Delineating the Physiological Roles of the PE and Catalytic Domains of LipY in Lipid Consumption in Mycobacterium-Infected Foamy Macrophages.

Within tuberculous granulomas, a subpopulation of Mycobacterium tuberculosis resides inside foamy macrophages (FM) that contain abundant cytoplasmic lipid bodies (LB) filled with triacylglycerol (TAG). Upon fusion of LB with M. tuberculosis-containing phagosomes, TAG is hydrolyzed and reprocessed by the bacteria into their own lipids, which accumulate as intracytosolic lipid inclusions (ILI). This phenomenon is driven by many mycobacterial lipases, among which LipY participates in the hydrolysis of host and bacterial TAG. However, the functional contribution of LipY's PE domain to TAG hydrolysis remains unclear. Here, enzymatic studies were performed to compare the lipolytic activities of recombinant LipY and its truncated variant lacking the N-terminal PE domain, LipY(ΔPE). Complementarily, an FM model was used where bone marrow-derived mouse macrophages were infected with M. bovis BCG strains either overexpressing LipY or LipY(ΔPE) or carrying a lipY deletion mutation prior to being exposed to TAG-rich very-low-density lipoprotein (VLDL). Results indicate that truncation of the PE domain correlates with increased TAG hydrolase activity. Quantitative electron microscopy analyses showed that (i) in the presence of lipase inhibitors, large ILI (ILI+3) were not formed because of an absence of LB due to inhibition of VLDL-TAG hydrolysis or inhibition of LB-neutral lipid hydrolysis by mycobacterial lipases, (ii) ILI+3 profiles in the strain overexpressing LipY(ΔPE) were reduced, and (iii) the number of ILI+3 profiles in the ΔlipY mutant was reduced by 50%. Overall, these results delineate the role of LipY and its PE domain in host and mycobacterial lipid consumption and show that additional mycobacterial lipases take part in these processes.

MAB_3551c encodes the primary triacylglycerol synthase involved in lipid accumulation in Mycobacterium abscessus.

Slow growing pathogenic mycobacteria utilize host-derived lipids and accumulate large amounts of triacylglycerol (TAG) in the form of intracytoplasmic lipid inclusions (ILI), serving as a source of carbon and energy during prolonged infection. Mycobacterium abscessus is an emerging and rapidly growing species capable to induce severe and chronic pulmonary infections. However, whether M. abscessus, like Mycobacterium tuberculosis, possesses the machinery to acquire and store host lipids, remains unaddressed. Herein, we aimed at deciphering the contribution of the seven putative M. abscessus TAG synthases (Tgs) in TAG synthesis/accumulation thanks to a combination of genetic and biochemical techniques and a well-defined foamy macrophage (FM) model along with electron microscopy. Targeted gene deletion and functional complementation studies identified the MAB_3551c product, Tgs1, as the major Tgs involved in TAG production. Tgs1 exhibits a preference for long acyl-CoA substrates and site-directed mutagenesis demonstrated that His144 and Gln145 are essential for enzymatic activity. Importantly, in the lipid-rich intracellular context of FM, M. abscessus formed large ILI in a Tgs1-dependent manner. This supports the ability of M. abscessus to assimilate host lipids and the crucial role of Tgs1 in intramycobacterial TAG production, which may represent important mechanisms for long-term storage of a rich energy supply.

Insights into the smooth-to-rough transitioning in Mycobacterium bolletii unravels a functional Tyr residue conserved in all mycobacterial MmpL family members.

In mycobacteria, MmpL proteins represent key components that participate in the biosynthesis of the complex cell envelope. Whole genome analysis of a spontaneous rough morphotype variant of Mycobacterium abscessus subsp. bolletii identified a conserved tyrosine that is crucial for the function of MmpL family proteins. Isogenic smooth (S) and rough (R) variants differed by a single mutation linked to a Y842H substitution in MmpL4a. This mutation caused a deficiency in glycopeptidolipid production/transport in the R variant and a gain in the capacity to produce cords in vitro. In zebrafish, increased virulence of the M. bolletii R variant over the parental S strain was found, involving massive production of serpentine cords, abscess formation and rapid larval death. Importantly, this finding allowed us to demonstrate an essential role of Tyr842 in several different MmpL proteins, including Mycobacterium tuberculosis MmpL3. Structural homology models of MmpL4a and MmpL3 identified two additional critical residues located in the transmembrane regions TM10 and TM4 that are facing each other. We propose that these central residues are part of the proton-motive force that supplies the energy for substrate transport. Hence, we provide important insights into mechanistic/structural aspects of MmpL proteins as lipid transporters and virulence determinants in mycobacteria.

Mycobacterium tuberculosis exploits asparagine to assimilate nitrogen and resist acid stress during infection.

Mycobacterium tuberculosis is an intracellular pathogen. Within macrophages, M. tuberculosis thrives in a specialized membrane-bound vacuole, the phagosome, whose pH is slightly acidic, and where access to nutrients is limited. Understanding how the bacillus extracts and incorporates nutrients from its host may help develop novel strategies to combat tuberculosis. Here we show that M. tuberculosis employs the asparagine transporter AnsP2 and the secreted asparaginase AnsA to assimilate nitrogen and resist acid stress through asparagine hydrolysis and ammonia release. While the role of AnsP2 is partially spared by yet to be identified transporter(s), that of AnsA is crucial in both phagosome acidification arrest and intracellular replication, as an M. tuberculosis mutant lacking this asparaginase is ultimately attenuated in macrophages and in mice. Our study provides yet another example of the intimate link between physiology and virulence in the tubercle bacillus, and identifies a novel pathway to be targeted for therapeutic purposes.

Reversible lipid accumulation and associated division arrest of Mycobacterium avium in lipoprotein-induced foamy macrophages may resemble key events during latency and reactivation of tuberculosis.

During the dormant phase of tuberculosis, Mycobacterium tuberculosis persists in lung granulomas by residing in foamy macrophages (FM) that contain abundant lipid bodies (LB) in their cytoplasm, allowing bacilli to accumulate lipids as intracytoplasmic lipid inclusions (ILI). An experimental model of FM is presented where bone marrow-derived mouse macrophages are infected with M. avium and exposed to very-low-density lipoprotein (VLDL) as a lipid source. Quantitative analysis of detailed electron microscope observations showed the following results. (i) Macrophages became foamy, and mycobacteria formed ILI, for which host triacylglycerides, rather than cholesterol, was essential. (ii) Lipid transfer occurred via mycobacterium-induced fusion between LB and phagosomes. (iii) Mycobacteria showed a thinned cell wall and became elongated but did not divide. (iv) Upon removal of VLDL, LB and ILI declined within hours, and simultaneous resumption of mycobacterial division restored the number of mycobacteria to the same level as that found in untreated control macrophages. This showed that the presence of ILI resulted in a reversible block of division without causing a change in the mycobacterial replication rate. Fluctuation between ILI either partially or fully extending throughout the mycobacterial cytoplasm was suggestive of bacterial cell cycle events. We propose that VLDL-driven FM constitute a well-defined cellular system in which to study changed metabolic states of intracellular mycobacteria that may relate to persistence and reactivation of tuberculosis.

Pathogenic brucellae replicate in human trophoblasts.

Brucellae replicate in a vacuole derived from the endoplasmic reticulum (ER) in epithelial cells, macrophages, and dendritic cells. In animals, trophoblasts are also key cellular targets where brucellae efficiently replicate in association with the ER. Therefore, we investigated the ability of Brucella spp. to infect human trophoblasts using both immortalized and primary trophoblasts. Brucella extensively proliferated within different subpopulations of trophoblasts, suggesting that they constitute an important niche in cases where the fetal-maternal barrier is breached. In extravillous trophoblasts (EVTs), B. abortus and B. suis replicated within single-membrane acidic lysosomal membrane-associated protein 1-positive inclusions, whereas B. melitensis replicated in the ER-derived compartment. Furthermore, B. melitensis but not B. abortus nor B. suis interfered with the invasive capacity of EVT-like cells in vitro. Because EVTs are essential for implantation during early stages of pregnancy, the nature of the replication niche may have a central role during Brucella-associated abortion in infected women.

Phosphorylation of mycobacterial PcaA inhibits mycolic acid cyclopropanation: consequences for intracellular survival and for phagosome maturation block.

Pathogenic mycobacteria survive within macrophages by residing in phagosomes, which they prevent from maturing and fusing with lysosomes. Although several bacterial components were seen to modulate phagosome processing, the molecular regulatory mechanisms taking part in this process remain elusive. We investigated whether the phagosome maturation block (PMB) could be modulated by signaling through Ser/Thr phosphorylation. Here, we demonstrated that mycolic acid cyclopropane synthase PcaA, but not MmaA2, was phosphorylated by mycobacterial Ser/Thr kinases at Thr-168 and Thr-183 both in vitro and in mycobacteria. Phosphorylation of PcaA was associated with a significant decrease in the methyltransferase activity, in agreement with the strategic structural localization of these two phosphoacceptors. Using a BCG ΔpcaA mutant, we showed that PcaA was required for intracellular survival and prevention of phagosome maturation in human monocyte-derived macrophages. The physiological relevance of PcaA phosphorylation was further assessed by generating PcaA phosphoablative (T168A/T183A) or phosphomimetic (T168D/T183D) mutants. In contrast to the wild-type and phosphoablative pcaA alleles, introduction of the phosphomimetic pcaA allele in the ΔpcaA mutant failed to restore the parental mycolic acid profile and cording morphotype. Importantly, the PcaA phosphomimetic strain, as the ΔpcaA mutant, exhibited reduced survival in human macrophages and was unable to prevent phagosome maturation. Our results add new insight into the importance of mycolic acid cyclopropane rings in the PMB and provide the first evidence of a Ser/Thr kinase-dependent mechanism for modulating mycolic acid composition and PMB.

The virulence protein SopD2 regulates membrane dynamics of Salmonella-containing vacuoles.

Salmonella enterica serovar Typhimurium is a Gram-negative bacterial pathogen causing gastroenteritis in humans and a systemic typhoid-like illness in mice. The capacity of Salmonella to cause diseases relies on the establishment of its intracellular replication niche, a membrane-bound compartment named the Salmonella-containing vacuole (SCV). This requires the translocation of bacterial effector proteins into the host cell by type three secretion systems. Among these effectors, SifA is required for the SCV stability, the formation of Salmonella-induced filaments (SIFs) and plays an important role in the virulence of Salmonella. Here we show that the effector SopD2 is responsible for the SCV instability that triggers the cytoplasmic release of a sifA(-) mutant. Deletion of sopD2 also rescued intra-macrophagic replication and increased virulence of sifA(-) mutants in mice. Membrane tubular structures that extend from the SCV are the hallmark of Salmonella-infected cells. Until now, these unique structures have not been observed in the absence of SifA. The deletion of sopD2 in a sifA(-) mutant strain re-established membrane trafficking from the SCV and led to the formation of new membrane tubular structures, the formation of which is dependent on other Salmonella effector(s). Taken together, our data demonstrate that SopD2 inhibits the vesicular transport and the formation of tubules that extend outward from the SCV and thereby contributes to the sifA(-) associated phenotypes. These results also highlight the antagonistic roles played by SopD2 and SifA in the membrane dynamics of the vacuole, and the complex actions of SopD2, SifA, PipB2 and other unidentified effector(s) in the biogenesis and maintenance of the Salmonella replicative niche.

The glyceraldehyde-3-phosphate dehydrogenase and the small GTPase Rab 2 are crucial for Brucella replication.

The intracellular pathogen Brucella abortus survives and replicates inside host cells within an endoplasmic reticulum (ER)-derived replicative organelle named the "Brucella-containing vacuole" (BCV). Here, we developed a subcellular fractionation method to isolate BCVs and characterize for the first time the protein composition of its replicative niche. After identification of BCV membrane proteins by 2 dimensional (2D) gel electrophoresis and mass spectrometry, we focused on two eukaryotic proteins: the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the small GTPase Rab 2 recruited to the vacuolar membrane of Brucella. These proteins were previously described to localize on vesicular and tubular clusters (VTC) and to regulate the VTC membrane traffic between the endoplasmic reticulum (ER) and the Golgi. Inhibition of either GAPDH or Rab 2 expression by small interfering RNA strongly inhibited B. abortus replication. Consistent with this result, inhibition of other partners of GAPDH and Rab 2, such as COPI and PKC iota, reduced B. abortus replication. Furthermore, blockage of Rab 2 GTPase in a GDP-locked form also inhibited B. abortus replication. Bacteria did not fuse with the ER and instead remained in lysosomal-associated membrane vacuoles. These results reveal an essential role for GAPDH and the small GTPase Rab 2 in B. abortus virulence within host cells.

Brucella evades macrophage killing via VirB-dependent sustained interactions with the endoplasmic reticulum.

The intracellular pathogen Brucella is the causative agent of brucellosis, a worldwide zoonosis that affects mammals, including humans. Essential to Brucella virulence is its ability to survive and replicate inside host macrophages, yet the underlying mechanisms and the nature of the replicative compartment remain unclear. Here we show in a model of Brucella abortus infection of murine bone marrow-derived macrophages that a fraction of the bacteria that survive an initial macrophage killing proceed to replicate in a compartment segregated from the endocytic pathway. The maturation of the Brucella-containing vacuole involves sustained interactions and fusion with the endoplasmic reticulum (ER), which creates a replicative compartment with ER-like properties. The acquisition of ER membranes by replicating Brucella is independent of ER-Golgi COPI-dependent vesicular transport. A mutant of the VirB type IV secretion system, which is necessary for intracellular survival, was unable to sustain interactions and fuse with the ER, and was killed via eventual fusion with lysosomes. Thus, we demonstrate that live intracellular Brucella evade macrophage killing through VirB-dependent sustained interactions with the ER. Moreover, we assign an intracellular function to the VirB system, as being required for late maturation events necessary for the biogenesis of an ER-derived replicative organelle.

Foamy macrophages from tuberculous patients' granulomas constitute a nutrient-rich reservoir for M. tuberculosis persistence.

Tuberculosis (TB) is characterized by a tight interplay between Mycobacterium tuberculosis and host cells within granulomas. These cellular aggregates restrict bacterial spreading, but do not kill all the bacilli, which can persist for years. In-depth investigation of M. tuberculosis interactions with granuloma-specific cell populations are needed to gain insight into mycobacterial persistence, and to better understand the physiopathology of the disease. We have analyzed the formation of foamy macrophages (FMs), a granuloma-specific cell population characterized by its high lipid content, and studied their interaction with the tubercle bacillus. Within our in vitro human granuloma model, M. tuberculosis long chain fatty acids, namely oxygenated mycolic acids (MA), triggered the differentiation of human monocyte-derived macrophages into FMs. In these cells, mycobacteria no longer replicated and switched to a dormant non-replicative state. Electron microscopy observation of M. tuberculosis-infected FMs showed that the mycobacteria-containing phagosomes migrate towards host cell lipid bodies (LB), a process which culminates with the engulfment of the bacillus into the lipid droplets and with the accumulation of lipids within the microbe. Altogether, our results suggest that oxygenated mycolic acids from M. tuberculosis play a crucial role in the differentiation of macrophages into FMs. These cells might constitute a reservoir used by the tubercle bacillus for long-term persistence within its human host, and could provide a relevant model for the screening of new antimicrobials against non-replicating persistent mycobacteria.

Mycobacterium requires an all-around closely apposing phagosome membrane to maintain the maturation block and this apposition is re-established when it rescues itself from phagolysosomes.

Pathogenic mycobacteria survive in macrophages of the host organism by residing in phagosomes which they prevent from undergoing maturation and fusion with lysosomes. Several molecular mechanisms have been associated with the phagosome maturation block. Here we show for Mycobacterium avium in mouse bone marrow-derived macrophages that the maturation block required an all-around close apposition between the mycobacterial surface and the phagosome membrane. When small (0.1 microm) latex beads were covalently attached to the mycobacterial surface to act as a spacer that interfered with a close apposition, phagosomes rapidly acquired lysosomal characteristics as indicators for maturation and fusion with lysosomes. As a result, several mycobacteria were delivered into single phagolysosomes. Detailed electron-microscope observations of phagosome morphology over a 7-day post-infection period showed a linear correlation between bead attachment and phagosome-lysosome fusion. After about 3 days post infection, conditions inside phagolysosomes caused a gradual release of beads. This allowed mycobacteria to re-establish a close apposition with the surrounding membrane and sequester themselves into individual, non-maturing phagosomes which had lost lysosomal characteristics. By rescuing themselves from phagolysosomes, mycobacteria remained fully viable and able to multiply at the normal rate. In order to unify the present observations and previously reported mechanisms for the maturation block, we discuss evidence that they may act synergistically to interfere with 'Phagosome Membrane Economics' by causing relative changes in incoming and outgoing endocytic membrane fluxes.

Dictyostelium cell death: early emergence and demise of highly polarized paddle cells.

Cell death in the stalk of Dictyostelium discoideum, a prototypic vacuolar cell death, can be studied in vitro using cells differentiating as a monolayer. To identify early events, we examined potentially dying cells at a time when the classical signs of Dictyostelium cell death, such as heavy vacuolization and membrane lesions, were not yet apparent. We observed that most cells proceeded through a stereotyped series of differentiation stages, including the emergence of "paddle" cells showing high motility and strikingly marked subcellular compartmentalization with actin segregation. Paddle cell emergence and subsequent demise with paddle-to-round cell transition may be critical to the cell death process, as they were contemporary with irreversibility assessed through time-lapse videos and clonogenicity tests. Paddle cell demise was not related to formation of the cellulose shell because cells where the cellulose-synthase gene had been inactivated underwent death indistinguishable from that of parental cells. A major subcellular alteration at the paddle-to-round cell transition was the disappearance of F-actin. The Dictyostelium vacuolar cell death pathway thus does not require cellulose synthesis and includes early actin rearrangements (F-actin segregation, then depolymerization), contemporary with irreversibility, corresponding to the emergence and demise of highly polarized paddle cells.

Functional role of the PE domain and immunogenicity of the Mycobacterium tuberculosis triacylglycerol hydrolase LipY.

PE and PPE proteins appear to be important for virulence and immunopathogenicity in mycobacteria, yet the functions of the PE/PPE domains remain an enigma. To decipher the role of these domains, we have characterized the triacylglycerol (TAG) hydrolase LipY from Mycobacterium tuberculosis, which is the only known PE protein expressing an enzymatic activity. The overproduction of LipY in mycobacteria resulted in a significant reduction in the pool of TAGs, consistent with the lipase activity of this enzyme. Unexpectedly, this reduction was more pronounced in mycobacteria overexpressing LipY lacking the PE domain [LipY(deltaPE)], suggesting that the PE domain participates in the modulation of LipY activity. Interestingly, Mycobacterium marinum contains a protein homologous to LipY, termed LipY(mar), in which the PE domain is substituted by a PPE domain. As for LipY, overexpression of LipY(mar) in Mycobacterium smegmatis significantly reduced the TAG pool, and this was further pronounced when the PPE domain of LipY(mar) was removed. Fractionation studies and Western blot analysis demonstrated that both LipY and LipY(deltaPE) were mainly present in the cell wall, indicating that the PE domain was not required for translocation to this site. Furthermore, electron microscopy immunolabeling of LipY(deltaPE) clearly showed a cell surface localization, thereby suggesting that the lipase may interact with the host immune system. Accordingly, a strong humoral response against LipY and LipY(deltaPE) was observed in tuberculosis patients. Together, our results suggest for the first time that both PE and PPE domains can share similar functional roles and that LipY represents a novel immunodominant antigen.

EM analysis of phagosomes.

Electron microscopy (EM) is the only technique that can combine sensitive protein-detection methods with detailed information on the substructure of cellular compartments. The purpose of this chapter is to describe some of the methods at the EM level that can be used to analyze the spatial organization of cell organelles with respect to phagosomes or vacuoles in which pathogens are sequestered, characterize the compartment in which pathogens are harbored, ie immature phagosomes, phagolysosomes, autophagosomes, and ER-derived vacuoles, to cite a few, and decipher the molecular mechanisms involved in survival of pathogens within infected host cells.

Brucella discriminates between mouse dendritic cell subsets upon in vitro infection.

Brucella is a Gram-negative bacterium responsible for brucellosis, a worldwide re-emerging zoonosis. Brucella has been shown to infect and replicate within Granulocyte macrophage colony-stimulating factor (GMCSF) in vitro grown bone marrow-derived dendritic cells (BMDC). In this cell model, Brucella can efficiently control BMDC maturation. However, it has been shown that Brucella infection in vivo induces spleen dendritic cells (DC) migration and maturation. As DCs form a complex network composed by several subpopulations, differences observed may be due to different interactions between Brucella and DC subsets. Here, we compare Brucella interaction with several in vitro BMDC models. The present study shows that Brucella is capable of replicating in all the BMDC models tested with a high infection rate at early time points in GMCSF-IL15 DCs and Flt3l DCs. GMCSF-IL15 DCs and Flt3l DCs are more activated than the other studied DC models and consequently intracellular bacteria are not efficiently targeted to the ER replicative niche. Interestingly, GMCSF-DC and GMCSF-Flt3l DC response to infection is comparable. However, the key difference between these 2 models concerns IL10 secretion by GMCSF DCs observed at 48 h post-infection. IL10 secretion can explain the weak secretion of IL12p70 and TNFα in the GMCSF-DC model and the low level of maturation observed when compared to GMCSF-IL15 DCs and Flt3l DCs. These models provide good tools to understand how Brucella induce DC maturation in vivo and may lead to new therapeutic design using DCs as cellular vaccines capable of enhancing immune response against pathogens.

Experimental Models of Foamy Macrophages and Approaches for Dissecting the Mechanisms of Lipid Accumulation and Consumption during Dormancy and Reactivation of Tuberculosis.

Despite a slight decline since 2014, tuberculosis (TB) remains the major deadly infectious disease worldwide with about 1.5 million deaths each year and with about one-third of the population being latently infected with Mycobacterium tuberculosis, the etiologic agent of TB. During primo-infection, the recruitment of immune cells leads to the formation of highly organized granulomas. Among the different cells, one outstanding subpopulation is the foamy macrophage (FM), characterized by the abundance of triacylglycerol-rich lipid bodies (LB). M. tuberculosis can reside in FM, where it acquires, from host LB, the neutral lipids which are subsequently processed and stored by the bacilli in the form of intracytosolic lipid inclusions (ILI). Although host LB can be viewed as a reservoir of nutrients for the pathogen during latency, the molecular mechanisms whereby intraphagosomal mycobacteria interact with LB and assimilate the LB-derived lipids are only beginning to be understood. Past studies have emphasized that these physiological processes are critical to the M. tuberculosis infectious-life cycle, for propagation of the infection, establishment of the dormancy state and reactivation of the disease. In recent years, several animal and cellular models have been developed with the aim of dissecting these complex processes and of determining the nature and contribution of their key players. Herein, we review some of the in vitro and in vivo models which allowed to gain significant insight into lipid accumulation and consumption in M. tuberculosis, two important events that are directly linked to pathogenicity, granuloma formation/maintenance and survival of the tubercle bacillus under non-replicative conditions. We also discuss the advantages and limitations of each model, hoping that this will serve as a guide for future investigations dedicated to persistence and innovative therapeutic approaches against TB.

The distinct fate of smooth and rough Mycobacterium abscessus variants inside macrophages.

Mycobacterium abscessus is a pathogenic, rapidly growing mycobacterium responsible for pulmonary and cutaneous infections in immunocompetent patients and in patients with Mendelian disorders, such as cystic fibrosis (CF). Mycobacterium abscessus is known to transition from a smooth (S) morphotype with cell surface-associated glycopeptidolipids (GPL) to a rough (R) morphotype lacking GPL. Herein, we show that M. abscessus S and R variants are able to grow inside macrophages and are present in morphologically distinct phagosomes. The S forms are found mostly as single bacteria within phagosomes characterized by a tightly apposed phagosomal membrane and the presence of an electron translucent zone (ETZ) surrounding the bacilli. By contrast, infection with the R form leads to phagosomes often containing more than two bacilli, surrounded by a loose phagosomal membrane and lacking the ETZ. In contrast to the R variant, the S variant is capable of restricting intraphagosomal acidification and induces less apoptosis and autophagy. Importantly, the membrane of phagosomes enclosing the S forms showed signs of alteration, such as breaks or partial degradation. Although not frequently encountered, these events suggest that the S form is capable of provoking phagosome-cytosol communication. In conclusion, M. abscessus S exhibits traits inside macrophages that are reminiscent of slow-growing mycobacterial species.