A novel live-cell imaging assay reveals regulation of endosome maturation.

Cell-cell communication is an essential process in life, with endosomes acting as key organelles for regulating uptake and secretion of signaling molecules. Endocytosed material is accepted by the sorting endosome where it either is sorted for recycling or remains in the endosome as it matures to be degraded in the lysosome. Investigation of the endosome maturation process has been hampered by the small size and rapid movement of endosomes in most cellular systems. Here, we report an easy versatile live-cell imaging assay to monitor endosome maturation kinetics, which can be applied to a variety of mammalian cell types. Acute ionophore treatment led to enlarged early endosomal compartments that matured into late endosomes and fused with lysosomes to form endolysosomes. Rab5-to-Rab7 conversion and PI(3)P formation and turn over were recapitulated with this assay and could be observed with a standard widefield microscope. We used this approach to show that Snx1 and Rab11-positive recycling endosome recruitment occurred throughout endosome maturation and was uncoupled from Rab conversion. In contrast, efficient endosomal acidification was dependent on Rab conversion. The assay provides a powerful tool to further unravel various aspects of endosome maturation.

Regulation of endo-lysosomal pathway and autophagic flux by broad-spectrum antipathogen inhibitor ABMA.

The endo-lysosome system is involved in endocytosis, protein sorting, and degradation as well as autophagy. Numerous toxins and pathogens exploit this system to enter host cells and exert their deleterious effects. Modulation of host endo-lysosome pathway may restrict multiple toxins intoxication as well as pathogen infection. ABMA, selected from a high-throughput screening against the cytotoxicity of ricin toxin, exhibits a broad-spectrum antitoxin and antipathogen activity. Here, we show that ABMA selectively retains endocytosed protein and toxin to late endosomes and thus delaying their intracellular trafficking. It also impairs the autophagic flux by excessive fusion of late endosomes and autophagosomes. Its exclusive action on late endosomes and corresponding consequences on the endo-lysosomal pathway and autophagic flux are distinct from known inhibitors such as bafilomycin A1, EGA, or chloroquine. Hence, besides being a broad-spectrum inhibitor of endocytosed toxins and pathogens, ABMA may serve as a molecular tool to dissect endo-lysosome system-related cellular physiology and mechanisms of pathogenesis.

The Endosomal Network: Mediators and Regulators of Endosome Maturation.

Endocytosis is a means for the cell to sample its environment for nutrients and regulate plasma membrane (PM) composition and area. Whereas the majority of internalized cargo is recycled back to the cell surface, select material is sent to the lysosome for degradation. Endosomes further play major roles in central cell activities as diverse as establishment of cell polarity and signaling, lysosomal storage and immunity. The complexity of endosomal functions is reflected by the extensive changes to endosome properties as they mature. The identity of individual endosomes is influenced by the presence of specific Rab GTPases and phosphoinositides (PIPs), which coordinate membrane traffic and facilitate endosomal functions. Motors and tethers direct the endosomes to the required locations and moderate fusion with other organelles. The maintenance of the elaborate endosomal network is supported by the ER and the trans-Golgi network (TGN), which promote the exchange of membrane components, provide enzymes, and assist with signaling. Additionally, V-ATPase is emerging as an underappreciated coordinator of endosome maturation and cell signaling. The inputs of the various mediators of endosome maturation are tightly regulated and coordinated to ensure appropriate maintenance and functioning of endosomes at each stage of the maturation process. Perturbations in endosome maturation are implicated in devastating diseases, such as neurodegeneration and cancer, and the endosome maturation processes are manipulated and exploited by intracellular pathogens to meet their own needs. A greater understanding of coordination and fine-tuning of endosome maturation will help us address various pathologies more effectively.

Flow Cytometric Quantification of Fatty Acid Uptake by Mycobacterium tuberculosis in Macrophages.

Mycobacterium tuberculosis (Mtb) has evolved to assimilate fatty acids from its host. However, until recently, there was no reliable way to quantify fatty acid uptake by the bacteria during host cell infection. Here we describe a new method to quantify fatty acid uptake by intracellular bacilli. We infect macrophages with Mtb constitutively expressing mCherry and then metabolically label them with Bodipy-palmitate. Following the labeling procedure, we isolate Mtb-containing phagosomes on a sucrose cushion and disrupt the phagosomes with detergent. After extensive washes, the isolated bacteria are analyzed by flow cytometry to determine the level of Bodipy-palmitate signal associated with the bacteria. Using a Mtb mutant strain defective in fatty acid uptake in liquid culture we determined that this mutant assimilated 10-fold less Bodipy-palmitate than the wild type strain during infection in macrophages. This quantitative method of fatty acid uptake can be used to further identify pathways involved in lipid uptake by intracellular Mtb and possibly other bacteria.

Detection and quantification of microbial manipulation of phagosomal function.

Macrophages are phagocytic cells that constitute the primary barrier against microbial infection. However, the frequency of this interaction has likely led to the selection and retention of specific strategies to subvert the antimicrobial behavior of these cells. Many intracellular pathogens manipulate their host macrophages, in order to survive, avoid degradation and immune recognition. Much of the antimicrobial defenses mobilized by the macrophage function through the lysosomal compartment and it is the subversion of these mechanisms that are the target of many pathogenic microbes. To investigate the potential manipulation of lysosomal function by infectious agents, the protocols provided here describe a wide range of techniques developed to study various lysosome functions, ranging from phagosome-lysosome fusion to a panel of hydrolytic activities. The suitability of various protocols for specific contexts is addressed. In addition to providing detailed methods for the elucidation of lysosome function and their applications to microbial infections, approaches are also discussed for developing new protocols that would complement our knowledge of microbial manipulation of lysosome functions. The continued technical evolution of these methods is central to increasing our understanding of host-pathogen interactions and cell biology in general.

Leishmania and the macrophage: a multifaceted interaction.

Leishmania, the causative agent of leishmaniases, is an intracellular parasite of macrophages, transmitted to humans via the bite of its sand fly vector. This protozoan organism has evolved strategies for efficient uptake into macrophages and is able to regulate phagosome maturation in order to make the phagosome more hospitable for parasite growth and to avoid destruction. As a result, macrophage defenses such as oxidative damage, antigen presentation, immune activation and apoptosis are compromised whereas nutrient availability is improved. Many Leishmania survival factors are involved in shaping the phagosome and reprogramming the macrophage to promote infection. This review details the complexity of the host-parasite interactions and summarizes our latest understanding of key events that make Leishmania such a successful intracellular parasite.

A novel method for non-transferrin-bound iron quantification by chelatable fluorescent beads based on flow cytometry.

The reliable measurement of non-transferrin-bound iron (NTBI) in serum has proved to be difficult and generally time consuming. We have sought a simple and fast method for such a determination. We adopted a fluorescence assay and designed a fluorescent dye with a chelating agent attached to sense iron. To avoid autofluorescence from serum samples, the iron probes were linked to beads and the autofluorescence could be separated and excluded from the measurement by flow cytometry due to the size difference between beads and serum proteins. Fluorescent beads containing both fluorescent and chelating moieties have been synthesized. The nature of the chelating function has been systematically investigated using four different chelators: bidentate hydroxypyranone, bidentate hydroxypyridinone, hexadentate hydroxypyranone and hexadentate hydroxypyridinone, each with different iron affinity constants. Competition studies demonstrate that the hexadentate hydroxypyridinone-based beads are capable of scavenging most of low molecular mass and albumin-bound iron but negligible amounts of iron from transferrin and ferritin. Serum samples from 30 patients with different types of disease and normal volunteers were measured. The concentrations of NTBI fall in the range -0.41 to +6.5 µM. The data have been compared with those obtained from the traditional 'NTA' method.

The Sculpting of the Mycobacterium tuberculosis Genome by Host Cell-Derived Pressures.

Mycobacterium tuberculosis is an incredibly successful pathogen with an extraordinary penetrance of its target host population. The ability to infect many yet cause disease in few is undoubtedly central to this success. This ability relies on sensing and responding to the changing environments encountered during the course of disease in the human host. This chapter discusses these environmental cues and stresses, and explores how the genome of Mtb has evolved under the purifying selections that they exert. In analyzing the response of Mtb to a broad range of intracellular pressures it is clear that, despite genome down-sizing, Mtb has retained an extraordinary flexibility in central carbon metabolism. We believe that it is this metabolic plasticity, more than any of the virulence factors, that is the foundation for Mtb's qualities of endurance.

Infection of macrophages with Mycobacterium tuberculosis induces global modifications to phagosomal function.

The phagosome is a central mediator of both the homeostatic and microbicidal functions of a macrophage. Following phagocytosis, Mycobacterium tuberculosis (Mtb) is able to establish infection through arresting phagosome maturation and avoiding the consequences of delivery to the lysosome. The infection of a macrophage by Mtb leads to marked changes in the behaviour of both the macrophage and the surrounding tissue as the bacterium modulates its environment to promote its survival. In this study, we use functional physiological assays to probe the biology of the phagosomal network in Mtb-infected macrophages. The resulting data demonstrate that Mtb modifies phagosomal function in a TLR2/TLR4-dependent manner, and that most of these modifications are consistent with an increase in the activation status of the cell. Specifically, superoxide burst is enhanced and lipolytic activity is decreased upon infection. There are some species- or cell type-specific differences between human and murine macrophages in the rates of acidification and the degree of proteolysis. However, the most significant modification is the marked reduction in intra-phagosomal lipolysis because this correlates with the marked increase in the retention of host lipids in the infected macrophage, which provides a potential source of nutrients that can be accessed by Mtb.

Dynamic quantitative assays of phagosomal function.

Much of the activity of the macrophage as an effector cell is performed within its phagocytic compartment. This ranges from the degradation of tissue debris as part of its homeostatic function to the generation of the superoxide burst as part of its microbicidal response to infection. We have developed a range of real-time readouts of phagosomal function that enable these activities to be rigorously quantified. This unit contains descriptions of several of these assays assessed by different methods of quantitation, including a fluorescence resonance emission transfer (FRET) assay for phagosome/lysosome fusion measured by spectrofluorometry, a fluorogenic assay for the superoxide burst measured by flow cytometry, and a fluorogenic assay for bulk proteolysis measured by confocal microscopy. These assays illustrate both the range of parameters that can be quantified and the flexibility of instrumentation that can be exploited for their quantitation.

Fluorescent 3-hydroxy-4-pyridinone hexadentate iron chelators: intracellular distribution and the relevance to antimycobacterial properties.

We report the synthesis and characterization of a fluorescent iron chelator (4), shown to be effective in inhibiting the growth of Mycobacterium avium in macrophages, together with the synthesis and characterization of two unsuccessful analogues selected to facilitate identification of the molecular properties responsible for the antimicrobial activity. Partition of the chelators in liposomes was investigated and the compounds were assessed with respect to uptake by macrophages, responsiveness to iron overload/iron deprivation and intracellular distribution by flow cytometry and confocal microscopy. The synthesis of the hexadentate chelators is based on a tetrahedral structure to which three bidentate 3-hydroxy-4-pyridinone chelating units are linked via amide bonds. The structure is synthetically versatile, allowing further addition of functional groups such as fluorophores. Here, we analyse the non-functionalized hexadentate unit (3) and the corresponding rhodamine B (4) and fluorescein (5) labelled chelators. The iron(III) stability constant was determined for 3 and the values log beta = 34.4 and pFe(3+) = 29.8 indicate an affinity for iron of the same order of magnitude as that of mycobacteria siderophores. Fluorescence properties in the presence of liposomes show that 4 strongly interacts with the lipid phase, whereas 5 does not. Such different behaviour may explain their distinct intracellular localization as revealed by confocal microscopy. The flow cytometry and confocal microscopy studies indicate that 4 is readily engulfed by macrophages and targeted to cytosol and vesicles of the endolysosomal continuum, whereas 5 is differentially distributed and only partially colocalizes with 4 after prolonged incubation. Differential distribution of the compounds is likely to account for their different efficacy against mycobacteria.

Proteophosophoglycans regurgitated by Leishmania-infected sand flies target the L-arginine metabolism of host macrophages to promote parasite survival.

All natural Leishmania infections start in the skin; however, little is known of the contribution made by the sand fly vector to the earliest events in mammalian infection, especially in inflamed skin that can rapidly kill invading parasites. During transmission sand flies regurgitate a proteophosphoglycan gel synthesized by the parasites inside the fly midgut, termed promastigote secretory gel (PSG). Regurgitated PSG can exacerbate cutaneous leishmaniasis. Here, we show that the amount of Leishmania mexicana PSG regurgitated by Lutzomyia longipalpis sand flies is proportional to the size of its original midgut infection and the number of parasites transmitted. Furthermore, PSG could exacerbate cutaneous L. mexicana infection for a wide range of doses (10-10,000 parasites) and enhance infection by as early as 48 hours in inflamed dermal air pouches. This early exacerbation was attributed to two fundamental properties of PSG: Firstly, PSG powerfully recruited macrophages to the dermal site of infection within 24 hours. Secondly, PSG enhanced alternative activation and arginase activity of host macrophages, thereby increasing L-arginine catabolism and the synthesis of polyamines essential for intracellular parasite growth. The increase in arginase activity promoted the intracellular growth of L. mexicana within classically activated macrophages, and inhibition of macrophage arginase completely ablated the early exacerbatory properties of PSG in vitro and in vivo. Thus, PSG is an essential component of the infectious sand fly bite for the early establishment of Leishmania in skin, which should be considered when designing and screening therapies against leishmaniasis.

Monitoring intracellular labile iron pools: A novel fluorescent iron(III) sensor as a potential non-invasive diagnosis tool.

The physiological and pathophysiological importance of intracellular redox active "labile" iron has created a significant need for improved noninvasive diagnostic tools to reliably monitor iron metabolism in living cells. In this context, fluorescent iron-sensitive chemosensors in combination with digital fluorescence spectroscopic methods have proven to be highly sensitive and indispensable tools to determine cellular iron homeostasis. Recently, application of fluorescent iron sensors has led to the identification of a complex sub-cellular iron compartmentation. Cell organelle-specific iron sensors will significantly contribute to enhance fundamental knowledge of cellular iron trafficking, representing a crucial prerequisite for the future development of therapeutic strategies in iron dysregulatory diseases. Here we present physicochemical characterization and functional investigation of a new 3-hydroxypyridin-4-one based fluorescent iron(III) sensor, exclusively monitoring labile iron pools in the endosomal/lysosomal compartments. In vitro studies of the fluorescein labeled probe were carried out in murine bone marrow derived macrophages. Endosomal/lysosomal accumulation of the probe was revealed by confocal microscopy. Flow cytometry analyses demonstrated high sensitivity of the probe towards exogenous alterations of intracellular iron concentrations as well as in response to the chelation potency of iron chelators, clinically approved for treatment of iron-overload related diseases.

Targeting the lysosome: fluorescent iron(III) chelators to selectively monitor endosomal/lysosomal labile iron pools.

Iron-sensitive fluorescent chemosensors in combination with digital fluorescence spectroscopy have led to the identification of a distinct subcellular compartmentation of intracellular redox-active "labile" iron. To investigate the distribution of labile iron, our research has been focused on the development of fluorescent iron sensors targeting the endosomal/lysosomal system. Following the recent introduction of a series of 3-hydroxypyridin-4-one (HPO) based fluorescent probes we present here two novel HPO sensors capable of accumulating and monitoring iron exclusively in endosomal/lysosomal compartments. Flow cytometric and confocal microscopy studies in murine macrophages revealed endosomal/lysosomal sequestration of the probes and high responsiveness toward alterations of vesicular labile iron concentrations. This allowed assessment of cellular iron status with high sensitivity in response to the clinically applied medications desferrioxamine, deferiprone, and deferasirox. The probes represent a powerful class of sensors for quantitative iron detection and clinical real-time monitoring of subcellular labile iron levels in health and disease.