Showing papers on "Phagosome published in 2003"

Genomic transcriptional profiling of the developmental cycle of Chlamydia trachomatis.

Abstract: Chlamydia trachomatis is one of the most common bacterial pathogens and is the etiological agent of debilitating sexually transmitted and ocular diseases in humans. The organism is an obligate intracellular prokaryote characterized by a highly specialized biphasic developmental cycle. We have performed genomic transcriptional analysis of the chlamydial developmental cycle. This approach has led to the identification of a small subset of genes that control the primary (immediate-early genes) and secondary (late genes) differentiation stages of the cycle. Immediate-early gene products initiate bacterial metabolism and potentially modify the bacterial phagosome to escape fusion with lysosomes. One immediate early gene (CT147) is a homolog of the human early endosomal antigen-1 that is localized to the chlamydial phagosome; suggesting a functional role for CT147 in establishing the parasitophorous vacuole in a nonfusogenic pathway. Late gene products terminate bacterial cell division and constitute structural components and remodeling activities involved in the formation of the highly disulfide cross-linked outer-membrane complex that functions in attachment and invasion of new host cells. Many of the genes expressed during the immediate-early and late differentiation stages are Chlamydia-specific and have evolutionary origins in eukaryotic lineages.

Phagosomes fuse with late endosomes and/or lysosomes by extension of membrane protrusions along microtubules: role of Rab7 and RILP.

Abstract: Nascent phagosomes must undergo a series of fusion and fission reactions to acquire the microbicidal properties required for the innate immune response. Here we demonstrate that this maturation process involves the GTPase Rab7. Rab7 recruitment to phagosomes was found to precede and to be essential for their fusion with late endosomes and/or lysosomes. Active Rab7 on the phagosomal membrane associates with the effector protein RILP (Rab7-interacting lysosomal protein), which in turn bridges phagosomes with dynein-dynactin, a microtubule-associated motor complex. The motors not only displace phagosomes in the centripetal direction but, strikingly, promote the extension of phagosomal tubules toward late endocytic compartments. Fusion of tubules with these organelles was documented by fluorescence and electron microscopy. Tubule extension and fusion with late endosomes and/or lysosomes were prevented by expression of a truncated form of RILP lacking the dynein-dynactin-recruiting domain. We conclude that full maturation of phagosomes requires the retrograde emission of tubular extensions, which are generated by activation of Rab7, recruitment of RILP, and consequent association of phagosomes with microtubule-associated motors.

Early phagosomes in dendritic cells form a cellular compartment sufficient for cross presentation of exogenous antigens

Abstract: Conventionally, MHC class I-restricted antigen (Ag) processing requires the action of the multimolecular peptide-loading complex within the endoplasmic reticulum (ER). Here we show that early phagosomes from human dendritic cells (DCs) contain the peptide-loading complex, incorporating MHC class I, β2 microglobulin, transporter associated with Ag processing (TAP), calreticulin, tapasin, and ERp57. Antigenic peptides could be translocated into purified phagosomes by TAP and loaded onto cognate class I molecules, inducing their specific dissociation from the loading complex. Endoglycosidase H-sensitive class I molecules were detected at the DC cell surface, suggesting that these molecules traffic there directly from phagosomes. Macropinocytosis also allowed internalized soluble Ags access to an ER-like compartment containing the class I loading complex. Blockade of TAP by endocytosis of a soluble derivative of human cytomegalovirus protein US6 confirmed that, although retrotranslocation into the cytosol is critical for processing, efficient association of class I molecules with peptides derived from exogenous Ags occurs within a compartment directly accessible to internalized proteins. Together, this evidence suggests that early phagosomes and pinosomes facilitate cross presentation of exogenous Ags by DCs.

Critical role of galectin-3 in phagocytosis by macrophages

Abstract: Galectin-3 is a member of a large family of animal lectins. This protein is expressed abundantly by macrophages, but its function in this cell type is not well understood. We have studied the effect of galectin-3 gene targeting on phagocytosis, a major function of macrophages. Compared with wild-type macrophages, galectin-3-deficient (gal3-/-) cells exhibited reduced phagocytosis of IgG-opsonized erythrocytes and apoptotic thymocytes in vitro. In addition, gal3-/- mice showed attenuated phagocytic clearance of apoptotic thymocytes by peritoneal macrophages in vivo. These mice also exhibited reduced IgG-mediated phagocytosis of erythrocytes by Kupffer cells in a murine model of autoimmune hemolytic anemia. Additional experiments indicate that extracellular galectin-3 does not contribute appreciably to the phagocytosis-promoting function of this protein. Confocal microscopic analysis of macrophages containing phagocytosed erythrocytes revealed localization of galectin-3 in phagocytic cups and phagosomes. Furthermore, gal3-/- macrophages exhibited a lower degree of actin rearrangement upon Fcgamma receptor crosslinkage. These results indicate that galectin-3 contributes to macrophage phagocytosis through an intracellular mechanism. Thus, galectin-3 may play an important role in both innate and adaptive immunity by contributing to phagocytic clearance of microorganisms and apoptotic cells.

Tuberculosis toxin blocking phagosome maturation inhibits a novel Ca2+/calmodulin-PI3K hVPS34 cascade.

Abstract: The capacity of Mycobacterium tuberculosis to infect latently over one billion people and cause two million fatalities annually rests with its ability to block phagosomal maturation into the phagolysosome in infected macrophages. Here we describe how M. tuberculosis toxin lipoarabinomannan (LAM) causes phagosome maturation arrest, interfering with a new pathway connecting intracellular signaling and membrane trafficking. LAM from virulent M. tuberculosis, but not from avirulent mycobacteria, blocked cytosolic Ca2+ increase. Ca2+ and calmodulin were required for a newly uncovered Ca2+/calmodulin phosphatidylinositol (PI)3 kinase hVPS34 cascade, essential for production of PI 3 phosphate (PI3P) on liposomes in vitro and on phagosomes in vivo. The interference of the trafficking toxin LAM with the calmodulin-dependent production of PI3P described here ensures long-term M. tuberculosis residence in vacuoles sequestered away from the bactericidal and antigen-processing organelles in infected macrophages.

Modulation of Rab5 and Rab7 recruitment to phagosomes by phosphatidylinositol 3-kinase.

Abstract: Phagosomal biogenesis is central for microbial killing and antigen presentation by leukocytes. However, the molecular mechanisms governing phagosome maturation are poorly understood. We analyzed the role and site of action of phosphatidylinositol 3-kinases (PI3K) and of Rab GTPases in maturation using both professional and engineered phagocytes. Rab5, which is recruited rapidly and transiently to the phagosome, was found to be essential for the recruitment of Rab7 and for progression to phagolysosomes. Similarly, functional PI3K is required for successful maturation. Remarkably, inhibition of PI3K did not preclude Rab5 recruitment to phagosomes but instead enhanced and prolonged it. Moreover, in the presence of PI3K inhibitors Rab5 was found to be active, as deduced from measurements of early endosome antigen 1 binding and by photobleaching recovery determinations. Though their ability to fuse with late endosomes and lysosomes was virtually eliminated by wortmannin, phagosomes nevertheless recruited a sizable amount of Rab7. Moreover, Rab7 recruited to phagosomes in the presence of PI3K antagonists retained the ability to bind its effector, Rab7-interacting lysosomal protein, suggesting that it is functionally active. These findings imply that (i) dissociation of Rab5 from phagosomes requires products of PI3K, (ii) PI3K-dependent effectors of Rab5 are not essential for the recruitment of Rab7 by phagosomes, and (iii) recruitment and activation of Rab7 are insufficient to induce fusion of phagosomes with late endosomes and lysosomes. Accordingly, transfection of constitutively active Rab7 did not bypass the block of phagolysosome formation exerted by wortmannin. We propose that Rab5 activates both PI3K-dependent and PI3K-independent effectors that act in parallel to promote phagosome maturation.

An Attenuated Strain of the Facultative Intracellular Bacterium Francisella tularensis Can Escape the Phagosome of Monocytic Cells

Abstract: The facultative intracellular bacterium Francisella tularensis is a highly virulent and contagious organism, and little is known about its intracellular survival mechanisms. We studied the intracellular localization of the attenuated human vaccine strain, F. tularensis LVS, in adherent mouse peritoneal cells, in mouse macrophage-like cell line J774A.1, and in human macrophage cell line THP-1. Confocal microscopy of infected J774A.1 cells indicated that during the first hour of infection the bacteria colocalized with the late endosomal-lysosomal glycoprotein LAMP-1, but within 3 h this colocalization decreased significantly from approximately 60% to 30%. Transmission electron microscopy revealed that >90% of bacteria were not enclosed by a phagosomal membrane after 2 h of infection, and some bacteria were in vacuoles that were only partially surrounded by a limiting membrane. Similar findings were obtained with all three host cell types. Immunoelectron microscopy performed with an F. tularensis LVS-specific polyclonal rabbit antiserum showed that the antiserum stained a thick, evenly distributed capsule-like material in bacteria grown in broth. In contrast, intracellular F. tularensis LVS cells were only marginally stained with this antiserum. Instead, most of the immunoreactive material was diffusely localized in the phagosomes or was associated with the phagosomal membrane. Our findings indicate that F. tularensis LVS is able to escape from the phagosomes of macrophages via a mechanism that may involve degradation of the phagosomal membrane.

Phagocytosis and the Inflammatory Response

Abstract: Macrophages are a cornerstone of the innate immune system. They detect infectious organisms via a plethora of receptors, phagocytose them, and orchestrate an appropriate host response. Phagocytosis is extraordinarily complex: numerous receptors stimulate particle internalization, the cytoskeletal elements mediating internalization differ by receptor system and the nature of the pathogen being internalized, and the outcome can differ by bacterium. After generating a panel of 150 monoclonal antibodies that recognizes proteins recruited to the phagosome, analysis of novel phagocytic proteins was prioritized by focusing on those that behave differently during the internalization of virulent and avirulent bacteria. Several novel proteins that have roles in membrane extension were characterized. Although the inflammatory pathways leading to appropriate host response are reasonably well defined, it is not clear how macrophages define the threat precisely. Recent work indicates that Toll-like receptors play a key role in reading a bar code on invading microorganisms and in eliciting a specific immune response. The mechanisms and coupling to the phagocytic response are discussed.

Phagocytosis and intracellular fate of Aspergillus fumigatus conidia in alveolar macrophages.

Abstract: Aspergillus fumigatus is the most prevalent airborne fungal pathogen responsible for fatal invasive aspergillosis in immunocompromised patients. Upon arrival in the lung alveolus, conidia of A. fumigatus are phagocytosed by alveolar macrophages, the major phagocytic cells of the lung. Engulfment and intracellular trafficking of A. fumigatus conidia in alveolar macrophages of two different origins, the murine cell line MH-S and human pulmonary alveolar macrophages, were analyzed by electron microscopy and immunofluorescence. Phagocytosis of A. fumigatus conidia required actin polymerization and phosphatidylinositol 3-kinase activity. Fusion of A. fumigatus phagosomes with early and late endosomes was shown by immunolabeling with specific markers for the transferrin receptor, early endosome antigen, and Rab7. Maturation of A. fumigatus phagolysosomes was monitored by using a fixable acidotropic probe, LysoTracker Red DND-99, and an anti-cathepsin D antibody. Bafilomycin A-induced inhibition of lysosomal acidification abolished the conidial killing by the macrophages. These data suggest that the maturation of A. fumigatus phagosomes results from fusion with the compartments of the endocytic pathway and that the killing of conidia depends on phagolysosome acidification. A model for the phagocytosis of A. fumigatus conidia by alveolar macrophages is proposed on the basis of these results.

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

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

Mycobacterium tuberculosis Phagosome Maturation Arrest: Mycobacterial Phosphatidylinositol Analog Phosphatidylinositol Mannoside Stimulates Early Endosomal Fusion

Abstract: Mycobacterium tuberculosis is a facultative intracellular pathogen that parasitizes macrophages by modulating properties of the Mycobacterium-containing phagosome. Mycobacterial phagosomes do not fuse with late endosomal/lysosomal organelles but retain access to early endosomal contents by an unknown mechanism. We have previously reported that mycobacterial phosphatidylinositol analog lipoarabinomannan (LAM) blocks a trans-Golgi network-to-phagosome phosphatidylinositol 3-kinase-dependent pathway. In this work, we extend our investigations of the effects of mycobacterial phosphoinositides on host membrane trafficking. We present data demonstrating that phosphatidylinositol mannoside (PIM) specifically stimulated homotypic fusion of early endosomes in an ATP-, cytosol-, and N-ethylmaleimide sensitive factor-dependent manner. The fusion showed absolute requirement for small Rab GTPases, and the stimulatory effect of PIM increased upon partial depletion of membrane Rabs with RabGDI. We found that stimulation of early endosomal fusion by PIM was higher when phosphatidylinositol 3-kinase was inhibited by wortmannin. PIM also stimulated in vitro fusion between model phagosomes and early endosomes. Finally, PIM displayed in vivo effects in macrophages by increasing accumulation of plasma membrane-endosomal syntaxin 4 and transferrin receptor on PIM-coated latex bead phagosomes. In addition, inhibition of phagosomal acidification was detected with PIM-coated beads. The effects of PIM, along with the previously reported action of LAM, suggest that M. tuberculosis has evolved a two-prong strategy to modify its intracellular niche: its products block acquisition of late endosomal/lysosomal constituents, while facilitating fusion with early endosomal compartments.

Cytoplasmic bacteria can be targets for autophagy.

Abstract: Autophagy is an important constitutive cellular process involved in size regulation, protein turnover and the removal of malformed or superfluous subcellular components. The process involves the sequestration of cytoplasm and organelles into double-membrane autophagic vacuoles for subsequent breakdown within lysosomes. In this work, we demonstrate that the intracellular pathogen Listeria monocytogenes can also be a target for autophagy. If infected macrophages are treated with chloramphenicol after phagosome lysis, the bacteria are internalized from the cell cytoplasm into autophagic vacuoles. The autophagic vacuoles appear to form by fusion of small cytoplasmic vesicles around the bacteria. These vesicular structures immunolabel with antibodies to protein disulphide isomerase, a marker for the rough ER. Internalization of metabolically arrested cytoplasmic L. monocytogenes represents an autophagic process as the vacuoles have double membranes and the process can be inhibited by the autophagy inhibitors 3-methyladenine and wortmannin. Additionally, the rate of internalization can be accelerated under starvation conditions and the vacuoles fuse with the endocytic pathway. Metabolic inhibition of cytoplasmic bacteria prevents them from adapting to the intracellular niche and reveals a host mechanism utilizing the autophagic pathway as a defence against invading pathogens by providing a route for their removal from the cytoplasm and subsequent delivery to the endocytic pathway for degradation.

Abnormal phagocytosis by retinal pigmented epithelium that lacks myosin VIIa, the Usher syndrome 1B protein

Abstract: Mutations in the myosin VIIa gene (MYO7A) cause Usher syndrome type 1B (USH1B), a major type of the deaf-blind disorder, Usher syndrome. We have studied mutant phenotypes in the retinas of Myo7a mutant mice (shaker1), with the aim of elucidating the role(s) of myosin VIIa in the retina and what might underlie photoreceptor degeneration in USH1B patients. A photoreceptor defect has been described. Here, we report that the phagocytosis of photoreceptor outer segment disks by the retinal pigment epithelium (RPE) is abnormal in Myo7a null mice. Both in vivo and in primary cultures of RPE cells, the transport of ingested disks out of the apical region is inhibited in the absence of Myo7a. The results with the cultured RPE cells were the same, irrespective of whether the disks came from wild-type or mutant mice, thus demonstrating that the RPE is the source of this defect. The inhibited transport seems to delay phagosome–lysosomal fusion, as the degradation of ingested disks was slower in mutant RPE. Moreover, fewer packets of disk membranes were ingested in vivo, possibly because retarded removal of phagosomes from the apical processes inhibited the ingestion of additional disk membranes. We conclude that Myo7a is required for the normal processing of ingested disk membranes in the RPE, primarily in the basal transport of phagosomes into the cell body where they then fuse with lysosomes. Because the phagocytosis of photoreceptor disks by the RPE has been shown to be critical for photoreceptor cell viability, this defect likely contributes to the progressive blindness in USH1B.

Phagocytosis by neutrophils induces an amino acid deprivation response in Saccharomyces cerevisiae and Candida albicans

Abstract: The transcriptional profiles of yeast cells that have been phagocytosed by either human neutrophils or monocytes were compared by using whole genome arrays. After phagocytosis by neutrophils, both Saccharomyces cerevisiae and Candida albicans respond by inducing genes of the methionine and arginine pathways. Neither of these pathways is induced upon phagocytosis by monocytes. Both fungi show a similar induction of these pathways when transferred from amino acid-rich medium to amino acid-deficient medium. These data suggest that the internal phagosome of the neutrophil is an amino acid-deficient environment.

Cutting Edge: Mycobacterium tuberculosis Blocks Ca2+ Signaling and Phagosome Maturation in Human Macrophages Via Specific Inhibition of Sphingosine Kinase

Abstract: One-third of the world's population is infected with Mycobacterium tuberculosis (Mtb), and three million people die of tuberculosis each year. Following its ingestion by macrophages (MPs), Mtb inhibits the maturation of its phagosome, preventing progression to a bactericidal phagolysosome. Phagocytosis of Mtb is uncoupled from the elevation in MP cytosolic Ca(2+) that normally accompanies microbial ingestion, resulting in inhibition of phagosome-lysosome fusion and increased intracellular viability. This study demonstrates that the mechanism responsible for this failure of Ca(2+)-dependent phagosome maturation involves mycobacterial inhibition of MP sphingosine kinase. Thus, inhibition of sphingosine kinase directly contributes to survival of Mtb within human MPs and represents a novel molecular mechanism of pathogenesis.

Phosphoinositide-3-kinase-independent contractile activities associated with Fcγ-receptor-mediated phagocytosis and macropinocytosis in macrophages

Abstract: Previous studies have shown that Fcgamma receptor (FcR)-mediated phagocytosis and macropinocytosis in macrophages consist of two dissociable activities: a phosphoinositide 3-kinase (PI3K)-independent extension of phagocytic cups and a PI3K-dependent contractile mechanism that closes phagosomes and ruffles into intracellular organelles. Here, we identify an additional contractile activity that persists in the presence of the PI3K inhibitor wortmannin. ML-7, an inhibitor of myosin-light-chain kinase (MLCK), inhibited FcR-mediated phagocytosis, macropinocytosis and cell movements associated with ruffling. Scanning electron microscopy demonstrated a striking difference in morphology between phagocytic cups in the different inhibitors: whereas phagocytic cups of control cells and wortmannin-treated cells conformed closely to particles and appeared to have constricted them, the phagocytic cups in cells treated with ML-7 were more open. Video microscopy of macrophages expressing green-fluorescent-protein (GFP)-actin fusions revealed that bound IgG-opsonized erythrocytes were squeezed during phagosome formation and closure. In ML-7, GFP-actin-rich protrusions extended outward but failed to squeeze particles. Moreover, in contrast to the effects of PI3K inhibitors, ML-7 markedly reduced ruffle movement, and perturbed circular ruffle formation. These PI3K-independent myosin-II-based contractile activities that squeeze phagocytic cups and curve ruffles therefore represent a third component activity of the actin cytoskeleton during phagocytosis and macropinocytosis.

ADP ribosylation factor 6 is activated and controls membrane delivery during phagocytosis in macrophages.

Abstract: Engulfment of particles by phagocytes is induced by their interaction with specific receptors on the cell surface, which leads to actin polymerization and the extension of membrane protrusions to form a closed phagosome. Membrane delivery from internal pools is considered to play an important role in pseudopod extension during phagocytosis. Here, we report that endogenous ADP ribosylation factor 6 (ARF6), a small GTP-binding protein, undergoes a sharp and transient activation in macrophages when phagocytosis was initiated via receptors for the Fc portion of immunoglobulins (FcRs). A dominant-negative mutant of ARF6 (T27N mutation) dramatically affected FcR-mediated phagocytosis. Expression of ARF6-T27N lead to a reduction in the focal delivery of vesicle-associated membrane protein 3+ endosomal recycling membranes at phagocytosis sites, whereas actin polymerization was unimpaired. This resulted in an early blockade in pseudopod extension and accumulation of intracellular vesicles, as observed by electron microscopy. We conclude that ARF6 is a major regulator of membrane recycling during phagocytosis.

Helicobacter pylori strains expressing the vacuolating cytotoxin interrupt phagosome maturation in macrophages by recruiting and retaining TACO (coronin 1) protein

Abstract: Summary Recent evidence suggests that persistence of Helicobacter pylori can be explained, at least in part, by the failure of macrophages to kill bacteria. The fate of type 1 H. pylori strain LC11, which expresses the cag pathogenicity island (PAI) and the vacuolating cytotoxin, and type 2 strain LC20, which lacks both these virulence factors, was determined following infection of the murine macrophage cell line RAW 264.7 or the human macrophage-like cell line THP-1. Helicobacter pylori strain LC11 displayed enhanced survival in macrophages in comparison with strain LC20 (4.0 ± 0.2 versus 2.1 ± 0.6 log CFU ml−1, P < 0.01) at 24 h. Phagosomes containing strain LC11 showed reduced co-localization with LysoTracker Red, higher levels of expression of the early endosome marker EEA1 expression and lower expression of the late endosome/lysosome marker LAMP1 relative to internalized strain LC20, both at 2 h and 24 h. These findings indicate that, in contrast to strain LC20, strain LC11 resides in a compartment with early endosome properties and does not fuse with lysosomes. In addition, phagosomes containing LC11 recruited and retained a higher percentage of TACO (coronin 1) protein in comparison with phagosomes containing strain LC20. Furthermore, IFN-γ stimulation facilitated maturation of phagosomes containing strain LC11 in association with the release of TACO and a reduction in bacterial survival. We have demonstrated through the use of isogenic cagA-, cagE-/picB- and vacA- mutant strains, that VacA plays a significant role in the interruption of the phagosome maturation. Taken together, these results indicate that, following phagocytosis, H. pylori strains expressing the vacuolating cytotoxin arrest phagosome maturation in association with the retention of TACO.

Constrained intracellular survival of Mycobacterium tuberculosis in human dendritic cells

Abstract: Dendritic cells (DCs) are likely to play a key role in immunity against Mycobacterium tuberculosis , but the fate of the bacterium in these cells is still unknown. Here we report that, unlike macrophages (Mφs), human monocyte-derived DCs are not permissive for the growth of virulent M. tuberculosis H37Rv. Mycobacterial vacuoles are neither acidic nor fused with host cell lysosomes in DCs, in a mode similar to that seen in mycobacterial infection of Mφs. However, uptake of the fluid phase marker dextran, and of transferrin, as well as accumulation of the recycling endosome-specific small GTPase Rab11 onto the mycobacterial phagosome, are almost abolished in infected DCs, but not in Mφs. Moreover, communication between mycobacterial phagosomes and the host-cell biosynthetic pathway is impaired, given that M. tuberculosis vacuoles in DCs stained for the endoplasmic reticulum-specific proteins Grp78/BiP and calnexin. This correlates with the absence of the fusion factor N -ethylmaleimide-sensitive factor onto the vacuolar membrane in this cell type. Trafficking between the vacuoles and the host cell recycling and biosynthetic pathways is strikingly reduced in DCs, which is likely to impair access of intracellular mycobacteria to essential nutrients and may thus explain the absence of mycobacterial growth in this cell type. This unique location of M. tuberculosis in DCs is compatible with their T lymphocyte-stimulating functions, because M. tuberculosis -infected DCs have the ability to specifically induce cytokine production by autologous T lymphocytes from presensitized individuals. DCs have evolved unique subcellular trafficking mechanisms to achieve their Ag-presenting functions when infected by intracellular mycobacteria.

Taking possession: biogenesis of the Salmonella-containing vacuole.

Abstract: The Gram-negative pathogen Salmonella enterica can survive and replicate within a variety of mammalian cells. Regardless of the cell type, internalized bacteria survive and replicate within the Salmonella-containing vacuole, the biogenesis of which is dependent on bacterially encoded virulence factors. In particular, Type III secretion systems translocate bacterial effector proteins into the eukaryotic cell where they can specifically interact with a variety of targets. Salmonella has two distinct Type III secretion systems that are believed to have completely different functions. The SPI2 system is induced intracellularly and is required for intracellular survival in macrophages; it plays no role in invasion but is categorized as being required for Salmonella-containing vacuole biogenesis. In contrast, the SPI1 Type III secretion system is induced extracellularly and is essential for invasion of nonphagocytic cells. Its role in post-invasion processes has not been well studied. Recent studies indicate that Salmonella-containing vacuole biogenesis may be more dependent on SPI1 than previously believed. Other non-SPI2 virulence factors and the host cell itself may play critical roles in determining the intracellular environment of this facultative intracellular pathogen. In this review we discuss the recent advances in determining the mechanisms by which Salmonella regulate Salmonella-containing vacuole biogenesis and the implications of these findings.

Novel PI 3-kinase-dependent mechanisms of trypanosome invasion and vacuole maturation

Abstract: Mammalian cell invasion by the protozoan parasite, Trypanosoma cruzi , is facilitated by the activation of host cell phosphatidylinositol 3 (PI 3)-kinases. We demonstrate that the well-characterized Ca 2+ -regulated lysosome-mediated parasite entry pathway is abolished by wortmannin pretreatment. In addition, we have characterized a novel route of T. cruzi invasion unexpectedly revealed in the course of this study. For over a decade, targeted exocytosis of lysosomes at the host cell plasma membrane was considered as the primary mechanism for T. cruzi entry into non-professional phagocytic cells. We now provide evidence that a significant fraction (50% or greater) of invading T. cruzi trypomastigotes exploit an alternate actin-independent entry pathway that involves formation of a tightly associated host cell plasma membrane-derived vacuole enriched in the lipid products of class I PI 3-kinases, PtdIns P 3 /PtdIns(3,4) P 2 . Initially devoid of lysosomal markers, the resultant parasite-containing vacuoles gradually acquire lysosome associated membrane protein 1 (lamp-1) and fluid phase endocytic tracer from the lysosomal compartment. In striking contrast to latex bead phagosomes, few T. cruzi vacuoles associate with the early endosomal marker, EEA1 and the 9maturation9 process becomes refractory to PI 3-kinase inhibition immediately following parasite internalization. Jointly, these data provide a new paradigm for T. cruzi invasion of non-professional phagocytic cells and reveal a novel vacuole maturation process that appears to bypass the requirement for EEA1.

Role of the Brucella suis lipopolysaccharide O antigen in phagosomal genesis and in inhibition of phagosome-lysosome fusion in murine macrophages.

Abstract: Brucella species are gram-negative, facultative intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment inside professional and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both cell types. However, the molecular mechanisms and the microbial factors involved are poorly understood. Smooth lipopolysaccharide (LPS) of Brucella has been reported to be an important virulence factor, although its precise role in pathogenesis is not yet clear. In this study, we show that the LPS O side chain is involved in inhibition of the early fusion between Brucella suis-containing phagosomes and lysosomes in murine macrophages. In contrast, the phagosomes containing rough mutants, which fail to express the O antigen, rapidly fuse with lysosomes. In addition, we show that rough mutants do not enter host cells by using lipid rafts, contrary to smooth strains. Thus, we propose that the LPS O chain might be a major factor that governs the early behavior of bacteria inside macrophages.

Deletion of the gene encoding p60 in Listeria monocytogenes leads to abnormal cell division and loss of actin-based motility.

Abstract: Protein p60 encoded by the iap gene is regarded as an essential gene product of Listeria monocytogenes. Here we report, however, the successful construction of a viable iap deletion mutant of L. monocytogenes EGD. The mutant, which produces no p60, shows abnormal septum formation and tends to form short filaments and hooked forms during logarithmic growth. These abnormal bacterial cells break into almost normal sized single bacteria in the late-stationary-growth phase. The iap mutant is strongly attenuated in a mouse model after intravenous injection, demonstrating the importance of p60 during infection, and the invasiveness of the Δiap mutant for 3T6 fibroblasts and Caco-2 epithelial cells is slightly reduced. Upon uptake by epithelial cells and macrophages, the iap mutant escapes from the phagosome into the cytosol with the same efficiency as the wild-type strain, and the mutant bacteria also grow intracellularly at a rate similar to that of the wild-type strain. Intracellular movement and cell-to-cell spread are drastically reduced in various cell lines, since the iap-negative bacteria fail to induce the formation of actin tails. However, the bacteria are covered with actin filaments. Most intracellular bacteria show a nonpolar and uneven distribution of ActA around the cell, in contrast to that for the wild-type strain, where ActA is concentrated at the old pole. In an iap+ revertant strain that produces wild-type levels of p60, intracellular movement, cell-to-cell spread, and polar distribution of ActA are fully restored. In vitro analysis of ActA distribution on the filaments of the Δiap strain shows that the loss of bacterial septum formation leads to ActA accumulation at the presumed division sites. In the light of data presented here and elswhere, we propose to rename iap (invasion-associated protein) cwhA (cell wall hydrolase A).

Phagosome Maturation: A Few Bugs in the System

Abstract: Cells of the innate immune system ingest and destroy invading microorganisms by initially engulfing them into a specialized vacuole, known as the phagosome. The membrane of the forming phagosome is similar to the plasmalemma and its contents resemble the extracellular milieu. As such, the nascent phagosome is not competent to kill and eliminate the ingested microorganisms. However, shortly after sealing, the phagosome undergoes a series of rapid and extensive changes in its composition, the result of a sophisticated sequence of membrane fusion and fission reactions. Understanding the molecular basis of these events is of particular importance, since they are often the target of disruption by intracellular parasites such as Mycobacterium, Salmonella and Legionella. The objective of this review is to summarize the current knowledge of the molecular mechanisms underlying phagosomal maturation and its subversion by parasitic microorganisms.

Listeria monocytogenes Mutants That Fail To Compartmentalize Listerolysin O Activity Are Cytotoxic, Avirulent, and Unable To Evade Host Extracellular Defenses

Abstract: Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from a phagosome and grows in the host cell cytosol. Escape of the bacterium from the phagosome to the cytosol is mediated by the bacterial pore-forming protein listeriolysin O (LLO). LLO has multiple mechanisms that optimize activity in the phagosome and minimize activity in the host cytosol. Mutants that fail to compartmentalize LLO activity are cytotoxic and have reduced virulence. We sought to determine why cytotoxic bacteria have attenuated virulence in the mouse model of listeriosis. In this study, we constructed a series of strains with mutations in LLO and with various degrees of cytotoxicity. We found that the more cytotoxic the strain in cell culture, the less virulent it was in mice. Induction of neutropenia increased the relative virulence of the cytotoxic strains 100-fold in the spleen and 10-fold in the liver. The virulence defect was partially restored in neutropenic mice by adding gentamicin, an antibiotic that kills extracellular bacteria. Additionally, L. monocytogenes grew more slowly in extracellular fluid (mouse serum) than within tissue culture cells. We concluded that L. monocytogenes controls the cytolytic activity of LLO to maintain its nutritionally rich intracellular niche and avoid extracellular defenses of the host.

Cellular prion protein promotes Brucella infection into macrophages.

Abstract: The products of the Brucella abortus virB gene locus, which are highly similar to conjugative DNA transfer system, enable the bacterium to replicate within macrophage vacuoles. The replicative phagosome is thought to be established by the interaction of a substrate of the VirB complex with macrophages, although the substrate and its host cellular target have not yet been identified. We report here that Hsp60, a member of the GroEL family of chaperonins, of B. abortus is capable of interacting directly or indirectly with cellular prion protein (PrPC) on host cells. Aggregation of PrPC tail-like formation was observed during bacterial swimming internalization into macrophages and PrPC was selectively incorporated into macropinosomes containing B. abortus. Hsp60 reacted strongly with serum from human brucellosis patients and was exposed on the bacterial surface via a VirB complex–associated process. Under in vitro and in vivo conditions, Hsp60 of B. abortus bound to PrPC. Hsp60 of B. abortus, expressed on the surface of Lactococcus lactis, promoted the aggregation of PrPC but not PrPC tail formation on macrophages. The PrPC deficiency prevented swimming internalization and intracellular replication of B. abortus, with the result that phagosomes bearing the bacteria were targeted into the endocytic network. These results indicate that signal transduction induced by the interaction between bacterial Hsp60 and PrPC on macrophages contributes to the establishment of B. abortus infection.