Elsa Anes

Selected lipids activate phagosome actin assembly and maturation resulting in killing of pathogenic mycobacteria

Pathogenic mycobacteria such as Mycobacterium tuberculosis and Mycobacterium avium facilitate disease by surviving intracellularly within a potentially hostile environment: the macrophage phagosome. They inhibit phagosome maturation processes, including fusion with lysosomes, acidification and, as shown here, membrane actin assembly. An in vitro assay developed for latex bead phagosomes (LBPs) provided insights into membrane signalling events that regulate phagosome actin assembly, a process linked to membrane fusion. Different lipids were found to stimulate or inhibit actin assembly by LBPs and mycobacterial phagosomes in vitro. In addition, selected lipids activated actin assembly and phagosome maturation in infected macrophages, resulting in a significant killing of M. tuberculosis and M. avium. In contrast, the polyunsaturated σ-3 lipids behaved differently and stimulated pathogen growth. Thus, lipids can be involved in both stimulatory and inhibitory signalling networks in the phagosomal membrane.

Mycobacterium tuberculosis protein ESAT-6 is a potent activator of the NLRP3/ASC inflammasome.

Interleukin‐1β (IL‐1β) represents one of the most important mediators of inflammation and host responses to infection. Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, induces IL‐1β secretion at the site of infection, but the underlying mechanism(s) are poorly understood. In this work we show that Mtb infection of macrophages stimulates caspase‐1 activity and promotes the secretion of IL‐1β. This stimulation requires live intracellular bacteria expressing a functional ESX‐1 secretion system. ESAT‐6, an ESX‐1 substrate implicated in membrane damage, is both necessary and sufficient for caspase‐1 activation and IL‐1β secretion. ESAT‐6 promotes the access of other immunostimulatory agents such as AG85 into the macrophage cytosol, indicating that this protein may contribute to caspase‐1 activation largely by perturbing host cell membranes. Using a high‐throughput shRNA‐based screen we found that numerous NOD‐like receptors (NLRs) and CARD domain‐containing proteins (CARDs) were important for IL‐1β secretion upon Mtb infection. Most importantly, NLRP3, ASC and caspase‐1 form an infection‐inducible inflammasome complex that is essential for IL‐1β secretion. In summary, we show that recognition of Mtb infection by the NLRP3 inflammasome requires the activity of the bacterial virulence factor ESAT‐6, and the subsequent IL‐1β response is regulated by a number of NLR/CARD proteins.

Anti-inflammatory effects of phosphatidylcholine

We recently showed that mucus from patients with ulcerative colitis, a chronic inflammatory disorder of the colon, is characterized by a low level of phosphatidylcholine (PC) while clinical studies reveal that therapeutic addition of PC using slow release preparations is beneficial. The positive role of PC in this disease is still elusive. Here we tested the hypothesis that exogenous application of PC has anti-inflammatory properties using three model systems. First, human Caco-2 cells were treated with tumor necrosis factor-α (TNF-α) to induce a pro-inflammatory response via activation of NF-κB. Second, latex bead phagosomes were analyzed for their ability to assemble actin in vitro, a process linked to pro-inflammatory signaling and correlating with the growth versus killing of mycobacteria in macrophages. The third system used was the rapid assembly of plasma membrane actin in macrophages in response to sphingosine 1-phosphate. TNF-α induced a pro-inflammatory response in Caco-2 cells, including 1) assembly of plasma membrane actin; 2) activation of both MAPKs ERK and p38; 3) transport of NF-κB subunits to the nucleus; and 4) subsequent up-regulation of the synthesis of pro-inflammatory gene products. Exogenous addition of most PCs tested significantly inhibited these processes. Other phospholipids like sphingomyelin or phosphatidylethanolamine showed no effects in these assays. PC also inhibited latex bead phagosome actin assembly, the killing of Mycobacterium tuberculosis in macrophages, and the sphingosine 1-phosphate-induced actin assembly in macrophages. TNF-α induces the activation of signaling molecules and the reorganization of the actin cytoskeleton in human intestinal cells. Exogenous application of PC blocks pro-inflammatory signaling in Caco-2 cells, in phagosomes in vitro and facilitates intracellular survival of mycobacteria. We provide further evidence that actin assembly by membranes is part of the pro-inflammatory response. Collectively, these results provide a molecular foundation for the clinical studies showing a beneficial effect of PC therapy in ulcerative colitis.

On the killing of mycobacteria by macrophages

Both pathogenic and non‐pathogenic mycobacteria are internalized into macrophage phagosomes. Whereas the non‐pathogenic types are invariably killed by all macrophages, the pathogens generally survive and grow. Here, we addressed the survival, production of nitrogen intermediates (RNI) and intracellular trafficking of the non‐pathogenic Mycobacterium smegmatis, the pathogen‐like, BCG and the pathogenic M. bovis in different mouse, human and bovine macrophages. The bacteriocidal effects of RNI were restricted for all bacterial species to the early stages of infection. EM analysis showed clearly that all the mycobacteria remained within phagosomes even at late times of infection. The fraction of BCG and M. bovis found in mature phagolysosomes rarely exceeded 10% of total, irrespective of whether bacteria were growing, latent or being killed, with little correlation between the extent of phagosome maturation and the degree of killing. Theoretical modelling of our data identified two different potential sets of explanations that are consistent with our results. The model we favour is one in which a small but significant fraction of BCG is killed in an early phagosome, then maturation of a small fraction of phagosomes with both live and killed bacteria, followed by extremely rapid killing and digestion of the bacteria in phago‐lysosomes.

Dynamic life and death interactions between Mycobacterium smegmatis and J774 macrophages

After internalization into macrophages non‐pathogenic mycobacteria are killed within phagosomes. Pathogenic mycobacteria can block phagosome maturation and grow inside phagosomes but under some conditions can also be killed by macrophages. Killing mechanisms are poorly understood, although phago‐lysosome fusion and nitric oxide (NO) production are implicated. We initiated a systematic analysis addressing how macrophages kill ‘non‐pathogenic’Mycobacterium smegmatis. This system was dynamic, involving periods of initial killing, then bacterial multiplication, followed by two additional killing stages. NO synthesis represented the earliest killing factor but its synthesis stopped during the first killing period. Phagosome actin assembly and fusion with late endocytic organelles coincided with the first and last killing phase, while recycling of phagosome content and membrane coincided with bacterial growth. Phagosome acidification and acquisition of the vacuolar (V) ATPase followed a different pattern coincident with later killing phases. Moreover, V‐ATPase localized to vesicles distinct from classical late endosomes and lysosomes. Map kinase p38 is a crucial regulator of all processes investigated, except NO synthesis, that facilitated the host for some functions while being usurped by live bacteria for others. A mathematical model argues that periodic high and low cellular killing activity is more effective than is a continuous process.

NF-κB Activation Controls Phagolysosome Fusion-Mediated Killing of Mycobacteria by Macrophages

Macrophages can potentially kill all mycobacteria by poorly understood mechanisms. In this study, we explore the role of NF-κB in the innate immune response of macrophages against Mycobacterium smegmatis, a nonpathogenic mycobacterium efficiently killed by macrophages, and Mycobacterium avium which survives within macrophages. We show that infection of macrophages with M. smegmatis induces an activation of NF-κB that is essential for maturation of mycobacterial phagosomes and bacterial killing. In contrast, the pathogenic M. avium partially represses NF-κB activation. Using microarray analysis, we identified many lysosomal enzymes and membrane-trafficking regulators, including cathepsins, LAMP-2 and Rab34, were regulated by NF-κB during infection. Our results argue that NF-κB activation increases the synthesis of membrane trafficking molecules, which may be rate limiting for regulating phagolysosome fusion during infection. The direct consequence of NF-κB inhibition is the impaired delivery of lysosomal enzymes to M. smegmatis phagosomes and reduced killing. Thus, the established role of NF-κB in the innate immune response can now be expanded to include regulation of membrane trafficking during infection.

Exosomal Hsp70 induces a pro-inflammatory response to foreign particles including mycobacteria

Background Exosomes are endosome-derived vesicles that are released when multi-vesicular bodies (MVBs) fuse with the plasma membrane. Exosomes released from mycobacteria-infected cells have recently been shown to be pro-inflammatory. A prominent host molecule that is found within these exosomes is Hsp70, a member of the heat-shock family of proteins. Methodology/Principal Findings We first characterized the exosomes purified from control and mycobacteria-infected cells. We found that relative to uninfected cells, macrophages infected with M. smegmatis and M. avium release more exosomes and the exosomes they released had more Hsp70 on their surface. Both exosomes and exogenous Hsp70 treatment of macrophages led to NF-κB activation and TNFα release in uninfected macrophages; Hsp70 levels were elevated in mycobacteria-infected cells. Macrophage treatment with Hsp70 also led to increase in the phagocytosis and maturation of latex-bead phagosomes. Finally, Hsp70 pre-incubation of M. smegmatis- and M. avium-infected cells led to increased phago-lysosome fusion, as well as more killing of mycobacteria within macrophages. Conclusions/Significance Our results fit into an emerging concept whereby exosomes-containing Hsp70 are effective inducers of inflammation, also in response to mycobacterial infection.

Antimycobacterial evaluation and preliminary phytochemical investigation of selected medicinal plants traditionally used in Mozambique.

ETHNOPHARMACOLOGICAL RELEVANCE Several medicinal plants are traditionally used in Mozambique to treat tuberculosis and related symptoms. AIMS OF THE STUDY It was aimed to assess the in vitro antimycobacterial activity of crude extracts from fifteen medicinal plants and to reveal main classes of compounds which may account for the activity of extracts. METHODS AND MATERIALS The plant materials were sequentially extracted by n-hexane, dichloromethane, ethyl acetate, and 70% ethanol. Decoction of each plant material was also prepared according to traditional use. Broth microdilution method was employed to screen extracts against two mycobacterial species: Mycobacterium smegmatis ATCC 607 and Mycobacterium tuberculosis H37Rv. The extracts with minimum inhibitory concentration(s) (MIC) below 125 μg/mL were considered active and further tested against different mycobacterial species and strains, namely Mycobacterium tuberculosis H37Ra, Mycobacterium bovis BCG ATCC 35734, Mycobacterium smegmatis mc(2) 155, Mycobacterium avium DSM 44156 and DSM 44157. Cytotoxic effect was evaluated against human macrophages from the monocytic THP-1 cells. Main classes of compounds in these active extracts were proposed from their (1)H NMR spectroscopic characterizations. RESULTS n-Hexane extracts of Maerua edulis and Securidaca longepedunculata, ethyl acetate extract of Tabernaemontana elegans and dichloromethane extract of Zanthoxylum capense were found to possess considerable activity against Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Ra with MIC 15.6-62.5 μg/mL. Tabernaemontana elegans ethyl acetate extract displayed strong activity against Mycobacterium tuberculosis H37Rv (MIC 15.6 μg/mL). Except for Tabernaemontana elegans ethyl acetate extract which presented potent cytotoxic effects in THP-1 cells (IC(50)<4 μg/mL), the other three plant extracts showed moderate to none toxicity. Based on (1)H NMR spectroscopic analysis, major components in both Maerua edulis and Securidaca longepedunculata n-hexane extracts were linear chain unsaturated fatty acids. Zanthoxylum capense dichloromethane extract contained more complex constituents (mostly phenolic compounds). In the most potent extract, Tabernaemontana elegans ethyl acetate extract, the prominent compounds were identified as indole alkaloids. CONCLUSIONS The pronounced antimycobacterial activity of the medicinal plants Maerua edulis, Securidaca longepedunculata, Zanthoxylum capense, and Tabernaemontana elegans suggested that they might provide compounds which could be potential anti-TB drug leads.

cAMP synthesis and degradation by phagosomes regulate actin assembly and fusion events: consequences for mycobacteria.

We showed recently that actin assembly by phagosomal membranes facilitates fusion with late endocytic organelles in macrophages. Moreover, lipids that induced phagosomal actin also stimulated this fusion process. In macrophages infected with pathogenic mycobacteria actin-stimulatory lipids led to an increase in pathogen destruction, whereas inhibitors facilitated their growth. A model was proposed whereby phagosomal membrane actin assembly provides tracks for lysosomes to move towards phagosomes, thereby facilitating fusion. Here, we investigated how cAMP affected phagosomal actin assembly in vitro, and phagosomal actin, acidification and late fusion events in J774 macrophages. Latex bead phagosomes are shown to possess adenylyl cyclase activity, which synthesizes cAMP, and phosphodiesterase activity, which degrades cAMP. The system is regulated by protein kinase A (PKA). Increasing cAMP levels inhibited, whereas decreasing cAMP levels stimulated, actin assembly in vitro and within cells. Increasing cAMP levels also inhibited phagosome-lysosome fusion and acidification in cells, whereas reducing cAMP had the opposite effect. High cAMP levels induced an increase in intraphagosomal growth in macrophages of both the non-pathogenic Mycobacterium smegmatis and the pathogenic Mycobacterium tuberculosis, whereas low cAMP levels or inhibition of PKA correlated with increased bacterial destruction. We argue that the phagosome cAMP-PKA system behaves as a molecular switch that regulates phagosome actin and maturation in macrophages.

The site-specific recombination locus of mycobacteriophage Ms6 determines DNA integration at the tRNAAlagene of Mycobacterium spp.

Summary: Genetic determinants of the temperate mycobacteriophage Ms6 required for chromosomal integration were identified. DNA sequence analysis of an attP-containing fragment revealed an ORF encoding a protein of 372 amino acid residues with a C-terminus similar to other conserved C-terminal regions typical of the phage integrase family. Comparison of the sequences of attP, attB and bacteria-prophage junctions attL and attR showed a 26 bp common core sequence, where recombination takes place, near the 5′ end of the integrase gene. Nucleotide sequence analysis of the attB chromosomal region showed that the core site overlaps the 3′ end of the tRNAAlagene. An integration-proficient plasmid vector was constructed and efficiently inserted at the tRNAAlagene of Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Ra. It was demonstrated that Ms6 and D29 integrative systems can be used in conjunction for inserting genes at multiple loci. The site-specific integration system of mycobacteriophage Ms6 is a new tool for mycobacterial genetic analysis and is poorly related to those of the L5 bacteriophage family.