Toxoplasma gondii GRA7-Targeted ASC and PLD1 Promote Antibacterial Host Defense via PKCα

Abstract

Tuberculosis is a global health problem and at least one-third of the world’s population is infected with Mycobacterium tuberculosis (MTB). MTB is a successful pathogen that enhances its own intracellular survival by inhibiting inflammation and arresting phago-lysosomal fusion. We previously demonstrated that Toxoplasma gondii (T. gondii) dense granule antigen (GRA) 7 interacts with TNF receptor-associated factor 6 via Myeloid differentiation primary response gene 88, enabling innate immune responses in macrophages. To extend these studies, we found that GRA7 interacts with host proteins involved in antimicrobial host defense mechanisms as a therapeutic strategy for tuberculosis. Here, we show that protein kinase C (PKC)α-mediated phosphorylation of T. gondii GRA7-I (Ser52) regulates the interaction of GRA7 with PYD domain of apoptosis-associated speck-like protein containing a carboxy-terminal CARD, which is capable of oligomerization and inflammasome activation can lead to antimicrobial defense against MTB. Furthermore, GRA7-III interacted with the PX domain of phospholipase D1, facilitating its enzyme activity, phago-lysosomal maturation, and subsequent antimicrobial activity in a GRA7-III (Ser135) phosphorylation-dependent manner via PKCα. Taken together, these results underscore a previously unrecognized role of GRA7 in modulating antimicrobial host defense mechanism during mycobacterial infection.

Toxoplasma gondii GRA8-derived peptide immunotherapy improves tumor targeting of colorectal cancer

“…Thus, we designed the ATRAM-conjugated multifunctional GRA8 peptide for targeting tumors and activating mitochondrial metabolism ( Figure 2C). To assess the function of the ATRAM-conjugated multifunctional GRA8 peptide in cancer cells, we produced His-tagged rATRAM-GRA8-M/AS and its mutants in bacteria and purified by affinity chromatography as previously described [8,19]. The purified rATRAM-GRA8-M/AS (10 kDa) was confirmed by SDS-PAGE and immunoblotting ( Figure 2D).…”

“…Wild-type C57BL/6 and BALB/c mice were purchased from Orient Bio (Gyeonggi-do, Korea). Primary bone marrow-derived macrophages (BMDMs) were isolated from C57BL/6 mice and cultured in DMEM for 3-5 d in the presence of M-CSF (R&D Systems, 416-ML), as described previously [19]. HEK293T cells (ATCC-11268; American Type Culture Collection), human colon cancer cell line HCT116 (ATCC-CCL247), HT-29 (ATCC-HTB-38), HepG2 (ATCC-HB-8065), Hep3B (ATCC-HB-8064), MCF7 (ATCC-HTB-22), and MDA-MB-231 (ATCC-HTB-26) were maintained in DMEM (Invitrogen) containing 10% FBS (Invitrogen), sodium pyruvate, nonessential amino acids, penicillin G (100 IU/ml), and streptomycin (100 μg/ml).…”

“…(ATATTTTTTR), and ATP5A1/SIRT3 binding seq. (YRFTVQTTQN) were cloned with an N-terminal 6xHis tag into the pRSFDuet-1 Vector (Novagen) and induced, harvested, and purified from Escherichia coli expression strain BL21(DE3) pLysS as described previously [8,19], in accordance with the standard protocols recommended by Novagen. rGRA8 was dialyzed with permeable cellulose membrane and tested for lipopolysaccharide contamination with a Limulus amebocyte lysate assay (BioWhittaker) and contained < 20 pg/ml at the concentrations of rGRA7 protein used in the experiments described here.…”

“…GST pulldown, immunoprecipitation, and immunoblot assays were performed as described previously [8,19]. For GST pulldown, cells were harvested and lysed in NP-40 buffer supplemented with a complete protease inhibitor cocktail (Roche).…”

“…In addition, enhanced selectivity has the potential to lower toxicity and side effects. Inasmuch as it is possible to design peptides to affect a wide range of targets, there are numerous possibilities of applying peptide therapeutics in several fields including endocrinology, immunology, oncology, and infectious disease [8,18,19]. In this study, we built upon the discoveries of the minimal peptide moieties for mitochondrial targeting and ATP5A1/SIRT3 binding, previously reported to be important in therapeutic approaches [8].…”