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.

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.

Legionella pneumophila Infection of Human Macrophages Retains Golgi Structure but Reduces O-Glycans.

Legionella pneumophila is an accidental pathogen that replicates intracellularly within the Legionella-containing vacuole (LCV) in macrophages. Within an hour of infection, L. pneumophila secretes effectors to manipulate Rab1 and intercept ER-derived vesicles to the LCV. The downstream consequences of interrupted ER trafficking on the Golgi of macrophages are not clear. We examined the Golgi structure and function in L. pneumophila-infected human U937 macrophages. Intriguingly, the size of the Golgi in infected macrophages remained similar to uninfected macrophages. Furthermore, TEM analysis also did not reveal any significant changes in the ultrastructure of the Golgi in L. pneumophila-infected cells. Drug-induced Golgi disruption impacted bacterial replication in human macrophages, suggesting that an intact organelle is important for bacteria growth. To probe for Golgi functionality after L. pneumophila infection, we assayed glycosylation levels using fluorescent lectins. Golgi O-glycosylation levels, visualized by the fluorescent cis-Golgi lectin, Helix pomatia agglutinin (HPA), significantly decreased over time as infection progressed, compared to control cells. N-glycosylation levels in the Golgi, as measured by L-PHA lectin staining, were not impacted by L. pneumophila infection. To understand the mechanism of reduced O-glycans in the Golgi we monitored UDP-GalNAc transporter levels in infected macrophages. The solute carrier family 35 membrane A2 (SLC35A2) protein levels were significantly reduced in L. pneumophila-infected U937 and HeLa cells and L. pneumophila growth in human macrophages benefitted from GalNAc supplementation. The pronounced reduction in Golgi HPA levels was dependent on the translocation apparatus DotA expression in bacteria and occurred in a ubiquitin-independent manner. Thus, L. pneumophila infection of human macrophages maintains and requires an intact host Golgi ultrastructure despite known interference of ER-Golgi trafficking. Finally, L. pneumophila infection blocks the formation of O-linked glycans and reduces SLC35A2 protein levels in infected human macrophages.

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.

Rab1b-GBF1-ARF1 Secretory Pathway Axis Is Required for Birnavirus Replication.

Birnaviruses are members of the Birnaviridae family, responsible for major economic losses to poultry and aquaculture. The family is composed of nonenveloped viruses with a segmented double-stranded RNA (dsRNA) genome. Infectious bursal disease virus (IBDV), the prototypic family member, is the etiological agent of Gumboro disease, a highly contagious immunosuppressive disease in the poultry industry worldwide. We previously demonstrated that IBDV hijacks the endocytic pathway for establishing the viral replication complexes on endosomes associated with the Golgi complex (GC). Here, we report that IBDV reorganizes the GC to localize the endosome-associated replication complexes without affecting its secretory functionality. By analyzing crucial proteins involved in the secretory pathway, we showed the essential requirement of Rab1b for viral replication. Rab1b comprises a key regulator of GC transport and we demonstrate that transfecting the negative mutant Rab1b N121I or knocking down Rab1b expression by RNA interference significantly reduces the yield of infectious viral progeny. Furthermore, we showed that the Rab1b downstream effector Golgi-specific BFA resistance factor 1 (GBF1), which activates the small GTPase ADP ribosylation factor 1 (ARF1), is required for IBDV replication, since inhibiting its activity by treatment with brefeldin A (BFA) or golgicide A (GCA) significantly reduces the yield of infectious viral progeny. Finally, we show that ARF1 dominant negative mutant T31N overexpression hampered IBDV infection. Taken together, these results demonstrate that IBDV requires the function of the Rab1b-GBF1-ARF1 axis to promote its replication, making a substantial contribution to the field of birnavirus-host cell interactions. IMPORTANCE Birnaviruses are unconventional members of the dsRNA viruses, with the lack of a transcriptionally active core being the main differential feature. This structural trait, among others that resemble those of the plus single-stranded (+ssRNA) viruses features, suggests that birnaviruses might follow a different replication program from that conducted by prototypical dsRNA members and the hypothesis that birnaviruses could be evolutionary links between +ssRNA and dsRNA viruses has been argued. Here, we present original data showing that IBDV-induced GC reorganization and the cross talk between IBDV and the Rab1b-GBF1-ARF1 mediate the intracellular trafficking pathway. The replication of several +ssRNA viruses depends on the cellular protein GBF1, but its role in the replication process is not clear. Thus, our findings make a substantial contribution to the field of birnavirus-host cell interactions and provide further evidence supporting the proposed evolutionary connection role of birnaviruses, an aspect which we consider especially relevant for researchers working in the virology field.

Aggregation and Size Attributes Analysis of Unadsorbed and Adjuvant-adsorbed Antigens using a Multispectral Imaging Flow Cytometer Platform.

Various vaccine quality attributes should be monitored to ensure consistency, potency, purity, and safety of vaccine products prior to lot release. Vaccine particle size and protein antigen aggregation are two important considerations for particle-adsorbed vaccines. In this study, we evaluated the use of imaging flow cytometry as a potential all-in-one platform to measure adjuvant particle size and to detect protein aggregates through a combination of brightfield microscopy, side scatter detection, and fluorescence microscopy. An aluminum phosphate adjuvant was analyzed for size using the brightfield function, and the size measurement was compared against laser diffraction. Heat-induced protein aggregates of either unadsorbed antigens or aluminum phosphate adjuvant-adsorbed antigens were stained with the fluorescent ProteoStat aggregation dye, followed by detection and analysis using a combination of the brightfield and fluorescence microscopy functions. The change in aggregation of unadsorbed antigens was confirmed using dynamic light scattering. These results demonstrate the versatility of the imaging flow cytometry platform for the evaluation of multiple vaccine quality characteristics.

Phagosome resolution regenerates lysosomes and maintains the degradative capacity in phagocytes.

Phagocytes engulf unwanted particles into phagosomes that then fuse with lysosomes to degrade the enclosed particles. Ultimately, phagosomes must be recycled to help recover membrane resources that were consumed during phagocytosis and phagosome maturation, a process referred to as "phagosome resolution." Little is known about phagosome resolution, which may proceed through exocytosis or membrane fission. Here, we show that bacteria-containing phagolysosomes in macrophages undergo fragmentation through vesicle budding, tubulation, and constriction. Phagosome fragmentation requires cargo degradation, the actin and microtubule cytoskeletons, and clathrin. We provide evidence that lysosome reformation occurs during phagosome resolution since the majority of phagosome-derived vesicles displayed lysosomal properties. Importantly, we show that clathrin-dependent phagosome resolution is important to maintain the degradative capacity of macrophages challenged with two waves of phagocytosis. Overall, our work suggests that phagosome resolution contributes to lysosome recovery and to maintaining the degradative power of macrophages to handle multiple waves of phagocytosis.

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.

A Novel Single Domain Antibody Targeting FliC Flagellin of Salmonella enterica for Effective Inhibition of Host Cell Invasion.

The enteric pathogen, Salmonella enterica is a major cause of human gastroenteritis globally and with increasing bacterial resistance to antibiotics, alternative solutions are urgently needed. Single domain antibodies (sdAbs), the smallest antibody fragments that retain antigen binding specificity and affinity, are derived from variable heavy-chain only fragments (VHH) of camelid heavy-chain-only immunoglobulins. SdAbs typically contain a single disulfide bond simplifying recombinant protein production in microbial systems. These factors make sdAbs ideally suited for the development of effective anti-bacterial therapeutics. To this end, we generated an anti-Salmonella VHH library from which we screened for high affinity sdAbs. We present a novel sdAb (Abi-Se07) that targets the Salmonella virulence factor, FliC, required for bacterial motility and invasion of host cells. We demonstrate that Abi-Se07 bound FliC with a K D of 16.2 ± 0.1 nM. In addition, Abi-Se07 exhibited cross-serovar binding to whole cells of S. enterica serovar Typhimurium, Heidelberg, and Hadar. Abi-Se07 significantly inhibited bacterial motility and significantly reduced S. enterica colonization in a more native environment of chicken jejunum epithelium. Taken together, we have identified a novel anti-Salmonella sdAb and discuss future efforts toward therapeutic development.

Polymorphisms of a Collagen-Like Adhesin Contributes to Legionella pneumophila Adhesion, Biofilm Formation Capacity and Clinical Prevalence.

Legionellosis is a severe respiratory illness caused by the inhalation of aerosolized water droplets contaminated with the opportunistic pathogen Legionella pneumophila. The ability of L. pneumophila to produce biofilms has been associated with its capacity to colonize and persist in human-made water reservoirs and distribution systems, which are the source of legionellosis outbreaks. Nevertheless, the factors that mediate L. pneumophila biofilm formation are largely unknown. In previous studies we reported that the adhesin Legionella collagen-like protein (Lcl), is required for auto-aggregation, attachment to multiple surfaces and the formation of biofilms. Lcl structure contains three distinguishable regions: An N-terminal region with a predicted signal sequence, a central region containing tandem collagen-like repeats (R-domain) and a C-terminal region (C-domain) with no significant homology to other known proteins. Lcl R-domain encodes tandem repeats of the collagenous tripeptide Gly-Xaa-Yaa (GXY), a motif that is key for the molecular organization of mammalian collagen and mediates the binding of collagenous proteins to different cellular and environmental ligands. Interestingly, Lcl is polymorphic in the number of GXY tandem repeats. In this study, we combined diverse biochemical, genetic, and cellular approaches to determine the role of Lcl domains and GXY repeats polymorphisms on the structural and functional properties of Lcl, as well as on bacterial attachment, aggregation and biofilm formation. Our results indicate that the R-domain is key for assembling Lcl collagenous triple-helices and has a more preponderate role over the C-domain in Lcl adhesin binding properties. We show that Lcl molecules oligomerize to form large supramolecular complexes to which both, R and C-domains are required. Furthermore, we found that the number of GXY tandem repeats encoded in Lcl R-domain correlates positively with the binding capabilities of Lcl and with the attachment and biofilm production capacity of L. pneumophila strains. Accordingly, the number of GXY tandem repeats in Lcl influences the clinical prevalence of L. pneumophila strains. Therefore, the number of Lcl tandem repeats could be considered as a potential predictor for virulence in L. pneumophila isolates.

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.

Phagocytosis: what's on the menu? 1.

Phagocytosis is an evolutionarily conserved process. In Protozoa, phagocytosis fulfills a feeding mechanism, while in Metazoa, phagocytosis diversified to play multiple organismal roles, including immune defence, tissue homeostasis, and remodeling. Accordingly, phagocytes display a high level of plasticity in their capacity to recognize, engulf, and process targets that differ in composition and morphology. Here, we review how phagocytosis adapts to its multiple roles and discuss in particular the effect of target morphology in phagocytic uptake and phagosome maturation.

Metabolic control of cytosolic-facing pools of diacylglycerol in budding yeast.

Diacylglycerol (DAG) is a key signaling lipid and intermediate in lipid metabolism. Our knowledge of DAG distribution and dynamics in cell membranes is limited. Using live-cell fluorescence microscopy we investigated the localization of yeast cytosolic-facing pools of DAG in response to conditions where lipid homeostasis and DAG levels were known to be altered. Two main pools were monitored over time using DAG sensors. One pool was associated with vacuolar membranes and the other localized to sites of polarized growth. Dynamic changes in DAG distribution were observed during resumption of growth from stationary phase, when DAG is used to support phospholipid synthesis for membrane proliferation. Vacuolar membranes experienced constant morphological changes displaying DAG enriched microdomains coexisting with liquid-disordered areas demarcated by Vph1. Formation of these domains was dependent on triacylglycerol (TAG) lipolysis. DAG domains and puncta were closely connected to lipid droplets. Lack of conversion of DAG to phosphatidate in growth conditions dependent on TAG mobilization, led to the accumulation of DAG in a vacuolar-associated compartment, impacting the polarized distribution of DAG at budding sites. DAG polarization was also regulated by phosphatidylserine synthesis/traffic and sphingolipid synthesis in the Golgi.

Biosensors for the Detection of Interaction between Legionella pneumophila Collagen-Like Protein and Glycosaminoglycans.

The adhesin Legionella collagen-like (Lcl) protein can bind to extracellular matrix components and mediate the binding of Legionella pneumophila to host cells. In this study, electrochemical impedance spectroscopy (EIS) and surface plasmon resonance (SPR)-based biosensors were employed to characterize these interactions between glycosaminoglycans (GAGs) and the adhesin Lcl protein. Fucoidan displayed a high affinity (KD 18 nM) for Lcl protein. Chondroitin sulfate A and dermatan sulfate differ in the position of a carboxyl group replacing D-glucuronate with D-iduronate. Our results indicated that the presence of D-iduronate in dermatan sulfate strongly hindered its interaction with Lcl. These biophysical studies provided valuable information in our understanding of adhesin-ligand interactions related to Legionella pneumophila infections.

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.

Legionella pneumophila: homeward bound away from the phagosome.

The intracellular pathogen Legionella pneumophila (Lp) survives and replicates inside a specialized vacuolar compartment that evades canonical phagosomal maturation. Through the action of a large number of effectors translocated into the host cytosol via the Dot/Icm type IV secretion system, Lp subverts host cell pathways to convert its nascent phagosome into an ER-derived compartment, the Legionella containing vacuole (LCV), which serves as bacterial replication niche.

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.