Добірка наукової літератури з теми "Phagosomal acidification"

Phagocytosis holds a central position in the development of a successful innate immune response and in the initiation of the corresponding adaptive response. The destruction of invading pathogens and the presentation of their antigens to lymphoid cells require acidification of the phagosomal lumen. The present review discusses the mechanism of phagosome acidification, with particular reference to the two components of the protonmotive force: the chemical (pH) gradient and the electrical potential across the phagosomal membrane. A method for the in situ measurement of the electrical potential across the phagosomal membrane is described. In addition, we discuss the finding that acidification is not only a consequence, but also a critical determinant of phagosome maturation. Luminal acidification appears to function as a timing device controlling the transition between early and late phagosomes.

ABSTRACT Following uptake, Francisella tularensis enters a phagosome that acquires limited amounts of lysosome-associated membrane glycoproteins and does not acquire cathepsin D or markers of secondary lysosomes. With additional time after uptake, F. tularensis disrupts its phagosomal membrane and escapes into the cytoplasm. To assess the role of phagosome acidification in phagosome escape, we followed acidification using the vital stain LysoTracker red and acquisition of the proton vacuolar ATPase (vATPase) using immunofluorescence within the first 3 h after uptake of live or killed F. tularensis subsp. holarctica live vaccine strain (LVS) by human macrophages. Whereas 90% of the phagosomes containing killed LVS stained intensely for the vATPase and were acidified, only 20 to 30% of phagosomes containing live LVS stained intensely for the vATPase and were acidified. To determine whether transient acidification might be required for phagosome escape, we assessed the impact on phagosome permeabilization of the proton pump inhibitor bafilomycin A. Using electron microscopy and an adenylate cyclase reporter system, we found that bafilomycin A did not prevent phagosomal permeabilization by F. tularensis LVS or virulent type A strains (F. tularensis subsp. tularensis strain Schu S4 and a recent clinical isolate) or by “F. tularensis subsp. novicida,” indicating that F. tularensis disrupts its phagosomal membrane by a mechanism that does not require acidification.

Methicillin-resistantStaphylococcus aureus(MRSA) causes invasive, drug-resistant skin and soft tissue infections. Reports thatS. aureusbacteria survive inside macrophages suggest that the intramacrophage environment may be a niche for persistent infection; however, mechanisms by which the bacteria might evade macrophage phagosomal defenses are unclear. We examined the fate of theS. aureus-containing phagosome in THP-1 macrophages by evaluating bacterial intracellular survival and phagosomal acidification and maturation and by testing the impact of phagosomal conditions on bacterial viability. Multiple strains ofS. aureussurvived inside macrophages, and in studies using the MRSA USA300 clone, the USA300-containing phagosome acidified rapidly and acquired the late endosome and lysosome protein LAMP1. However, fewer phagosomes containing live USA300 bacteria than those containing dead bacteria associated with the lysosomal hydrolases cathepsin D and β-glucuronidase. Inhibiting lysosomal hydrolase activity had no impact on intracellular survival of USA300 or otherS. aureusstrains, suggesting thatS. aureusperturbs acquisition of lysosomal enzymes. We examined the impact of acidification onS. aureusintramacrophage viability and found that inhibitors of phagosomal acidification significantly impaired USA300 intracellular survival. Inhibition of macrophage phagosomal acidification resulted in a 30-fold reduction in USA300 expression of the staphylococcal virulence regulatoragrbut had little effect on expression ofsarA,saeR, orsigB. Bacterial exposure to acidic pHin vitroincreasedagrexpression. Together, these results suggest thatS. aureussurvives inside macrophages by perturbing normal phagolysosome formation and that USA300 may sense phagosomal conditions and upregulate expression of a key virulence regulator that enables its intracellular survival.

ABSTRACT Francisella tularensis is an intracellular pathogen that can survive and replicate within macrophages. Following phagocytosis and transient interactions with the endocytic pathway, F. tularensis rapidly escapes from its original phagosome into the macrophage cytoplasm, where it eventually replicates. To examine the importance of the nascent phagosome for the Francisella intracellular cycle, we have characterized early trafficking events of the F. tularensis subsp. tularensis strain Schu S4 in a murine bone marrow-derived macrophage model. Here we show that early phagosomes containing Schu S4 transiently interact with early and late endosomes and become acidified before the onset of phagosomal disruption. Inhibition of endosomal acidification with the vacuolar ATPase inhibitor bafilomycin A1 or concanamycin A prior to infection significantly delayed but did not block phagosomal escape and cytosolic replication, indicating that maturation of the early Francisella-containing phagosome (FCP) is important for optimal phagosomal escape and subsequent intracellular growth. Further, Francisella pathogenicity island (FPI) protein expression was induced during early intracellular trafficking events. Although inhibition of endosomal acidification mimicked the early phagosomal escape defects caused by mutation of the FPI-encoded IglCD proteins, it did not inhibit the intracellular induction of FPI proteins, demonstrating that this response is independent of phagosomal pH. Altogether, these results demonstrate that early phagosomal maturation is required for optimal phagosomal escape and that the early FCP provides cues other than intravacuolar pH that determine intracellular induction of FPI proteins.

We analyzed the distribution, fate, and functional role of phosphatidylinositol 4-phosphate (PtdIns4P) during phagosome formation and maturation. To this end, we used genetically encoded probes consisting of the PtdIns4P-binding domain of the bacterial effector SidM. PtdIns4P was found to undergo complex, multiphasic changes during phagocytosis. The phosphoinositide, which is present in the plasmalemma before engagement of the target particle, is transiently enriched in the phagosomal cup. Soon after the phagosome seals, PtdIns4P levels drop precipitously due to the hydrolytic activity of Sac2 and phospholipase C, becoming undetectable for ∼10 min. PtdIns4P disappearance coincides with the emergence of phagosomal PtdIns3P. Conversely, the disappearance of PtdIns3P that signals the transition from early to late phagosomes is accompanied by resurgence of PtdIns4P, which is associated with the recruitment of phosphatidylinositol 4-kinase 2A. The reacquisition of PtdIns4P can be prevented by silencing expression of the kinase and can be counteracted by recruitment of a 4-phosphatase with a heterodimerization system. Using these approaches, we found that the secondary accumulation of PtdIns4P is required for proper phagosomal acidification. Defective acidification may be caused by impaired recruitment of Rab7 effectors, including RILP, which were shown earlier to displace phagosomes toward perinuclear lysosomes. Our results show multimodal dynamics of PtdIns4P during phagocytosis and suggest that the phosphoinositide plays important roles during the maturation of the phagosome.

ABSTRACT Phagosome acidification is an important component of the microbicidal response by infected eukaryotic cells. Thus, intracellular pathogens that reside within phagosomes must either block phagosome acidification or be able to survive at low pH. In this work, we studied the effect of phagosomal acidification on the survival of intracellular Salmonella enterica serovar Typhimurium in different cell types. Bafilomycin A1, a specific inhibitor of the vacuolar proton-ATPases, was used to block acidification of salmonella-containing vacuoles. We found that in several epithelial cell lines, treatment with bafilomycin A1 had no effect on intracellular survival or replication. Furthermore, although acidification was essential for Salmonella intracellular survival in J774 cultured macrophages, as reported previously (13), it is not essential in other macrophage cell lines. These data suggest that vacuolar acidification may play a role in intracellular survival of salmonellae only under certain conditions and in specific cell types.

Defects in the innate immune system in the lung with attendant bacterial infections contribute to lung tissue damage, respiratory insufficiency, and ultimately death in the pathogenesis of cystic fibrosis (CF). Professional phagocytes, including alveolar macrophages (AMs), have specialized pathways that ensure efficient killing of pathogens in phagosomes. Phagosomal acidification facilitates the optimal functioning of degradative enzymes, ultimately contributing to bacterial killing. Generation of low organellar pH is primarily driven by the V-ATPases, proton pumps that use cytoplasmic ATP to load H+ into the organelle. Critical to phagosomal acidification are various channels derived from the plasma membrane, including the anion channel cystic fibrosis transmembrane conductance regulator, which shunt the transmembrane potential generated by movement of protons. Here we show that the transient receptor potential canonical-6 (TRPC6) calcium-permeable channel in the AM also functions to shunt the transmembrane potential generated by proton pumping and is capable of restoring microbicidal function to compromised AMs in CF and enhancement of function in non-CF cells. TRPC6 channel activity is enhanced via translocation to the cell surface (and then ultimately to the phagosome during phagocytosis) in response to G-protein signaling activated by the small molecule (R)-roscovitine and its derivatives. These data show that enhancing vesicular insertion of the TRPC6 channel to the plasma membrane may represent a general mechanism for restoring phagosome activity in conditions, where it is lost or impaired.

The mechanisms underlying the survival of intracellular parasites such as mycobacteria in host macrophages remain poorly understood. In mice, mutations at the Nramp1 gene (for natural resistance-associated macrophage protein), cause susceptibility to mycobacterial infections. Nramp1 encodes an integral membrane protein that is recruited to the phagosome membrane in infected macrophages. In this study, we used microfluorescence ratio imaging of macrophages from wild-type and Nramp1 mutant mice to analyze the effect of loss of Nramp1 function on the properties of phagosomes containing inert particles or live mycobacteria. The pH of phagosomes containing live Mycobacterium bovis was significantly more acidic in Nramp1- expressing macrophages than in mutant cells (pH 5.5 ± 0.06 versus pH 6.6 ± 0.05, respectively; P <0.005). The enhanced acidification could not be accounted for by differences in proton consumption during dismutation of superoxide, phagosomal buffering power, counterion conductance, or in the rate of proton “leak”, as these were found to be comparable in wild-type and Nramp1-deficient macrophages. Rather, after ingestion of live mycobacteria, Nramp1-expressing cells exhibited increased concanamycin-sensitive H+ pumping across the phagosomal membrane. This was associated with an enhanced ability of phagosomes to fuse with vacuolar-type ATPase–containing late endosomes and/or lysosomes. This effect was restricted to live M. bovis and was not seen in phagosomes containing dead M. bovis or latex beads. These data support the notion that Nramp1 affects intracellular mycobacterial replication by modulating phagosomal pH, suggesting that Nramp1 plays a central role in this process.

We identify here a novel class of loss-of-function alleles of uncoordinated locomotion(unc)-108, which encodes the Caenorhabditis elegans homologue of the mammalian small guanosine triphosphatase Rab2. Like the previously isolated dominant-negative mutants, unc-108 loss-of-function mutant animals are defective in locomotion. In addition, they display unique defects in the removal of apoptotic cells, revealing a previously uncharacterized function for Rab2. unc-108 acts in neurons and engulfing cells to control locomotion and cell corpse removal, respectively, indicating that unc-108 has distinct functions in different cell types. Using time-lapse microscopy, we find that unc-108 promotes the degradation of engulfed cell corpses. It is required for the efficient recruitment and fusion of lysosomes to phagosomes and the acidification of the phagosomal lumen. In engulfing cells, UNC-108 is enriched on the surface of phagosomes. We propose that UNC-108 acts on phagosomal surfaces to promote phagosome maturation and suggest that mammalian Rab2 may have a similar function in the degradation of apoptotic cells.

Mycobacterium tuberculosis (Mtb) is an obligate intra-cellular pathogen that causes the disease tuberculosis (TB) in its human hosts. An estimated 1% of the world population is reported to get infected with the disease every year. The capacity of Mtb to tolerate multiple antibiotics, particularly within its host, represents a major problem in TB management. Moreover, patients co-infected with Mtb and another global pathogen human immunodeficiency virus (HIV) showed high rates of anti-TB therapy failure as compared to patients infected with only Mtb. The protracted therapy time for a cocktail of antibiotics often leads to non-adherence among patients resulting in ineffectiveness of the regimen. Also, prolonged exposure to antibiotics paves the way for the acquisition of mutations that generate genetically drug-resistant Mtb strains. Heterogeneity within Mtb populations has long been associated with refractoriness to antibiotic therapy during growth in vitro and inside host cells/tissues [Chapter 1]. It has been reported that the micro-environment faced by Mtb inside host phagocytes promotes tolerance towards clinically-relevant anti-TB drugs, possibly contributing to reduced clearance of Mtb from patient lesions. During chronic phase of infection, exposure to host immune pressures induces metabolic quiescence, which contributes to a drug-tolerant phenotype. Additionally, tolerance to antibiotics has recently also been attributed to replicating Mtb in unstimulated macrophages. Expansion of this drug-tolerant Mtb population within lesions could lead to dissemination of tolerant bacteria to new sites, ultimately reducing the efficacy of antibiotics in eradicating of Mtb. Association of host immune pressures in mobilizing drug tolerance indicates an active crosstalk between host and pathogen. In this regard, it is crucial that we identify host-specific cues and Mtb’s adaptation program in response to environmental signals for mechanistic dissection of phenotypic drug tolerance in replicating Mtb during infection. The major aim of this study was to characterize the cross-talk between host immune pressures and bacterial genetic determinants that sense these pressures to mediate realignment of bacterial metabolism for survival. Upon uptake by naïve macrophages, Mtb is exposed to host stresses such as limited acidification of the phagosome (~ pH 6.2) and superoxide (O2.) stress by recruitment of vacuolar ATPases (V-ATPases) and phagocyte oxidases (NOX2) on the phagosomal membrane, respectively. Since these stresses are known to induce redox imbalance, Mtb induces several protective mechanisms to maintain redox homeostasis for survival. A major technological advance in the redox field emerged by the development of a fluorescent biosensor (Mrx1-roGFP2) that facilitates real-time quantification of the redox potential of the major cytosolic antioxidant in Mtb (mycothiol/MSH; EMSH) during infection. Using this tool, it was reported that Mtb population localized within macrophages exhibits heterogeneity in EMSH as compared to uniform EMSH displayed by broth-grown bacteria. Further, redox-diverse bacterial sub-populations demonstrated variable susceptibility towards anti-TB drugs, suggesting a link between redox physiology and drug tolerance in Mtb. On this basis, we sought to comprehensively characterize redox-diverse fractions of intra-phagosomal Mtb to understand the underlying mechanism of phenotypic drug tolerance. Our RNAsequencing (RNA-seq) of redox-altered Mtb provided distinct transcriptional signatures, which aided in identification of bacterial determinants of antibiotic tolerance [Chapter 2]. Phagosomal acidification is possibly one of the earliest host stresses that Mtb faces upon internalization by macrophages. Previous reports suggest that maintenance of intrabacterial pH homeostasis, upon exposure to an acidified environment, could affect Mtb’s redox physiology. In this regard, we sought to dissect the link between phagosomal acidification, heterogeneity in EMSH and multi-class drug tolerance in Mtb during infection [Chapter 3]. We identified that redox-diverse Mtb sub-populations within macrophages faced different degrees of phagosomal acidification. Blocking phagosomal acidification using lysosomotropic agents subverted heterogeneity in EMSH and reversed drug tolerance to anti-TB drugs isoniazid (Inh) and rifampicin (Rif). We also show that the pH and redox-dependent drug tolerance of Mtb is significantly higher when the pathogen infects macrophages with actively replicating HIV-1, suggesting that it could contribute to high rates of TB therapy failure during HIV-TB coinfection. Our data emphasizes upon the crucial role played by host acidification in generating redox-based heterogeneity and drug tolerance in intra-macrophage Mtb. The role of the lysosomotropic agent chloroquine (CQ), in blocking acidification of sub-cellular compartments, is well-established in anti-malarial therapy as well as in autophagyrelated studies. Based on our results in Mtb-infected macrophages, where CQ consistently decreased tolerance to Inh and Rif, we attempted to assess the effectiveness of CQ in reversing Mtb drug tolerance in vivo [Chapter 4]. In chronically-infected BALB/c mice, coadministration of CQ with Inh or Rif dramatically reduced the fraction of drug-tolerant Mtb, ameliorated lung pathology, and reduced post-chemotherapeutic relapse. Similar decrease inbacterial lung burden and infection-mediated tissue damage was observed in Mtb-infected guinea pigs treated with a combination of CQ and Inh. The pharmacokinetic profile of CQ exhibited no significant drug-drug interaction with first line anti-TB drugs, making it a robust candidate to be considered for host-directed therapy in TB management. In summation, our data delineate a functional link between phagosomal pH, redox metabolism and multi-drug tolerance in replicating Mtb [Chapter 5]. We have attempted to investigate the mechanistic underpinnings of how intra-phagosomal Mtb senses acidification as a cue to generate phenotypic variants of redox state which consequently exhibit differential sensitivity to anti-TB drugs. We also propose the repositioning of CQ as host-directed therapy based on its role in blocking phagosomal acidification and redox heterogeneity to potentiate antibiotic efficacy. Our findings, along with the high oral bioavailability and long half-life in patient sera, makes CQ a promising candidate to be added to current treatment regimens for shortening TB therapy and achieving a relapse-free cure.

Specialized cells of the innate immune system, such as macrophages, employ lysosomal enzymes, together with cationic peptides and reactive oxygen intermediates, to eliminate invading microorganisms ensnared within phagosomes. The effectiveness of this impressive armamentarium is potentiated by the acid pH generated by the vacuolar-type ATPase (V-ATPase). The determinants of the luminal pH of phagosomes and of the lysosomes they fuse with are not completely understood, but the V-ATPase is known to be electrogenic and net accumulation of protons requires charge compensation. For this reason, counter-ion pathways are thought to serve a central role in the control of acidification. It has generally been assumed that a parallel anion influx accompanies proton pumping to dissipate the voltage that tends to build up. In fact, impaired chloride channel activity in cystic fibrosis has been proposed to underlie the defective phagolysosome acidification and microbial killing reported in lung macrophages. In the first part of this thesis, I devised methods to dialyze the lumen of lysosomes in intact cells, while monitoring lysosomal pH, in order to assess the individual contribution of counter-ions to acidification. Surprisingly, anions were found to be completely dispensable for proton pumping, whereas the presence of permeant cations in the lysosomal lumen was essential. Accordingly, defects in lysosomal anion permeability cannot explain the impaired microbicidal capacity of phagocytes in cystic fibrosis. Even though counter-ion permeation pathways exist, dissipation of the electrical contribution of the V-ATPase may not be complete. If present, a transmembrane potential would alter the rate and extent of proton accumulation in phagosomes and lysosomes. However, no estimates of the voltage across the phagosomes were available. To overcome this deficiency, in the second part of this thesis, I describe a noninvasive procedure to estimate the voltage across the phagosome using fluorescence resonance energy transfer. This novel approach, in combination with organellar pH measurements, demonstrated that proton pumping is not limited by counter-ion permeability.

Phagocytosis is an innate immune response that is paramount in the clearance of pathogenic particles. Recognition of target particles by phagocytic receptors expressed on phagocytes induces modifications in the underlying actin cytoskeleton to form pseudopods that encircle and internalize the target particle into a membrane bound organelle called the phagosome. The nascent phagosome undergoes a maturation sequence that is characterized by substantial remodeling of the membrane and its luminal contents through interactions with components of the endocytic pathway, culminating in an acidic and hydrolytic organelle capable of digesting and elminating pathogens. Phagosome maturation is a complicated pathway that involves many protein and lipid signaling molecules. Several factors that influence phagosome maturation particularly the participation of pH, lysosome-associated membrane proteins-1 and –2, cholesterol, in addition to the survival and escape mechanisms used by, Burkholderica cenocepacia were explored. All three tenets are essential for phagosome maturation, although each factor has different mechanistic consequences. Acidification alters Rab5 activation, while ablation of LAMPs and accumulation of cholesterol interferes with various aspects of Rab 7 turnover in phagosomes and/or endosome membranes. Moreover, Burkholderia cenocepacia, an intracellular pathogen, inactivates Rab7 on phagosome membranes from within the vacuole lumen. Herein, mechanisms that govern phagosome maturation are explored and several molecules are added to the long list of essential players in this complicated pathway.

Mycobacterium tuberculosis is the causal agent of human tuberculosis. The initial events of the establishment of the infection include the phagocytosis by several innate immune response cells. This chapter will discuss the immune cells involved, the phagocytic pattern recognition receptors (PPRs) that recognize and mediate bacteria phagocytosis (such as C-type lectin receptors, Toll-like receptors, complement receptors, and scavenger receptors), and the outcome of this initial interaction. Additionally, the bacterial strategies to evade the immune response—which includes the inhibition of the phagosome maturation and arresting of phagosome acidification, the mechanisms to survive to the reactive nitrogen species and reactive oxygen species, and finally, the resistance to the apoptosis and autophagy—will be reviewed. Finally, the host-pathogen interaction of M. tuberculosis with the phagocytic human cells during the primary events of the tuberculosis infection will also be reviewed.