Biology subjects

Kegawa, Y.

Publications and source records attributed to Kegawa, Y..

3 recordsLinked to original sources

bioRxiv2024

The invasion pore induced by Toxoplasma gondii

Obligate intracellular parasites invade host cells to survive. Following host cell contact, the apicomplexan Toxoplasma gondii injects proteins required for invasion into the host cell. Here, electrophysiological recordings of host cells acquired at sub- 200 ms resolution allowed detection and analysis of a transient increase in host membrane conductance following exposure to Toxoplasma gondii. Transients always preceded invasion but parasites depleted of the moving junction protein RON2 generated transients without invading, ruling out a direct structural role for RON2 in generating the conductance pathway or restricting the diffusion of its components. Time-series analysis developed for transients and applied to the entire transient dataset (910,000 data points) revealed multiple quantal conductance changes in the parasite-induced transient, consistent with a rapid insertion, then slower removal, blocking, or inactivation of pore-like conductance steps. Quantal steps for RH had a principal mode with Gaussian mean of 0.26 nS, similar in step size to the apicomplexan protein translocon EXP2. Without RON2 the quantal mean was significantly different (0.19 nS). Because no invasion occurs without poration, the term invasion pore is proposed.

biophysics↗

bioRxiv2024

Perforation of the host cell plasma membrane during Toxoplasma gondii invasion requires rhoptry exocytosis

Toxoplasma gondii is an obligate intracellular parasite, and the delivery of effector proteins from the parasite into the host cell during invasion is critical for invasion itself and for parasite virulence. The effector proteins are released from specialized apical secretory organelles known as rhoptries. While much has been learned recently about the structure and composition of the rhoptry exocytic machinery and the function of individual rhoptry effector proteins that are exocytosed, virtually nothing is known about how the released proteins are translocated across the host cell plasma membrane. Previous electrophysiology experiments reported an unanticipated observation that invasion by T. gondii is preceded by a transient increase in host cell plasma membrane conductance. Here, we confirm this electrophysiological observation and propose that the conductance transient represents a parasite-induced perforation in the host cell plasma membrane through which rhoptry proteins are delivered. As a first step towards testing this hypothesis, and to provide higher throughput than patch clamp electrophysiology, we developed an alternative assay to detect the perforation. This assay utilizes high-speed, multi-wavelength fluorescence imaging to enable simultaneous visualization of host cell perforation and parasite invasion. Using this assay, we interrogated a panel of mutant parasites conditionally depleted of key invasion-related proteins. Parasites lacking signaling proteins involved in triggering rhoptry secretion (e.g., CLAMP) or components of the rhoptry exocytic machinery (e.g., Nd9, RASP2) are defective in their ability to induce the perforation. These data are consistent with a model in which the perforating agents that disrupt host cell membrane integrity during invasion - and may thereby provide the conduit for delivery of rhoptry effector proteins - are stored within the rhoptries themselves and released upon contact with the host cell.

microbiology↗

bioRxiv2022

PIEZO1-dependent erythrocyte dehydration as the mechanism for selection of an allele protecting from severe malaria.

PIEZO1 is a cation specific mechanoreceptor channel implicated in red blood cell (RBC) volume homeostasis. Several PIEZO1 gain of function (GoF) variants demonstrate delayed channel inactivation and can cause hereditary xerocytosis (HX), a disease characterized by hemolytic anemia, RBC dehydration, and shape distortion. The milder PIEZO1E756del GoF variant, prevalent in populations of African descent, protects carriers from severe malaria caused by Plasmodium falciparum and ameliorate disease in a rodent malaria model. To explore the mechanism of this malaria protection, P. falciparum infection of human PIEZO1E756del RBC was analyzed in shear-stressed and static cultures with and without Yoda1, a PIEZO1 agonist. RBC dehydration was a common pathophysiological factor affecting parasite replication in both culture conditions. PIEZO1 channel opening by either Yoda1 or shear stress produced dehydration-dependent cell hemolysis, inhibiting P. falciparum infection. Since the physiological activator of PIEZO1 in circulating RBC is shear stress, we propose that shear stress-induced dehydration, disproportionally affecting RBC of GoF PIEZO1 E756del carriers, makes erythrocytes less habitable for P. falciparum to the point of hemolysis, and thus ameliorates malaria in GoF PIEZO1E756del carriers. More generally, RBC dehydration processes may be a pathway for protection from the severe form of malaria common to several hematological disorders, including sickle cell trait. Key pointsO_LIPIEZO1E756del activation in African American donor RBC provokes dehydration-dependent cell hemolysis, impairing P. falciparum replication. C_LIO_LIRBC dehydration could be a malaria ameliorating factor in several known RBC hematological disorders, including sickle cell trait. C_LI

biophysics↗