SummaryWe have used the cryosection immunogold technique to study the composition of the Mycobacterium tuberculosis phagosome. We have used quantitative immunogold staining to determine the distribution of several known markers of the endosomal-lysosomal pathway in human monocytes after ingestion of either M. tuberculosis, Legionella pneumophila, or polystyrene beads. Compared with the other phagocytic particles studied, the M. tuberculosis phagosome exhibits delayed clearance of major histocompatibility complex (MHC) class I molecules, relatively intense staining for MHC class II molecules and the endosomal marker transferrin receptor, and relatively weak staining for the lysosomal membrane glycoproteins, CD63, LAMP-l, and LAMP-2 and the lysosomal acid protease, cathepsin D. In contrast to M. tuberculosis, the L. pneumophila phagosome rapidly dears MHC class I molecules and excludes all endosomal-lysosomal markers studied. In contrast to both live M. tuberculosis and L. pneumophih phagosomes, phagosomes containing either polystyrene beads or heat-killed M. tuberculosis stain intensely for lysosomal membrane glycoproteins and cathepsin D. These findings suggest that (a) M. tuberculosis retards the maturation of its phagosome along the endosomal-lysosomal pathway and resides in a compartment with endosomal, as opposed to lysosomal, characteristics; and (b) the intraphagosomal pathway, i.e., the pathway followed by several intracellular parasites that inhibit phagosome-lysosome fusion, is heterogeneous.

Francisella tularensis, the agent of tularemia, is an intracellular pathogen, but little is known about the compartment in which it resides in human macrophages. We have examined the interaction of a recent virulent clinical isolate of F. tularensis subsp. tularensis and the live vaccine strain with human macrophages by immunoelectron and confocal immunofluorescence microscopy. We assessed the maturation of the F. tularensis phagosome by examining its acquisition of the lysosome-associated membrane glycoproteins (LAMPs) CD63 and LAMP1 and the acid hydrolase cathepsin D. Two to four hours after infection, vacuoles containing live F. tularensis cells acquired abundant staining for LAMPs but little or no staining for cathepsin D. However, after 4 h, the colocalization of LAMPs with live F. tularensis organisms declined dramatically. In contrast, vacuoles containing formalin-killed bacteria exhibited intense staining for all of these late endosomal/lysosomal markers at all time points examined (1 to 16 h). We examined the pH of the vacuoles 3 to 4 h after infection by quantitative immunogold staining and by fluorescence staining for lysosomotropic agents. Whereas phagosomes containing killed bacteria stained intensely for these agents, indicating a marked acidification of the phagosomes (pH 5.5), phagosomes containing live F. tularensis did not concentrate these markers and thus were not appreciably acidified (pH 6.7). An ultrastructural analysis of the F. tularensis compartment revealed that during the first 4 h after uptake, the majority of F. tularensis bacteria reside within phagosomes with identifiable membranes. The cytoplasmic side of the membranes of ϳ50% of these phagosomes was coated with densely staining fibrils of ϳ30 nm in length. In many cases, these coated phagosomal membranes appeared to bud, vesiculate, and fragment. By 8 h after infection, the majority of live F. tularensis bacteria lacked any ultrastructurally discernible membrane separating them from the host cell cytoplasm. These results indicate that F. tularensis initially enters a nonacidified phagosome with LAMPs but without cathepsin D and that the phagosomal membrane subsequently becomes morphologically disrupted, allowing the bacteria to gain direct access to the macrophagic cytoplasm. The capacity of F. tularensis to alter the maturation of its phagosome and to enter the cytoplasm is likely an important element of its capacity to parasitize macrophages and has major implications for vaccine development.

SummaryPrevious studies have demonstrated that the Mycobacterium tuberculosis phagosome in human monocyte-derived macrophages acquires markers of early and late endosomes, but direct evidence of interaction of the M. tuberculosis phagosome with the endosomal compartment has been lacking. Using the cryosection immunogold technique, we have found that the M. tuberculosis phagosome acquires exogenously added transferrin in a time-dependent fashion. Nearmaximal acquisition of transferrin occurs within 15 rain, kinetics of acquisition consistent with interaction of the M. tuberculosis phagosome with early endosomes. Transferrin is chased out of the M. tuberculosis phagosome by incubation of the infected macrophages in culture medium lacking human transferrin. Phagosomes containing latex beads or heat-killed M. tuberculosis, on the other hand, do not acquire staining for transferrin. These and other findings demonstrate that M. tuberculosis arrests the maturation of its phagosome at a stage at which the phagosome interacts with early and late endosomes, but not with lysosomes. The transferrin endocytic pathway potentially provides a novel route for targeting antimicrobials to the M. tuberculosis phagosome. ~pObacterium tuberculosis is an intracellular pathogen that arasitizes human mononuclear phagocytes. Throughout its life cycle in these host cells, this bacterium resides and multiplies in a membrane-bound phagosome that resists acidification and fusion with lysosomes (1-4). In previous studies, using quantitative immunoelectron microscopy, we have examined the composition and maturation of the M. tuberculosis phagosome, focusing on known markers of the endocytic pathway (4). These studies revealed that the M. tuberculosis phagosome exhibits delayed clearance of MHC class I molecules, relatively intense staining for MHC class II molecules and the endosomal marker transfemn receptor, and relatively weak staining for the lysosome-associated membrane glycoproteins CD63, LAMP-l, and LAMP-2 and the lysosomal acid protease cathepsin D. Like M. tuberculosis, wild-type Legionella pneumophila inhibits phagosomelysosome fusion and phagosome acidification (5, 6). However, in contrast to the M. tuberculosis phagosome, the L. pneumophila phagosome rapidly clears MHC class I molecules and excludes MHC class II molecules as well as all endosomal-lysosornal markers studied (4,7,8). In contrast to phagosomes containing either live M. tuberculosis or L. pneumophila, phagosomes containing heat-killed M. tuberculosis or polystyrene beads fuse with lysosomes and stain intensely for lysosome-associated membrane glycoproteins and cathepsin D (4). These findings suggested that M. tuberculosis retards the maturation of its phagosome along the endocytic pathway and resides in a compartment with endosomal, as opposed to lysosomal, characteristics. However, direct evidence in support of this hypothesis, and more specifically, direct evidence that the M. tuberculosis phagosome acquires endosoreal markers by virtue of interaction with endosomes ...

Intracellular bacterial pathogens employ a variety of strategies to invade their eukaryotic host cells. From an ultrastructural standpoint, the processes that bacteria employ to invade their host cells include conventional phagocytosis, coiling phagocytosis, and ruffling/triggered macropinocytosis. In this paper, we describe a novel process by which Francisella tularensis, the agent of tularemia, enters host macrophages. F. tularensis is a remarkably infectious facultative intracellular bacterial parasite-as few as 10 bacteria can cause lifethreatening disease in humans. However, the ultrastructure of its uptake and the receptor mechanisms that mediate its uptake have not been reported previously. We have used fluorescence microscopy and electron microscopy to examine the adherence and uptake of a virulent recent clinical isolate of F. tularensis, subspecies tularensis, and the live vaccine strain (LVS), subspecies holarctica, by human macrophages. We show here that both strains of F. tularensis enter human macrophages by a novel process of engulfment within asymmetric, spacious pseudopod loops, a process that differs ultrastructurally from all previously described uptake mechanisms. We demonstrate also that adherence and uptake of F. tularensis by macrophages is strongly dependent upon complement receptors and upon serum with intact complement factor C3 and that uptake requires actin microfilaments. These findings have significant implications for understanding the intracellular biology and virulence of this extremely infectious pathogen.

We have characterized the CD1b-mediated presentation pathway for the mycobacterial lipoglycan lipoarabinomannan (LAM) in monocyte-derived antigen-presenting cells. The macrophage mannose receptor (MR) was responsible for uptake of LAM. Antagonism of MR function inhibited both the internalization of LAM and the presentation of this antigen to LAM-reactive T cells. Intracellular MRs were most abundant in early endosomes, but they also were located in the compartment for MHC class II antigen loading (MIIC). Internalized LAM was transported to late endosomes, lysosomes, and MIICs. MRs colocalized with CD1b molecules, suggesting that the MR could deliver LAM to late endosomes for loading onto CD1b. LAM and CD1b colocalized in organelles that may be sites of lipoglycan antigen loading. This pathway links recognition of microbial antigens by a receptor of the innate immune system to the induction of adaptive T cell responses.

Summary Type VI secretion systems (T6SSs) are newly identified contractile nanomachines that translocate effector proteins across bacterial membranes. The Francisella pathogenicity island, required for bacterial phagosome escape, intracellular replication and virulence, was presumed to encode a T6SS-like apparatus. Here, we experimentally confirm the identity of this T6SS and, by cryo electron microscopy (cryoEM), show the structure of its post-contraction sheath at 3.7 Å resolution. We demonstrate the assembly of this T6SS by IglA/IglB and secretion of its putative effector proteins in response to environmental stimuli. The sheath has a quaternary structure with handedness opposite that of contracted sheath of T4 phage tail and is organized in an interlaced two-dimensional array by means of β sheet augmentation. By structure-based mutagenesis, we show that this interlacing is essential to secretion, phagosomal escape, and intracellular replication. Our atomic model of the T6SS will facilitate design of drugs targeting this highly prevalent secretion apparatus.

We report a high-throughput platform for delivering large cargo into 100,000 cells in 1 min. An array of micro-cavitation bubbles explode in response to laser pulsing, forming pores in adjacent cell membranes, and immediately thereafter, pressurized flows drive slow diffusing cargo through these pores into cells. The platform delivers large cargo including bacteria, enzymes, antibodies, and nanoparticles into diverse cell types with high efficiency and cell viability. We used this platform to explore the intracellular lifestyle of Francisella novicida and discovered that the iglC gene is unexpectedly required for intracellular replication even after phagosome escape into the cell cytosol.

We have investigated the activity and extra- (9), Legionella pneumophila Philadelphia 1 (10), Bacillus cereus (ATCC 14579), and Bacillus subtilis (ATCC 6051) were grown as described. For comparative glutamine synthetase assays, all bacteria were grown in 7H9 medium at pH 6.7 and 7.5 or in Sauton's medium (11).Purification of Glutamine Synthetase. Supernatant from 18 liters of M. tuberculosis Erdman strain cultures was filtered through Tuifryn 0.45-and 0.22-,um filters (Gelman) and concentrated by tangential flow through a polyethersulfone membrane (Filtron Technology, Northborough, MA). Proteins in this concentrate were precipitated with ammonium sulfate at 100% saturation, pelleted by centrifugation, and dialyzed against sorbitol buffer (10%6 sorbitol/10 mM potassium phosphate, pH 7.0/5 mM 2-mercaptoethanol/0.2 mM EDTA). The proteins were applied to DEAE-Sepharose CL-6B (Pharmacia) and glutamine synthetase was eluted at 0.5-1 M NaCl. The enzyme was further chromatographed on thiopropyl-Sepharose 6B (Pharmacia), eluted at 150-250 mM 2-mercaptoethanol, concentrated to 2.5 ml in a Diaflo unit (Amicon), and finally size fractionated on Sepharose 6B (Pharmacia). Enzymatically active fractions were pooled and stored at 40C. Protein concentrations were determined by the bicinchoninic acid reagent (Pierce). Proteins in the active fractions were analyzed by SDS/10% PAGE and stained with Coomassie brilliant blue R or silver nitrate. The N-terminal sequence of glutamine synthetase was determined on poly-(vinylidene difluoride) membranes at the University of California, Los Angeles, protein microsequencing facility with a Porton 2090 E amino acid sequencer.Assays of Glutamine Synthetase Activity. The enzyme was assayed both in the biosynthetic (forward) assay (glutamate + ATP + ammonia --glutamine + ADP + Pj) and in the transfer assay (glutamine + hydroxylamine y-glutamylhydroxamate + ammonia) as described (12). One unit of glutamine synthetase was defined as the amount of enzyme producing 1 pmol of Pi per min in the biosynthetic reaction or 1 pnmol of -glutamylhydroxamate per min in the transfer reaction.The pH optima of glutamine synthetase were determined for both assay systems for the range pH 6.0-9.0. The enzyme's cation requirements were also examined for both reactions. Cobalt(II) chloride, magnesium chloride, manganese chloride, or zinc(II) chloride was added at 50 mM for the biosynthetic reaction and at 3 mM for the transfer reaction.*To whom reprint requests should be addressed. 9342The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.