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

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.     

Molecular Pathogenesis of Infections Caused by Legionella pneumophila

Summary: The genus Legionella contains more than 50 species, of which at least 24 have been associated with human infection. The best-characterized member of the genus, Legionella pneumophila, is the major causative agent of Legionnaires'' disease, a severe form of acute pneumonia. L. pneumophila is an intracellular pathogen, and as part of its pathogenesis, the bacteria avoid phagolysosome fusion and replicate within alveolar macrophages and epithelial cells in a vacuole that exhibits many characteristics of the endoplasmic reticulum (ER). The formation of the unusual L. pneumophila vacuole is a feature of its interaction with the host, yet the mechanisms by which the bacteria avoid classical endosome fusion and recruit markers of the ER are incompletely understood. Here we review the factors that contribute to the ability of L. pneumophila to infect and replicate in human cells and amoebae with an emphasis on proteins that are secreted by the bacteria into the Legionella vacuole and/or the host cell. Many of these factors undermine eukaryotic trafficking and signaling pathways by acting as functional and, in some cases, structural mimics of eukaryotic proteins. We discuss the consequences of this mimicry for the biology of the infected cell and also for immune responses to L. pneumophila infection.     

Role of cholesterol and the ganglioside GM(1) in entry and short-term survival of Brucella suis in murine macrophages

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 types of cells. We have previously shown that the maturation inhibition of the Brucella-containing phagosome appears to be restricted at the phagosomal membrane, but the precise molecular mechanisms and factors involved in this inhibition have yet to be identified. Interestingly, recent studies have revealed that caveolae or lipid rafts are implicated in the entry of some microorganisms into host cells and mediate an endocytic pathway avoiding fusion with lysosomes. In this study, we investigated the role of cholesterol and the ganglioside GM(1), two components of lipid rafts, in entry and short-term survival of Brucella suis in murine macrophages, by using cholesterol-sequestering (filipin and beta-methyl cyclodextrin) and GM(1)-binding (cholera toxin B) molecules. Our results suggest that lipid rafts may provide a portal for entry of Brucella into murine macrophages under nonopsonic conditions, thus allowing phagosome-lysosome fusion inhibition, and provide further evidence to support the idea that the phagosome maturation inhibition is restricted at the phagosomal membrane.     

Do microtubules around the Toxoplasma gondii-containing parasitophorous vacuole in skeletal muscle cells form a barrier for the phagolysosomal fusion?

The intracellular fate of Toxoplasma gondii was studied in primary cultures of skeletal muscle cells (SMC). The labelling of secondary lysosomes with BSA-Au particles showed no phagolysosomal fusion with the vacuole containing the parasite. After internalization of the parasites, the parasitophorous vacuole became involved by closely apposed endoplasmic reticulum (ER) and mitochondria; within 18 h of interaction, microtubules were visualized in association with the parasitophorous vacuole, suggesting that they could form a barrier for the phagolysosomal fusion.     

Toxoplasma gondii : an ultrastructural study of host-cell invasion by the bradyzoite stage

The invasion of Toxoplasma gondii tachyzoites and bradyzoites was followed in bovine kidney cells via electron microscopy. The process of invasion differed between bradyzoites and tachyzoites. In the early stages of entry there was evidence of localised formation of membrane projections in the host cell adjacent to the parasite. Parasite reorientation and rhoptry release appeared to be necessary for invasion; however, the tight junction could not be clearly discerned and there was no evidence of constriction or of any membrane shedding from the parasite. The resulting parasitophorous vacuole was smaller than the tachyzoite vacuole and parasites were frequently found to lie immediately under the host cell membrane. The vacuole was rapidly adapted by the release and formation of an intra-phagosomal membrane network, while the parasitophorous vacuole formed a relationship with host-cell endoplasmic reticulum. Received: 28 March 1998 / Accepted: 28 April 1998     

Immunodominant, protective response to the parasite Toxoplasma gondii requires antigen processing in the endoplasmic reticulum

The parasite Toxoplasma gondii replicates in a specialized intracellular vacuole and causes disease in many species. Protection from toxoplasmosis is mediated by CD8(+) T cells, but the T. gondii antigens and host genes required for eliciting protective immunity are poorly defined. Here we identified GRA6, a polymorphic protein secreted in the parasitophorous vacuole, as the source of the immunodominant and protective decapeptide HF10 presented by the H-2L(d) major histocompatibility complex class I molecule. Presentation of the HF10-H-2L(d) ligand required proteolysis by ERAAP, the endoplasmic reticulum aminopeptidase associated with antigen processing. Consequently, expansion of protective CD8(+) T cell populations was impaired in T. gondii-infected ERAAP-deficient mice, which were more susceptible to toxoplasmosis. Thus, endoplasmic reticulum proteolysis is critical for eliciting protective immunity to a vacuolar parasite.     

Recombinase-deficient T cell development by selective accumulation of CD3 into lipid rafts

The pre-T cell receptor (pre-TCR) promotes the development of thymocytes with productive rearrangement at the TCR beta chain locus by signaling in a ligand-independent fashion. The TCR beta chain associates with the invariant pre-Talpha (pTalpha) chain, which bears specific charged residues in the extracellular portion mediating pre-TCR self-oligomerization. In recombinase-deficient thymocytes, calnexin (CNX) associated with CD3 chains is inefficiently retained in the endoplasmic reticulum (ER) and weakly expressed in the plasma membrane. Deliberate cross-linking of CNX/CD3 complexes mimics pre-TCR signaling. Here, we show that, analogously to the pTalpha chain, surface CNX is palmitoylated and that CD3 prominently accumulated in lipid rafts upon cross-linking. Mutant CNX isoforms devoid of ER retention determined pre-TCR-like signaling and simulated beta selection only when stably translocating CD3 to lipid rafts. Inclusion of the palmitoylated cytoplasmic tail from the pTalpha chain in recombinant CNX strikingly improved the pre-TCR-like signaling efficiency of CNX/CD3 in rafts. This study indicates that lipid rafts in the plasma membrane represent proficient microdomains for the initiation of pre-TCR signaling, and supports the view that beta selection by oligomerized pre-TCR is implemented by the pTalpha cytoplasmic tail.     

In vitro studies of rickettsia-host cell interactions: ultrastructural changes induced by Rickettsia rickettsii infection of chicken embryo fibroblasts.

Secondary chicken embryo fibroblasts infected with the Sheila Smith strain of Rickettsia rickettsii and grown in monolayer culture undergo rapid morphological alterations. Transmission electron microscopic examination of cells at intervals after infection showed several progressive host cell lesions, including widespread dilatation of the rough endoplasmic reticulum and outer nuclear envelope and the accumulation of electron-dense material within the cisternae of intracellular membranes. Dilatation of the rough endoplasmic reticulum is a common, early reversible manifestation of other forms of cell injury. However, the severity of the damage to the host cell resulting from the progressive distention of intracellular membranes and the subsequent formation of small segments of membrane-bound host cytoplasm within the cisternae of these membranes is unknown. Early in the infection cycle, the rickettsiae were found free in the host cell cytoplasm, within invaginations of the nuclear envelope, occasionally free in the space between the outer and inner nuclear membranes, and in the host nucleoplasm, but not within cisternae formed by swollen endoplasmic reticulum. As a consequence of intracisternal swelling and fusion of intracellular membranes later in the infection cycle, the majority of the rickettsiae were found surrounded by host cytoplasm bound by host-derived internal membranes and appeared to persist in this state until cell lysis. The overall cytopathological changes in cells infected with R. richettsii appear dramatic and, from other studies in our laboratory, are significantly different from those observed in cells infected with Rickettsia prowazekii.     

Cholesterol depletion reduces entry of Campylobacter jejuni cytolethal distending toxin and attenuates intoxication of host cells

Campylobacter jejuni is a common cause of pediatric diarrhea worldwide. Cytolethal distending toxin, produced by Campylobacter jejuni, is a putative virulence factor that induces cell cycle arrest and apoptosis in eukaryotic cells. Cellular cholesterol, a major component of lipid rafts, has a pivotal role in regulating signaling transduction and protein trafficking as well as pathogen internalization. In this study, we demonstrated that cell intoxication by Campylobacter jejuni cytolethal distending toxin is through the association of cytolethal distending toxin subunits and membrane cholesterol-rich microdomains. Cytolethal distending toxin subunits cofractionated with detergent-resistant membranes, while the distribution reduced upon the depletion of cholesterol, suggesting that cytolethal distending toxin subunits are associated with lipid rafts. The disruption of cholesterol using methyl-β-cyclodextrin not only reduced the binding activity of cytolethal distending toxin subunits on the cell membrane but also impaired their delivery and attenuated toxin-induced cell cycle arrest. Accordingly, cell intoxication by cytolethal distending toxin was restored by cholesterol replenishment. These findings suggest that membrane cholesterol plays a critical role in the Campylobacter jejuni cytolethal distending toxin-induced pathogenesis of host cells.     

Legionella pneumophila replication vacuole formation involves rapid recruitment of proteins of the early secretory system

Legionella pneumophila vacuole biogenesis was analyzed by using a cell-free system. We show that calnexin, Sec22b, and Rab1 are recruited to the vacuole very shortly after bacterial uptake, and we have identified Rab1 as a potential host factor involved in the endoplasmic reticulum recruitment process.     

A unique Rab GTPase, EhRabA, is involved in motility and polarization of Entamoeba histolytica cells

Entamoeba histolytica, an enteric protozoan parasite, infects 10% of the world's population leading to 50 million cases of invasive amoebiasis annually. Motility, which requires cell polarization, is important to the virulence of this pathogen, as it may result in destruction of host tissues and invasion. To gain insight into these processes in Entamoeba, a unique Rab GTPase, EhRabA, which localizes to the leading edge of cells, was characterized. Cell lines expressing a dominant negative version of EhRabA (EhRabA-DN) were generated. These mutant cells exhibited alterations in cell shape, polarity, and motility, supporting a role for this Rab in the regulation of these processes. Consistent with the notion that a dynamic actin cytoskeleton is crucial to cell polarity and motility, these mutants also exhibited alterations in the actin cytoskeleton. Cells expressing EhRabA-DN also displayed defects in several virulence functions including the ability to adhere to host cells, destroy host cells, and release cysteine proteases. Mislocalization of a prominent adhesion molecule, the galactose/N-acetylgalactosamine (Gal/GalNAc) adherence lectin and reorganization of ordered lipid domains, known as lipid rafts, also accompanied expression of EhRabA-DN. Interestingly, several endocytic processes were unaffected by expression of EhRabA-DN. Together, these data suggest that EhRabA may be involved in the regulation of polarization, motility and actin cytoskeletal dynamics: functions that participate in the pathogenicity of Entamoeba.     

Attenuated endocytosis and toxicity of a mutant cholera toxin with decreased ability to cluster ganglioside GM1 molecules

Cholera toxin (CT) moves from the plasma membrane (PM) of host cells to the endoplasmic reticulum (ER) by binding to the lipid raft ganglioside GM(1). The homopentomeric B-subunit of the toxin can bind up to five GM(1) molecules at once. Here, we examined the role of polyvalent binding of GM(1) in CT action by producing chimeric CTs that had B-subunits with only one or two normal binding pockets for GM(1). The chimeric toxins had attenuated affinity for binding to host cell PM, as expected. Nevertheless, like wild-type (wt) CT, the CT chimeras induced toxicity, fractionated with detergent-resistant membranes extracted from toxin-treated cells, displayed restricted diffusion in the plane of the PM in intact cells, and remained bound to GM(1) when they were immunoprecipitated. Thus, binding normally to two or perhaps only one GM(1) molecule is sufficient for association with lipid rafts in the PM and toxin action. The chimeric toxins, however, were much less potent than wt toxin, and they entered the cell by endocytosis more slowly, suggesting that clustering of GM(1) molecules by the B-subunit enhances the efficiency of toxin uptake and perhaps also trafficking to the ER.     

The broad-range phospholipase C and a metalloprotease mediate listeriolysin O-independent escape of Listeria monocytogenes from a primary vacuole in human epithelial cells.

Intracellular growth of Listeria monocytogenes begins after lysis of the primary vacuole formed upon bacterial entry into a host cell. Listeriolysin O (LLO), a pore-forming hemolysin encoded by hly, is essential for vacuolar lysis in most cell types. However, in human epithelial cells, LLO- mutants are capable of growth, suggesting that gene products other than LLO are capable of mediating escape from a vacuole. In this study, we investigated the role of other bacterial gene products in lysis of the primary vacuole in the human epithelial cell line Henle 407. Double internal in-frame deletion mutants were constructed by introducing a mutated hly allele into strains harboring deletions in either of the phospholipase C (PLC)-encoding genes or a metalloprotease-encoding gene. Bacterial escape from the primary vacuole, intracellular growth, and cell-to-cell spread were evaluated in Henle 407 cells. The results indicated that, in the absence of LLO, the broad-range PLC and the metalloprotease were both required for lysis of the primary vacuole in Henle 407 cells. Although phosphatidylinositol-specific PLC was not required, the efficiency of escape was reduced in an LLO phosphatidylinositol-specific PLC double mutant. These observations suggest that the relative importance of LLO, the phospholipases, and the metalloprotease may vary in different cell types or in cells from different species. In addition, these studies provide insight into the mechanisms of action of virulence determinants involved in the lysis of vacuolar membranes.     

The nature of the phagosomal membrane: endoplasmic reticulum versus plasmalemma

For decades, the vacuole that surrounds particles engulfed by phagocytosis was believed to originate from the plasma membrane. Conversion of the nascent phagosome into a microbicidal organelle was thought to result from the subsequent, orderly fusion of early endosomes, late endosomes, and ultimately, lysosomes with the original plasma membrane-derived vacuole. This conventional model has been challenged, if not superseded, by a revolutionary model that regards phagosome formation as resulting from the particle sliding into the endoplasmic reticulum via an opening at the base of the phagocytic cup. The merits and implications of these two hypotheses are summarized here and analyzed in light of recent results.     

High-polarity Mycobacterium avium-derived lipids interact with murine macrophage lipid rafts

Cholesterol- and sphingolipid-rich membrane microdomains (lipid rafts) are widely recognized as portals for pathogenic micro-organisms. A growing body of evidence demonstrates mobilization of host plasma cell membrane lipid rafts towards the site of contact with several pathogens as well as a strict dependence on cholesterol for appropriate internalization. The fate of lipid rafts once the pathogen has been internalized and the nature of the pathogen components that interact with them is however less understood. To address both these issues, infection of the J774 murine cell line with Mycobacterium avium was used as a model. After demonstrating that M. avium induces lipid raft mobilization and that M. avium infects J774 by a cholesterol-dependent mechanism, it is shown here that mycobacterial phagosomes harbour lipid rafts, which are, at least in part, of plasma cell membrane origin. On the other hand, by using latex microbeads coated with any of the three fractions of M. avium-derived lipids of different polarity, we provide evidence that high-polarity, in contrast to low-polarity and intermediate-polarity, mycobacterial lipids or uncoated latex beads have a strong capacity to induce lipid raft mobilization. These results suggest that high-polarity mycobacterial lipid(s) interact with host cell cholesterol-enriched microdomains which may in turn influence the course of infection.     

Evidence for pore formation in host cell membranes by ESX-1-secreted ESAT-6 and its role in Mycobacterium marinum escape from the vacuole

The ESX-1 secretion system plays a critical role in the virulence of M. tuberculosis and M. marinum, but the precise molecular and cellular mechanisms are not clearly defined. Virulent M. marinum is able to escape from the Mycobacterium-containing vacuole (MCV) into the host cell cytosol, polymerize actin, and spread from cell to cell. In this study, we have examined nine M. marinum ESX-1 mutants and the wild type by using fluorescence and electron microscopy detecting MCV membranes and actin polymerization. We conclude that ESX-1 plays an essential role in M. marinum escape from the MCV. We also show that the ESX-1 mutants acquire the ability to polymerize actin after being artificially delivered into the macrophage cytosol by hypotonic shock treatment, indicating that ESX-1 is not directly involved in initiation of actin polymerization. We provide evidence that M. marinum induces membrane pores ~4.5 nm in diameter, and this activity correlates with ESAT-6 secretion. Importantly, purified ESAT-6, but not the other ESX-1-secreted proteins, is able to cause dose-dependent pore formation in host cell membranes. These results suggest that ESAT-6 secreted by M. marinum ESX-1 could play a direct role in producing pores in MCV membranes, facilitating M. marinum escape from the vacuole and cell-to-cell spread. Our study provides new insight into the mechanism by which ESX-1 secretion and ESAT-6 enhance the virulence of mycobacterial infection.     

脂筏与感染性疾病

脂筏是生物膜中含有特殊脂质和蛋白质的微区,不仅是跨膜信号转导等多种生命活动的重要参与者,而且在感染性疾病的发生和发展中扮演了重要的角色。脂筏是多种病原体进入宿主细胞的位点,支持病毒粒子的组装和出芽,其信号转导功能一方面可以启动宿主细胞的保护性免疫应答,另一方面也被病原体利用,以利于病原体的传播和疾病发生。此外,脂筏在朊蛋白感染、HCV基因组复制、多种细菌毒素作用以及维持疟原虫的胞内寄生生活等方面也都有重要作用。脂筏在感染性疾病中的重要作用提示,它很有可能成为扭转这些疾病发生和进程以及治疗这些疾病的重要入手点。     

Three Antagonistic Cyclic di-GMP-Catabolizing Enzymes Promote Differential Dot/Icm Effector Delivery and Intracellular Survival at the Early Steps of Legionella pneumophila Infection

Legionella pneumophila is an intracellular pathogen which replicates within protozoan cells and can accidently infect alveolar macrophages, causing an acute pneumonia in humans. The second messenger cyclic di-GMP (c-di-GMP) has been shown to play key roles in the regulation of various bacterial processes, including virulence. While investigating the function of the 22 potential c-di-GMP-metabolizing enzymes of the L. pneumophila Lens strain, we found three that directly contribute to its ability to infect both protozoan and mammalian cells. These three enzymes display diguanylate cyclase (Lpl0780), phosphodiesterase (Lpl1118), and bifunctional diguanylate cyclase/phosphodiesterase (Lpl0922) activities, which are all required for the survival and intracellular replication of L. pneumophila. Mutants with deletions of the corresponding genes are efficiently taken up by phagocytic cells but are partially defective for the escape of the Legionella-containing vacuole (LCV) from the host degradative endocytic pathway and result in lower survival. In addition, Lpl1118 is required for efficient endoplasmic reticulum recruitment to the LCV. Trafficking and biogenesis of the LCV are dependent upon the orchestrated actions of several type 4 secretion system Dot/Icm effectors proteins, which exhibit differentially altered translocation in the three mutants. While translocation of some effectors remained unchanged, others appeared over- and undertranslocated. A general translocation offset of the large repertoire of Dot/Icm effectors may be responsible for the observed defects in the trafficking and biogenesis of the LCV. Our results suggest that L. pneumophila uses cyclic di-GMP signaling to fine-tune effector delivery and ensure effective evasion of the host degradative pathways and establishment of a replicative vacuole.     

Floating cholera toxin into epithelial cells: functional association with caveolae-like detergent-insoluble membrane microdomains

In polarized cells, signal transduction by cholera toxin (CT) requires apical endocytosis and retrograde transport into Golgi cisternae and likely endoplasmic reticulum (ER) (Lencer et al., J. Cell Biol. 131, 951-962 (1995)). We have recently found that the toxin's apical membrane receptor ganglioside GM1 acts specifically in this signal transduction pathway, likely by coupling CT with caveolae or caveolae-related membrane domains (lipid rafts) (Wolf et al., J. Cell Biol. 141, 917-927 (1998)). Work in progress shows that 1) cholesterol depletion uncouples the CT-GM1 receptor complex from signal transduction, a characteristic of lipid rafts; 2) the GM1 acyl chains rather than the carbohydrate head groups appear to account for the structural basis of ganglioside specificity in toxin trafficking; and 3) intestinal epithelial cells obtained from normal adult humans exhibit lipid rafts which differentiate between CT-GM1 and LTIIb-GD1a complexes and which contain caveolin 1.     

Effect of cholesterol depletion on exocytosis of alveolar type II cells

Alveolar epithelial type II cells secrete lung surfactant via exocytosis. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) are implicated in this process. Lipid rafts, the cholesterol- and sphingolipid-rich microdomains, may offer a platform for protein organization on the cell membrane. We tested the hypothesis that lipid rafts organize exocytotic proteins in type II cells and are essential for the fusion of lamellar bodies, the secretory granules of type II cells, with the plasma membrane. The lipid rafts, isolated from type II cells using 1% Triton X-100 and a sucrose gradient centrifugation, contained the lipid raft markers, flotillin-1 and -2, whereas they excluded the nonraft marker, Na+-K+ ATPase. SNAP-23, syntaxin 2, and VAMP-2 were enriched in lipid rafts. When type II cells were depleted of cholesterol, the association of SNAREs with the lipid rafts was disrupted and the formation of fusion pore was inhibited. Furthermore, the cholesterol-depleted plasma membrane had less ability to fuse with lamellar bodies, a process mediated by annexin A2. The secretagogue-stimulated secretion of lung surfactant from type II cells was also reduced by methyl-beta-cyclodextrin. When the raft-associated cell surface protein, CD44, was cross-linked using anti-CD44 antibodies, the CD44 clusters were observed. Syntaxin 2, SNAP-23, and annexin A2 co-localized with the CD44 clusters, which were cholesterol dependent. Our results suggested that lipid rafts may form a functional platform for surfactant secretion in alveolar type II cells, and raft integrity was essential for the fusion between lamellar bodies with the plasma membrane.