Microscopy-Based Tracking and Quantification Methods to Study Phagosome Resolution.

Phagosome resolution is a newly defined, terminal stage in the process of phagocytosis. During this phase, phagolysosomes are fragmented into smaller vesicles, which we called phagosome-derived vesicles (PDVs). PDVs gradually accumulate within macrophages, while the phagosomes diminish in size until the organelles are no longer detectable. Although PDVs share the same maturation markers as phagolysosomes, they are heterogeneous in size and very dynamic, which makes PDVs difficult to track. Thus, to analyze PDV populations in cells, we developed methods to differentiate PDVs from the phagosomes in which they were derived and further assess their characteristics. In this chapter, we describe two microscopy-based methods that can be used to quantify different aspects of phagosome resolution: volumetric analysis of phagosome shrinkage and PDV accumulation and co-occurrence analysis of various membrane markers with PDVs.

Aluminum hydroxide adjuvant diverts the uptake and trafficking of genetically detoxified pertussis toxin to lysosomes in macrophages.

Aluminum salts have been successfully utilized as adjuvants to enhance the immunogenicity of vaccine antigens since the 1930s. However, the cellular mechanisms behind the immune adjuvanticity effect of these materials in antigen-presenting cells are poorly understood. In this study, we investigated the uptake and trafficking of aluminum oxy-hydroxide (AlOOH), in RAW 264.7 murine and U-937 human macrophages-like cells. Furthermore, we determined the impact that the adsorption to AlOOH particulates has on the trafficking of a Bordetella pertussis vaccine candidate, the genetically detoxified pertussis toxin (gdPT). Our results indicate that macrophages internalize AlOOH by constitutive macropinocytosis assisted by the filopodial protrusions that capture the adjuvant particles. Moreover, we show that AlOOH has the capacity to nonspecifically adsorb IgG, engaging opsonic phagocytosis, which is a feature that may allow for more effective capture and uptake of adjuvant particles by antigen-presenting cells (APCs) at the site of vaccine administration. We found that AlOOH traffics to endolysosomal compartments that hold degradative properties. Importantly, while we show that gdPT escapes degradative endolysosomes and traffics toward the retrograde pathway, as reported for the wild-type pertussis toxin, the adsorption to AlOOH diverts gdPT to traffic to the adjuvant's lysosome-type compartments, which may be key for MHC-II-driven antigen presentation and activation of CD4+ T cell. Thus, our findings establish a direct link between antigen adsorption to AlOOH and the intracellular trafficking of antigens within antigen-presenting cells and bring to light a new potential mechanism for aluminum adjuvancy. Moreover, the in-vitro single-cell approach described herein provides a general framework and tools for understanding critical attributes of other vaccine formulations.

Phosphatidylinositol 3-Phosphate Mediates the Establishment of Infectious Bursal Disease Virus Replication Complexes in Association with Early Endosomes.

Infectious bursal disease virus (IBDV) is the archetypal member of the family Birnaviridae and the etiological agent of Gumboro disease, a highly contagious immunosuppressive infection of concern to the global poultry sector for its adverse health effects in chicks. Unlike most double-stranded RNA (dsRNA) viruses, which enclose their genomes within specialized cores throughout their viral replication cycle, birnaviruses organize their bisegmented dsRNA genome in ribonucleoprotein (RNP) structures. Recently, we demonstrated that IBDV exploits endosomal membranes for replication. The establishment of IBDV replication machinery on the cytosolic leaflet of endosomal compartments is mediated by the viral protein VP3 and its intrinsic ability to target endosomes. In this study, we identified the early endosomal phosphatidylinositol 3-phosphate [PtdIns(3)P] as a key host factor of VP3 association with endosomal membranes and consequent establishment of IBDV replication complexes in early endosomes. Indeed, our data reveal a crucial role for PtdIns(3)P in IBDV replication. Overall, our findings provide new insights into the replicative strategy of birnaviruses and strongly suggest that it resembles those of positive-strand RNA (+ssRNA) viruses, which replicate in association with host membranes. Furthermore, our findings support the role of birnaviruses as evolutionary intermediaries between +ssRNA and dsRNA viruses and, importantly, demonstrate a novel role for PtdIns(3)P in the replication of a dsRNA virus.IMPORTANCEInfectious bursal disease virus (IBDV) infects chicks and is the causative agent of Gumboro disease. During IBDV outbreaks in recent decades, the emergence of very virulent variants and the lack of effective prevention/treatment strategies to fight this disease have had devastating consequences for the poultry industry. IBDV belongs to the peculiar family Birnaviridae Unlike most dsRNA viruses, birnaviruses organize their genomes in ribonucleoprotein complexes and replicate in a core-independent manner. We recently demonstrated that IBDV exploits host cell endosomes as platforms for viral replication, a process that depends on the VP3 viral protein. In this study, we delved deeper into the molecular characterization of IBDV-endosome association and investigated the role of host cell phosphatidylinositide lipids in VP3 protein localization and IBDV infection. Together, our findings demonstrate that PtdIns(3)P serves as a scaffold for the association of VP3 to endosomes and reveal its essential role for IBDV replication.

Pseudobutyrivibrio xylanivorans adhesion to epithelial cells.

The ruminal bacteria Pseudobutyrivibrio xylanivorans strain 2 (P. xylanivorans 2), that mediates the digestion of plant fiber, is considered an attractive candidate for probiotics. Adherence to the epithelium of the digestive tract of the host is one of the major requirements for probiotics. In this study, we assessed the adhesion of P. xylanivorans 2 to SW480 cells and characterized this process utilizing multiple microscopy approaches. Our results indicate that a multiplicity of infection of 200 CFU/cell allows the highest bacteria to cell binding ratio, with a lower percentage of auto-agglutination events. The comparison of the adherence capacity subjected heat-shock treatment (100 °C, 1 min), which produces the denaturalization of proteins at the bacterial surface, as opposed untreated P. xylanivorans, suggested that this bacteria may attach to SW480 cells utilizing a proteinaceous structure. Confocal microscopy analyses indicate that P. xylanivorans 2 attachment induces the formation of F-actin-enriched areas on the surface of SW480 cells. Transmission electron microscopy (TEM) revealed the formation of a structure similar to a pedestal in the area of the epithelial cell surface, where the bacterium rests. Finally, a casual finding of TEM analysis of transverse and longitudinal thin-sections of P. xylanivorans 2, revealed irregular intra-cytoplasmic structures compatibles with the so-called bacterial microcompartments. This is the first ultrastructural description of bacterial microcompartments-like structures in the genus Pseudobutyrivibrio.

Small Rho GTPases and the Effector VipA Mediate the Invasion of Epithelial Cells by Filamentous Legionella pneumophila.

Legionella pneumophila (Lp) exhibits different morphologies with varying degrees of virulence. Despite their detection in environmental sources of outbreaks and in respiratory tract secretions and lung autopsies from patients, the filamentous morphotype of Lp remains poorly studied. We previously demonstrated that filamentous Lp invades lung epithelial cells (LECs) and replicates intracellularly in a Legionella containing vacuole. Filamentous Lp activates ß1integrin and E-cadherin receptors at the surface of LECs leading to the formation of actin-rich cell membrane structures we termed hooks and membrane wraps. These structures entrap segments of an Lp filament on host cell surface and mediate bacterial internalization. Here we investigated the molecular mechanisms responsible for the actin rearrangements needed for the formation and elongation of these membrane wraps and bacterial internalization. We combined genetic and pharmacological approaches to assess the contribution of signaling downstream of ß1integrin and E-cadherin receptors, and Lp Dot/Icm secretion system- translocated effectors toward the invasion process. Our studies demonstrate a multi-stage mechanism of LEC invasion by filamentous Lp. Bacterial attachment to host cells depends on signaling downstream of ß1integrin and E-cadherin activation, leading to Rho GTPases-dependent activation of cellular actin nucleating proteins, Arp2/3 and mDia. This mediates the formation of primordial membrane wraps that entrap the filamentous bacteria on the cell surface. Following this, in a second phase of the invasion process the Dot/Icm translocated effector VipA mediates rapid membrane wrap elongation, leading to the engulfment of the filamentous bacteria by the LECs. Our findings provide the first description of Rho GTPases and a Dot/Icm effector VipA regulating the actin dynamics needed for the invasion of epithelial cells by Lp.

Infectious Bursal Disease Virus Hijacks Endosomal Membranes as the Scaffolding Structure for Viral Replication.

Birnaviruses are unconventional members of the group of double-stranded RNA (dsRNA) viruses that are characterized by the lack of a transcriptionally active inner core. Instead, the birnaviral particles organize their genome in ribonucleoprotein complexes (RNPs) composed by dsRNA segments, the dsRNA-binding VP3 protein, and the virally encoded RNA-dependent RNA polymerase (RdRp). This and other structural features suggest that birnaviruses may follow a completely different replication program from that followed by members of the Reoviridae family, supporting the hypothesis that birnaviruses are the evolutionary link between single-stranded positive RNA (+ssRNA) and dsRNA viruses. Here we demonstrate that infectious bursal disease virus (IBDV), a prototypical member of the Birnaviridae family, hijacks endosomal membranes of infected cells through the interaction of a viral protein, VP3, with the phospholipids on the cytosolic leaflet of these compartments for replication. Employing a mutagenesis approach, we demonstrated that VP3 domain PATCH 2 (P2) mediates the association of VP3 with the endosomal membranes. To determine the role of VP3 P2 in the context of the virus replication cycle, we used avian cells stably overexpressing VP3 P2 for IBDV infection. Importantly, the intra- and extracellular virus yields, as well as the intracellular levels of VP2 viral capsid protein, were significantly diminished in cells stably overexpressing VP3 P2. Together, our results indicate that the association of VP3 with endosomes has a relevant role in the IBDV replication cycle. This report provides direct experimental evidence for membranous compartments such as endosomes being required by a dsRNA virus for its replication. The results also support the previously proposed role of birnaviruses as an evolutionary link between +ssRNA and dsRNA viruses.IMPORTANCE Infectious bursal disease (IBD; also called Gumboro disease) is an acute, highly contagious immunosuppressive disease that affects young chickens and spreads worldwide. The etiological agent of IBD is infectious bursal disease virus (IBDV). This virus destroys the central immune organ (bursa of Fabricius), resulting in immunosuppression and reduced responses of chickens to vaccines, which increase their susceptibility to other pathogens. IBDV is a member of Birnaviridae family, which comprises unconventional members of dsRNA viruses, whose replication strategy has been scarcely studied. In this report we show that IBDV hijacks the endosomes of the infected cells for establishing viral replication complexes via the association of the ribonucleoprotein complex component VP3 with the phospholipids in the cytosolic leaflet of endosomal membranes. We show that this interaction is mediated by the VP3 PATCH 2 domain and demonstrate its relevant role in the context of viral infection.

pH of endophagosomes controls association of their membranes with Vps34 and PtdIns(3)P levels.

Phagocytosis of filamentous bacteria occurs through tubular phagocytic cups (tPCs) and takes many minutes to engulf these filaments into phagosomes. Contravening the canonical phagocytic pathway, tPCs mature by fusing with endosomes. Using this model, we observed the sequential recruitment of early and late endolysosomal markers to the elongating tPCs. Surprisingly, the regulatory early endosomal lipid phosphatidylinositol-3-phosphate (PtdIns(3)P) persists on tPCs as long as their luminal pH remains neutral. Interestingly, by manipulating cellular pH, we determined that PtdIns(3)P behaves similarly in canonical phagosomes as well as endosomes. We found that this is the product of a pH-based mechanism that induces the dissociation of the Vps34 class III phosphatidylinositol-3-kinase from these organelles as they acidify. The detachment of Vps34 stops the production of PtdIns(3)P, allowing for the turnover of this lipid by PIKfyve. Given that PtdIns(3)P-dependent signaling is important for multiple cellular pathways, this mechanism for pH-dependent regulation of Vps34 could be at the center of many PtdIns(3)P-dependent cellular processes.

Filamentous Bacteria as Targets to Study Phagocytosis.

Filamentous targets are internalized via phagocytic cups that last for several minutes before closing to form a phagosome. This characteristic offers the possibility to study key events in phagocytosis with greater spatial and temporal resolution than is possible to achieve using spherical particles, for which the transition from a phagocytic cup to an enclosed phagosome occurs within a few seconds after particle attachment. In this chapter, we provide methodologies to prepare filamentous bacteria and describe how they can be used as targets to study different aspects of phagocytosis.

PIKfyve inhibition interferes with phagosome and endosome maturation in macrophages.

Macrophages eliminate pathogens and cell debris through phagocytosis, a process by which particulate matter is engulfed and sequestered into a phagosome. Nascent phagosomes are innocuous organelles resembling the plasma membrane. However, through a maturation process, phagosomes are quickly remodeled by fusion with endosomes and lysosomes to form the phagolysosome. Phagolysosomes are highly acidic and degradative leading to particle decomposition. Phagosome maturation is intimately dependent on the endosomal pathway, during which diverse cargoes are sorted for recycling to the plasma membrane or for degradation in lysosomes. Not surprisingly, various regulators of the endosomal pathway are also required for phagosome maturation, including phosphatidylinositol-3-phosphate, an early endosomal regulator. However, phosphatidylinositol-3-phosphate can be modified by the lipid kinase PIKfyve into phosphatidylinositol-3,5-bisphosphate, which controls late endosome/lysosome functions. The role of phosphatidylinositol-3,5-bisphosphate in macrophages and phagosome maturation remains basically unexplored. Using Fcγ receptor-mediated phagocytosis as a model, we describe our research showing that inhibition of PIKfyve hindered certain steps of phagosome maturation. In particular, PIKfyve antagonists delayed removal of phosphatidylinositol-3-phosphate and reduced acquisition of LAMP1 and cathepsin D, both common lysosomal proteins. Consistent with this, the degradative capacity of phagosomes was reduced but phagosomes appeared to still acidify. We also showed that trafficking to lysosomes and their degradative capacity was reduced by PIKfyve inhibition. Overall, we provide evidence that PIKfyve, likely through phosphatidylinositol-3,5-bisphosphate synthesis, plays a significant role in endolysosomal and phagosome maturation in macrophages.

Filamentous morphology of bacteria delays the timing of phagosome morphogenesis in macrophages.

Although filamentous morphology in bacteria has been associated with resistance to phagocytosis, our understanding of the cellular mechanisms behind this process is limited. To investigate this, we followed the phagocytosis of both viable and dead Legionella pneumophila filaments. The engulfment of these targets occurred gradually and along the longitudinal axis of the filament, therefore defining a long-lasting phagocytic cup stage that determined the outcome of phagocytosis. We found that these phagocytic cups fused with endosomes and lysosomes, events linked to the maturation of phagosomes according to the canonical pathway, and not with the remodeling of phagocytic cups. Nevertheless, despite acquiring phagolysosomal features these phagocytic cups failed to develop hydrolytic capacity before their sealing. This phenomenon hampered the microbicidal activity of the macrophage and enhanced the capacity of viable filamentous L. pneumophila to escape phagosomal killing in a length-dependent manner. Our results demonstrate that key aspects in phagocytic cup remodeling and phagosomal maturation could be influenced by target morphology.

Chlamydia trachomatis vacuole maturation in infected macrophages.

Chlamydia trachomatis is an obligate intracellular bacterium responsible for one of the most common sexually transmitted diseases. In epithelial cells, C. trachomatis resides in a modified membrane-bound vacuole known as an inclusion, which is isolated from the endocytic pathway. However, the maturation process of C. trachomatis within immune cells, such as macrophages, has not been studied extensively. Here, we demonstrated that RAW macrophages effectively suppressed C. trachomatis growth and prevented Golgi stack disruption, a hallmark defect in epithelial cells after C. trachomatis infection. Next, we systematically examined association between C. trachomatis and various endocytic pathway markers. Spinning disk confocal time-lapse studies revealed significant and rapid association between C. trachomatis with Rab7 and LAMP1, markers of late endosomes and lysosomes. Moreover, pretreatment with an inhibitor of lysosome acidification led to significant increases in C. trachomatis growth in macrophages. At later stages of infection, C. trachomatis associated with the autophagy marker LC3. TEM analysis confirmed that a significant portion of C. trachomatis resided within double-membrane-bound compartments, characteristic of autophagosomes. Together, these results suggest that macrophages can suppress C. trachomatis growth by targeting it rapidly to lysosomes; moreover, autophagy is activated at later stages of infection and targets significant numbers of the invading bacteria, which may enhance subsequent chlamydial antigen presentation.

Mechanism of invasion of lung epithelial cells by filamentous Legionella pneumophila.

Legionella, the aetiological agent responsible for Legionellosis, is an opportunistic pathogen that infects humans upon the inhalation of contaminated aerosolized water droplets. Legionella is pleomorphic and its different morphotypes exhibit varying degrees of virulence. While the filamentous forms of Legionella pneumophila (Lp) have been reported in patient samples since the first description of legionellosis, their role in disease has not been studied. Our results show that both E-cadherin and ß1 integrin receptors mediate filamentous Lp (FLp) attachment to lung epithelial cells (LECs). The activation of these receptors induces the formation of actin enriched membrane surface structures that we designated 'hooks' and 'membrane wraps'. These structures entrap the filaments on the cell surface leading to their gradual internalization through a zipper mechanism of phagocytosis dependent on actomyosin activity. The supply of E-cadherin receptors from the recycling pathway and ß1 integrins released from focal adhesion turnover are required to sustain this process. Intracellular FLp inhabits a vacuolar compartment where filaments differentiate into short rods and replicate to produce infective progeny. Here we are reporting a first description of the invasion mechanism used by FLp to invade LECs. Therefore, filamentous morphotype of Lp can induce its own uptake by LECs and has the potential ability to cause disease.

Vacuolating cytotoxin and variants in Atg16L1 that disrupt autophagy promote Helicobacter pylori infection in humans.

BACKGROUND & AIMS: The Helicobacter pylori toxin vacuolating cytotoxin (VacA) promotes gastric colonization, and its presence (VacA(+)) is associated with more-severe disease. The exact mechanisms by which VacA contributes to infection are unclear. We previously found that limited exposure to VacA induces autophagy of gastric cells, which eliminates the toxin; we investigated whether autophagy serves as a defense mechanism against H pylori infection. METHODS: We investigated the effect of VacA on autophagy in human gastric epithelial cells and primary gastric cells from mice. Expression of p62, a marker of autophagy, was also assessed in gastric tissues from patients infected with toxigenic (VacA(+)) or nontoxigenic strains. We analyzed the effect of VacA on autophagy in peripheral blood monocytes obtained from subjects with different genotypes of ATG16L1, which regulates autophagy. We performed genotyping for ATG16L1 in 2 cohorts of infected and uninfected subjects. RESULTS: Prolonged exposure of human gastric epithelial cells and mouse gastric cells to VacA disrupted induction of autophagy in response to the toxin, because the cells lacked cathepsin D in autophagosomes. Loss of autophagy resulted in the accumulation of p62 and reactive oxygen species. Gastric biopsy samples from patients infected with VacA(+), but not nontoxigenic strains of H pylori, had increased levels of p62. Peripheral blood monocytes isolated from individuals with polymorphisms in ATG16L1 that increase susceptibility to Crohn's disease had reduced induction of autophagy in response to VacA(+) compared to cells from individuals that did not have these polymorphisms. The presence of the ATG16L1 Crohn's disease risk variant increased susceptibility to H pylori infection in 2 separate cohorts. CONCLUSIONS: Autophagy protects against infection with H pylori; the toxin VacA disrupts autophagy to promote infection, which could contribute to inflammation and eventual carcinogenesis.

Helicobacter pylori cytotoxin-associated gene A activates the signal transducer and activator of transcription 3 pathway in vitro and in vivo.

Persistent infection with Helicobacter pylori confers an increased risk for the development of gastric cancer. However, the exact mechanisms whereby this bacterium causes carcinogenesis have not been completely elucidated. Recent evidence indicates that aberrant activation of the signal transducers and activators of transcription 3 (STAT3) signaling pathway may play a role in gastric carcinogenesis. Therefore, we hypothesized that H. pylori infection modulates STAT3 signaling, favoring gastric cancer development. In epithelial cells infected with H. pylori, STAT3 was activated, as assessed by immunoblotting for phosphorylated STAT3, immunofluorescence of translocated STAT3, fluorescence recovery after photobleaching, and luciferase activation in transfected cells. Activation was dependent on translocation but not phosphorylation of cytotoxin-associated gene A (CagA) in host cells. Activation seemed to be receptor-mediated because preincubation of cells with the interleukin-6 (IL-6) receptor superantagonist sant7 or inhibition of gp130 by a monoclonal antibody prevented H. pylori-mediated STAT3 activation. However, activation was not related to autocrine activation by IL-6 or IL-11. CagA+ wild-type H. pylori, but not the noncarcinogenic cagA- mutant, activated STAT3 in gastric epithelial cells in vivo in the gerbil model of H. pylori-mediated gastric carcinogenesis. Collectively, these results indicate that H. pylori CagA activates the STAT3 signaling pathway in vitro and in vivo, providing a potential mechanism by which chronic H. pylori infection promotes the development of gastric cancer.

Effect of Helicobacter pylori's vacuolating cytotoxin on the autophagy pathway in gastric epithelial cells.

Host cell responses to Helicobacter pylori infection are complex and incompletely understood. Here, we report that autophagy is induced within human-derived gastric epithelial cells (AGS) in response to H. pylori infection. These autophagosomes were distinct and different from the large vacuoles induced during H. pylori infection. Autophagosomes were detected by transmission electron microscopy, conversion of LC3-I to LC3-II, GFP-LC3 recruitment to autophagosomes, and depended on Atg5 and Atg12. The induction of autophagy depended on the vacuolating cytotoxin (VacA) and, moreover, VacA was sufficient to induce autophagosome formation. The channel-forming activity of VacA was necessary for inducing autophagy. Intracellular VacA partially co-localized with GFP-LC3, indicating that the toxin associates with autophagosomes. The inhibition of autophagy increased the stability of intracellular VacA, which in turn resulted in enhanced toxin-mediated cellular vacuolation. These findings suggest that the induction of autophagy by VacA may represent a host mechanism to limit toxin-induced cellular damage.

SopB promotes phosphatidylinositol 3-phosphate formation on Salmonella vacuoles by recruiting Rab5 and Vps34.

Salmonella colonizes a vacuolar niche in host cells during infection. Maturation of the Salmonella-containing vacuole (SCV) involves the formation of phosphatidylinositol 3-phosphate (PI(3)P) on its outer leaflet. SopB, a bacterial virulence factor with phosphoinositide phosphatase activity, was proposed to generate PI(3)P by dephosphorylating PI(3,4)P2, PI(3,5)P2, and PI(3,4,5)P3. Here, we examine the mechanism of PI(3)P formation during Salmonella infection. SopB is required to form PI(3,4)P2/PI(3,4,5)P3 at invasion ruffles and PI(3)P on nascent SCVs. However, we uncouple these events experimentally and reveal that SopB does not dephosphorylate PI(3,4)P2/PI(3,4,5)P3 to produce PI(3)P. Instead, the phosphatase activity of SopB is required for Rab5 recruitment to the SCV. Vps34, a PI3-kinase that associates with active Rab5, is responsible for PI(3)P formation on SCVs. Therefore, SopB mediates PI(3)P production on the SCV indirectly through recruitment of Rab5 and its effector Vps34. These findings reveal a link between phosphoinositide phosphatase activity and the recruitment of Rab5 to phagosomes.

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.

Alteration of epithelial structure and function associated with PtdIns(4,5)P2 degradation by a bacterial phosphatase.

Elucidation of the role of PtdIns(4,5)P(2) in epithelial function has been hampered by the inability to selectively manipulate the cellular content of this phosphoinositide. Here we report that SigD, a phosphatase derived from Salmonella, can effectively hydrolyze PtdIns(4,5)P(2), generating PtdIns(5)P. When expressed by microinjecting cDNA into epithelial cells forming confluent monolayers, wild-type SigD induced striking morphological and functional changes that were not mimicked by a phosphatase-deficient SigD mutant (C462S). Depletion of PtdIns(4,5)P(2) in intact SigD-injected cells was verified by detachment from the membrane of the pleckstrin homology domain of phospholipase Cdelta, used as a probe for the phosphoinositide by conjugation to green fluorescent protein. Single-cell measurements of cytosolic pH indicated that the Na(+)/H(+) exchange activity of epithelia was markedly inhibited by depletion of PtdIns(4,5)P(2). Similarly, anion permeability, measured using two different halide-sensitive probes, was depressed in cells expressing SigD. Depletion of PtdIns(4,5)P(2) was associated with marked alterations in the actin cytoskeleton and its association with the plasma membrane. The junctional complexes surrounding the injected cells gradually opened and the PtdIns(4,5)P(2)-depleted cells eventually detached from the monolayer, which underwent rapid restitution. Similar observations were made in intestinal and renal epithelial cultures. In addition to its effects on phosphoinositides, SigD has been shown to convert inositol 1,3,4,5,6-pentakisphosphate (IP(5)) into inositol 1,4,5,6-tetrakisphosphate (IP(4)), and the latter has been postulated to mediate the diarrhea caused by Salmonella. However, the effects of SigD on epithelial cells were not mimicked by microinjection of IP(4). In contrast, the cytoskeletal and ion transport effects were replicated by hydrolyzing PtdIns(4,5)P(2) with a membrane-targeted 5-phosphatase or by occluding the inositide using high-avidity tandem PH domain constructs. We therefore suggest that opening of the tight junctions and inhibition of Na(+)/H(+) exchange caused by PtdIns(4,5)P(2) hydrolysis combine to account, at least in part, for the fluid loss observed during Salmonella-induced diarrhea.

Receptor activation alters inner surface potential during phagocytosis.

The surface potential of biological membranes varies according to their lipid composition. We devised genetically encoded probes to assess surface potential in intact cells. These probes revealed marked, localized alterations in the charge of the inner surface of the plasma membrane of macrophages during the course of phagocytosis. Hydrolysis of phosphoinositides and displacement of phosphatidylserine accounted for the change in surface potential at the phagosomal cup. Signaling molecules such as K-Ras, Rac1, and c-Src that are targeted to the membrane by electrostatic interactions were rapidly released from membrane subdomains where the surface charge was altered by lipid remodeling during phagocytosis.

Quantitative and dynamic assessment of the contribution of the ER to phagosome formation.

Phagosomes were traditionally thought to originate from an invagination and scission of the plasma membrane to form a distinct intracellular vacuole. An alternative model implicating the endoplasmic reticulum (ER) as a major component of nascent and maturing phagosomes was recently proposed (Gagnon et al., 2002). To reconcile these seemingly disparate hypotheses, we used a combination of biochemical, fluorescence imaging, and electron microscopy techniques to quantitatively and dynamically assess the contribution of the plasmalemma and of the ER to phagosome formation and maturation. We could not verify even a transient physical continuity between the ER and the plasma membrane, nor were we able to detect a significant contribution of the ER to forming or maturing phagosomes in either macrophages or dendritic cells. Instead, our data indicate that the plasma membrane is the main constituent of nascent and newly formed phagosomes, which are progressively remodeled by fusion with endosomal and eventually lysosomal compartments as phagosomes mature into acidic, degradative organelles.