The Rv2626c protein of Mycobacterium tuberculosis is a promising vaccine candidate owing to its strong serum antibody response in patients with tuberculosis. However, there is limited information regarding the intracellular response induced by Rv2626c in macrophages. In this study, we demonstrated that Rv2626c interacts with the RING domain of TRAF6 and inhibits lysine (K) 63-linked polyubiquitination of TRAF6 (E3 ubiquitin ligase activity); this results in the suppression of TLR4 inflammatory signaling in macrophages. Furthermore, we showed that the C-terminal 123-131-amino acid Rv2626c motif promotes macrophage recruitment, phagocytosis, M2 macrophage polarization, and subsequent bacterial clearance. We developed rRv2626c-CA, a conjugated peptide containing the Cterminal 123-131-amino acid Rv2626c that targets macrophages, penetrates the cell membrane, and has demonstrated significant therapeutic effects in a mouse model of cecal ligation and puncture-induced sepsis. This multifunctional rRv2626c-CA has considerably improved potency, with an IC 50 that is 250-fold (in vitro) or 1,000-fold (in vivo) lower than that of rRv2626c-WT. We provide evidence for new peptide-based drugs with anti-inflammatory and antibacterial properties for the treatment of sepsis.

Targeted tumor and efficient, specific biological drug delivery in vivo has been one of the main challenges in protein-based cancer-targeted therapies. Mitochondria are potential therapeutic targets for various anti-cancer drugs. We have previously reported that protein kinase Cα-mediated phosphorylation of Toxoplasma gondii GRA8 is required for mitochondrial trafficking and regulating the interaction of the C-terminal of GRA8 with ATP5A1/SIRT3 in mitochondria. Furthermore, SIRT3 facilitates ATP5A1 deacetylation, mitochondrial activation, and subsequent antiseptic activity in vivo. Herein we developed a recombinant acidity-triggered rational membrane (ATRAM)-conjugated multifunctional GRA8 peptide (rATRAM-G8-M/AS) comprising ATRAM as the cancer-targeting cell-penetrating peptide, and essential/ minimal residues for mitochondrial targeting or ATP5A1/SIRT3 binding. This peptide construct showed considerably improved potency about cancer cell death via mitochondria activity and biogenesis compared with rGRA8 alone in HCT116 human carcinoma cells, reaching an IC 50 value of up to 200-fold lower in vitro and 500-fold lower in vivo. Notably, rATRAM-G8-M/AS treatment showed significant therapeutic effects in a mouse xenograft model through mitochondrial metabolic resuscitation, and it produced negligible immunogenicity and immune responses in vivo. Thus, these results demonstrate that rATRAM-G8-M/AS represents a useful therapeutic strategy against tumors, particularly colon cancer. This strategy represents an urgently needed paradigm shift for therapeutic intervention.

Dense granule proteins (GRAs) are essential components in Toxoplasma gondii, which are suggested to be promising serodiagnostic markers in toxoplasmosis. In this study, we investigated the function of GRA9 in host response and the associated regulatory mechanism, which were unknown. We found that GRA9 interacts with NLR family pyrin domain containing 3 (NLRP3) involved in inflammation by forming the NLRP3 inflammasome. The C-terminal of GRA9 (GRA9C) is essential for GRA9–NLRP3 interaction by disrupting the NLRP3 inflammasome through blocking the binding of apoptotic speck-containing (ASC)-NLRP3. Notably, Q200 of GRA9C is essential for the interaction of NLRP3 and blocking the conjugation of ASC. Recombinant GRA9C (rGRA9C) showed an anti-inflammatory effect and the elimination of bacteria by converting M1 to M2 macrophages. In vivo, rGRA9C increased the anti-inflammatory and bactericidal effects and subsequent anti-septic activity in CLP- and E. coli- or P. aeruginosa-induced sepsis model mice by increasing M2 polarization. Taken together, our findings defined a role of T. gondii GRA9 associated with NLRP3 in host macrophages, suggesting its potential as a new candidate therapeutic agent for sepsis.

Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB), avoids the host immune system through its virulence factors. MPT63 and MPT64 are the virulence factors secreted by MTB which regulate host proteins for the survival and proliferation of MTB in the host. Here, we found that MPT63 bound directly with TBK1 and p47phox, whereas MPT64 interacted with TBK1 and HK2. We constructed a MPT63/64-derived multifunctional recombinant protein (rMPT) that was able to interact with TBK1, p47phox, or HK2. rMPT was shown to regulate IFN-β levels and increase inflammation and concentration of reactive oxygen species (ROS), while targeting macrophages and killing MTB, both in vitro and in vivo. Furthermore, the identification of the role of rMPT against MTB was achieved via vaccination in a mouse model. Taken together, we here present rMPT, which, by regulating important immune signaling systems, can be considered an effective vaccine or therapeutic agent against MTB.

Mycobacterium tuberculosis (Mtb) is the causative pathogen of tuberculosis (TB), which manipulates the host immunity to ensure survival and colonization in the host. Mtb possess a unique family of proteins, named PE_PGRS, associated with Mtb pathogenesis. Thus, elucidation of the functions of PE_PGRS proteins is necessary to understand TB pathogenesis. Here, we investigated the role of PE_PGRS38 binding to herpesvirus-associated ubiquitin-specific protease (HAUSP, USP7) in regulating the activity of various substrate proteins by modulating their state of ubiquitination. We constructed the recombinant PE_PGRS38 expressed in M. smegmatis (Ms_PE_PGRS38) to investigate the role of PE_PGRS38. We found that Ms_PE_PGRS38 regulated the cytokine levels in murine bone marrow-derived macrophages by inhibiting the deubiquitination of tumor necrosis factor receptor-associated factor (TRAF) 6 by HAUSP. Furthermore, the PE domain in PE_PGRS38 was identified as essential for mediating TRAF6 deubiquitination. Ms_PE_PGRS38 increased the intracellular burden of bacteria by manipulating cytokine levels in vitro and in vivo. Overall, we revealed that the interplay between HAUSP and PE_PGRS38 regulated the inflammatory response to increase the survival of mycobacteria.