19 research outputs found
Chimeric antigen receptors that trigger phagocytosis
Chimeric antigen receptors (CARs) are synthetic receptors that reprogram T cells to kill cancer. The success of CAR-T cell therapies highlights the promise of programmed immunity and suggests that applying CAR strategies to other immune cell lineages may be beneficial. Here, we engineered a family of Chimeric Antigen Receptors for Phagocytosis (CAR-Ps) that direct macrophages to engulf specific targets, including cancer cells. CAR-Ps consist of an extracellular antibody fragment, which can be modified to direct CAR-P activity towards specific antigens. By screening a panel of engulfment receptor intracellular domains, we found that the cytosolic domains from Megf10 and FcRÉ£ robustly triggered engulfment independently of their native extracellular domain. We show that CAR-Ps drive specific engulfment of antigen-coated synthetic particles and whole human cancer cells. Addition of a tandem PI3K recruitment domain increased cancer cell engulfment. Finally, we show that CAR-P expressing murine macrophages reduce cancer cell number in co-culture by over 40%
Mutations in the bacterial cell division protein FtsZ highlight the role of GTP binding and longitudinal subunit interactions in assembly and function
Recommended from our members
The SidC and SidE families of Legionella pneumophila effectors differentially regulate ubiquitination, morphology, and stable capture of Rab5A at the bacterial vacuole surface
Intracellular bacterial pathogens have evolved to survive in a distinctly hostile environment. Intravacuolar pathogens such as Legionella pneumophila (L.p.) reside within a membrane bound compartment in their host cell, reshaping host materials into a new, protected organelle. The molecular mechanisms employed by L.p. during early infection to defend its vacuole are not entirely understood. As host cells uptake the bacterium by phagocytosis, L.p. must avoid classical trafficking of its phagosome through the endolysosomal pathway or attack by autophagy, which would ultimately lead to destruction of the bacterium in the lysosome. In this work, we set out to define how L.p. manipulates host regulatory proteins to defend its vacuole, termed the Legionella-containing vacuole (LCV), from these threats during early infection. First, we explore the interaction of Rab5, the master regulator of the early endosomal compartment which is required for phagosome maturation, with the LCV. We determine that Rab5 associates with the LCV and is both mono- and polyubiquitinated during infection. This ubiquitination is dependent on two families of secreted bacterial proteins, SidC/SdcA and the SidE family of effectors. Finally, we find that SidE family dependent polyubiquitination is necessary for retention of Rab5 at the LCV membrane. Next, we recognized the unique opportunity presented by Rab5, as this host protein localizes to both the WT and avirulent strain LCV. We determine that Rab5 associated with the WT LCV has a distinctive morphology and stability that is dependent on the SidE family of effectors rather than host regulators. Finally, we discover that L.p. infection induces a burst of ubiquitination of both host and bacterial proteins, and that SidC/SdcA play a central role in this burst. We propose a new role of SidC/SdcA as a metaeffector, which regulates the activity of other as yet unknown bacterial proteins. Taken together, our findings suggest a model in which ubiquitin is used by L.p. as a tool to retain and stabilize proteins at the LCV membrane, perhaps playing a role in containment of deleterious host proteins or participating in a physical blockade around the vacuole. This work illustrates the complex interplay between two fascinating families of L.p. effectors, and refines our understanding of the strategies utilized by L.p to protect its vacuole during early infection
Additional file 1: of Mutations in the bacterial cell division protein FtsZ highlight the role of GTP binding and longitudinal subunit interactions in assembly and function
zapA overexpression does not restore colony formation to ftsZ84 cells under nonpermissive conditions. (PDF 807 kb
Recommended from our members
Cross-family small GTPase ubiquitination by the intracellular pathogen Legionella pneumophila
The intracellular bacterial pathogen Legionella pneumophila (L.p.) manipulates eukaryotic host ubiquitination machinery to form its replicative vacuole. While nearly 10% of L.p.'s ∼330 secreted effector proteins are ubiquitin ligases or deubiquitinases, a comprehensive measure of temporally resolved changes in the endogenous host ubiquitinome during infection has not been undertaken. To elucidate how L.p. hijacks host cell ubiquitin signaling, we generated a proteome-wide analysis of changes in protein ubiquitination during infection. We discover that L.p. infection increases ubiquitination of host regulators of subcellular trafficking and membrane dynamics, most notably ∼40% of mammalian Ras superfamily small GTPases. We determine that these small GTPases undergo nondegradative ubiquitination at the Legionella-containing vacuole (LCV) membrane. Finally, we find that the bacterial effectors SidC/SdcA play a central role in cross-family small GTPase ubiquitination, and that these effectors function upstream of SidE family ligases in the polyubiquitination and retention of GTPases in the LCV membrane. This work highlights the extensive reconfiguration of host ubiquitin signaling by bacterial effectors during infection and establishes simultaneous ubiquitination of small GTPases across the Ras superfamily as a novel consequence of L.p. infection. Our findings position L.p. as a tool to better understand how small GTPases can be regulated by ubiquitination in uninfected contexts
Recommended from our members
Chimeric antigen receptors that trigger phagocytosis.
Chimeric antigen receptors (CARs) are synthetic receptors that reprogram T cells to kill cancer. The success of CAR-T cell therapies highlights the promise of programmed immunity and suggests that applying CAR strategies to other immune cell lineages may be beneficial. Here, we engineered a family of Chimeric Antigen Receptors for Phagocytosis (CAR-Ps) that direct macrophages to engulf specific targets, including cancer cells. CAR-Ps consist of an extracellular antibody fragment, which can be modified to direct CAR-P activity towards specific antigens. By screening a panel of engulfment receptor intracellular domains, we found that the cytosolic domains from Megf10 and FcRÉ£ robustly triggered engulfment independently of their native extracellular domain. We show that CAR-Ps drive specific engulfment of antigen-coated synthetic particles and whole human cancer cells. Addition of a tandem PI3K recruitment domain increased cancer cell engulfment. Finally, we show that CAR-P expressing murine macrophages reduce cancer cell number in co-culture by over 40%
Data from: Chimeric antigen receptors that trigger phagocytosis
Chimeric antigen receptors (CARs) are synthetic receptors that reprogram T cells to kill cancer. The success of CAR-T cell therapies highlights the promise of programmed immunity and suggests that applying CAR strategies to other immune cell lineages may be beneficial. Here, we engineered a family of Chimeric Antigen Receptors for Phagocytosis (CAR-Ps) that direct macrophages to engulf specific targets, including cancer cells. CAR-Ps consist of an extracellular antibody fragment, which can be modified to direct CAR-P activity towards specific antigens. By screening a panel of engulfment receptor intracellular domains, we found that the cytosolic domains from Megf10 and FcRÉ£ robustly triggered engulfment independently of their native extracellular domain. We show that CAR-Ps drive specific engulfment of antigen-coated synthetic particles and whole human cancer cells. Addition of a tandem PI3K recruitment domain increased cancer cell engulfment. Finally, we show that
CAR-P expressing murine macrophages reduce cancer cell number in co-culture by over 40%