Label-free proteomics and systems biology analysis of mycobacterial phagosomes in dendritic cells and macrophages

Proteomics has been applied to study intracellular bacteria and phagocytic vacuoles in different host cell lines, especially macrophages (Mφs). For mycobacterial phagosomes, few studies have identified over several hundred proteins for systems assessment of the phagosome maturation and antigen presentation pathways. More importantly, there has been a scarcity in publication on proteomic characterization of mycobacterial phagosomes in dendritic cells (DCs). In this work, we report a global proteomic analysis of Mφ and DC phagosomes infected with a virulent, an attenuated, and a vaccine strain of mycobacteria. We used label-free quantitative proteomics and bioinformatics tools to decipher the regulation of phagosome maturation and antigen presentation pathways in Mφs and DCs. We found that the phagosomal antigen presentation pathways are repressed more in DCs than in Mφs. The results suggest that virulent mycobacteria might co-opt the host immune system to stimulate granuloma formation for persistence while minimizing the antimicrobial immune response to enhance mycobacterial survival. The studies on phagosomal proteomes have also shown promise in discovering new antigen presentation mechanisms that a professional antigen presentation cell might use to overcome the mycobacterial blockade of conventional antigen presentation pathways.     

The sodium proton exchanger NHE9 regulates phagosome maturation and bactericidal activity in macrophages

Acidification of phagosomes is essential for the bactericidal activity of macrophages. Targeting machinery that regulates pH within the phagosomes is a prominent strategy employed by various pathogens that have emerged as major threats to public health. Nascent phagosomes acquire the machinery for pH regulation through a graded maturation process involving fusion with endolysosomes. Meticulous coordination between proton pumping and leakage mechanisms is crucial for maintaining optimal pH within the phagosome. However, relative to mechanisms involved in acidifying the phagosome lumen, little is known about proton leakage pathways in this organelle. Sodium proton transporter NHE9 is a known proton leakage pathway located on the endosomes. As phagosomes acquire proteins through fusions with endosomes during maturation, NHE9 seemed a promising candidate for regulating proton fluxes on the phagosome. Here, using genetic and biophysical approaches, we show NHE9 is an important proton leakage pathway associated with the maturing phagosome. NHE9 is highly expressed in immune cells, specifically macrophages; however, NHE9 expression is strongly downregulated upon bacterial infection. We show that compensatory ectopic NHE9 expression hinders the directed motion of phagosomes along microtubules and promotes early detachment from the microtubule tracks. As a result, these phagosomes have shorter run lengths and are not successful in reaching the lysosome. In accordance with this observation, we demonstrate that NHE9 expression levels negatively correlate with bacterial survival. Together, our findings show that NHE9 regulates lumenal pH to affect phagosome maturation, and consequently, microbicidal activity in macrophages.     

The phagosome proteome: insight into phagosome functions

Phagosomes are key organelles for the innate ability of macrophages to participate in tissue remodeling, clear apoptotic cells, and restrict the spread of intracellular pathogens. To understand the functions of phagosomes, we initiated the systematic identification of their proteins. Using a proteomic approach, we identified >140 proteins associated with latex bead-containing phagosomes. Among these were hydrolases, proton pump ATPase subunits, and proteins of the fusion machinery, validating our approach. A series of unexpected proteins not previously described along the endocytic/phagocytic pathways were also identified, including the apoptotic proteins galectin3, Alix, and TRAIL, the anti-apoptotic protein 14-3-3, the lipid raft-enriched flotillin-1, the anti-microbial molecule lactadherin, and the small GTPase rab14. In addition, 24 spots from which the peptide masses could not be matched to entries in any database potentially represent new phagosomal proteins. The elaboration of a two-dimensional gel database of >160 identified spots allowed us to analyze how phagosome composition is modulated during phagolysosome biogenesis. Remarkably, during this process, hydrolases are not delivered in bulk to phagosomes, but are instead acquired sequentially. The systematic characterization of phagosome proteins provided new insights into phagosome functions and the protein or groups of proteins involved in and regulating these functions.     

Unraveling the human dendritic cell phagosome proteome by organellar enrichment ranking

Dendritic cells (DC) take up pathogens through phagocytosis and process them into protein and lipid fragments for presentation to T cells. So far, the proteome of the human DC phagosome, a detrimental compartment for antigen processing and presentation as well as for DC activation, remains largely uncharacterized. Here we have analyzed the protein composition of phagosomes from human monocyte-derived DC. For LC-MS/MS analysis we purified phagosomes from DC using latex beads targeted to DC-SIGN, and quantified proteins using a label-free method. We used organellar enrichment ranking (OER) to select proteins with a high potential to be relevant for phagosome function. The method compares phagosome protein abundance with protein abundance in whole DC. Phagosome enrichment indicates specific recruitment to the phagosome rather than co-purification or passive incorporation. Using OER we extracted the most enriched proteins that we further complemented with functionally associated proteins to define a set of 90 phagosomal proteins that included many proteins with established relevance on DC phagosomes as well as high potential novel candidates. We already experimentally confirmed phagosomal recruitment of Galectin-9, which has not been previously associated with phagocytosis, to both bead and pathogen containing phagosomes, suggesting a role for Galectin-9 in DC phagocytosis.     

Association of a macrophage galactoside-binding protein with Mycobacterium-containing phagosomes

Mycobacteria reside intracellularly in a vacuole that allows it to circumvent the antimicrobial environment of the host macrophage. Although the mycobacterial phagosome exhibits selective fusion with vesicles of the endosomal system, identification of host and bacterial factors associated with phagosome bio-genesis is limited. To identify these potential factors, mAbs were generated to a membrane preparation of mycobacterial phagosomes isolated from M. tuberculosis -infected macrophages. A mAb recognizing a 32–35 kDa macrophage protein associated with the phagosomal membrane of Mycobacterium was identified. N-terminal sequence analysis identified this protein as Mac-2 or galectin-3, a galactoside-binding protein of macrophages. Galectin-3 (gal-3) was shown to accumulate in Mycobacterium -containing phagosomes during the course of infection. This accumu-lation was specific for phagosomes containing live mycobacteria and occurred primarily at the cytosolic face of the phagosome membrane. In addition, bind-ing of gal-3 to mycobacterial phosphatidylinositol mannosides (PIMs) demonstrated a novel interaction between host carbohydrate-binding proteins and released mycobacterial glycolipids. Infection of macrophages from gal-3-deficient mice indicated that the protein did not play a role in infection in vitro . In contrast, infection of gal-3-deficient mice revealed a reduced capacity to clear late but not early infection.     

The Tetrahymena thermophila phagosome proteome

In vertebrates, phagocytosis occurs mainly in specialized cells of the immune system and serves as a primary defense against invading pathogens, but it also plays a role in clearing apoptotic cells and in tissue remodeling during development. In contrast, unicellular eukaryotes, such as the ciliate Tetrahymena thermophila, employ phagocytosis to ingest and degrade other microorganisms to meet their nutritional needs. To learn more about the protein components of the multistep process of phagocytosis, we carried out an analysis of the Tetrahymena phagosome proteome. Tetrahymena cells were fed polystyrene beads, which allowed for the efficient purification of phagosomes. The protein composition of purified phagosomes was then analyzed by multidimensional separation coupled with tandem mass spectrometry. A total of 453 peptides were identified that resulted in the identification of 73 putative phagosome proteins. Twenty-eight of the proteins have been implicated in phagocytosis in other organisms, indicating that key aspects of phagocytosis were conserved during evolution. Other identified proteins have not previously been associated with phagocytosis, including some of unknown function. Live-cell confocal fluorescence imaging of Tetrahymena strains expressing green fluorescent protein-tagged versions of four of the identified phagosome proteins provided evidence that at least three of the proteins (including two with unknown functions) are associated with phagosomes, indicating that the bulk of the proteins identified in the analyses are indeed phagosome associated.     

Coronin is involved in uptake of Mycobacterium bovis BCG in human macrophages but not in phagosome maintenance

By applying density gradient electrophoresis (DGE) to human macrophages infected with Mycobacterium bovis BCG, we were able to separate three different bacterial fractions representing arrested phagosomes, phagolysosomes and mycobacterial clumps. After further purification of the phagosomal population, we found that isolated phagosomes containing live BCG were arrested in maturation as they exhibited only low amounts of the lysosomal glycoprotein LAMP-1 and processing of the lysosomal hydrolase cathepsin D was blocked. In addition, low amounts of MHC class I and class II molecules and the absence of HLA-DM suggest sequestration of mycobacterial phagosomes from antigen-processing pathways. We further investigated the involvement of the actin-binding protein coronin in intracellular survival of mycobacteria and showed that human coronin, as well as F-actin, were associated with early stages of mycobacterial phagocytosis but not with phagosome maintenance. Therefore, we conclude that the unique DGE migration pattern of arrested phagosomes is not as a result of retention of coronin, but that there are other proteins or lipids responsible for the block in maturation in human macrophages.     

The Mycobacterium bovis Bacille Calmette-Gu��rin Phagosome Proteome

Mycobacterium tuberculosis and Mycobacterium bovis bacille Calmette-Guérin (BCG) alter the maturation of their phagosomes and reside within a compartment that resists acidification and fusion with lysosomes. To define the molecular composition of this compartment, we developed a novel method for obtaining highly purified phagosomes from BCG-infected human macrophages and analyzed the phagosomes by Western immunoblotting and mass spectrometry-based proteomics. Our purification procedure revealed that BCG grown on artificial medium becomes less dense after growth in macrophages. By Western immunoblotting, LAMP-2, Niemann-Pick protein C1, and syntaxin 3 were readily detectable on the BCG phagosome but at levels that were lower than on the latex bead phagosome; flotillin-1 and the vacuolar ATPase were barely detectable on the BCG phagosome but highly enriched on the latex bead phagosome. Immunofluorescence studies confirmed the scarcity of flotillin on BCG phagosomes and demonstrated an inverse correlation between bacterial metabolic activity and flotillin on M. tuberculosis phagosomes. By mass spectrometry, 447 human host proteins were identified on BCG phagosomes, and a partially overlapping set of 289 human proteins on latex bead phagosomes was identified. Interestingly, the majority of the proteins identified consistently on BCG phagosome preparations were also identified on latex bead phagosomes, indicating a high degree of overlap in protein composition of these two compartments. It is likely that many differences in protein composition are quantitative rather than qualitative in nature. Despite the remarkable overlap in protein composition, we consistently identified a number of proteins on the BCG phagosomes that were not identified in any of our latex bead phagosome preparations, including proteins involved in membrane trafficking and signal transduction, such as Ras GTPase-activating-like protein IQGAP1, and proteins of unknown function, such as FAM3C. Our phagosome purification procedure and initial proteomics analyses set the stage for a quantitative comparative analysis of mycobacterial and latex bead phagosome proteomes.Mycobacterium tuberculosis, the etiological agent of tuberculosis, is a facultative intracellular bacterium. In human macrophages, M. tuberculosis resides in a membrane-bound phagosomal compartment that resists fusion with lysosomes and is only mildly acidified (1–5). In previous studies, using the cryosection immunogold technique, we have found that the M. tuberculosis phagosome exhibits delayed clearance of major histocompatability complex class I molecules and relatively weak staining for lysosomal membrane glycoproteins CD63, LAMP-1,¹ and LAMP-2 and the lysosomal acid protease cathepsin D (6–10). Studies by other investigators have also demonstrated that M. tuberculosis and other mycobacterial species, including Mycobacterium bovis BCG, reside in phagosomes that resist acidification, are less mature, and less fusogenic with lysosomes than phagosomes containing inert particles (11–13). These results are consistent with the hypothesis that M. tuberculosis and M. bovis BCG retard the maturation of their phagosomes along the endolysosomal pathway and reside in a compartment that has not matured fully to a phagolysosome (7). Although the phagosomes of latex beads have been subjected to detailed proteomics analysis by Desjardins and co-workers (14), a detailed proteomics study of the M. bovis BCG phagosome has not been reported previously.We describe in this study a novel method for the purification of the BCG phagosome from infected human macrophages, a detailed proteomics analysis of the BCG phagosome, and a comparison of the phagosome with latex bead phagosomes isolated from human macrophages. This study is the first comprehensive proteomics study of the M. bovis BCG phagosome and the first mass spectrometry-based proteomics study of the latex bead phagosome in human macrophages. We showed by Western immunoblotting that, relative to latex bead phagosomes, the BCG phagosome is relatively depleted in LAMP-2, NPC1, flotillin-1, vATPase, and syntaxin 3. Remarkably, by mass spectrometry, we documented a high degree of overlap in the set of proteins on BCG and latex bead phagosomes but also noteworthy differences. Novel proteins detected on the BCG phagosome but not on the latex bead phagosome include CD44, intercellular adhesion molecule 1, protein FAM3C, Ral-A/Ral-B, stress-induced phosphoprotein 1, band 4.1-like protein 3, septin-7, Ras GTPase-activating protein-like protein IQGAP1, Rab-6A, erlin-2, and tumor protein D54. Conversely, proteins identified on latex bead phagosomes but not on the BCG phagosome are β-galactosidase and sialate O-acetylesterase.     

Membrane trafficking along the phagocytic pathway

Phagosome maturation involves extensive remodelling of the phagosomal membrane as a result of intracellular transport events. Newly formed phagosomes exchange membrane-associated and soluble proteins with early endosomes by fusion. Budding of vesicles from the phagosome and fusion with Golgi-derived vesicles may also contribute to the remodelling of the phagosomal compartment. As a consequence of changes in membrane composition, phagosomes acquire the ability to fuse with late endocytic compartments. In vitro reconstitution and other studies suggest that the trafficking events underlying phagosome maturation require several GTP-binding proteins, including Rab5 and Galphas', NSF-SNAP-SNARE complexes and coatomers.     

Maturation of human neutrophil phagosomes includes incorporation of molecular chaperones and endoplasmic reticulum quality control machinery

A Human neutrophils are an essential component of the innate immune response. Although significant progress has been made toward understanding mechanisms of phagocytosis and microbicidal activity, a comprehensive analysis of proteins comprising neutrophil phagosomes has not been conducted. To that end, we used subcellular proteomics to identify proteins associated with human neutrophil phagosomes following receptor-mediated phagocytosis. Proteins (n = 411 spots) resolved from neutrophil phagosome fractions were identified by MALDI-TOF MS and/or LC-MS/MS analysis. Those associated with phagocytic vacuoles originated from multiple subcellular compartments, including the cytosol, plasma membrane, specific and azurophilic granules, and cytoskeleton. Unexpectedly several enzymes typically associated with mitochondria were identified in phagosome fractions. Furthermore proteins characteristic of the endoplasmic reticulum, including 11 molecular chaperones, were resolved from phagosome preparations. Confocal microscopy confirmed that proteins representing these major subcellular compartments were enriched on phagosomes of intact neutrophils. Notably calnexin and glucose-regulated protein 78 co-localized with gp91(phox) in human neutrophils and were thus likely delivered to phagosomes by fusion of specific granules. We conclude that neutrophil phagosomes have heretofore unrecognized complexity and function, which includes potential for antigen processing events.     

Intraphagosomal Measurement of the Magnitude and Duration of the Oxidative Burst

Generation of an oxidative burst within the phagosomes of neutrophils, dendritic cells and macrophages is an essential component of the innate immune system. To examine the kinetics of the oxidative burst in the macrophage phagosome, we developed two new assays using beads coated with oxidation-sensitive fluorochromes. These assays permitted quantification and temporal resolution of the oxidative burst within the phagosome. The macrophage phagosomal oxidative burst is short lived, with oxidation of bead-associated substrates reaching maximal activity within 30 min following phagocytosis. Additionally, the extent and rate of macrophage phagosomal substrate oxidation were subject to immunomodulation by activation with lipopolysaccharide and/or interferon-γ.     

Phagocyte meets prey: uptake, internalization, and killing of bacteria by Dictyostelium amoebae

Dictyostelium cells are professional phagocytes that avidly consume bacteria, their natural prey. Fluorescent probes have allowed us to monitor the initial steps in this process in living cells. Using probes that bind to F-actin, we have visualized the assembly and disassembly of actin filaments responsible for extending the phagocytic cup to engulf a bacterium, and, after the phagosome has sealed, the assembly of new actin filaments to propel the phagosome away from the site of uptake. Using bacteria expressing fluorescent proteins that are susceptible to proteolysis, we have monitored the loss of that fluorescent signal and the staining of the bacterial contents with neutral red, indicating permeabilization of the bacterial cell wall and acidification of the cytoplasm. We find that acidification occurs during a period of microtubule-based transport that promotes fusion of the phagosome with microtubule-associated acidic endosomes. Actin-powered phagosome internalization, transport of the phagosome along microtubules, proteolysis and acidification of bacterial contents, all typically occur within the first six or seven minutes after formation of the phagosome. Thus, tracking individual phagosomes has revealed that early steps in phagosome maturation occur much more rapidly than had been inferred from previous population studies.     

Mycobacterium tuberculosis-specific phagosome proteome and underlying signaling pathways

The phagosome is very important to host immunity and tissue homeostasis maintenance. The destiny of the phagosome is closely associated with the outcome of the pathogen within. Most pathogens are successfully delivered to the lysosome and destroyed via the fusion of the phagosome with the lysosome. Mycobacterium tuberculosis has evolved multiple tactics to deflect the normal fusion process, such as delaying the phagosome maturation and acidification, thereby evading the immune recognition and subsequent elimination. Identification of the specific constituents of M. tuberculosis phagosome and the underlying signaling pathways are pivotal to define the key molecular features of this process and better targets to control this recalcitrant pathogen. Proteomic profiling is a comprehensive approach to define the protein inventory. In this review, currently available mycobacteria-containing phagosome proteome data were compiled. Ten putative evolutionarily conserved phagosome proteins were summarized. Unique proteins of the M. tuberculosis-containing phagosome proteome were compiled via comparison with other phagosomes, especially the inert latex bead phagosome. Signaling events associated with these unique proteins, such as Rab GTPase and PI3P, were also found and discussed. The data will facilitate better characterization of the M. tuberculosis specific phagosome constituents and involved signaling, and host-derived targets for better tuberculosis control.     

Arrested maturation of Neisseria-containing phagosomes in the absence of the lysosome-associated membrane proteins, LAMP-1 and LAMP-2

Mature, microbicidal phagosomes are rich in the lysosome-associated membrane proteins, LAMP-1 and LAMP-2, two highly glycosylated proteins presumed to form a protective barrier lining the phagosomal membrane. Pathogenic Neisseria secrete a protease that selectively cleaves LAMP-1, suggesting a critical role for LAMP proteins in the microbicidal competence of phagosomes. To determine the requirement for LAMP proteins in bacterial phagocytosis, we employed embryonic fibroblasts isolated from knockout mice lacking lamp-1, lamp-2 or both genes, as well as small interfering RNA (siRNA)-mediated knockdown of LAMP expression in a human epithelial cell line. Like wild-type cells, those lacking either LAMP-1 or LAMP-2 alone formed phagosomes that gradually acquired microbicidal activity and curtailed bacterial growth. In contrast, LAMP-1 and LAMP-2 double-deficient fibroblasts failed to kill engulfed Neisseria gonorrhoeae. In these cells, maturation was arrested prior to the acquisition of Rab7. As a result, the Rab7-interacting lysosomal protein (RILP, a Rab7 effector) was not recruited to the phagosomes, which were consequently unable to undergo dynein/dynactin-mediated centripetal displacement along microtubules and remained in a predominantly peripheral location. The inability of such phagosomes to migrate towards lysosomes likely contributed to their incomplete maturation and inability to eliminate bacteria. These findings suggest that neisserial degradation of LAMP-1 is not sufficient to affect its survival within the phagosome, and establish LAMP proteins as critical components in the process whereby phagosomes acquire microbicidal capabilities.     

Neisseria gonorrhoeae phagosomes delay fusion with primary granules to enhance bacterial survival inside human neutrophils

Symptomatic infection with Neisseria gonorrhoeae (Gc) promotes inflammation driven by polymorphonuclear leucocytes (PMNs, neutrophils), yet some Gc survive PMN exposure during infection. Here we report a novel mechanism of gonococcal resistance to PMNs: Gc phagosomes avoid maturation into phagolysosomes by delayed fusion with primary (azurophilic) granules, which contain antimicrobial components including serine proteases. Reduced phagosome‐primary granule fusion was observed in gonorrheal exudates and human PMNs infected ex vivo. Delayed phagosome–granule fusion could be overcome by opsonizing Gc with immunoglobulin. Using bacterial viability dyes along with antibodies to primary granules revealed that Gc survival in PMNs correlated with early residence in primary granule‐negative phagosomes. However, when Gc was killed prior to PMN exposure, dead bacteria were also found in primary granule‐negative phagosomes. These results suggest that Gc surface characteristics, rather than active bacterial processes, influence phagosome maturation and that Gc death inside PMNs occurs after phagosome–granule fusion. Ectopically increasing primary granule–phagosome fusion, by immunoglobulin opsonization or PMN treatment with lysophosphatidylcholine, reduced intracellular Gc viability, which was attributed in part to serine protease activity. We conclude that one method for Gc to avoid PMN clearance in acute gonorrhoea is by delaying primary granule–phagosome fusion, thus preventing formation of a degradative phagolysosome.     

Phagocyte meets prey: Uptake, internalization, and killing of bacteria by Dictyostelium amoebae

Dictyostelium cells are professional phagocytes that avidly consume bacteria, their natural prey. Fluorescent probes have allowed us to monitor the initial steps in this process in living cells. Using probes that bind to F-actin, we have visualized the assembly and disassembly of actin filaments responsible for extending the phagocytic cup to engulf a bacterium, and, after the phagosome has sealed, the assembly of new actin filaments to propel the phagosome away from the site of uptake. Using bacteria expressing fluorescent proteins that are susceptible to proteolysis, we have monitored the loss of that fluorescent signal and the staining of the bacterial contents with neutral red, indicating permeabilization of the bacterial cell wall and acidification of the cytoplasm. We find that acidification occurs during a period of microtubule-based transport that promotes fusion of the phagosome with microtubule-associated acidic endosomes. Actin-powered phagosome internalization, transport of the phagosome along microtubules, proteolysis and acidification of bacterial contents, all typically occur within the first six or seven minutes after formation of the phagosome. Thus, tracking individual phagosomes has revealed that early steps in phagosome maturation occur much more rapidly than had been inferred from previous population studies.     

Macrophage activation downregulates the degradative capacity of the phagosome

The phagosome is key to most macrophage functions. It is the site of degradation of particulate material, of bacterial killing and the generation of peptides for antigen presentation. Despite its role at the fulcrum of the innate and acquired immune systems, little is known about the physiology of this organelle in activated macrophages. In this study, we utilize fluorometric techniques to characterize functional alterations in the lumenal environment of the maturing phagosome following stimulation of macrophages with interferon-gamma and/or lipopolysaccharide. In addition to modulating the kinetics of phagosomal acidification, activation results in a phagosome with diminished hydrolytic activities that varies markedly with the activation status of the cell. Differential levels of proteolytic, lipolytic and beta-galactosidase activities were observed in the phagosome but not in the total lysosomal extract, indicating selective delivery of enzymes to the developing phagosome. Despite the suppression of hydrolytic activities observed in early phagosomes, late phagosomes exhibit an enhanced and protracted accumulation of lysosomal cargo. The data are consistent with limiting proteolysis in the early phagosome to maximize epitope generation and antigen presentation while sequestering the degradative capacity in the late phagolysosome.     

The Francisella tularensis pathogenicity island protein IglC and its regulator MglA are essential for modulating phagosome biogenesis and subsequent bacterial escape into the cytoplasm

The Francisella tularensis subsp. novicida-containing phagosome (FCP) matures into a late endosome-like stage that acquires the late endosomal marker LAMP-2 but does not fuse to lysosomes, for the first few hours after bacterial entry. This modulation in phagosome biogenesis is followed by disruption of the phagosome and bacterial escape into the cytoplasm where they replicate. Here we examined the role of the Francisella pathogenicity island (FPI) protein IglC and its regulator MglA in the intracellular fate of F. tularensis subsp. novicida within human macrophages. We show that F. tularensis mglA and iglC mutant strains are defective for survival and replication within U937 macrophages and human monocyte-derived macrophages (hMDMs). The defect in intracellular replication of both mutants is associated with a defect in disruption of the phagosome and failure to escape into the cytoplasm. Approximately, 80-90% of the mglA and iglC mutants containing phagosomes acquire the late endosomal/lysosomal marker LAMP-2 similar to the wild-type (WT) strain. Phagosomes harbouring the mglA or iglC mutants acquire the lysosomal enzyme Cathepsin D, which is excluded from the phagosomes harbouring the WT strain. In hMDMs in which the lysosomes are preloaded with BSA-gold or Texas Red Ovalbumin, phagosomes harbouring the mglA or the iglC mutants acquire both lysosomal tracers. We conclude that the FPI protein IglC and its regulator MglA are essential for modulating phagosome biogenesis and subsequent bacterial escape into the cytoplasm. Therefore, acquisition of the FPI, within which iglC is contained, is essential for the pathogenic evolution of F. tularensis to evade lysosomal fusion within human macrophages and cause tularemia. This is the first example of specific virulence factors of F. tularensis that are essential for evasion of fusion of the FCP to lysosomes.     

The fbpA/sapM double knock out strain of Mycobacterium tuberculosis is highly attenuated and immunogenic in macrophages

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death due to bacterial infections in mankind, and BCG, an attenuated strain of Mycobacterium bovis, is an approved vaccine. BCG sequesters in immature phagosomes of antigen presenting cells (APCs), which do not fuse with lysosomes, leading to decreased antigen processing and reduced Th1 responses. However, an Mtb derived ΔfbpA attenuated mutant underwent limited phagosome maturation, enhanced immunogenicity and was as effective as BCG in protecting mice against TB. To facilitate phagosome maturation of ΔfbpA, we disrupted an additional gene sapM, which encodes for an acid phosphatase. Compared to the wild type Mtb, the ΔfbpAΔsapM (double knock out; DKO) strain was attenuated for growth in mouse macrophages and PMA activated human THP1 macrophages. Attenuation correlated with increased oxidants in macrophages in response to DKO infection and enhanced labeling of lysosomal markers (CD63 and rab7) on DKO phagosomes. An in vitro Antigen 85B peptide presentation assay was used to determine antigen presentation to T cells by APCs infected with DKO or other mycobacterial strains. This revealed that DKO infected APCs showed the strongest ability to present Ag85B to T cells (>2500 pgs/mL in 4 hrs) as compared to APCs infected with wild type Mtb or ΔfbpA or ΔsapM strain (<1000 pgs/mL in 4 hrs), indicating that DKO strain has enhanced immunogenicity than other strains. The ability of DKO to undergo lysosomal fusion and vacuolar acidification correlated with antigen presentation since bafilomycin, that inhibits acidification in APCs, reduced antigen presentation. Finally, the DKO vaccine elicited a better Th1 response in mice after subcutaneous vaccination than either ΔfbpA or ΔsapM. Since ΔfbpA has been used in mice as a candidate vaccine and the DKO (ΔfbpAΔsapM) mutant is more immunogenic than ΔfbpA, we propose the DKO is a potential anti-tuberculosis vaccine.     

Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages

Mycobacterium tuberculosis is an intracellular pathogen persisting within phagosomes through interference with phagolysosome biogenesis. Here we show that stimulation of autophagic pathways in macrophages causes mycobacterial phagosomes to mature into phagolysosomes. Physiological induction of autophagy or its pharmacological stimulation by rapamycin resulted in mycobacterial phagosome colocalization with the autophagy effector LC3, an elongation factor in autophagosome formation. Autophagy stimulation increased phagosomal colocalization with Beclin-1, a subunit of the phosphatidylinositol 3-kinase hVPS34, necessary for autophagy and a target for mycobacterial phagosome maturation arrest. Induction of autophagy suppressed intracellular survival of mycobacteria. IFN-gamma induced autophagy in macrophages, and so did transfection with LRG-47, an effector of IFN-gamma required for antimycobacterial action. These findings demonstrate that autophagic pathways can overcome the trafficking block imposed by M. tuberculosis. Autophagy, which is a hormonally, developmentally, and, as shown here, immunologically regulated process, represents an underappreciated innate defense mechanism for control of intracellular pathogens.