Colon-targeted S100A8/A9-specific peptide systems ameliorate colitis and colitis-associated colorectal cancer in mouse models.

The link between chronic inflammation and cancer development is well acknowledged. Inflammatory bowel disease including ulcerative colitis and Crohn's disease frequently promotes colon cancer development. Thus, control of intestinal inflammation is a therapeutic strategy to prevent and manage colitis-associated colorectal cancer (CRC). Recently, gut mucosal damage-associated molecular patterns S100A8 and S100A9, acting via interactions with their pattern recognition receptors (PRRs), especially TLR4 and RAGE, have emerged as key players in the pathogenesis of colonic inflammation. We found elevated serum levels of S100A8 and S100A9 in both colitis and colitis-associated CRC mouse models along with significant increases in their binding with PRR, TLR4, and RAGE. In this study we developed a dual PRR-inhibiting peptide system (rCT-S100A8/A9) that consisted of TLR4- and RAGE-inhibiting motifs derived from S100A8 and S100A9, and conjugated with a CT peptide (TWYKIAFQRNRK) for colon-specific delivery. In human monocyte THP-1 and mouse BMDMs, S100A8/A9-derived peptide comprising TLR4- and RAGE-interacting motif (0.01, 0.1, 1 µM) dose-dependently inhibited the binding of S100 to TLR4 or RAGE, and effectively inhibited NLRP3 inflammasome activation. We demonstrated that rCT-S100A8/A9 had appropriate drug-like properties including in vitro stabilities and PK properties as well as pharmacological activities. In mouse models of DSS-induced acute and chronic colitis, injection of rCT-S100A8/A9 (50 µg·kg-1·d-1, i.p. for certain consecutive days) significantly increased the survival rates and alleviated the pathological injuries of the colon. In AOM/DSS-induced colitis-associated colorectal cancer (CAC) mouse model, injection of rCT-S100A8/A9 (50 µg·kg-1·d-1, i.p.) increased the body weight, decreased tumor burden in the distal colon, and significantly alleviated histological colonic damage. In mice bearing oxaliplatin-resistant CRC xenografts, injection of rCT-S100A8/A9 (20 µg/kg, i.p., every 3 days for 24-30 days) significantly inhibited the tumor growth with reduced EMT-associated markers in tumor tissues. Our results demonstrate that targeting the S100-PRR axis improves colonic inflammation and thus highlight this axis as a potential therapeutic target for colitis and CRC.

Tumor-targeted liposomes with platycodin D2 promote apoptosis in colorectal cancer.

Conventional chemotherapy for colorectal cancer (CRC), though efficacious, is discouraging due to its limited targeting capability, lack of selectivity, and chemotherapy-associated side effects. With the advent of nanomedicines, a liposomal delivery system making use of a combination of anticancer phytochemicals is fast gaining popularity as one of the most promising nanoplatforms for CRC treatment. Rising evidence supports phytochemicals such as platycosides for their anticancer potency. To this end, a combination therapy including tumor-targeted liposomes along with phytochemicals might have a greater therapeutic potential against cancer. In this study, we developed acidity-triggered rational membrane (ATRAM) along with conjugated platycodin D2 (PCD2) and liposomes (PCD2-Lipo-ATRAM) as a tumor-targeting therapy. The PCD2-Lipo-ATRAM treatment demonstrated a successful tumor-targeting ability in the CRC xenografts, in which PCD2 not only exerted a potent antitumor effect by inducing apoptotic cell death and but also functioned as a liposome membrane stabilizer. Moreover, PCD2-Lipo-ATRAM suppressed antiapoptotic BCL-2 family proteins, resulting in enhanced cytotoxicity toward CRC cells by inducing intrinsic caspase-9/-3 mediated apoptosis. Thus, our data has shown that tumor-targeting PCD2-based liposomal systems represent a promising strategy for CRC therapy, since they directly target the tumors, unlike other therapies that can miss the target.

Multifaceted Evaluation of Antibiotic Therapy as a Factor Associated with Candidemia in Non-Neutropenic Patients.

We aimed to evaluate various aspects of antibiotic therapy as factors associated with candidemia in non-neutropenic patients. A retrospective, matched, case-control study was conducted in two teaching hospitals. Patients with candidemia (cases) were compared to patients without candidemia (controls), matched by age, intensive care unit admission, duration of hospitalization, and type of surgery. Logistic regression analyses were performed to identify factors associated with candidemia. A total of 246 patients were included in the study. Of 123 candidemia patients, 36% had catheter-related bloodstream infections (CRBSIs). Independent factors in the whole population included immunosuppression (adjusted odds ratio [aOR] = 2.195; p = 0.036), total parenteral nutrition (aOR = 3.642; p < 0.001), and anti-methicillin-resistant S. aureus (MRSA) therapy for ≥11 days (aOR = 5.151; p = 0.004). The antibiotic factor in the non-CRBSI population was anti-pseudomonal beta-lactam treatment duration of ≥3 days (aOR = 5.260; p = 0.008). The antibiotic factors in the CRBSI population included anti-MRSA therapy for ≥11 days (aOR = 10.031; p = 0.019). Antimicrobial stewardship that reduces exposure to these antibacterial spectra could help prevent the development of candidemia.

Inhibition of CD82 improves colitis by increasing NLRP3 deubiquitination by BRCC3.

CD82 is a transmembrane protein that is involved in cancer suppression and activates immune cells; however, information on the NLRP3 inflammasome is limited. Herein, we show that although CD82 suppressed the activation of the NLRP3 inflammasome in vivo and in vitro, CD82 deficiency decreased the severity of colitis in mice. Furthermore, two binding partners of CD82, NLRP3 and BRCC3, were identified. CD82 binding to these partners increased the degradation of NLRP3 by blocking BRCC3-dependent K63-specific deubiquitination. Previous studies have shown that CD82-specific bacteria in the colon microbiota called Bacteroides vulgatus (B. vulgatus) regulated the expression of CD82 and promoted the activation of the NLRP3 inflammasome. Accordingly, we observed that B. vulgatus administration increased mouse survival by mediating CD82 expression and activating NLRP3 in mice with colitis. Overall, this study showed that CD82 suppression reduced the pathogenesis of colitis by elevating the activation of the NLRP3 inflammasome through BRCC3-dependent K63 deubiquitination. Based on our findings, we propose that B. vulgatus is a novel therapeutic candidate for colitis.

Pathogen-derived peptides in drug targeting and its therapeutic approach.

Peptides, short stretches of amino acids or small proteins that occupy a strategic position between proteins and amino acids, are readily accessible by chemical and biological methods. With ideal properties for forming high-affinity and specific interactions with host target proteins, they have established an important niche in the drug development spectrum complementing small molecule and biological therapeutics. Among the most successful biomedicines in use today, peptide-based drugs show great promise. This, coupled with recent advances in synthetic and nanochemical biology, has led to the creation of tailor-made peptide therapeutics for improved biocompatibility. This review presents an overview of the latest research on pathogen-derived, host-cell-interacting peptides. It also highlights strategies for using peptide-based therapeutics that address cellular transport challenges through the introduction of nanoparticles that serve as platforms to facilitate the delivery of peptide biologics and therapeutics for treating various inflammatory diseases. Finally, this paper describes future perspectives, specific pathogen-based peptides that can enhance specificity, efficiency, and capacity in functional peptide-based therapeutics, which are in the spotlight as new treatment alternatives for various diseases, and also presents verified sequences and targets that can increase chemical and pharmacological value.

PE_PGRS38 Interaction With HAUSP Downregulates Antimycobacterial Host Defense via TRAF6.

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.

Discovery of Mycobacterium tuberculosis Rv3364c-Derived Small Molecules as Potential Therapeutic Agents to Target SNX9 for Sepsis.

The serine protease inhibitor Rv3364c of Mycobacterium tuberculosis (MTB) is highly expressed in cells during MTB exposure. In this study, we showed that the 12WLVSKF17 motif of Rv3364c interacts with the BAR domain of SNX9 and inhibits endosome trafficking to interact with p47phox, thereby suppressing TLR4 inflammatory signaling in macrophages. Derived from the structure of this Rv3364c peptide motif, 2,4-diamino-6-(4-tert-butylphenyl)-1,3,5-trazine, DATPT as a 12WLVSKF17 peptide-mimetic small molecule has been identified. DATPT can block the SNX9-p47phox interaction in the endosome and suppress reactive oxygen species and inflammatory cytokine production; it demonstrated significant therapeutic effects in a mouse model of cecal ligation and puncture-induced sepsis. DATPT has considerably improved potency, with an IC50 500-fold (in vitro) or 2000-fold (in vivo) lower than that of the 12WLVSKF17 peptide. Furthermore, DATPT shows potent antibacterial activities by reduction in ATP production and leakage of intracellular ATP out of bacteria. These results provide evidence for peptide-derived small molecule DATPT with anti-inflammatory and antibacterial functions for the treatment of sepsis.

Mito-TIPTP Increases Mitochondrial Function by Repressing the Rubicon-p22phox Interaction in Colitis-Induced Mice.

The run/cysteine-rich-domain-containing Beclin1-interacting autophagy protein (Rubicon) is essential for the regulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase by interacting with p22phox to trigger the production of reactive oxygen species (ROS) in immune cells. In a previous study, we demonstrated that the interaction of Rubicon with p22phox increases cellular ROS levels. The correlation between Rubicon and mitochondrial ROS (mtROS) is poorly understood. Here, we report that Rubicon interacts with p22phox in the outer mitochondrial membrane in macrophages and patients with human ulcerative colitis. Upon lipopolysaccharide (LPS) activation, the binding of Rubicon to p22phox was elevated, and increased not only cellular ROS levels but also mtROS, with an impairment of mitochondrial complex III and mitochondrial biogenesis in macrophages. Furthermore, increased Rubicon decreases mitochondrial metabolic flux in macrophages. Mito-TIPTP, which is a p22phox inhibitor containing a mitochondrial translocation signal, enhances mitochondrial function by inhibiting the association between Rubicon and p22phox in LPS-primed bone-marrow-derived macrophages (BMDMs) treated with adenosine triphosphate (ATP) or dextran sulfate sodium (DSS). Remarkably, Mito-TIPTP exhibited a therapeutic effect by decreasing mtROS in DSS-induced acute or chronic colitis mouse models. Thus, our findings suggest that Mito-TIPTP is a potential therapeutic agent for colitis by inhibiting the interaction between Rubicon and p22phox to recover mitochondrial function.

Procalcitonin as a predictor of early antibiotic treatment failure in patients with gram-negative bloodstream infections caused by urinary tract infections.

We retrospectively evaluated whether initial procalcitonin (PCT) levels can predict early antibiotic treatment failure (ATF) in patients with gram-negative bloodstream infections (GN-BSI) caused by urinary tract infections from January 2018 to November 2019. Early ATF was defined as the following: (1) hemodynamically unstable or febrile at Day 3; (2) the need for mechanical ventilation or continuous renal replacement therapy at Day 3; (3) patients who died within 3 days (date of blood culture: Day 0). The study included 189 patients; 42 showed early ATF. Independent risk factors for early ATF were initial admission to the intensive care unit (odds ratio: 7.735, 95% confidence interval: 2.567-23.311; P < 0.001) and PCT levels ≥30 ng/mL (odds ratio: 5.413, 95% confidence interval: 2.188-13.388; P < 0.001). Antibiotic factors were not associated with early ATF. Initial PCT levels may be helpful to predict early ATF in these patients.

Mycobacterium tuberculosis Rv2626c-derived peptide as a therapeutic agent for sepsis.

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 C-terminal 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 IC50 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.

Toxoplasma gondii GRA9 Regulates the Activation of NLRP3 Inflammasome to Exert Anti-Septic Effects in Mice.

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.

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

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 IC50 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.