubules in microbial pathogenesis.

Only two amino acids are essential for cytolytic toxin recognition of cholesterol at the membrane surface.

The recognition and binding of cholesterol is an important feature of many eukaryotic, viral, and prokaryotic proteins, but the molecular details of such interactions are understood only for a few proteins. The pore-forming cholesterol-dependent cytolysins (CDCs) contribute to the pathogenic mechanisms of a large number of Gram-positive bacteria. Cholesterol dependence of the CDC mechanism is a hallmark of these toxins, yet the identity of the CDC cholesterol recognition motif has remained elusive. A detailed analysis of membrane interactive structures at the tip of perfringolysin O (PFO) domain 4 reveals that a threonine-leucine pair mediates CDC recognition of and binding to membrane cholesterol. This motif is conserved in all known CDCs and conservative changes in its sequence or order are not well tolerated. Thus, the Thr-Leu pair constitutes a common structural basis for mediating CDC-cholesterol recognition and binding, and defines a unique paradigm for membrane cholesterol recognition by surface-binding proteins.

Intermedilysin-receptor interactions during assembly of the pore complex: assembly intermediates increase host cell susceptibility to complement-mediated lysis.

Intermedilysin (ILY) is an unusual member of the family of cholesterol-dependent cytolysins because it binds to human CD59 (hCD59) rather than directly to cholesterol-rich membranes. Binding of ILY to hCD59 initiates a series of conformational changes within the toxin that result in the conversion of the soluble monomer into an oligomeric membrane-embedded pore complex. In this study the association of ILY with its membrane receptor has been examined throughout the assembly and formation of the pore complex. Using ILY mutants trapped at various stages of pore assembly, we show ILY remains engaged with hCD59 throughout the assembly of the prepore oligomer, but it disengages from the receptor upon the conversion to the pore complex. We further show that the assembly intermediates increase the sensitivity of the host cell to lysis by its complement membrane attack complex, apparently by blocking the hCD59-binding site for complement proteins C8alpha and C9.