Acquisition of Host Cytosolic Protein by Toxoplasma gondii Bradyzoites

Abstract

Toxoplasma gondii is a protozoan parasite that persists in the central nervous system as intracellular chronic-stage bradyzoites that are encapsulated by a thick cyst wall. While the cyst wall separates bradyzoites from the host cytosol, it has been posited that small solutes can traverse the cyst wall to sustain bradyzoites. Recently, it was found that host cytosolic macromolecules can cross the parasitophorous vacuole and are ingested and digested by actively replicating acute-stage tachyzoites. However, the extent to which bradyzoites have an active ingestion pathway remained unknown. To interrogate this, we modified previously published protocols that look at tachyzoite acquisition and digestion of host proteins by measuring parasite accumulation of a host-expressed reporter protein after impairment of an endolysosomal protease (cathepsin protease L [CPL]). Using two cystogenic parasite strains (ME49 and Pru), we demonstrate that T. gondii bradyzoites can ingest host-derived cytosolic mCherry. Bradyzoites acquire host mCherry within 4 h of invasion and after cyst wall formation. This study provides direct evidence that host macromolecules can be internalized by T. gondii bradyzoites across the cyst wall in infected cells. IMPORTANCE Chronic infection of humans with Toxoplasma gondii is common, but little is known about how this intracellular parasite obtains the resources that it needs to persist indefinitely inside neurons and muscle cells. Here, we provide evidence that the chronic-stage form of T. gondii can internalize proteins from the cytosol of infected cells despite residing within an intracellular cyst that is surrounded by a cyst wall. We also show that accumulation of host-derived protein within the chronic-stage parasites is enhanced by disruption of a parasite protease, suggesting that such protein is normally degraded to generate peptides and amino acids. Taken together, our findings imply that chronic-stage T. gondii can ingest and digest host proteins, potentially to support its persistence.

Toxoplasma TgATG9 is critical for autophagy and long-term persistence in tissue cysts

“…We propose two possibilities, that bradyzoites uptake host proteins that are trafficked to the VAC for degradation via the endolysosomal system or that, in the absence of TgATG9, bradyzoites have an alternative mechanism of autophagy. The first hypothesis is supported by our recent work, which demonstrated that bradyzoites can ingest and degrade host-derived cytosolic proteins ( Kannan et al, 2021 ). In the second possibility, as discussed further below, we observed the formation of vesicles with two or more membranes in ∆atg9 bradyzoites by TEM and while we are currently unable to determine the nature of these vesicles, it remains possible that they represent alternative autophagosome-like structures that do not stain well with the cationic amphiphilic dye CytoID.…”

“…However, this also raises the question as to how T. gondii tissue cysts containing bradyzoites are able to persist within the host with potentially limited access to host material for sustenance. Although recent work reported that T. gondii bradyzoites can ingest and digest protein from the cytosol of infected cells in vitro ( Kannan et al, 2021 ), the extent to which this process helps to sustain bradyzoites remains unknown.…”

A Family of Toxoplasma gondii Genes Related to GRA12 Regulate Cyst Burdens and Cyst Reactivation

“…GRA2 and GRA6, through lipid binding (55), form membrane tubules that are involved in heterophagic ingestion of host cell proteins (56), Rab-positive host cell vesicles (57), and Rab7-positive host lipid droplets (20). Acquisition of host cell cargo and digestion are also likely to be essential for chronic infection (58), and recently published evidence supports the hypothesis that encysted bradyzoites have the capability to import host cell proteins and cargo (59).…”

Lessons from Toxoplasma: Host responses that mediate parasite control and the microbial effectors that subvert them

“…While long considered to be inert, it is now recognized that bradyzoites undergo episodic bursts of proliferation, internalize host-derived macromolecules, and export parasite effectors (Kannan et al, 2021;Watts et al, 2015). The cyst wall is an important interface between host and parasite that sits just below the PVM (Guevara et al, 2020;Tu et al, 2020;Young et al, 2020), and not every protein that can escape from the PVM in tachyzoites can cross the cyst wall (Krishnamurthy and Saeij, 2018).…”