Phagosome Acidification Research Articles

Trehalose polyphleates participate in Mycobacterium abscessus fitness and pathogenesis.

Mycobacteria produce a large repertoire of surface-exposed lipids with major biological functions. Among these lipids, trehalose polyphleates (TPPs) are instrumental in the infection of Mycobacterium abscessus by the therapeutic phage BPs. However, while the biosynthesis and transport of TPPs across the membrane by MmpL10 have been reported, the role of TPPs in host infection remains enigmatic. Here, we addressed whether the loss of TPPs influences interactions with macrophages and the virulence of M. abscessus. As anticipated, the deletion of mmpL10 in smooth (S) and rough (R) variants of M. abscessus abrogated TPP production, which was rescued upon gene complementation. Importantly, infection of human THP-1 cells with the mmpL10 mutants was associated with decreased intramacrophage survival and a reduced proportion of infected cells. The rough mmpL10 mutant showed an impaired capacity to block phagosomal acidification and was unable to co-localize with Galectin-3, a marker of phagosomal membrane damage. This suggests that TPPs participate, directly or indirectly, in phagolysosomal fusion and in phagosomal membrane damage to establish cytosolic communication. The TPP defect that affects the fitness and virulence of M. abscessus was further demonstrated in zebrafish embryos using a rough clinical strain resistant to phage BPs and harboring a frameshift mutation in mmpL10. Infection with this strain was correlated with a slight decrease in embryo survival and a reduced bacterial burden as compared to the corresponding parental and complemented derivatives. Together, these results indicate that TPPs are important surface lipids contributing to the pathogenicity of M. abscessus.IMPORTANCETrehalose polyphleates (TPPs) are complex lipids associated with the mycobacterial cell surface and were identified 50 years ago. While the TPP biosynthetic pathway has been described recently, the role of these lipids in the biology of mycobacteria remains yet to be established. The wide distribution of TPPs across mycobacterial species suggests that they may exhibit important functions in these actinobacteria. Here, we demonstrate that Mycobacterium abscessus, an emerging multidrug-resistant pathogen that causes severe lung diseases in cystic fibrosis patients, requires TPPs for survival in macrophages and virulence in a zebrafish model of infection. These findings support the importance of this underexplored family of lipids in mycobacterial pathogenesis.

Methodological approaches to the comparative assessment of cellular reactions of innate immunity in mollusk <I>Pomacea</I> sp. (<I>Caenogastropoda, Ampullariidae</I>) in experimental aseptic inflammation

One of the significant methodological problems that arise in the study of cells of the innate immunity of invertebrates is the unsuitability of culture media and even salt solutions intended for mammals and humans. Another important methodological point is the significantly pronounced reactions of coagulation, which play an important protective role in all invertebrates, but manifest themselves in vitro in cellular suspensions prepared even with optimal saline solutions for this animal. To solve this problem, researchers widely use an anticoagulation buffer with ethylenediaminetetraacetic acid (EDTA) even in functional tests. From the standpoint of traditional approaches of mammalian immunology, the use of EDTA is possible only for the study of cell morphology, but not their functions. The aim of the work is to adapt methodological approaches to the comparative assessment of cellular reactions of innate immunity in Pomacea sp mollusks in experimental aseptic inflammation. To evaluate the functions of phagocytic cells in mollusk Pomacea sp., we used a complete salt solution (CSS) with optimal concentrations of glucose, calcium ions, magnesium with addition to prevent coagulation, cellular aggregation and degranulation instead of EDTA sodium salt of heparin at a concentration of 100 IU/mL (CSS-hep). As a result of a specially conducted experiment, it was shown that during incubation of hemolymph cells in this solution, their viability is preserved, evaluated by flow laser cytometry during incubation with annexin V-PE (PE Annexin V) and 7-aminoactinomycin D (7- AAD), as well as in a test with trypan blue. The possibility of studying the phagocytic activity and a phagosome acidification of leukocytes from hemolymph, hematopoiesis organ (kidney) and the focus of inflammation induced by intramuscular injection of sterile suspension of zymosan using CSS-hep is shown. Unlike the Limnaea stagnalis and Biomphalaria glabrata mollusks, in which the production of reactive oxygen species by phagocytes in the luminol-dependent chemiluminescence reaction is well documented, we were unable to induce this reaction in Pomacea sp. mollusks. In a specially conducted experiment, we did not reveal the effect of the sodium salt of heparin at a concentration of 100 IU/mL on the production of reactive oxygen species by rat phagocytes.

Leukocyte kinetics and bacterial clearance during S. pneumoniae pneumonia and contributions of ICAM-1.

Streptococcus pneumoniae is a leading cause of community-acquired pneumonia. Intercellular adhesion molecule-1 (ICAM-1) is an adhesion molecule that is highly expressed on the pulmonary capillary endothelium, alveolar epithelium and other cell types within the lung. ICAM-1 plays important roles in leukocyte adhesion, migration, and motility. To determine the contributions of ICAM-1 to bacterial clearance and leukocyte kinetics during pneumonia, mice were inoculated with S. pneumoniae and evaluated 1, 4 and 7 days later. Our results show that Icam1-/-mice have a greater number of viable bacteria within the lung at each time point. The impaired clearance observed in Icam1-/- mice was not due to an impediment in leukocyte recruitment. In fact, Icam1-/- mice had a greater number of neutrophils and recruited inflammatory macrophages in the lung tissue and the alveoli/airways on day 7. In contrast, fewer alveolar macrophages were present in the BAL of Icam1-/-mice. The loss of body weight and the concentrations of inflammatory mediators in the BAL were also significantly greater in Icam1-/- mice. Mechanistic studies to understand the defect in clearance show that neutrophils and macrophage subpopulations had no defect in phagocytosis or acidification of phagosomes. RNA sequencing reveals many differences in gene expression, but no suggestion of a defect in phagocytosis or killing. Thus, that ICAM-1 is necessary for the clearance of S. pneumoniae and for the resolution of pneumonia, but is not required for the recruitment of neutrophils or inflammatory macrophages into the pneumonic lung parenchyma or the alveoli/airways during S. pneumoniae-induced pneumonia.

Differences in phagosome acidification of Manila clam hemocytes in response to two Perkinsus species with contrasting proliferation dynamics in the host: P. olseni and P. mediterraneus

Understanding the factors determining the host ranges of Perkinsus spp., a significant group of pathogenic protozoans affecting shellfish, is essential for preventing their spread and designing effective control measures. Considering that differences in the ability to proliferate within the host may influence the determination of host range, we first injected six Perkinsus spp. into Manila clams Ruditapes philippinarum and monitored the variations of trophozoite numbers. Although all six species were detected in the challenged clams 28 days post infection, the infection intensities varied among species, and particularly two species showed contrasting infection trends: P. mediterraneus showed a decreasing trend of infection, declining to the lowest intensity, whereas that of P. olseni continuously increased, reaching the highest intensity. In vitro exposure to Manila clam hemocytes revealed that the survival of P. mediterraneus trophozoites was suppressed, in contrast to P. olseni, which maintained their viability. Despite similar phagocytic indices for both species, the rate of phagosome acidification was significantly higher for hemocytes phagocytizing P. mediterraneus than those targeting P. olseni. Notably, phagosome acidification was significantly suppressed in hemocytes phagocytizing live P. olseni trophozoites, suggesting that P. olseni may secrete a substance that modulates phagosome acidification, and thereby evades intracellular digestion by the host’s hemocytes. Conversely, P. mediterraneus, with a lower affinity for infecting Manila clams, did not exhibit such modulation. Based on these results, we consider that the ability to modulate phagosome acidification in host hemocytes might be at least one factor in determining the host range of Perkinsus species.

Phosphatidylserine liposomes induce a phagosome acidification-dependent and ROS-mediated intracellular killing of Mycobacterium abscessus in human macrophages.

Mycobacterium abscessus (Mab) is an opportunistic nontuberculous mycobacterium responsible of difficult-to-treat pulmonary infections in vulnerable patients, such as those suffering from Cystic Fibrosis (CF), where it represents a major cause of morbidity and mortality. Additionally, due to the intrinsic extensive antimicrobial resistance spectrum displayed by this species and the side effects reported for some available antibiotics, the therapeutic management of such infections remains extremely difficult. In the present study, we show that phosphatidylserine liposomes (PS-L) enhance intracellular mycobacterial killing of Mab infected human macrophages with functional or pharmacologically inhibited cystic fibrosis conductance regulator (CFTR), by a mechanism involving phagosome acidification and reactive oxygen species (ROS) production. Additionally, PS-L significantly reduce proinflammatory response of Mab infected macrophages in terms of NF-kB activation and TNF-α production, irrespective of CFTR inhibition. Altogether, these results represent the proof of concept for a possible future development of PS-L as a therapeutic strategy against difficult-to-treat Mab infection.

The role of MNK1-mTORC1 pathway in modulating macrophage responses to Vibrio vulnificus infection.

Mitogen-activated protein kinase (MAPK)-interacting kinase (MNK) plays a role in promoting the production of tumor necrosis factor alpha and interleukin-6 in macrophages during Vibrio vulnificus (Vv) infection. Inhibition or knockout of MNK1 in J774A.1 cells resulted in reduced cytokine production without affecting their transcription levels. MNK1 also impairs phagocytosis, bacterial clearance, and phagosome acidification in Vv-infected cells through the MNK1-mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. The findings highlight the importance of the MNK1-mTORC1 pathway in modulating macrophage responses to Vv infection.

The different roles of V-ATPase a subunits in phagocytosis/endocytosis and autophagy

ABSTRACT Microglia are specialized macrophages responsible for the clearance of dead neurons and pathogens by phagocytosis and degradation. The degradation requires phagosome maturation and acidification provided by the vesicular- or vacuolar-type H+-translocating adenosine triphosphatase (V-ATPase), which is composed of the cytoplasmic V1 domain and the membrane-embedded Vo domain. The V-ATPase a subunit, an integral part of the Vo domain, has four isoforms in mammals. The functions of different isoforms on phagosome maturation in different cells/species remain controversial. Here we show that mutations of both the V-ATPase Atp6v0a1 and Tcirg1b/Atp6v0a3 subunits lead to the accumulation of phagosomes in zebrafish microglia. However, their mechanisms are different. The V-ATPase Atp6v0a1 subunit is mainly distributed in early and late phagosomes. Defects of this subunit lead to a defective transition from early phagosomes to late phagosomes. In contrast, The V-ATPase Tcirg1b/Atp6v0a3 subunit is primarily located on lysosomes and regulates late phagosome-lysosomal fusion. Defective Tcirg1b/Atp6v0a3, but not Atp6v0a1 subunit leads to reduced acidification and impaired macroautophagy/autophagy in microglia. We further showed that ATP6V0A1/a1 and TCIRG1/a3 subunits in mouse macrophages preferentially located in endosomes and lysosomes, respectively. Blocking these subunits disrupted early-to-late endosome transition and endosome-to-lysosome fusion, respectively. Taken together, our results highlight the essential and conserved roles played by different V-ATPase subunits in multiple steps of phagocytosis and endocytosis across various species. Abbrevations: Apoe: apolipoprotein E; ANXA5/annexin V: annexin A5; ATP6V0A1/a1: ATPase H+-transporting V0 subunit a1; ATP6V0A2/a2: ATPase H+-transporting V0 subunit a2; ATP6V0A4/a4: ATPase H+-transporting V0 subunit a4; dpf: days post-fertilization; EEA1: early endosome antigen 1; HOPS: homotypic fusion and protein sorting; LAMP1: lysosomal associated membrane protein 1; Lcp1: lymphocyte cytosolic protein 1 (L-plastin); Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; NR: neutral red; PBS: phosphate-buffered saline; PtdIns: phosphatidylinositol; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns(3,5)P2: phosphatidylinositol (3,5)-bisphosphate; RAB4: RAB4, member RAS oncogene family; RAB5: RAB5, member RAS oncogene family; RAB7: RAB7, member RAS oncogene family; TCIRG1/Atp6v0a3/a3: T cell immune regulator 1, ATPase H+-transporting V0 subunit a3; V-ATPase: vacuolar-type H+-translocating adenosine triphosphatase; Xla.Tubb2b/NBT: tubulin beta 2B class IIb.

A real-time antibody-dependent cellular phagocytosis assay by live cell imaging

Antibody-dependent cellular phagocytosis (ADCP) is a cellular process by which antibody-opsonized targets (pathogens or cells) activate the Fc receptors on the surface of phagocytes to induce phagocytosis, resulting in internalization and degradation of pathogens or target cells through phagosome acidification. Besides NK cells-mediated antibody-dependent cellular cytotoxicity (ADCC), tumor-infiltrated monocytes and macrophages can directly kill tumor cells in the presence of tumor antigen-specific antibodies through ADCP, representing another attractive strategy for cancer immunotherapy. Even though several methods have been developed to measure ADCP, an automated and high-throughput quantitative assay should offer highly desirable advantages for drug discovery. In this study we established a new ADCP assay to identify therapeutical monoclonal antibodies (mAbs) that facilitate macrophages phagocytosis of live target cells. We used Incucyte, an imaging system for live cell analysis. By labeling the live target cells with a pH sensitive dye (pHrodo), we successfully monitored the ADCP in real time. We demonstrated that our image-based assay is robust and quantitative, suitable for screening and characterization of therapeutical mAbs that directly kill target cells through ADCP. Furthermore, we found different subtypes of macrophages have distinct ADCP activities using both mouse and human primary macrophages differentiated in vitro. By studying various mAbs with mutations in their Fc regions using our assay, we showed that the variants with increased binding to Fc gamma receptors (FcγRs) have enhanced ADCP activities.

Abstract 2059: An image-based real time method to measure antibody-dependent cellular phagocytosis

Abstract Antibody-dependent cellular phagocytosis (ADCP) is an important mechanism of action of therapeutic antibodies. In vitro quantification of macrophage phagocytosis has been technically challenging. Flow cytometry assays were traditionally used for ADCP activity measurements, these methods were static measurements at certain time point, thus provide no information about engulfment kinetics. Moreover, the flow cytometry assays require lifting the macrophage cells from the labware which may affect macrophage activities. We have developed a real time ADCP assay using a live cell analysis system. The target cells labeled with a pH sensitive dye only fluoresce when they are engulfed in acidic phagosomes in the presence of specific antibodies. Our assay is a plate-based with a simple mix and read protocol without disturbing the macrophages. The new method is quantitative and robust, can be readily used for high-throughput screening for functional antibodies. By studying the ADCP activities of different subtypes of macrophages, we found M2 microphages showed the strongest ADCP activity compare to M0 and M1 macrophages, due to their different expression levels of FcgRs. The assay is highly sensitive to differentiate the antibody variants with enhanced Fc function. It can also be used to study the kinetics and mechanisms of ADCP. Citation Format: Jianyong Wang, Yongchang Shi, Yonglian Sun, James T. Koerber, Akiko Seki, Sascha Rutz. An image-based real time method to measure antibody-dependent cellular phagocytosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2059.

Rapid unleashing of macrophage efferocytic capacity via transcriptional pause release.

During development, inflammation or tissue injury, macrophages may successively engulf and process multiple apoptotic corpses via efferocytosis to achieve tissue homeostasis1. How macrophages may rapidly adapt their transcription to achieve continuous corpse uptake is incompletely understood. Transcriptional pause/release is an evolutionarily conserved mechanism, in which RNA polymerase(Pol) II initiates transcription for 20-60 nucleotides, is paused for minutes to hours and isthen released to make full-length mRNA2. Here we show that macrophages, within minutes of corpse encounter, use transcriptional pause/release to unleash a rapid transcriptional response. For human and mouse macrophages, the Pol II pause/release was required for continuous efferocytosis in vitro and in vivo. Interestingly, blocking Pol II pause/release did not impede Fc receptor-mediated phagocytosis, yeast uptake or bacterial phagocytosis. Integration of data from three genomic approaches-precision nuclear run-on sequencing, RNA sequencing, and assay for transposase-accessible chromatin using sequencing (ATAC-seq)-on efferocytic macrophages at different time points revealed that Pol II pause/release controls expression of select transcription factors and downstream target genes. Mechanistic studies on transcription factor EGR3, prominently regulated by pause/release, uncovered EGR3-related reprogramming of other macrophage genes involved in cytoskeleton and corpse processing. Using lysosomal probes and a new genetic fluorescent reporter, we identify a role for pause/release in phagosome acidification during efferocytosis. Furthermore, microglia from egr3-deficient zebrafish embryos displayed reduced phagocytosis of apoptotic neurons and fewer maturing phagosomes, supporting defective corpse processing. Collectively, these data indicate that macrophages use Pol II pause/release as a mechanism to rapidly alter their transcriptional programs for efficient processing of the ingested apoptotic corpses and for successive efferocytosis.

Gallic acid promotes macrophage phagosome acidification and phagolysosome formation by activating NLRP3/mTOR signaling pathway

IntroductionGallic acid (GA) has a good therapeutic effect in bacteriological inhibition and plays a variety of functions in maintaining the stability of the immune system. The aim of the present study was to investigate the effect of GA on the bactericidal activity of macrophages against Vibrio vulnificus (Vv). MethodsA cell counting kit-8 (CCK-8) assay was carried out to test the cytotoxicity of GA on J774A.1 cells. Concentration of proinflammatory cytokines in J774A.1 cells were evaluated by ELISA. The internalization and degradation of Vv in the phagosomes were observed by transmission electron microscopy (TEM). The phagosome acidification and phagolysosome formation were detected to evaluate the bacteria-clearing function of J774A.1 cells. The bactericidal activity of GA in vivo was also investigated by collecting the survival time of Vv infected mice and observing the inflammatory infiltration of organs. ResultsOur results demonstrated that GA at 50 μM significantly inhibited the proinflammatory cytokines levels, promoted phagosome acidification and phagolysosome formation in J774A.1 cells with Vv infection. This may be related to the activation of NLRP3/mTOR signaling pathway. Additionally, GA treatment improves the survival and bactericidal activity of mice infected with Vv. ConclusionsIn summary, GA exerts bactericidal activity against Vv infection by regulating the formation and acidification of phagocytic lysosomes in macrophages.

Macrophage subpopulation identity in Drosophila is modulated by apoptotic cell clearance and related signalling pathways.

In Drosophila blood, plasmatocytes of the haemocyte lineage represent the functional equivalent of vertebrate macrophages and have become an established in vivo model with which to study macrophage function and behaviour. However, the use of plasmatocytes as a macrophage model has been limited by a historical perspective that plasmatocytes represent a homogenous population of cells, in contrast to the high levels of heterogeneity of vertebrate macrophages. Recently, a number of groups have reported transcriptomic approaches which suggest the existence of plasmatocyte heterogeneity, while we identified enhancer elements that identify subpopulations of plasmatocytes which exhibit potentially pro-inflammatory behaviours, suggesting conservation of plasmatocyte heterogeneity in Drosophila. These plasmatocyte subpopulations exhibit enhanced responses to wounds and decreased rates of efferocytosis when compared to the overall plasmatocyte population. Interestingly, increasing the phagocytic requirement placed upon plasmatocytes is sufficient to decrease thesize of these plasmatocyte subpopulations in the embryo. However, the mechanistic basis for this response was unclear. Here, we examine how plasmatocyte subpopulations are modulated by apoptotic cell clearance (efferocytosis) demands and associated signalling pathways. We show that loss of the phosphatidylserine receptor Simu prevents an increased phagocytic burden from modulating specific subpopulation cells, while blocking other apoptotic cell receptors revealed no such rescue. This suggests that Simu-dependent efferocytosis is specifically involved in determining fate of particular subpopulations. Supportive of our original finding, mutations in amo (the Drosophila homolog of PKD2), a calcium-permeable channel which operates downstream of Simu, phenocopy simu mutants. Furthermore, we show that Amo is involved in the acidification of the apoptotic cell-containing phagosomes, suggesting that this reduction in pH may be associated with macrophage reprogramming. Additionally, our results also identify Ecdysone receptor signalling, a pathway related to control of cell death during developmental transitions, as a controller of plasmatocyte subpopulation identity. Overall, these results identify fundamental pathways involved in the specification of plasmatocyte subpopulations and so further validate Drosophila plasmatocytes as a heterogeneous population of macrophage-like cells within this important developmental and immune model.

Acidification of the phagosome orchestrates the motor forces directing its transport

Phagosomes are dynamic organelles formed by macrophages to capture and destroy microbial pathogens. Phagosome transport from the cell periphery to the perinuclear region, is essential for fusion with lysosomes and the elimination of pathogens. Molecular motors, kinesin and dynein, generate opposing forces, transporting the phagosome away from and towards the lysosome, respectively. Luminal acidification plays a crucial role in determining the net directional movement of the phagosome. The mechanics of this regulation are not known. In this study, we used the sodium proton exchanger NHE9 to selectively modulate phagosomal acidification in macrophages. We then investigated its impact on the mechanical properties of kinesin and dynein motors through optical trapping experiments. We observed a negative correlation between the tenacity of dynein motors and pH under high resistive forces. Reduced luminal acidification impaired generation of dynein cooperative forces, which are crucial for transporting the phagosome to the lysosome. Conversely, the kinesin-powered motility of phagosomes is enabled by a decrease in phagosomal acidification. Given the various methods pathogens employ to limit phagosomal acidification, our findings are highly significant in the context of host-pathogen interactions.

Differential survival of Staphylococcal species in macrophages.

Staphylococcus aureus is considered an extracellular pathogen, yet the bacterium is able to survive within and escape from host cells. An agr/sae mutant of strain USA300 is unable to escape from macrophages but can replicate and survive within. We questioned whether such "non-toxic" S. aureus resembles the less pathogenic coagulase-negative Staphylococcal (CoNS) species like S. epidermidis, S. carnosus, S. lugdunensis, S. capitis, S. warneri, or S. pettenkoferi. We show that the CoNS are more efficiently killed in macrophage-like THP-1 cells or in human primary macrophages. Mutations in katA, copL, the regulatory system graRS, or sigB did not impact bacterial survival in THP-1 cells. Deletion of the superoxide dismutases impaired S. aureus survival in primary macrophages but not in THP-1 cells. However, expression of the S. aureus-specific sodM in S. epidermidis was not sufficient to protect this species from being killed. Thus, at least in those cells, better bacterial survival of S. aureus could not be linked to higher protection from ROS. However, "non-toxic" S. aureus was found to be insensitive to pH, whereas most CoNS were protected when phagosomal acidification was inhibited. Thus, species differences are at least partially linked to differences in sensitivity to acidification.

Timing of Phagosome Maturation Depends on Their Transport Switching from Actin to Microtubule Tracks.

Phagosomes, specialized membrane compartments responsible for digesting internalized pathogens, undergo sequential dynamic and biochemical changes as they mature from nascent phagosomes to degradative phagolysosomes. Maturation of phagosomes depends on their transport along actin filaments and microtubules. However, the specific quantitative relationship between the biochemical transformation and transport dynamics remains poorly characterized. The autonomous nature of phagosomes, moving and maturing at different rates, makes understanding this relationship challenging. Addressing this challenge, in this study we engineered particle sensors to image and quantify single phagosomes' maturation. We found that as phagosomes move from the actin cortex to microtubule tracks, the timing of their actin-to-microtubule transition governs the duration of the early phagosome stage before acquiring degradative capacities. Prolonged entrapment of phagosomes in the actin cortex extends the early phagosome stage by delaying the dissociation of early endosome markers and phagosome acidification. Conversely, a shortened transition from actin- to microtubule-based movements causes the opposite effect on phagosome maturation. These results suggest that the actin- and microtubule-based transport of phagosomes functions like a "clock" to coordinate the timing of biochemical events during phagosome maturation, which is crucial for effective pathogen degradation.

Shikonin and Juglone Inhibit Mycobacterium tuberculosis Low-Molecular-Weight Protein Tyrosine Phosphatase a (Mt-PTPa).

Low-molecular-weight protein tyrosine phosphatases (LMW-PTPs) are involved in promoting the intracellular survival of Mycobacterium tuberculosis (Mtb), the causative organism of tuberculosis. These PTPs directly alter host signalling pathways to evade the hostile environment of macrophages and avoid host clearance. Among these, protein tyrosine phosphatase A (Mt-PTPa) is implicated in phagosome acidification failure, thereby inhibiting phagosome maturation to promote Mycobacterium tuberculosis (Mtb) survival. In this study, we explored Mt-PTPa as a potential drug target for treating Mtb. We started by screening a library of 502 pure natural compounds against the activities of Mt-PTPa in vitro, with a threshold of 50% inhibition of activity via a <500 µM concentration of the candidate drugs. The initial screen identified epigallocatechin, myricetin, rosmarinic acid, and shikonin as hits. Among these, the naphthoquinone, shikonin (5, 8-dihydroxy-2-[(1R)-1-hydroxy-4-methyl-3-pentenyl]-1,4-naphthoquinone), showed the strongest inhibition (IC50 33 µM). Further tests showed that juglone (5-hydroxy-1,4-naphthalenedione), another naphthoquinone, displayed similar potent inhibition of Mt-PTPa to shikonin. Kinetic analysis of the inhibition patterns suggests a non-competitive inhibition mechanism for both compounds, with inhibitor constants (Ki) of 8.5 µM and 12.5 µM for shikonin and juglone, respectively. Our findings are consistent with earlier studies suggesting that Mt-PTPa is susceptible to specific allosteric modulation via a non-competitive or mixed inhibition mechanism.

Mycobacterium tuberculosis and its clever approaches to escape the deadly macrophage.

Mycobacterium tuberculosis (Mt.b), a deadly disease causer, is a facultative parasite. This microorganism has developed several methods to defend itself, once internalized within specialised vacuoles in the macrophages. A wide array of receptors like the complement receptor mannose receptors, scavenger receptor assists the entry of the microbe within the phagocytic macrophages. However, Mt.b is clever enough to protect itself from the hostile environment of the macrophage thereby prevailing within it. The microbe can efficiently inhibit processes like phagosome-lysosome fusion, acidification of phagosomes, release of proinflammatory cytokines and stop crucial events like apoptosis. Additionally, it also adopts resistance to killing by reactive oxygen intermediates and reactive nitrogen intermediates. There are multiple genes both in host and the pathogen which are involved in this successful survival of Mt.b. The regulation of phagolysosome fusion is mediated by proteins such as Coronin, TlyA, SapM, PnkG, EsxH. The microbe has certain mechanisms to even acquire iron from the host cell, to withstand iron deprivation as a mode of host's defence mechanism. This review focuses on the various defensive adaptations acquired by Mt.b for fighting against the deprived conditions existing within the macrophages and their capability of proliferating successfully within it, thereby resulting in a diseased condition.

D-mannose acts as a V-ATPase inhibitor to suppress inflammatory cytokines generation and bacterial killing in macrophage

Vacuolar-type H+-ATPase (V-ATPase) critically controls phagosome acidification to promote pathogen digestion and clearance in macrophage. However, the specific subunits of V-ATPase have been evidenced to play contradictory functions in inflammatory cytokines generation and secretion exposure to external bacterial or LPS stimulation. Therefore, identifying the unique function of the separate subunit of V-ATPase is extremely important to regulate macrophage function. Here, we found that D-mannose, a C-2 epimer of glucose, suppressed ATP6V1B2 lysosomal translocation to inhibit V-ATPase activity in macrophages, thereby causing the scaffold protein axis inhibitor protein (AXIN) recruitment to lysosomal membrane and AMPK activation. Correspondingly, LPS-stimulated macrophage M1 polarization was significantly suppressed by D-mannose via down-regulating NF-κB signaling pathway in response to AMPK activation, while IL-4 induced macrophage M2 polarization were not affected. Furthermore, the failure of lysosomal localization of ATP6V1B2 caused by D-mannose also led to the acidification defects of lysosome. Therefore, D-mannose displayed a remarkable function in inhibiting macrophage phagocytosis and bacterial killing. Taken together, D-mannose acts a novel V-ATPase suppressor to attenuate macrophage inflammatory production but simultaneously prevent macrophage phagocytosis and bacterial killing.

Bi-functional particles for real-time phagosome acidification and proteolysis multiplex assay in macrophages.

Phagosome acidification and proteolysis are essential processes in the immune response to contain and eliminate pathogens. In recent years, there has been an increased desire for a rapid and accurate method of assessing these processes in real-time. Here, we outline the development of a multiplexed assay that allows simultaneous monitoring of phagosome acidification and proteolysis in the same sample using silica beads conjugated to pHrodo and DQ BSA. We describe in detail how to prepare the bi-functional particles and show proof of concept using differentially activated macrophages. This multiplexed spectrophotometric assay allows rapid and accurate assessment of phagosome acidification and proteolysis in real-time and could provide valuable information for understanding the immune response to pathogen invasion.

ClC-7 drives intraphagosomal chloride accumulation to support hydrolase activity and phagosome resolution.

Degradative organelles contain enzymes that function optimally at the acidic pH generated by the V-ATPase. The resulting transmembrane H+ gradient also energizes the secondary transport of several solutes, including Cl-. We report that Cl- influx, driven by the 2Cl-/H+ exchanger ClC-7, is necessary for the resolution of phagolysosomes formed by macrophages. Cl- transported via ClC-7 had been proposed to provide the counterions required for electrogenic H+ pumping. However, we found that deletion of ClC-7 had a negligible effect on phagosomal acidification. Instead, luminal Cl- was found to be required for activation of a wide range of phagosomal hydrolases including proteases, nucleases, and glycosidases. These findings argue that the primary role of ClC-7 is the accumulation of (phago)lysosomal Cl- and that the V-ATPases not only optimize the activity of degradative hydrolases by lowering the pH but, importantly, also play an indirect role in their activation by providing the driving force for accumulation of luminal Cl- that stimulates hydrolase activity allosterically.