Phagosome-lysosome Fusion Research Articles

Predictive Binding Affinity of Plant-Derived Natural Products Towards the Protein Kinase G Enzyme of Mycobacterium tuberculosis (MtPknG).

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a growing public health concern worldwide, especially with the emerging challenge of drug resistance to the current drugs. Efforts to discover and develop novel, more effective, and safer anti-TB drugs are urgently needed. Products from natural sources, such as medicinal plants, have played an important role in traditional medicine and continue to provide some inspiring templates for the design of new drugs. Protein kinase G, produced by M. tuberculosis (MtPKnG), is a serine/threonine kinase, that has been reported to prevent phagosome-lysosome fusion and help prolong M. tuberculosis survival within the host’s macrophages. Here, we used an in silico, target-based approach (docking) to predict the interactions between MtPknG and 84 chemical constituents from two medicinal plants (Pelargonium reniforme and Pelargonium sidoides) that have a well-documented historical use as natural remedies for TB. Docking scores for ligands towards the target protein were calculated using AutoDock Vina as the predicted binding free energies. Ten flavonoids present in the aerial parts of P. reniforme and/or P. sidoides showed docking scores ranging from −11.1 to −13.2 kcal/mol. Upon calculation of all ligand efficiency indices, we observed that the (−ΔG/MW) ligand efficiency index for flavonoids (4), (5) and (7) was similar to the one obtained for the AX20017 control. When taking all compounds into account, we observed that the best (−ΔG/MW) efficiency index was obtained for coumaric acid, coumaraldehyde, p-hydroxyphenyl acetic acid and p-hydroxybenzyl alcohol. We found that methyl gallate and myricetin had ligand efficiency indices superior and equal to the AX20017 control efficiency, respectively. It remains to be seen if any of the compounds screened in this study exert an effect in M. tuberculosis-infected macrophages.

Simvastatin induces autophagic flux to restore cerulein-impaired phagosome-lysosome fusion in acute pancreatitis

BackgroundDuring pancreatitis, autophagy is activated, but lysosomal degradation of dysfunctional organelles including mitochondria is impaired, resulting in acinar cell death. Retrospective cohort analyses demonstrated an association between simvastatin use and decreased acute pancreatitis incidence. MethodsWe examined whether simvastatin can protect cell death induced by cerulein and the mechanisms involved during acute pancreatitis. Mice were pretreated with DMSO or simvastatin (20 mg/kg) for 24 h followed by 7 hourly cerulein injections and sacrificed 1 h after last injection to harvest blood and tissue for analysis. ResultsPancreatic histopathology revealed that simvastatin reduced necrotic cell death, inflammatory cell infiltration and edema. We found that cerulein triggered mitophagy with autophagosome formation in acinar cells. However, autophagosome-lysosome fusion was impaired due to altered levels of LAMP-1, AMPK and ULK-1, resulting in autophagosome accumulation (incomplete autophagy). Simvastatin abrogated these effects by upregulating LAMP-1 and activating AMPK which phosphorylated ULK-1, resulting in increased formation of functional autolysosomes. In contrast, autophagosomes accumulated in control group during pancreatitis. The effects of simvastatin to promote autophagic flux were inhibited by chloroquine. Mitochondria from simvastatin-treated mice were resistant to calcium overload compared to control, suggesting that simvastatin induced mitochondrial quality control to eliminate susceptible mitochondria. Clinical specimens showed a significant increase in cell-free mtDNA in plasma during pancreatitis compared to normal controls. Furthermore, genetic deletion of parkin abrogated the benefits of simvastatin. ConclusionOur findings reveal the novel role of simvastatin in enhancing autophagic flux to prevent pancreatic cell injury and pancreatitis.

C646 modulates inflammatory response and antibacterial activity of macrophage

C646 is a newly discovered competitive p300/CREB-binding protein-specific inhibitor. Previous studies have shown its potential antitumor activity, but the immunomodulatory function of C646 remains largely unknown. In this study, we investigated the effects of C646 in cytokine expression and antibacterial activity in mouse macrophages. Results showed that C646 significantly reduced LPS-induced pro-inflammatory cytokines, which relied on suppression of JNK, ERK1/2, and NF-κB p65 signaling pathways. In addition, the inhibitory effects were not associated with modulating the expression of CD14/TLR4/MD2 complex or antagonizing its binding ability to LPS. Furthermore, C646 also down-regulated the levels of FcγR III/II and CR3 on macrophage, impaired the phagocytic ability against E. coli, and blocked phagosome-lysosome fusion. Consistent with this, C646 inhibited macrophage-associated bactericidal ability. Collectively, these data indicated that C646 exhibited potent immunomodulatory effects on macrophage both in the production of pro-inflammatory cytokines and bacterial phagocytosis.

ArhGAP12 plays dual roles in Stabilin-2 mediated efferocytosis: Regulates Rac1 basal activity and spatiotemporally turns off the Rac1 to orchestrate phagosome maturation

The rapid and precise clearance of apoptotic cells (efferocytosis) involves a series of phagocytic processes through which apoptotic cells are recognized, engulfed, and degraded within phagocytes. The Rho-family GTPases critically rearrange the cytoskeleton for these phagocytic processes, but we know little about the mechanisms by which regulatory proteins control the spatiotemporal activities of the Rho-family GTPases. Here, we identify ArhGAP12 as a functional GTPase-activating protein (GAP) of Rac1 during Stabilin-2 mediated efferocytosis. ArhGAP12 constitutively forms a complex with the phosphatidylserine receptor, Stabilin-2, via direct interaction with the downstream protein, GULP, but is released from the complex when Stabilin-2 interacts with apoptotic cells. When the phagocytic cup is closed and the apoptotic cell is surrounded by the phagosomal membrane, ArhGAP12 localizes to the phagocytic cup via a specific interaction with phosphatidylinositol-4,5-bisphosphate, which is transiently biosynthesized in the phagocytic cup. Down-regulation of ArhGAP12 results in sustained Rac1 activity, arrangement of F-actin, and delayed phagosome-lysosome fusion. Our results collectively suggest that ArhGAP12 carries dual roles in Stabilin-2 mediated efferocytosis: it binds to GULP/Stabilin-2 and switches off Rac1 basal activity and switches on the Rac1 by releasing itself from the complex. In addition, the spatiotemporal membrane targeting of ArhGAP12 inactivates Rac1 in a time-specific and spatially coordinated manner to orchestrate phagosome maturation. This may shed light on how other RhoGAPs spatiotemporally inactivate Rac or Cdc42 during phagocytosis by various cells, in different circumstances.

Alpha-1-Antitrypsin Enhances Primary Human Macrophage Immunity Against Non-tuberculous Mycobacteria.

Rationale: The association between non-tuberculous mycobacterial lung disease and alpha-1-antitrypsin (AAT) deficiency is likely due, in part, to underlying emphysema or bronchiectasis. But there is increasing evidence that AAT itself enhances host immunity against microbial pathogens and thus deficiency could compromise host protection.Objectives: The goal of this project is to determine if AAT could augment macrophage activity against non-tuberculous mycobacteria.Methods: We compared the ability of monocyte-derived macrophages cultured in autologous plasma that were obtained immediately before and soon after AAT infusion—given to individuals with AAT deficiency—to control an ex vivo Mycobacterium intracellulare infection.Measurements and Main Results: We found that compared to pre-AAT infused monocyte-derived macrophages plus plasma, macrophages, and contemporaneous plasma obtained after a session of AAT infusion were significantly better able to control M. intracellulare infection; the reduced bacterial burden was linked with greater phagosome-lysosome fusion and increased autophagosome formation/maturation, the latter due to AAT inhibition of both M. intracellulare–induced nuclear factor-kappa B activation and A20 expression. While there was a modest increase in apoptosis in the M. intracellulare-infected post-AAT infused macrophages and plasma, inhibiting caspase-3 in THP-1 cells, monocyte-derived macrophages, and alveolar macrophages unexpectedly reduced the M. intracellulare burden, indicating that apoptosis impairs macrophage control of M. intracellulare and that the host protective effects of AAT occurred despite inducing apoptosis.Conclusion: AAT augments macrophage control of M. intracellulare infection through enhancing phagosome-lysosome fusion and autophagy.

Restoration of cytosolic calcium inhibits Mycobacterium tuberculosis intracellular growth: Theoretical evidence and experimental observation

Mycobacterium tuberculosis (Mtb) is a highly successful intracellular pathogen because of its ability to modulate host’s anti-microbial pathways. Phagocytosis acts as the first line of defence against microbial infection. However, Mtb inhibits Phosphatidylinositol 3-phosphate (PI3P) oscillations which is required for phagolysosomal fusion. Here we attempted to understand the mechanisms by which Mtb eliminates phagosome-lysosome fusion. To address this, we built a four dimensional ordinary differential equation model and explored the contribution of PI3P during Mtb phagocytosis. Using this model, we identified some sensitive parameters that influence the dynamics of host-pathogen interactions. We observed that PI3P dynamics can be controlled by regulating the intracellular calcium oscillations. Some plausible methods to restore PI3P oscillations are ER flux rate, recruitment rate of proteins, like Rab GTPase, and cooperativity coefficient of calcium dependent consumption of calmodulin. Further, we investigated whether modulation of these pathways is a potential therapeutic intervention strategy. Here we showed that RyR2 agonist caffeine stimulated calcium influx and inhibited growth of intracellular Mtb in macrophages. Taken together, we demonstrate that modulation of host calcium level is a plausible strategy for killing of intracellular Mtb.

The Lung Mucosa Environment in the Elderly Increases Host Susceptibility to Mycobacterium tuberculosis Infection.

As we age, there is an increased risk for the development of tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) infection. Few studies consider that age-associated changes in the alveolar lining fluid (ALF) may increase susceptibility by altering soluble mediators of innate immunity. We assessed the impact of adult or elderly human ALF during Mtb infection in vitro and in vivo. We identified amplification of pro-oxidative and proinflammatory pathways in elderly ALF and decreased binding capability of surfactant-associated surfactant protein A (SP-A) and surfactant protein D (SP-D) to Mtb. Human macrophages infected with elderly ALF-exposed Mtb had reduced control and fewer phagosome-lysosome fusion events, which was reversed when elderly ALF was replenished with functional SP-A/SP-D. In vivo, exposure to elderly ALF exacerbated Mtb infection in young mice. Our studies demonstrate how the pulmonary environment changes as we age and suggest that Mtb may benefit from declining host defenses in the lung mucosa of the elderly.

The role of SARM in the control of immune response driven by Klebsiella pneumonia infection

Introduction Klebsiella pneumonia is a Gram-negative, capsulated bacteria, which is an important cause of community-acquired and nosocomial pneumonia. Klebsiella co-opts cellular functions dedicated to control immune balance to limit the activation of inflammatory responses. SARM (Sterile α-and armadillo-motif containing protein), the fifth identified member of the TIR (Toll-interleukin 1 receptor (1LR)) adaptor family, negatively regulates IRF and NF-kB activation by affecting TLR4 and TLR3 TRIF-dependent signalling. It is currently unclear the role, if any, of SARM in bacterial infections. Here, we aim to dissect the contribution of SARM in Klebsiella infections, and, specifically, to investigate whether Klebsiella may exploit SARM as part of the pathogen’s portfolio immune evasion strategies. Results SARM contributed to Klebsiella anti-inflammation strategies in macrophages through by increasing AKT phosphorylation (to limit phagosome-lysosome fusion), and preventing the activation of NF-kB (to control inflammatory responses). SARM also negatively regulated type I IFN regulatory factor (IRF3) by decreasing IRF3 phosphorylation. Notably, SARM played a role as inflammasome inhibitor, as observed by increased IL-1β secretion in the supernatants of infected sarm-/-. SARM also inhibited ASC oligomerization in Klebsiella-infected macrophages as seen by the increased ASC monomers release by sarm-/- BMDMs. SARM was also required for pyroptosis following Klebsiella infection. Interestingly, Klebsiella induced the expression of SARM in a TLR4-TRAM-TRIF-IRF3-IFNAR dependent manner, demonstrating that Klebsiella exploits type I IFN to trigger SARM to control inflammasome activation, and the activation of inflammatory responses. Conclusions These findings have uncovered how Klebsiella manipulates the TLR adaptor SARM to dampen the activation of host defences.

A DNA-based fluorescent reporter maps HOCl production in the maturing phagosome.

Phagocytes destroy pathogens by trapping them in a transient organelle called the phagosome, where they are bombarded with reactive oxygen species (ROS) and reactive nitrogen species (RNS). Imaging reactive species within the phagosome would directly reveal the chemical dynamics underlying pathogen destruction. Here we introduce a fluorescent, DNA-based combination reporter, cHOClate, which simultaneously images hypochlorous acid (HOCl) and pH quantitatively. Using cHOClate targeted to phagosomes in live cells, we successfully map phagosomal production of a specific ROS, HOCl, as a function of phagosome maturation. We found that phagosomal acidification was gradual in macrophages and upon completion, HOCl was released in a burst. This revealed that phagosome-lysosome fusion was essential not only for phagosome acidification, but also for providing the chloride necessary for myeloperoxidase activity. This method can be expanded to image several kinds of ROS and RNS and be readily applied to identify how resistant pathogens evade phagosomal killing.

Histophilus somni Survives in Bovine Macrophages by Interfering with Phagosome-Lysosome Fusion but Requires IbpA for Optimal Serum Resistance.

Histophilus somni is capable of intracellular survival within professional phagocytic cells, but the mechanism of survival is not understood. The Fic motif within the direct repeat (DR1)/DR2 domains of the IbpA fibrillary network protein of H. somni is cytotoxic to epithelial and phagocytic cells, which may interfere with the bactericidal activity of these cells. To determine the contribution of IbpA and Fic to resistance to host defenses, H. somni strains and mutants that lacked all or a region of ibpA (including the DR1/DR2 regions) were tested for survival in bovine monocytic cells and for serum susceptibility. An H. somni mutant lacking IbpA, but not the DR1/DR2 region within ibpA, was more susceptible to killing by antiserum than the parent, indicating that the entire protein was associated with serum resistance. H. somni strains expressing IbpA replicated in bovine monocytes for at least 72 h and were toxic for these cells. Virulent strain 2336 mutants lacking the entire ibpA gene or both DR1 and DR2 were not toxic to the monocytes but still survived within the monocytes for at least 72 h. Monitoring of intracellular trafficking of H. somni with monoclonal antibodies to phagosomal markers indicated that the early phagosomal marker early endosome antigen 1 colocalized with all isolates tested, but only strains that could survive intracellularly did not colocalize with the late lysosomal marker lysosome-associated membrane protein 2 and prevented the acidification of phagosomes. These results indicated that virulent isolates of H. somni were capable of surviving within phagocytic cells through interference in phagosome-lysosome maturation. Therefore, H. somni may be considered a permissive intracellular pathogen.

PIKfyve accelerates phagosome acidification through activation of TRPML1 while arrests aberrant vacuolation independent of the Ca2+ channel.

PIKfyve phosphorylates PtdIns(3)P to PtdIns(3, 5)P2. One of the best characterized effector downstream of PtdIns(3, 5)P2 is a lysosomal Ca2+ channel, TRPML1. Although it has been reported that TRPML1 is involved in phagosome-lysosome fusion, the relevance of the Ca2+ channel in phagosome acidification has been denied. In this article, however, we demonstrated that the phagosome acidification was dependent on TRPML1. Based on the classical idea that Fluorescein isothiocyanate (FITC)-fluorescence is highly sensitive to acidic pH, we could estimate the phagosome acidification by time laps imaging. FITC-zymosan fluorescence that was engulfed by macrophages, decreased immediately after the uptake while the extinction of FITC-zymosan fluorescence was delayed in PIKfyve-deficient cells. The acidification arrest was completely rescued in the presence of Ca2+ ionophore A23187. Cells treated with a PIKfyve inhibitor, apilimod, also showed delayed phagosome acidification but were rescued by the overexpression of TRPML1. Additionally, TRPML1 agonist, ML-SA1 was effective to acidify the phagosome in PIKfyve-deficient cells. Another phenotype observed in PIKfyve-deficient cells is vacuole formation. Unexpectedly, enlarged vacuole formation in PIKfyve-deficient cells was not rescued by Ca2+ or over expression of TRPML1. It is likely that the acidification and vacuolation arrest is bifurcating downstream of PIKfyve.

Identification of Novel Mycobacterial Inhibitors Against Mycobacterial Protein Kinase G.

Protein kinase G (PknG) is a eukaryotic-like serine/threonine kinase that is expressed by Mycobacterium tuberculosis and promotes survival of mycobacteria in host macrophages by suppressing phagosome-lysosome fusion. Thus, compounds showing inhibitory activity against PknG are promising anti-mycobacterial agents. We therefore aimed to develop anti-mycobacterial agents by identifying new PknG inhibitors. A luciferase-based PknG kinase assay was used to screen potential inhibitors of PknG. We found that four compounds, namely AZD7762, R406, R406-free base, and CYC116, inhibited PknG activities. AZD7762, R406, and R406-free base promoted transfer of mycobacteria to lysosomes. These compounds also inhibited survival of M. bovis Bacillus Calmette–Guérin (BCG) inside human macrophages. Furthermore, R406 and R406-free base showed bactericidal activity against BCG in infected human macrophages without cytotoxicity. The PknG inhibitors identified in this study by the luciferase-based PknG kinase assay may be promising leads for the development of anti-mycobacterial agents.

Phosphorylation of SNAP-23 at Ser95 causes a structural alteration and negatively regulates Fc receptor-mediated phagosome formation and maturation in macrophages.

SNAP-23 is a plasma membrane-localized soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNARE) involved in Fc receptor (FcR)-mediated phagocytosis. However, the regulatory mechanism underlying its function remains elusive. Using phosphorylation-specific antibodies, SNAP-23 was found to be phosphorylated at Ser95 in macrophages. To understand the role of this phosphorylation, we established macrophage lines overexpressing the nonphosphorylatable S95A or the phosphomimicking S95D mutation. The efficiency of phagosome formation and maturation was severely reduced in SNAP-23-S95D–overexpressing cells. To examine whether phosphorylation at Ser95 affected SNAP-23 structure, we constructed intramolecular Förster resonance energy transfer (FRET) probes of SNAP-23 designed to evaluate the approximation of the N termini of the two SNARE motifs. Interestingly, a high FRET efficiency was detected on the membrane when the S95D probe was used, indicating that phosphorylation at Ser95 caused a dynamic structural shift to the closed form. Coexpression of IκB kinase (IKK) 2 enhanced the FRET efficiency of the wild-type probe on the phagosome membrane. Furthermore, the enhanced phagosomal FRET signal in interferon-γ–activated macrophages was largely dependent on IKK2, and this kinase mediated a delay in phagosome-lysosome fusion. These results suggested that SNAP-23 phosphorylation at Ser95 played an important role in the regulation of SNARE-dependent membrane fusion during FcR-mediated phagocytosis.

Morphological and functional characterization of hemocytes from two deep-sea vesicomyid clams Phreagena okutanii and Abyssogena phaseoliformis

Deep-sea vesicomyid clams harboring intracellular symbiotic sulfur-oxidizing bacteria are often dominant in chemosynthetic animal communities. Although they are known to have erythrocytes, little is known about other hemocytes. To investigate the types and roles of various hemocytes in vesicomyid clams, we performed morphological, histochemical and functional characterization of the hemocytes in two species, Phreagena okutanii, collected from 873 to 978 m depth, and Abyssogena phaseoliformis, from 5199 to 5355 m. Both were found to have three types of hemocytes: erythrocytes (ERCs), eosinophilic granulocytes (EGs), and basophilic granulocytes (BGs). The ERCs contain hemoglobin in the cytoplasm, with basophilic vacuoles containing acid polysaccharide, neutral lipids, and peroxidase. The EGs were found to contain acid polysaccharides and eosinophilic granules containing lysosomal enzymes, acid and alkaline phosphatases, chloroacetate esterase, and peroxidase. Although BGs had some basophilic granules with alkaline phosphatase, they lacked acid phosphatase and acid polysaccharides. The EGs and BGs were shown to have phagocytic ability, while the ERCs exhibited no phagocytosis. The EGs showed higher phagocytic activity as well as a higher phagosome-lysosome fusion rate than BGs. The hemocytes of the two vesicomyid species differed in the intracellular structures. In A. phaseoliformis, ERCs additionally contained neutral polysaccharides in vacuoles and had vesicles with acinus-like acidic mucus in the cytoplasm, neither of which were observed in P. okutanii. The eosinophilic granules in the EGs had heteromorphically-elongated shapes containing homogeneously electron-dense material in P. okutanii, but were more spherical and composed of fibrous structures in A. phaseoliformis. The difference in hemocytes between the two clams seems to be reflective of phylogenetically differentiated lineages adapting to differing conditions in their respective deep-sea environments, such as dissolved oxygen, hydrogen sulfide concentration, and hydrostatic pressure. In the view of phylogeny of veneroida clams including two vesicomyids, their hemocytes appear to be categorizable into three basic types, with the first containing ERCs and agranulocytes, the second including EGs, and the third comprised of BGs, small eosinophilic granulocytes, and other granulocytes. The present data showed no phagocytic activity of ERCs and a lack of agranulocytes in both vesicomyid species, and when combined with previous reports that other veneroid clams show low or no phagocytic activity, this suggests that ERCs have become evolutionarily differentiated from agranulocytes in the ancestral vesicomyid clam.

HDAC6 controls innate immune and autophagy responses to TLR-mediated signalling by the intracellular bacteria Listeria monocytogenes.

Recent evidence on HDAC6 function underlines its role as a key protein in the innate immune response to viral infection. However, whether HDAC6 regulates innate immunity during bacterial infection remains unexplored. To assess the role of HDAC6 in the regulation of defence mechanisms against intracellular bacteria, we used the Listeria monocytogenes (Lm) infection model. Our data show that Hdac6-/- bone marrow-derived dendritic cells (BMDCs) have a higher bacterial load than Hdac6+/+ cells, correlating with weaker induction of IFN-related genes, pro-inflammatory cytokines and nitrite production after bacterial infection. Hdac6-/- BMDCs have a weakened phosphorylation of MAPK signalling in response to Lm infection, suggesting altered Toll-like receptor signalling (TLR). Compared with Hdac6+/+ counterparts, Hdac6-/- GM-CSF-derived and FLT3L-derived dendritic cells show weaker pro-inflammatory cytokine secretion in response to various TLR agonists. Moreover, HDAC6 associates with the TLR-adaptor molecule Myeloid differentiation primary response gene 88 (MyD88), and the absence of HDAC6 seems to diminish the NF-κB induction after TLR stimuli. Hdac6-/- mice display low serum levels of inflammatory cytokine IL-6 and correspondingly an increased survival to a systemic infection with Lm. The impaired bacterial clearance in the absence of HDAC6 appears to be caused by a defect in autophagy. Hence, Hdac6-/- BMDCs accumulate higher levels of the autophagy marker p62 and show defective phagosome-lysosome fusion. These data underline the important function of HDAC6 in dendritic cells not only in bacterial autophagy, but also in the proper activation of TLR signalling. These results thus demonstrate an important regulatory role for HDAC6 in the innate immune response to intracellular bacterial infection.

Inducible nitric oxide synthase in innate immune cells is important for restricting cyst formation of Toxoplasma gondii in the brain but not required for the protective immune process to remove the cysts

Significantly larger numbers of Toxoplasma gondii cysts were detected in the brains of RAG1−/−NOS2−/− than RAG1−/− mice following infection. In contrast, the cyst numbers markedly decreased in a same manner in both strains of mice after receiving CD8+ immune T cells. Thus, NOS2-mediated innate immunity is important for inhibiting formation of cysts in the brain but not required for the T cell-initiated cyst removal, which is associated with phagocyte accumulation. Treatment with chloroquine, an inhibitor of endolysosomal acidification, partially but significantly inhibited the T cell-mediated cyst removal, suggesting that phagosome–lysosome fusion could be involved in the T. gondii cyst elimination.

Sequential actions of phosphatidylinositol phosphates regulate phagosome-lysosome fusion.

Phagosomes mature into phagolysosomes by sequential fusion with early endosomes, late endosomes, and lysosomes. Phagosome-with-lysosome fusion (PLF) results in the delivery of lysosomal hydrolases into phagosomes and in digestion of the cargo. The machinery that drives PLF has been little investigated. Using a cell-free system, we recently identified the phosphoinositide lipids (PIPs) phosphatidylinositol 3-phosphate (PI(3)P) and phosphatidylinositol 4-phosphate (PI(4)P) as regulators of PLF. We now report the identification and the PIP requirements of four distinct subreactions of PLF. Our data show that (i) PI(3)P and PI(4)P are dispensable for the disassembly and activation of (phago)lysosomal soluble N-ethylmaleimide-sensitive factor attachment protein receptors, that (ii) PI(3)P is required only after the tethering step, and that (iii) PI(4)P is required during and after tethering. Moreover, our data indicate that PI(4)P is needed to anchor Arl8 (Arf-like GTPase 8) and its effector homotypic fusion/vacuole protein sorting complex (HOPS) to (phago)lysosome membranes, whereas PI(3)P is required for membrane association of HOPS only. Our study provides a first link between PIPs and established regulators of membrane fusion in late endocytic trafficking.

Transglutaminase type 2 plays a key role in the pathogenesis of Mycobacterium tuberculosis infection.

Mycobacterium tuberculosis (MTB), the aetiological agent of tuberculosis (TB), is capable of interfering with the phagosome maturation pathway, by inhibiting phagosome-lysosome fusion and the autophagic process to ensure survival and replication in macrophages. Thus, it has been proposed that the modulation of autophagy may represent a therapeutic approach to reduce MTB viability by enhancing its clearance. The aim of this study was to investigate whether transglutaminase type 2 (TG2) is involved in the pathogenesis of MTB. We have shown that either genetic or pharmacological inhibition of TG2 leads to a marked reduction in MTB replicative capacity. Infection of TG2 knockout mice demonstrated that TG2 is required for MTB intracellular survival in macrophages and host tissues. The same inhibitory effect can be reproduced in vitro using Z-DON, a specific inhibitor of the transamidating activity of TG2. Massive cell death observed in macrophages that properly express TG2 is hampered by the absence of the enzyme and can be largely reduced by the treatment of wild-type macrophages with the TG2 inhibitor. Our data suggest that reduced MTB replication in cells lacking TG2 is due to the impairment of LC3/autophagy homeostasis. Finally, we have shown that treatment of MTB-infected murine and human primary macrophages with cystamine, a TG2 inhibitor already tested in clinical studies, causes a reduction in intracellular colony-forming units in human macrophages similar to that achieved by the anti-TB drug capreomycin. These results suggest that inhibition of TG2 activity is a potential novel approach for the treatment of TB.

Development and application of a recombination-based library versus library high- throughput yeast two-hybrid (RLL-Y2H) screening system.

Protein-protein interaction (PPI) network maintains proper function of all organisms. Simple high-throughput technologies are desperately needed to delineate the landscape of PPI networks. While recent state-of-the-art yeast two-hybrid (Y2H) systems improved screening efficiency, either individual colony isolation, library preparation arrays, gene barcoding or massive sequencing are still required. Here, we developed a recombination-based ‘library vs library’ Y2H system (RLL-Y2H), by which multi-library screening can be accomplished in a single pool without any individual treatment. This system is based on the phiC31 integrase-mediated integration between bait and prey plasmids. The integrated fragments were digested by MmeI and subjected to deep sequencing to decode the interaction matrix. We applied this system to decipher the trans-kingdom interactome between Mycobacterium tuberculosis and host cells and further identified Rv2427c interfering with the phagosome–lysosome fusion. This concept can also be applied to other systems to screen protein–RNA and protein–DNA interactions and delineate signaling landscape in cells.

Suppression of Toll-like receptor 2-mediated proinflammatory responses by Mycobacterium tuberculosis protein Rv3529c.

Microorganisms are known to devise various strategies to thwart protective responses by the host. One such strategy is to incorporate sequences and domains in their genes/proteins that have similarity to various domains of the host proteins. In this study, we report that Mycobacterium tuberculosis protein Rv3529c exhibits significant similarity to the death domain of the TLR pathway adaptor protein MyD88. Incubation of macrophages with Rv3529c specifically inhibited TLR2-mediated proinflammatory responses. This included attenuated oxidative burst, reduced phosphorylation of MAPK-ERK, reduced activation of transcription factor NF-κB and reduced secretion of proinflammatory cytokines IFN-γ, IL-6, and IL-17A with a concomitant increased secretion of suppressor cytokines IL-10 and TGF-β. Importantly, Rv3529c significantly inhibited TLR2-induced association of MyD88 with IRAK1 by competitively binding with IRAK1. Further, Rv3529c mediated inhibition of apoptosis and phagosome-lysosome fusion. Lastly, incubation of macrophages with Rv3529c increased bacterial burden inside macrophages. The data presented show another strategy evolved by M. tuberculosis toward immune evasion that centers on incorporating sequences in proteins that are similar to crucial proteins in the innate immune system of the host.