Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular interaction that is not clearly understood is non-lytic exocytosis. In this process, yeast cells are released into the extracellular space by a poorly understood mechanism that leaves both the macrophage and Cn viable. Here, we describe how to follow a large number of individually infected macrophages for a 24 hr infection period by time-lapsed microscopy. Infected macrophages are housed in a heating chamber with a CO₂ atmosphere attached to a microscope that provides the same conditions as a cell-culture incubator. Live digital microscopy can provide information about the dynamic interactions between a host and pathogen that is not available from static images. Being able to visualize each infected cell can provide clues as to how macrophages handle fungal infections, and vice versa. This technique is a powerful tool in studying the dynamics that are behind a complex phenomenon.     

Identification of Candida glabrata Genes Involved in pH Modulation and Modification of the Phagosomal Environment in Macrophages

Candida glabrata currently ranks as the second most frequent cause of invasive candidiasis. Our previous work has shown that C. glabrata is adapted to intracellular survival in macrophages and replicates within non-acidified late endosomal-stage phagosomes. In contrast, heat killed yeasts are found in acidified matured phagosomes. In the present study, we aimed at elucidating the processes leading to inhibition of phagosome acidification and maturation. We show that phagosomes containing viable C. glabrata cells do not fuse with pre-labeled lysosomes and possess low phagosomal hydrolase activity. Inhibition of acidification occurs independent of macrophage type (human/murine), differentiation (M1-/M2-type) or activation status (vitamin D₃ stimulation). We observed no differential activation of macrophage MAPK or NFκB signaling cascades downstream of pattern recognition receptors after internalization of viable compared to heat killed yeasts, but Syk activation decayed faster in macrophages containing viable yeasts. Thus, delivery of viable yeasts to non-matured phagosomes is likely not triggered by initial recognition events via MAPK or NFκB signaling, but Syk activation may be involved. Although V-ATPase is abundant in C. glabrata phagosomes, the influence of this proton pump on intracellular survival is low since blocking V-ATPase activity with bafilomycin A1 has no influence on fungal viability. Active pH modulation is one possible fungal strategy to change phagosome pH. In fact, C. glabrata is able to alkalinize its extracellular environment, when growing on amino acids as the sole carbon source in vitro. By screening a C. glabrata mutant library we identified genes important for environmental alkalinization that were further tested for their impact on phagosome pH. We found that the lack of fungal mannosyltransferases resulted in severely reduced alkalinization in vitro and in the delivery of C. glabrata to acidified phagosomes. Therefore, protein mannosylation may play a key role in alterations of phagosomal properties caused by C. glabrata.     

Cystic fibrosis transmembrane conductance regulator-independent phagosomal acidification in macrophages

It was reported recently that the cystic fibrosis transmembrane conductance regulator (CFTR) is required for acidification of phagosomes in alveolar macrophages (Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., Huang, P., Tong, J., Naren, A. P., Bindokas, V., Palfrey, H. C., and Nelson, D. J. (2006) Nat. Cell Biol. 8, 933-944). Here we determined whether the CFTR chloride channel is a generalized pathway for chloride entry into phagosomes in macrophages and whether mutations in CFTR could contribute to alveolar macrophage dysfunction. The pH of mature phagolysosomes in macrophages was measured by fluorescence ratio imaging using a zymosan conjugate containing Oregon Green(R) 488 and tetramethylrhodamine. Acidification of phagolysosomes in J774A.1 macrophages (pH approximately 5.1 at 45 min), murine alveolar macrophages (pH approximately 5.3), and human alveolar macrophages (pH approximately 5.3) was insensitive to CFTR inhibition by the thiazolidinone CFTR(inh)-172. Acidification of phagolysosomes in alveolar macrophages isolated from mice homozygous for DeltaF508-CFTR, the most common mutation in cystic fibrosis, was not different compared with that in alveolar macrophages isolated from wild-type mice. We also measured the kinetics of phagosomal acidification in J774A.1 and murine alveolar macrophages using a zymosan conjugate containing fluorescein and tetramethylrhodamine. Phagosomal acidification began within 3 min of zymosan binding and was complete within approximately 15 min of internalization. The rate of phagosomal acidification in J774A.1 cells was not slowed by CFTR(inh)-172 and was not different in alveolar macrophages from wild-type versus DeltaF508-CFTR mice. Our data indicate that phagolysosomal acidification in macrophages is not dependent on CFTR channel activity and do not support a proposed mechanism for cystic fibrosis lung disease involving defective phagosomal acidification and bacterial killing in alveolar macrophages.     

CFTR regulates phagosome acidification in macrophages and alters bactericidal activity

Acidification of phagosomes has been proposed to have a key role in the microbicidal function of phagocytes. Here, we show that in alveolar macrophages the cystic fibrosis transmembrane conductance regulator Cl- channel (CFTR) participates in phagosomal pH control and has bacterial killing capacity. Alveolar macrophages from Cftr-/- mice retained the ability to phagocytose and generate an oxidative burst, but exhibited defective killing of internalized bacteria. Lysosomes from CFTR-null macrophages failed to acidify, although they retained normal fusogenic capacity with nascent phagosomes. We hypothesize that CFTR contributes to lysosomal acidification and that in its absence phagolysosomes acidify poorly, thus providing an environment conducive to bacterial replication.     

Vomocytosis: What we know so far

Vomocytosis, or nonlytic exocytosis, has been reported for Cryptococcus neoformans since 2006. Since then, the repertoire of vomocytosing pathogens and host cells has increased and so have the molecular components linked to vomocytosis occurrence. Nonetheless, the mechanism underlying this phenomenon, whether it is triggered by the host or the pathogen, and how it affects disease progression are still unresolved. This review contains a summary of the main findings regarding vomocytosis and the outstanding questions puzzling scientists to this day.     

Phagocytosis of Cryptococcus neoformans by,and Nonlytic Exocytosis from,Acanthamoeba castellanii

Cryptococcus neoformans, an encapsulated, pathogenic yeast, is endowed with a variety of virulence factors, including a polysaccharide capsule. During mammalian infection, the outcome of the interaction between C. neoformans and macrophages is central to determining the fate of the host. Previous studies have shown similarities between the interaction of C. neoformans with macrophages and with amoebae, resulting in the proposal that fungal virulence for mammals originated from selection by amoeboid predators. In this study, we investigated the interaction of C. neoformans with the soil amoeba Acanthamoeba castellanii. Comparison of phagocytic efficiency of the wild type, nonencapsulated mutants, and complemented strains showed that the capsule was antiphagocytic for amoebae. Capsular enlargement was associated with a significant reduction in phagocytosis, suggesting that this phenomenon protects against ingestion by phagocytic predators. C. neoformans var. neoformans cells were observed to exit amoebae several hours after ingestion, in a process similar to the recently described nonlytic exocytosis from macrophages. Cryptococcal exocytosis from amoebae was dependent on the strain and on actin and required fungal viability. Additionally, the presence of a capsule was inversely correlated with the likelihood of extrusion in certain strains. In summary, nonlytic exocytosis from amoebae provide another parallel to observations in fungus-macrophage interactions. These results provide additional support for the notion that some mechanisms of virulence observed during mammalian infection originated, and were selected for, by environmental interactions.The encapsulated yeast Cryptococcus neoformans is an environmental organism that is capable of causing human disease. This fungus is a facultative intracellular pathogen with a unique pathogenic strategy, despite no obvious need for replication in an animal host as part of its life cycle (10). C. neoformans is known to interact with protozoa, some of which have been shown to be effective predators for this fungus (6, 26), and amoebae appear to be important for the control of C. neoformans in the environment (28). Previously, we reported that the interaction of C. neoformans with Acanthamoeba castellanii directly paralleled the interaction with human macrophages (33). Similarities between C. neoformans interactions with amoebae and macrophages included intracellular replication in a phagosome and the release of polysaccharide-containing vesicles into the cytoplasm (33). Furthermore, passage of avirulent C. neoformans and Histoplasma capsulatum through slime mold and amoebae was shown to increase virulence in mice (31, 32). On the basis of these observations, it was proposed that the capacity for mammalian virulence emerged from interactions with phagocytic predators, such as amoebae and slime mold, in the environment (7, 17, 30). Consequently, single-cell protists have emerged as important systems for the study of C. neoformans virulence, and subsequent studies have investigated the interaction of this fungus with slime mold and paramecia (9, 31). Additional evidence for this concept comes from studies of insect fungal pathogens, which suggest that the capacity for insect pathogenicity may follow preadaptation from interactions with amoebae in the environment (4). Understanding the mechanisms by which virulence emerges in environmental microbes is important considering that global warming has been hypothesized to bring about new fungal diseases in the coming century (13).Recent work in our laboratory and in that of Robin May simultaneously uncovered a novel strategy of avoiding macrophage killing whereby yeast cells were expulsed without lysis of the host cell (2, 19). The process is remarkable in that extrusion of the C. neoformans-filled phagosome is accompanied by the survival of both the host cells and the yeast cells. Phagosome extrusion or fungal exocytosis appears to be a C. neoformans-dictated event that is dependent on both the presence of the polysaccharide capsule and on the depolymerization of actin. A corollary of the hypothesis that C. neoformans virulence emerged from interactions with environmental predators is that phenomena observed with mammalian cells are likely to have a counterpart in free-living phagocytic cells. Consequently, the observation of an apparently unique event such as phagosomal extrusion from mammalian macrophages suggested a need to search for similar events in C. neoformans interactions with environmental phagocytic predators.In this study, we investigated parallels between the intracellular pathogenic strategy of C. neoformans in both macrophages and A. castellanii, focusing on characterizing the impact of the capsule on protozoan phagocytosis and on ascertaining whether fungal cells could also exit amoebae, including the role of the capsule in that possible mechanism. Using time-lapse microscopy, we observed the exocytosis of C. neoformans from A. castellanii. While there are significant differences in the nonlytic exocytosis process when comparing amoebae and macrophages, the observation of this phenomenon in amoebae provides additional support for the idea that the virulence of C. neoformans was selected for, and is maintained, by interactions in the environment with other soil organisms.(This research was conducted by Cara Chrisman in partial fulfillment of the requirements for a Ph.D. from the Sue Golding Graduate Division of Medical Science, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY [awarded in 2010].)     

The fungal pathogen Cryptococcus neoformans manipulates macrophage phagosome maturation

Phagocytosis by cells of the innate immune system, such as macrophages, and the subsequent successful maturation of the phagosome, is key for the clearance of pathogens. The fungal pathogen Cryptococcus neoformans is known to overcome killing by host phagocytes and both replicate within these cells and also escape via a non‐lytic process termed vomocytosis. Here we demonstrate that, during intracellular growth, cryptococci modify phagolysosome maturation. Live cryptococci, but not heat‐killed pathogens or inert targets, induce the premature removal of the early phagosome markers Rab5 and Rab11. In addition, significant acidification of the phagosome, calcium flux and protease activity is hindered, thus rendering the phagosome permissive for cryptococcal proliferation. Interestingly, several attenuated cryptococcal mutants retain this ability to subvert phagosomal maturation, suggesting that hitherto unidentified pathogen mechanisms regulate this process.     

Bafilomycin A1 inhibits lysosomal,phagosomal, and plasma membrane H+-ATPase and induces lysosomal enzyme secretion in macrophages

Bafilomycin A₁, a specific inhibitor of H⁺-ATPases of the vacuolar type, was in the present study shown, at similar concentrations, to induce secretion of lysosomal enzyme and to elevate lysosomal pH in mouse macrophages. These results lend support to the previous suggestion of a triggering role for an increase in lysosomal pH and a permissive role for cytosolic pH in the exocytosis of macrophage lysosomal enzyme. Vacuolar H⁺-ATPases are present in the macrophage plasma membrane as well as in intracellular membranes, for example, those of the lysosomal and phagosomal compartments. Phagosomal acidification was shown to be achieved in part by a mechanism with a similar sensitivity to bafilomycin A₁ as lysosomal H⁺ transport and in part by an early, bafilomycin A₁-insensitive mechanism. We found a lesser sensitivity towards bafilomycin A₁ of the lysosomal and phagosomal H⁺-ATPase than that localized in the plasma membrane, indicating differences among H⁺-ATPases at the subcellular level. Also, by attempts to mobilize lysosomal H⁺-ATPase to the plasma membrane, support was obtained for the notion that subcellular H⁺-ATPase populations differ and thus possibly could be differentially regulated. © 1995 Wiley-Liss, Inc.     

The Cch1-Mid1 High-Affinity Calcium Channel Contributes to the Virulence of Cryptococcus neoformans by Mitigating Oxidative Stress

Pathogenic fungi have developed mechanisms to cope with stresses imposed by hosts. For Cryptococcus spp., this implies active defense mechanisms that attenuate and ultimately overcome the onslaught of oxidative stresses in macrophages. Among cellular pathways within Cryptococcus neoformans'' arsenal is the plasma membrane high-affinity Cch1-Mid1 calcium (Ca²⁺) channel (CMC). Here we show that CMC has an unexpectedly complex and disparate role in mitigating oxidative stress. Upon inhibiting the Ccp1-mediated oxidative response pathway with antimycin, strains of C. neoformans expressing only Mid1 displayed enhanced growth, but this was significantly attenuated upon H₂O₂ exposure in the absence of Mid1, suggesting a regulatory role for Mid1 acting through the Ccp1-mediated oxidative stress response. This notion is further supported by the interaction detected between Mid1 and Ccp1 (cytochrome c peroxidase). In contrast, Cch1 appears to have a more general role in promoting cryptococci survival during oxidative stress. A strain lacking Cch1 displayed a growth defect in the presence of H₂O₂ without BAPTA [(1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, cesium salt] or additional stressors such as antimycin. Consistent with a greater contribution of Cch1 to oxidative stress tolerance, an intracellular growth defect was observed for the cch1Δ strain in the macrophage cell line J774A.1. Interestingly, while the absence of either Mid1 or Cch1 significantly compromises the ability of C. neoformans to tolerate oxidative stress, the absence of both Mid1 and Cch1 has a negligible effect on C. neoformans growth during H₂O₂ stress, suggesting the existence of a compensatory mechanism that becomes active in the absence of CMC.     

Fungal-Induced Cell Cycle Impairment, Chromosome Instability and Apoptosis via Differential Activation of NF-��B

Microbial pathogens have developed efficient strategies to compromise host immune responses. Cryptococcus neoformans is a facultative intracellular pathogen, recognised as the most common cause of systemic fungal infections leading to severe meningoencephalitis, mainly in immunocompromised patients. This yeast is characterized by a polysaccharide capsule, which inhibits its phagocytosis. Whereas phagocytosis escape and macrophage intracellular survival have been intensively studied, extracellular survival of this yeast and restraint of host innate immune response are still poorly understood. In this study, we have investigated whether C. neoformans affected macrophage cell viability and whether NF-κB (nuclear factor-κB), a key regulator of cell growth, apoptosis and inflammation, was involved. Using wild-type (WT) as well as mutant strains of C. neoformans for the pathogen side, and WT and mutant cell lines with altered NF-κB activity or signalling as well as primary macrophages for the host side, we show that C. neoformans manipulated NF-κB-mediated signalling in a unique way to regulate macrophage cell fate and viability. On the one hand, serotype A strains reduced macrophage proliferation in a capsule-independent fashion. This growth decrease, which required a critical dosage of NF-κB activity, was caused by cell cycle disruption and aneuploidy, relying on fungal-induced modification of expression of several cell cycle checkpoint regulators in S and G2/M phases. On the other hand, C. neoformans infection induced macrophage apoptosis in a capsule-dependent manner with a differential requirement of the classical and alternative NF-κB signalling pathways, the latter one being essential. Together, these findings shed new light on fungal strategies to subvert host response through uncoupling of NF-κB activity in pathogen-controlled apoptosis and impairment of cell cycle progression. They also provide the first demonstration of induction of aneuploidy by a fungal pathogen, which may have wider implications for human health as aneuploidy is proposed to promote tumourigenesis.     

Phagosomal acidification is mediated by a vacuolar-type H(+)-ATPase in murine macrophages

The mechanism underlying phagosomal acidification was studied in thioglycolate-elicited murine macrophages. The pH of the phagosomal compartment (pHp) was measured fluorimetrically in macrophage suspensions following ingestion of fluorescein isothiocyanate-labeled Staphylococcus aureus. At 37 degrees C, pHp decreased rapidly, reaching a steady state value of 5.8-6.1, while the cytoplasmic pH remained near neutrality, pH 7.1. The phagosome to cytosol pH gradient could be collapsed by addition of nigericin, monensin, or weak bases. The substrate dependence and inhibitor sensitivity profile of phagosomal acidification were investigated in intact and permeabilized cells. Phagosomal acidification was inhibited when ATP was depleted using metabolic inhibitors or permeabilizing the plasma membrane by electroporation. In permeabilized cells, acidification could be initiated by readdition of both Mg2+ and ATP. Neither adenosine 5'-(beta,gamma-imido)triphosphate nor adenosine 5'-(gamma-thio)triphosphate supported phagosomal acidification. Inhibitors of F1F0-type H(+)-ATPase such as oligomycin and azide, and the E1E2-type H(+)-ATPase inhibitor vanadate had no effect on phagosomal acidification. In contrast, the rate of phagosomal acidification was reduced by micromolar concentrations of N-ethylmaleimide and N,N'-dicyclohexylcarbodiimide. In permeabilized cells, nitrate inhibited the acidification with an apparent Ki of 25 mM. Phagosomal acidification was also effectively blocked by the macrolide antibiotic bafilomycin A1, with an apparent Ki of approximately 3 mM in both intact and electroporated cells. In this concentration range, bafilomycin A1 selectively inhibits vacuolar H(+)-ATPases. The substrate requirement and inhibitor susceptibility profile of phagosomal acidification strongly suggest that proton translocation across the phagosomal membrane is mediated by a vacuolar-type H(+)-ATPase.     

Phagosomal Acidification Prevents Macrophage Inflammatory Cytokine Production to Malaria,and Dendritic Cells Are the Major Source at the Early Stages of Infection: IMPLICATION FOR MALARIA PROTECTIVE IMMUNITY DEVELOPMENT*

Inflammatory cytokines produced at the early stages of malaria infection contribute to shaping protective immunity and pathophysiology. To gain mechanistic insight into these processes, it is important to understand the cellular origin of cytokines because both cytokine input and cytokine-producing cells play key roles. Here, we determined cytokine responses by monocytes, macrophages, and dendritic cells (DCs) to purified Plasmodium falciparum and Plasmodium berghei ANKA, and by spleen macrophages and DCs from Plasmodium yoelii 17NXL-infected and P. berghei ANKA-infected mice. The results demonstrate that monocytes and macrophages do not produce inflammatory cytokines to malaria parasites and that DCs are the primary source early in infection, and DC subsets differentially produce cytokines. Importantly, blocking of phagosomal acidification by inhibiting vacuolar-type H⁺-ATPase enabled macrophages to elicit cytokine responses. Because cytokine responses to malaria parasites are mediated primarily through endosomal Toll-like receptors, our data indicate that the inability of macrophages to produce cytokines is due to the phagosomal acidification that disrupts endosomal ligand-receptor engagement. Macrophages efficiently produced cytokines to LPS upon simultaneously internalizing parasites and to heat-killed Escherichia coli, demonstrating that phagosomal acidification affects endosomal receptor-mediated, but not cell surface receptor-mediated, recognition of Toll-like receptor agonists. Enabling monocytes/macrophages to elicit immune responses to parasites by blocking endosomal acidification can be a novel strategy for the effective development of protective immunity to malaria. The results have important implications for enhancing the efficacy of a whole parasite-based malaria vaccine and for designing strategies for the development of protective immunity to pathogens that induce immune responses primarily through endosomal receptors.     

Cytosolic Access of Mycobacterium tuberculosis: Critical Impact of Phagosomal Acidification Control and Demonstration of Occurrence In Vivo

Mycobacterium tuberculosis (Mtb) uses efficient strategies to evade the eradication by professional phagocytes, involving—as recently confirmed—escape from phagosomal confinement. While Mtb determinants, such as the ESX-1 type VII secretion system, that contribute to this phenomenon are known, the host cell factors governing this important biological process are yet unexplored. Using a newly developed flow-cytometric approach for Mtb, we show that macrophages expressing the phagosomal bivalent cation transporter Nramp-1, are much less susceptible to phagosomal rupture. Together with results from the use of the phagosome acidification inhibitor bafilomycin, we demonstrate that restriction of phagosomal acidification is a prerequisite for mycobacterial phagosomal rupture and cytosolic contact. Using different in vivo approaches including an enrichment and screen for tracking rare infected phagocytes carrying the CD45.1 hematopoietic allelic marker, we here provide first and unique evidence of M. tuberculosis-mediated phagosomal rupture in mouse spleen and lungs and in numerous phagocyte types. Our results, linking the ability of restriction of phagosome acidification to cytosolic access, provide an important conceptual advance for our knowledge on host processes targeted by Mtb evasion strategies.     

Macrophage Interaction with Paracoccidioides brasiliensis Yeast Cells Modulates Fungal Metabolism and Generates a Response to Oxidative Stress

Macrophages are key players during Paracoccidioides brasiliensis infection. However, the relative contribution of the fungal response to counteracting macrophage activity remains poorly understood. In this work, we evaluated the P. brasiliensis proteomic response to macrophage internalization. A total of 308 differentially expressed proteins were detected in P. brasiliensis during infection. The positively regulated proteins included those involved in alternative carbon metabolism, such as enzymes involved in gluconeogenesis, beta-oxidation of fatty acids and amino acids catabolism. The down-regulated proteins during P. brasiliensis internalization in macrophages included those related to glycolysis and protein synthesis. Proteins involved in the oxidative stress response in P. brasiliensis yeast cells were also up-regulated during macrophage infection, including superoxide dismutases (SOD), thioredoxins (THX) and cytochrome c peroxidase (CCP). Antisense knockdown mutants evaluated the importance of CCP during macrophage infection. The results suggested that CCP is involved in a complex system of protection against oxidative stress and that gene silencing of this component of the antioxidant system diminished the survival of P. brasiliensis in macrophages and in a murine model of infection.     

Glucosylceramide Administration as a Vaccination Strategy in Mouse Models of Cryptococcosis

Cryptococcus neoformans is an opportunistic fungal pathogen and the causative agent of the disease cryptococcosis. Cryptococcosis is initiated as a pulmonary infection and in conditions of immune deficiency disseminates to the blood stream and central nervous system, resulting in life-threatening meningoencephalitis. A number of studies have focused on the development of a vaccine against Cryptococcus, primarily utilizing protein-conjugated components of the Cryptococcus polysaccharide capsule as antigen. However, there is currently no vaccine against Cryptococcus in the clinic. Previous studies have shown that the glycosphingolipid, glucosylceramide (GlcCer), is a virulence factor in C. neoformans and antibodies against this lipid inhibit fungal growth and cell division. In the present study, we have investigated the possibility of using GlcCer as a therapeutic agent against C. neoformans infections in mouse models of cryptococcosis. GlcCer purified from a non-pathogenic fungus, Candida utilis, was administered intraperitoneally, prior to infecting mice with a lethal dose of C. neoformans. GlcCer administration prevented the dissemination of C. neoformans from the lungs to the brain and led to 60% mouse survival. GlcCer administration did not cause hepatic injury and elicited an anti-GlcCer antibody response, which was observed independent of the route of administration and the strains of mouse. Taken together, our results suggest that fungal GlcCer can protect mice against lethal doses of C. neoformans infection and can provide a viable vaccination strategy against Cryptococcus.     

Mycobacterium tuberculosis WhiB3 Responds to Vacuolar pH-induced Changes in Mycothiol Redox Potential to Modulate Phagosomal Maturation and Virulence

The ability of Mycobacterium tuberculosis to resist intraphagosomal stresses, such as oxygen radicals and low pH, is critical for its persistence. Here, we show that a cytoplasmic redox sensor, WhiB3, and the major M. tuberculosis thiol, mycothiol (MSH), are required to resist acidic stress during infection. WhiB3 regulates the expression of genes involved in lipid anabolism, secretion, and redox metabolism, in response to acidic pH. Furthermore, inactivation of the MSH pathway subverted the expression of whiB3 along with other pH-specific genes in M. tuberculosis. Using a genetic biosensor of mycothiol redox potential (E_MSH), we demonstrated that a modest decrease in phagosomal pH is sufficient to generate redox heterogeneity in E_MSH of the M. tuberculosis population in a WhiB3-dependent manner. Data indicate that M. tuberculosis needs low pH as a signal to alter cytoplasmic E_MSH, which activates WhiB3-mediated gene expression and acid resistance. Importantly, WhiB3 regulates intraphagosomal pH by down-regulating the expression of innate immune genes and blocking phagosomal maturation. We show that this block in phagosomal maturation is in part due to WhiB3-dependent production of polyketide lipids. Consistent with these observations, MtbΔwhiB3 displayed intramacrophage survival defect, which can be rescued bypharmacological inhibition of phagosomal acidification. Last, MtbΔwhiB3 displayed marked attenuation in the lungs of guinea pigs. Altogether, our study revealed an intimate link between vacuolar acidification, redox physiology, and virulence in M. tuberculosis and discovered WhiB3 as crucial mediator of phagosomal maturation arrest and acid resistance in M. tuberculosis.     

Yersinia pestis Requires Host Rab1b for Survival in Macrophages

Yersinia pestis is a facultative intracellular pathogen that causes the disease known as plague. During infection of macrophages Y. pestis actively evades the normal phagosomal maturation pathway to establish a replicative niche within the cell. However, the mechanisms used by Y. pestis to subvert killing by the macrophage are unknown. Host Rab GTPases are central mediators of vesicular trafficking and are commonly targeted by bacterial pathogens to alter phagosome maturation and killing by macrophages. Here we demonstrate for the first time that host Rab1b is required for Y. pestis to effectively evade killing by macrophages. We also show that Rab1b is specifically recruited to the Yersinia containing vacuole (YCV) and that Y. pestis is unable to subvert YCV acidification when Rab1b expression is knocked down in macrophages. Furthermore, Rab1b knockdown also altered the frequency of association between the YCV with the lysosomal marker Lamp1, suggesting that Rab1b recruitment to the YCV directly inhibits phagosome maturation. Finally, we show that Rab1b knockdown also impacts the pH of the Legionella pneumophila containing vacuole, another pathogen that recruits Rab1b to its vacuole. Together these data identify a novel role for Rab1b in the subversion of phagosome maturation by intracellular pathogens and suggest that recruitment of Rab1b to the pathogen containing vacuole may be a conserved mechanism to control vacuole pH.     

Contribution of Fdh3 and Glr1 to Glutathione Redox State,Stress Adaptation and Virulence in Candida albicans

The major fungal pathogen of humans, Candida albicans, is exposed to reactive nitrogen and oxygen species following phagocytosis by host immune cells. In response to these toxins, this fungus activates potent anti-stress responses that include scavenging of reactive nitrosative and oxidative species via the glutathione system. Here we examine the differential roles of two glutathione recycling enzymes in redox homeostasis, stress adaptation and virulence in C. albicans: glutathione reductase (Glr1) and the S-nitrosoglutathione reductase (GSNOR), Fdh3. We show that the NADPH-dependent Glr1 recycles GSSG to GSH, is induced in response to oxidative stress and is required for resistance to macrophage killing. GLR1 deletion increases the sensitivity of C. albicans cells to H₂O₂, but not to formaldehyde or NO. In contrast, Fdh3 detoxifies GSNO to GSSG and NH₃, and FDH3 inactivation delays NO adaptation and increases NO sensitivity. C. albicans fdh3⎔ cells are also sensitive to formaldehyde, suggesting that Fdh3 also contributes to formaldehyde detoxification. FDH3 is induced in response to nitrosative, oxidative and formaldehyde stress, and fdh3Δ cells are more sensitive to killing by macrophages. Both Glr1 and Fdh3 contribute to virulence in the Galleria mellonella and mouse models of systemic infection. We conclude that Glr1 and Fdh3 play differential roles during the adaptation of C. albicans cells to oxidative, nitrosative and formaldehyde stress, and hence during the colonisation of the host. Our findings emphasise the importance of the glutathione system and the maintenance of intracellular redox homeostasis in this major pathogen.     

Phthiocerol Dimycocerosates of M. tuberculosis Participate in Macrophage Invasion by Inducing Changes in the Organization of Plasma Membrane Lipids

Phthiocerol dimycocerosates (DIM) are major virulence factors of Mycobacterium tuberculosis (Mtb), in particular during the early step of infection when bacilli encounter their host macrophages. However, their cellular and molecular mechanisms of action remain unknown. Using Mtb mutants deleted for genes involved in DIM biosynthesis, we demonstrated that DIM participate both in the receptor-dependent phagocytosis of Mtb and the prevention of phagosomal acidification. The effects of DIM required a state of the membrane fluidity as demonstrated by experiments conducted with cholesterol-depleting drugs that abolished the differences in phagocytosis efficiency and phagosome acidification observed between wild-type and mutant strains. The insertion of a new cholesterol-pyrene probe in living cells demonstrated that the polarity of the membrane hydrophobic core changed upon contact with Mtb whereas the lateral diffusion of cholesterol was unaffected. This effect was dependent on DIM and was consistent with the effect observed following DIM insertion in model membrane. Therefore, we propose that DIM control the invasion of macrophages by Mtb by targeting lipid organisation in the host membrane, thereby modifying its biophysical properties. The DIM-induced changes in lipid ordering favour the efficiency of receptor-mediated phagocytosis of Mtb and contribute to the control of phagosomal pH driving bacilli in a protective niche.