Biology subjects

Waldman, B. S.

Publications and source records attributed to Waldman, B. S..

4 recordsLinked to original sources

bioRxiv2023

Functional profiling of the Toxoplasma genome during acute mouse infection

Within a host, pathogens encounter a diverse and changing landscape of cell types, nutrients, and immune responses. Examining host-pathogen interactions in animal models can therefore reveal aspects of infection absent from cell culture. We use CRISPR-based screens to functionally profile the entire genome of the model apicomplexan parasite Toxoplasma gondii during mouse infection. Barcoded gRNAs were used to track mutant parasite lineages, enabling detection of bottlenecks and mapping of population structures. We uncovered over 300 genes that modulate parasite fitness in mice with previously unknown roles in infection. These candidates span multiple axes of host-parasite interaction, including determinants of tropism, host organelle remodeling, and metabolic rewiring. We mechanistically characterized three novel candidates, including GTP cyclohydrolase I, against which a small-molecule inhibitor could be repurposed as an antiparasitic compound. This compound exhibited antiparasitic activity against T. gondii and Plasmodium falciparum, the most lethal agent of malaria. Taken together, we present the first complete survey of an apicomplexan genome during infection of an animal host, and point to novel interfaces of host-parasite interaction that may offer new avenues for treatment.

microbiology↗

bioRxiv2022

A positive feedback loop controls Toxoplasma chronic differentiation

Successful infection strategies must balance pathogen amplification and persistence. In Toxoplasma gondii, this is accomplished through differentiation into dedicated cyst-forming chronic stages that avoid clearance by the host immune system. The transcription factor BFD1 is both necessary and sufficient for stage conversion; however, its regulation is not understood. We examine five factors transcriptionally activated by BFD1. One of these is a cytosolic RNA-binding protein of the CCCH-type zinc finger family, which we name BFD2. Parasites lacking BFD2 fail to induce BFD1 and are consequently unable to fully differentiate in culture or in mice. BFD2 interacts with the BFD1 transcript in a stress-dependent manner. Deletion of BFD2 reduces BFD1 protein levels, but not mRNA abundance. The reciprocal effects on BFD2 transcription and BFD1 translation outline a positive feedback loop that enforces commitment to differentiation. BFD2 helps explain how parasites commit to the chronic gene-expression program and elucidates how the balance between proliferation and persistence is achieved over the course of infection.

microbiology↗

bioRxiv2021

Toxoplasma gondii subverts the host ESCRT machinery for parasite uptake of host cytosolic proteins

Toxoplasma gondii is a master manipulator capable of effectively siphoning the resources from the host cell for its intracellular subsistence. However, the molecular underpinnings of how the parasite gains resources from its host remain largely unknown. Residing within a non-fusogenic parasitophorous vacuole, the parasite must acquire resources across the limiting membrane of its replicative niche, which is decorated with parasite proteins including those secreted from dense granules. We discovered a role for the Endosomal Sorting Complex Required for Transport (ESCRT) machinery in host cytosolic protein uptake by T. gondii by disrupting host ESCRT function. We identified the transmembrane dense granule protein TgGRA14, which contains motifs homologous to the late domain motifs of HIV-1 Gag, as a candidate for the recruitment of the host ESCRT machinery to the PV membrane. Using an HIV virus-like particle (VLP) release assay, we found that the motif-containing portion of TgGRA14 is sufficient to substitute for HIV Gag late domain to mediate ESCRT-dependent VLP budding. We also show that TgGRA14 is proximal to and interacts with host ESCRT components and other dense granule proteins during infection. Furthermore, analysis of GRA14-deficient parasites revealed a marked reduction in ingestion of a host cytosolic protein compared to WT parasites. Thus, we propose a model in which T. gondii recruits the host ESCRT machinery to the PV where it can interact with TgGRA14 for the internalization of host cytosolic proteins across the PVM. These findings provide new insight into how T. gondii accesses contents of the host cytosol by exploiting a key pathway for vesicular budding and membrane scission. Author summaryIntracellular pathogens exploit their host to gain the resources necessary to sustain infection; however, precisely how the intracellular parasite Toxoplasma gondii acquires essential nutrients from its host remains poorly understood. Previous work showed that T. gondii is capable of internalizing host derived cytosolic proteins and delivering them to its lysosome-like compartment for degradation. However, the mechanism by which the material is trafficked across the membrane delimiting the replicative vacuole in which the parasite resides remained unclear. Here, we report a role for the parasite effector protein TgGRA14 in the recruitment of the host ESCRT machinery for the uptake of host cytosolic proteins. Important human pathogens have developed strategies for exploiting the host ESCRT machinery for intracellular subsistence. Our study sheds lights on the strategy used by a eukaryotic pathogen in subverting the host ESCRT machinery for the internalization of resources from its host cells.

microbiology↗

bioRxiv2019

Identification of a master regulator of differentiation in Toxoplasma

Toxoplasma gondii chronically infects a quarter of the worlds population, and its recrudescence can cause life-threatening disease in immunocompromised individuals and recurrent ocular lesions in the immunocompetent. Chronic stages are established by differentiation of rapidly replicating tachyzoites into slow-growing bradyzoites, which form intracellular cysts resistant to immune clearance and existing therapies. Despite its central role in infection, the molecular basis of chronic differentiation is not understood. Through Cas9-mediated genetic screening and single-cell transcriptional profiling, we identify and characterize a putative transcription factor (BFD1) as necessary and sufficient for differentiation. Translation of BFD1 appears to be stress regulated, and its constitutive expression elicits differentiation in the absence of stress. As a Myb-like factor, BFD1 provides a counterpoint to the ApiAP2 factors which dominate our current view of parasite gene regulation. Overall, BFD1 provides a genetic switch to study and control Toxoplasma differentiation, and will inform prevention and treatment of chronic infection.

microbiology↗