TBK-1 promotes autophagy-mediated antimicrobial defense by controlling autophagosome maturation.

Autophagy is a fundamental biological process of the eukaryotic cell contributing to diverse cellular and physiological functions including cell-autonomous defense against intracellular pathogens. Here, we screened the Rab family of membrane trafficking regulators for effects on autophagic elimination of Mycobacterium tuberculosis var. bovis BCG and found that Rab8b and its downstream interacting partner, innate immunity regulator TBK-1, are required for autophagic elimination of mycobacteria in macrophages. TBK-1 was necessary for autophagic maturation. TBK-1 coordinated assembly and function of the autophagic machinery and phosphorylated the autophagic adaptor p62 (sequestosome 1) on Ser-403, a residue essential for its role in autophagic clearance. A key proinflammatory cytokine, IL-1ß, induced autophagy leading to autophagic killing of mycobacteria in macrophages, and this IL-1ß activity was dependent on TBK-1. Thus, TBK-1 is a key regulator of immunological autophagy and is responsible for the maturation of autophagosomes into lytic bactericidal organelles.

Deficit of female sex hormones desensitizes rat cardiac mitophagy.

Long-term deprivation of female sex hormones has been shown to mediate accumulation of damaged mitochondria in ventricular muscle leading to cardiovascular dysfunction. Therefore, the roles of female sex hormones in mitochondrial quality control are closely focused. In the present study, depletion of female sex hormones impairing mitochondrial autophagy in the heart was hypothesized. Cardiac mitophagy was therefore investigated in the heart of 10-week ovariectomized (OVX) and sham-operated (SHAM) rats. By using isolated mitochondria preparation, results demonstrated an increase in mitochondrial PTEN-induced kinase 1 accumulation in the sample of OVX rats indicating mitochondrial outer membrane dysfunction. However, no change in p62 and LC3-II translocation to mitochondria was observed between two groups indicating unresponsiveness of mitophagosome formation in the OVX rat heart. This loss might be resulted from significant decreases in Parkin and Bcl2l13 expression, but not Bnip3 activation. In summary, results suggest that mitochondrial abnormality in the heart after deprivation of female sex hormones could consequently be due to desensitization of mitophagy process.

Caspase-4 Mediates Restriction of Burkholderia pseudomallei in Human Alveolar Epithelial Cells.

Melioidosis is an infectious disease with a high mortality rate responsible for community-acquired sepsis in Southeast Asia and Northern Australia. The causative agent of this disease is Burkholderia pseudomallei, a Gram-negative bacterium that resides in soil and contaminated natural water. After entering into host cells, the bacteria escape into the cytoplasm, which has numerous cytosolic sensors, including the noncanonical inflammatory caspases. Although the noncanonical inflammasome (caspase-11) has been investigated in a murine model of B. pseudomallei infection, its role in humans, particularly in lung epithelial cells, remains unknown. We, therefore, investigated the function of caspase-4 (ortholog of murine caspase-11) in intracellular killing of B. pseudomallei The results showed that B. pseudomallei induced caspase-4 activation at 12 h postinfection in human alveolar epithelial A549 cells. The number of intracellular B. pseudomallei bacteria was increased in the absence of caspase-4, suggesting its function in intracellular bacterial restriction. In contrast, a high level of caspase-4 processing was observed when cells were infected with lipopolysaccharide (LPS) mutant B. pseudomallei The enhanced bacterial clearance in LPS-mutant-infected cells is also correlated with a higher degree of caspase-4 activation. These results highlight the susceptibility of the LPS mutant to caspase-4-mediated intracellular bacterial killing.

Delivery of cytosolic components by autophagic adaptor protein p62 endows autophagosomes with unique antimicrobial properties.

Autophagy allows cells to self-digest portions of their own cytoplasm for a multitude of physiological purposes, including innate and adaptive immunity functions. In one of its innate immunity manifestations, autophagy, is known to contribute to the killing of intracellular microbes, including Mycobacterium tuberculosis, although the molecular mechanisms have been unclear. Here, we delineated sequential steps of the autophagic pathway necessary to control intracellular M. tuberculosis and found that in addition to autophagy initiation and maturation, an accessory autophagy-targeting molecule p62 (A170 or SQSTM1) was required for mycobactericidal activity. The p62 adaptor protein delivered specific ribosomal and bulk ubiquitinated cytosolic proteins to autolysosomes where they were proteolytically converted into products capable of killing M. tuberculosis. Thus, p62 brings cytosolic proteins to autolysosomes where they are processed from innocuous precursors into neo-antimicrobial peptides, explaining in part the unique bactericidal properties of autophagic organelles.

LAP-like process as an immune mechanism downstream of IFN-γ in control of the human malaria Plasmodium vivax liver stage.

IFN-γ is a major regulator of immune functions and has been shown to induce liver-stage Plasmodium elimination both in vitro and in vivo. The molecular mechanism responsible for the restriction of liver-stage Plasmodium downstream of IFN-γ remains uncertain, however. Autophagy, a newly described immune defense mechanism, was recently identified as a downstream pathway activated in response to IFN-γ in the control of intracellular infections. We thus hypothesized that the killing of liver-stage malarial parasites by IFN-γ involves autophagy induction. Our results show that whereas IFN-γ treatment of human hepatocytes activates autophagy, the IFN-γ-mediated restriction of liver-stage Plasmodium vivax depends only on the downstream autophagy-related proteins Beclin 1, PI3K, and ATG5, but not on the upstream autophagy-initiating protein ULK1. In addition, IFN-γ enhanced the recruitment of LC3 onto the parasitophorous vacuole membrane (PVM) and increased the colocalization of lysosomal vesicles with P. vivax compartments. Taken together, these data indicate that IFN-γ mediates the control of liver-stage P. vivax by inducing a noncanonical autophagy pathway resembling that of LC3-associated phagocytosis, in which direct decoration of the PVM with LC3 promotes the fusion of P. vivax compartments with lysosomes and subsequent killing of the pathogen. Understanding the hepatocyte response to IFN-γ during Plasmodium infection and the roles of autophagy-related proteins may provide an urgently needed alternative strategy for the elimination of this human malaria.

Synthesis and Immunological Studies of the Lipomannan Backbone Glycans Found on the Surface of Mycobacterium tuberculosis.

Investigations into novel bacterial drug targets and vaccines are necessary to overcome tuberculosis. Lipomannan (LM), found on the surface of Mycobacterium tuberculosis (Mtb), is actively involved in the pathogenesis and survival of Mtb. Here, we report for the first time a rapid synthesis and biological activities of an LM glycan backbone, α(1-6)mannans. The rapid synthesis is achieved via a regio- and stereoselective ring opening polymerization to generate multiple glycosidic bonds in one simple chemical step, allowing us to finish assembling the defined polysaccharides of 5-20 units within days rather than years. Within the same pot, the polymerization is terminated by a thiol-linker to serve as a conjugation point to carrier proteins and surfaces for immunological experiments. The synthetic glycans are found to have adjuvant activities in vivo. The interactions with DC-SIGN demonstrated the significance of α(1-6)mannan motif present in LM structure. Moreover, surface plasmon resonance (SPR) showed that longer chain of synthetic α(1-6)mannans gain better lectin's binding affinity. The chemically defined components of the bacterial envelope serve as important tools to reveal the interactions of Mtb with mammalian hosts and facilitate the determination of the immunologically active molecular components.

Differentiation in pyroptosis induction by Burkholderia pseudomallei and Burkholderia thailandensis in primary human monocytes, a possible cause of sepsis in acute melioidosis patients.

Melioidosis caused by Burkholderia pseudomallei is an infectious disease with a high mortality rate. In acute melioidosis, sepsis is a major cause of death among patients. Once the bacterium enters the bloodstream, immune system dysregulation ensues, leading to cytokine storms. In contrast to B. pseudomallei, a closely related but non-virulent strain B. thailandensis has rarely been reported to cause cytokine storms or death in patients. However, the mechanisms in which the virulent B. pseudomallei causes sepsis are not fully elucidated. It is well-documented that monocytes play an essential role in cytokine production in the bloodstream. The present study, therefore, determined whether there is a difference in the innate immune response to B. pseudomallei and B. thailandensis during infection of primary human monocytes and THP-1 monocytic cells by investigating pyroptosis, an inflammatory death pathway known to play a pivotal role in sepsis. Our results showed that although both bacterial species exhibited a similar ability to invade human monocytes, only B. pseudomallei can significantly increase the release of cytosolic enzyme lactate dehydrogenase (LDH) as well as the increases in caspase-1 and gasdermin D activations in both cell types. The results were consistent with the significant increase in IL-1ß and IL-18 production, key cytokines involved in pyroptosis. Interestingly, there was no significant difference in other cytokine secretion, such as IL-1RA, IL-10, IL-12p70, IL-15, IL-8, and IL-23 in cells infected by both bacterial species. Furthermore, we also demonstrated that ROS production played a crucial role in controlling pyroptosis activation during B. pseudomallei infection in primary human monocytes. These findings suggested that pyroptosis induced by B. pseudomallei in the human monocytes may contribute to the pathogenesis of sepsis in acute melioidosis patients.

Therapeutic Implications of Ceritinib in Cholangiocarcinoma beyond ALK Expression and Mutation.

Cholangiocarcinoma (CCA) is a difficult-to-treat cancer, with limited therapeutic options and surgery being the only curative treatment. Standard chemotherapy involves gemcitabine-based therapies combined with cisplatin, oxaliplatin, capecitabine, or 5-FU with a dismal prognosis for most patients. Receptor tyrosine kinases (RTKs) are aberrantly expressed in CCAs encompassing potential therapeutic opportunity. Hence, 112 RTK inhibitors were screened in KKU-M213 cells, and ceritinib, an approved targeted therapy for ALK-fusion gene driven cancers, was the most potent candidate. Ceritinib's cytotoxicity in CCA was assessed using MTT and clonogenic assays, along with immunofluorescence, western blot, and qRT-PCR techniques to analyze gene expression and signaling changes. Furthermore, the drug interaction relationship between ceritinib and cisplatin was determined using a ZIP synergy score. Additionally, spheroid and xenograft models were employed to investigate the efficacy of ceritinib in vivo. Our study revealed that ceritinib effectively killed CCA cells at clinically relevant plasma concentrations, irrespective of ALK expression or mutation status. Ceritinib modulated multiple signaling pathways leading to the inhibition of the PI3K/Akt/mTOR pathway and activated both apoptosis and autophagy. Additionally, ceritinib and cisplatin synergistically reduced CCA cell viability. Our data show ceritinib as an effective treatment of CCA, which could be potentially explored in the other cancer types without ALK mutations.

Autophagy and pattern recognition receptors in innate immunity.

Autophagy is a physiologically and immunologically controlled intracellular homeostatic pathway that sequesters and degrades cytoplasmic targets including macromolecular aggregates, cellular organelles such as mitochondria, and whole microbes or their products. Recent advances show that autophagy plays a role in innate immunity in several ways: (i) direct elimination of intracellular microbes by digestion in autolysosomes, (ii) delivery of cytosolic microbial products to pattern recognition receptors (PRRs) in a process referred to as topological inversion, and (iii) as an anti-microbial effector of Toll-like receptors and other PRR signaling. Autophagy eliminates pathogens in vitro and in vivo but, when aberrant due to mutations, contributes to human inflammatory disorders such as Crohn's disease. In this review, we examine these relationships and propose that autophagy is one of the most ancient innate immune defenses that has possibly evolved at the time of alpha-protobacteria-pre-eukaryote relationships, leading up to modern eukaryotic cell-mitochondrial symbiosis, and that during the metazoan evolution, additional layers of immunological regulation have been superimposed and integrated with this primordial innate immunity mechanism.

BORC complex specific components and Kinesin-1 mediate autophagy evasion by the autophagy-resistant Mycobacterium tuberculosis Beijing strain.

Autophagy induction by starvation has been shown to enhance lysosomal delivery to mycobacterial phagosomes, resulting in the restriction of the Mycobacterium tuberculosis reference strain H37Rv. In contrast to H37Rv, our previous study showed that strains belonging to the notorious M. tuberculosis Beijing genotype could evade autophagic elimination. Our recent RNA-Seq analysis also discovered that the autophagy-resistant M. tuberculosis Beijing strain (BJN) evaded autophagic control by upregulating the expression of Kxd1, a BORC complex component, and Plekhm2, both of which function in lysosome positioning towards the cell periphery in host macrophages, thereby suppressing enhanced lysosomal delivery to its phagosome and sparing the BJN from elimination as a result. In this work, we further characterised the other specific components of the BORC complex, BORC5-8, and Kinesin proteins in autophagy resistance by the BJN. Depletion of BORCS5-8 and Kinesin-1, but not Kinesin-3, reverted autophagy avoidance by the BJN, resulting in increased lysosomal delivery to the BJN phagosomes. In addition, the augmented lysosome relocation towards the perinuclear region could now be observed in the BJN-infected host cells depleted in BORCS5-8 and Kinesin-1 expressions. Taken together, the data uncovered new roles for BORCS5-8 and Kinesin-1 in autophagy evasion by the BJN.

Novel Potent Autophagy Inhibitor Ka-003 Inhibits Dengue Virus Replication.

Every year, dengue virus (DENV) affects millions of people. Currently, there are no approved drugs for the treatment of DENV infection. Autophagy is a conserved degradation process that was shown to be induced by DENV infection and required for optimal DENV replication. The modulation of autophagy is, therefore, considered an attractive target to treat DENV infection. This study carried out a high-content image screen analysis using Crispr-Cas9 GFP-LC3 knocked-in HeLa cells of a compound library synthesized from or inspired by natural products and their biocongener precursors to discover novel autophagy inhibitors. The screen identified Ka-003 as the most effective compound for decreasing the number of autophagic vacuoles inside cells upon autophagy induction. Ka-003 could inhibit autophagy in a dose-dependent manner at low micromolar concentrations. More importantly, Ka-003 demonstrated the concentration-dependent inhibition of DENV production in Crispr-Cas9 GFP-LC3 knocked-in THP-1 monocytes. The core structure of Ka-003, which is a methyl cyclohexene derivative, resembles those found in mulberry plants, and could be synthetically prepared in a bioinspired fashion. Taken together, data indicate that Ka-003 hampered autophagy and limited DENV replication. The low cytotoxicity of Ka-003 suggests its therapeutic potential, which warrants further studies for the lead optimization of the compound for dengue treatment.

ECDD-S16 targets vacuolar ATPase: A potential inhibitor compound for pyroptosis-induced inflammation.

BACKGROUND: Cleistanthin A (CA), extracted from Phyllanthus taxodiifolius Beille, was previously reported as a potential V-ATPase inhibitor relevant to cancer cell survival. In the present study, ECDD-S16, a derivative of cleistanthin A, was investigated and found to interfere with pyroptosis induction via V-ATPase inhibition. OBJECTIVE: This study examined the ability of ECDD-S16 to inhibit endolysosome acidification leading to the attenuation of pyroptosis in Raw264.7 macrophages activated by both surface and endosomal TLR ligands. METHODS: To elucidate the activity of ECDD-S16 on pyroptosis-induced inflammation, Raw264.7 cells were pretreated with the compound before stimulation with surface and endosomal TLR ligands. The release of lactate dehydrogenase (LDH) was determined by LDH assay. Additionally, the production of cytokines and the expression of pyroptosis markers were examined by ELISA and immunoblotting. Moreover, molecular docking was performed to demonstrate the binding of ECDD-S16 to the vacuolar (V-)ATPase. RESULTS: This study showed that ECDD-S16 could inhibit pyroptosis in Raw264.7 cells activated with surface and endosomal TLR ligands. The attenuation of pyroptosis by ECDD-S16 was due to the impairment of endosome acidification, which also led to decreased Reactive Oxygen Species (ROS) production. Furthermore, molecular docking also showed the possibility of inhibiting endosome acidification by the binding of ECDD-S16 to the vacuolar (V-)ATPase in the region of V0. CONCLUSION: Our findings indicate the potential of ECDD-S16 for inhibiting pyroptosis and prove that vacuolar H+ ATPase is essential for pyroptosis induced by TLR ligands.

A rapid and scalable density gradient purification method for Plasmodium sporozoites.

BACKGROUND: Malaria remains a major human health problem, with no licensed vaccine currently available. Malaria infections initiate when infectious Plasmodium sporozoites are transmitted by Anopheline mosquitoes during their blood meal. Investigations of the malaria sporozoite are, therefore, of clear medical importance. However, sporozoites can only be produced in and isolated from mosquitoes, and their isolation results in large amounts of accompanying mosquito debris and contaminating microbes. METHODS: Here is described a discontinuous density gradient purification method for Plasmodium sporozoites that maintains parasite infectivity in vitro and in vivo and greatly reduces mosquito and microbial contaminants. RESULTS: This method provides clear advantages over previous approaches: it is rapid, requires no serum components, and can be scaled to purify >107 sporozoites with minimal operator involvement. Moreover, it can be effectively applied to both human (Plasmodium falciparum, Plasmodium vivax) and rodent (Plasmodium yoelii) infective species with excellent recovery rates. CONCLUSIONS: This novel method effectively purifies viable malaria sporozoites by greatly reducing contaminating mosquito debris and microbial burdens associated with parasite isolation. Large-scale preparations of purified sporozoites will allow for enhanced in vitro infections, proteomics, and biochemical characterizations. In conjunction with aseptic mosquito rearing techniques, this purification technique will also support production of live attenuated sporozoites for vaccination.

TLR9 Negatively Regulates Intracellular Bacterial Killing by Pyroptosis in Burkholderia pseudomallei-Infected Mouse Macrophage Cell Line (Raw264.7).

Melioidosis is a serious infectious disease caused by Burkholderia pseudomallei. This bacterium is able to survive and multiply inside the immune cells such as macrophages. It is well established that Toll-like receptors (TLRs), particularly surface TLRs such as TLR2, TLR4, and TLR5, play an essential role in defending against this bacterial infection. However, the involvement of endosomal TLRs in the infection has not been elucidated. In this study, we demonstrated that the number of intracellular bacteria is reduced in TLR9-depleted RAW264.7 cells infected with B. pseudomallei, suggesting that TLR9 is involved in intracellular bacterial killing in macrophages. As several reports have previously demonstrated that pyroptosis is essential for restricting intracellular bacterial killing, particularly in B. pseudomallei infection, we also observed an increased release of cytosolic enzyme lactate dehydrogenase (LDH) in TLR9-depleted cells infected with B. pseudomallei, suggesting TLR9 involvement in pyroptosis in this context. Consistently, the increases in caspase-11 and gasdermind D (GSDMD) activations, which are responsible for the LDH release, were also detected. Moreover, we demonstrated that the increases in pyroptosis and bacterial killing in B. pseudomallei-infected TLR9-depleted cells were due to the augmentation of the IFN-ß, one of the key cytokines known to regulate caspase-11. Altogether, this finding showed that TLR9 suppresses macrophage killing of B. pseudomallei by regulating pyroptosis. This information provides a novel mechanism of TLR9 in the regulation of intracellular bacterial killing by macrophages, which could potentially be leveraged for therapeutic intervention. IMPORTANCE Surface TLRs have been well established to play an essential role in Burkholderia pseudomallei infection. However, the role of endosomal TLRs has not been elucidated. In the present study, we demonstrated that TLR9 plays a crucial role by negatively regulating cytokine production, particularly IFN-ß, a vital cytokine to control pyroptosis via caspase-11 activation. By depletion of TLR9, the percentage of pyroptosis was significantly increased, leading to suppression of intracellular survival in B. pseudomallei-infected macrophages. These findings provide a new role of TLR9 in macrophages.

Host cell transcriptomic response to the multidrug-resistant Mycobacterium tuberculosis clonal outbreak Beijing strain reveals its pathogenic features.

The upsurge of multidrug-resistant infections has rendered tuberculosis the principal cause of death among infectious diseases. A clonal outbreak multidrug-resistant triggering strain of Mycobacterium tuberculosis was identified in Kanchanaburi Province, labelled "MKR superspreader," which was found to subsequently spread to other regions, as revealed by prior epidemiological reports in Thailand. Herein, we showed that the MKR displayed a higher growth rate upon infection into host macrophages in comparison with the H37Rv reference strain. To further elucidate MKR's biology, we utilized RNA-Seq and differential gene expression analyses to identify host factors involved in the intracellular viability of the MKR. A set of host genes function in the cellular response to lipid pathway was found to be uniquely up-regulated in host macrophages infected with the MKR, but not those infected with H37Rv. Within this set of genes, the IL-36 cytokines which regulate host cell cholesterol metabolism and resistance against mycobacteria attracted our interest, as our previous study revealed that the MKR elevated genes associated with cholesterol breakdown during its growth inside host macrophages. Indeed, when comparing macrophages infected with the MKR to H37Rv-infected cells, our RNA-Seq data showed that the expression ratio of IL-36RN, the negative regulator of the IL-36 pathway, to that of IL-36G was greater in macrophages infected with the MKR. Furthermore, the MKR's intracellular survival and increased intracellular cholesterol level in the MKR-infected macrophages were diminished with decreased IL-36RN expression. Overall, our results indicated that IL-36RN could serve as a new target against this emerging multidrug-resistant M. tuberculosis strain.

The autophagy-resistant Mycobacterium tuberculosis Beijing strain upregulates KatG to evade starvation-induced autophagic restriction.

Mycobacterium tuberculosis utilizes several mechanisms to block phagosome-lysosome fusion to evade host cell restriction. However, induction of host cell autophagy by starvation was shown to overcome this block, resulting in enhanced lysosomal delivery to mycobacterial phagosomes and the killing of the M. tuberculosis reference strain H37Rv. Nevertheless, our previous studies found that strains belonging to the M. tuberculosis Beijing genotype can resist starvation-induced autophagic elimination, though the mycobacterial factors involved remain unclear. In this study, we showed that KatG expression is upregulated in the autophagy-resistant M. tuberculosis Beijing strain (BJN) during autophagy induction by the starvation of host macrophages, while such increase was not observed in the H37Rv. KatG depletion using the CRISPR-dCas9 interference system in the BJN resulted in increased lysosomal delivery to its phagosome and decreased its survival upon autophagy induction by starvation. As KatG functions by catabolizing ROS, we determined the source of ROS contributing to the starvation-induced autophagic elimination of mycobacteria. Using siRNA-mediated knockdown, we found that Superoxide dismutase 2, which generates mitochondrial ROS but not NADPH oxidase 2, is important for the starvation-induced lysosomal delivery to mycobacterial phagosomes. Taken together, these findings showed that KatG is vital for the BJN to evade starvation-induced autophagic restriction.

Lysosome repositioning as an autophagy escape mechanism by Mycobacterium tuberculosis Beijing strain.

Induction of host cell autophagy by starvation was shown to enhance lysosomal delivery to mycobacterial phagosomes, resulting in the restriction of Mycobacterium tuberculosis reference strain H37Rv. Our previous study showed that strains belonging to M. tuberculosis Beijing genotype resisted starvation-induced autophagic elimination but the factors involved remained unclear. Here, we conducted RNA-Seq of macrophages infected with the autophagy-resistant Beijing strain (BJN) compared to macrophages infected with H37Rv upon autophagy induction by starvation. Results identified several genes uniquely upregulated in BJN-infected macrophages but not in H37Rv-infected cells, including those encoding Kxd1 and Plekhm2, which function in lysosome positioning towards the cell periphery. Unlike H37Rv, BJN suppressed enhanced lysosome positioning towards the perinuclear region and lysosomal delivery to its phagosome upon autophagy induction by starvation, while depletion of Kxd1 and Plekhm2 reverted such effects, resulting in restriction of BJN intracellular survival upon autophagy induction by starvation. Taken together, these data indicated that Kxd1 and Plekhm2 are important for the BJN strain to suppress lysosome positioning towards the perinuclear region and lysosomal delivery into its phagosome during autophagy induction by starvation to evade starvation-induced autophagic restriction.

Transcriptional response to the host cell environment of a multidrug-resistant Mycobacterium tuberculosis clonal outbreak Beijing strain reveals its pathogenic features.

Tuberculosis is a global public health problem with emergence of multidrug-resistant infections. Previous epidemiological studies of tuberculosis in Thailand have identified a clonal outbreak multidrug-resistant strain of Mycobacterium tuberculosis in the Kanchanaburi province, designated "MKR superspreader", and this particular strain later was found to also spread to other regions. In this study, we elucidated its biology through RNA-Seq analyses and identified a set of genes involved in cholesterol degradation to be up-regulated in the MKR during the macrophage cell infection, but not in the H37Rv reference strain. We also found that the bacterium up-regulated genes associated with the ESX-1 secretion system during its intracellular growth phase, while the H37Rv did not. All results were confirmed by qRT-PCR. Moreover, we showed that compounds previously shown to inhibit the mycobacterial ESX-1 secretion system and cholesterol utilisation, and FDA-approved drugs known to interfere with the host cholesterol transportation were able to decrease the intracellular survival of the MKR when compared to the untreated control, while not that of the H37Rv. Altogether, our findings suggested that such pathways are important for the MKR's intracellular growth, and potentially could be targets for the discovery of new drugs against this emerging multidrug-resistant strain of M. tuberculosis.

Autophagy in immunity against mycobacterium tuberculosis: a model system to dissect immunological roles of autophagy.

The recognition of autophagy as an immune mechanism has been affirmed in recent years. One of the model systems that has helped in the development of our current understanding of how autophagy and more traditional immunity systems cooperate in defense against intracellular pathogens is macrophage infection with Mycobacterium tuberculosis. M. tuberculosis is a highly significant human pathogen that latently infects billions of people and causes active disease in millions of patients worldwide. The ability of the tubercle bacillus to persist in human populations rests upon its macrophage parasitism. One of the initial reports on the ability of autophagy to act as a cell-autonomous innate immunity mechanism capable of eliminating intracellular bacteria was on M. tuberculosis. This model system has further contributed to the recognition of multiple connections between conventional immune regulators and autophagy. In this chapter, we will review how these studies have helped to establish the following principles: (1) autophagy functions as an innate defense mechanism against intracellular microbes; (2) autophagy is under the control of pattern recognition receptors (PRR) such as Toll-like receptors (TLR), and it acts as one of the immunological output effectors of PRR and TLR signaling; (3) autophagy is one of the effector functions associated with the immunity-regulated GTPases, which were initially characterized as molecules involved in cell-autonomous defense, but whose mechanism of function was unknown until recently; (4) autophagy is an immune effector of Th1/Th2 T cell response polarization-autophagy is activated by Th1 cytokines (which act in defense against intracellular pathogens) and is inhibited by Th2 cytokines (which make cells accessible to intracellular pathogens). Collectively, the studies employing the M. tuberculosis autophagy model system have contributed to the development of a more comprehensive view of autophagy as an immunological process. This work and related studies by others have led us to propose a model of how autophagy, an ancient innate immunity defense, became integrated over the course of evolution with other immune mechanisms of ever-increasing complexity.

A novel potent autophagy inhibitor ECDD-S27 targets vacuolar ATPase and inhibits cancer cell survival.

Autophagy is a conserved lysosomal-dependent cellular degradation process and its dysregulation has been linked to numerous diseases including neurodegeneration, infectious diseases, and cancer. Modulation of autophagy is therefore considered as an attractive target for disease intervention. We carried out a high-content image analysis screen of natural product-derived compounds to discover novel autophagy modulating molecules. Our screen identified ECDD-S27 as the most effective compound for increasing the number of autophagic vacuoles inside cells. The structure of ECDD-S27 revealed that it is a derivative of cleistanthin A, a natural arylnaphthalene lignan glycoside found in plants. ECDD-S27 increases the number of autophagic vacuoles by inhibiting the autophagic flux and is able to restrict the survival of different cancer cells at low nanomolar concentrations. Molecular docking and SERS analysis showed that ECDD-S27 may potentially target the V-ATPase. Upon treatment of various cancer cells with ECDD-S27, the V-ATPase activity is potently inhibited thereby resulting in the loss of lysosomal acidification. Taken together, these data indicated that ECDD-S27 retards the autophagy pathway by targeting the V-ATPase and inhibits cancer cell survival. The observed antitumor activity without cytotoxicity to normal cells suggests the therapeutic potential warranting further studies on lead optimization of the compound for cancer treatment.