Phagosomal Lumen Research Articles

Phagosome Neutrality in Host Defense

Activation of proteases in the phagosomes of neutrophils occurs by neutralization of the phagosomal pH by NADPH oxidase. In this issue, Savina et al. (2006) show that dendritic cell phagosomes also recruit NADPH oxidase but with different results. Here, the neutralization of phagosomal pH reduces protease activity, which preserves antigens for crosspresentation on class I MHC molecules.

NOX2 Controls Phagosomal pH to Regulate Antigen Processing during Crosspresentation by Dendritic Cells

To initiate adaptative cytotoxic immune responses, proteolytic peptides derived from phagocytosed antigens are presented by dendritic cells (DCs) to CD8+ T lymphocytes through a process called antigen "crosspresentation." The partial degradation of antigens mediated by lysosomal proteases in an acidic environment must be tightly controlled to prevent destruction of potential peptides for T cell recognition. We now describe a specialization of the phagocytic pathway of DCs that allows a fine control of antigen processing. The NADPH oxidase NOX2 is recruited to the DC's early phagosomes and mediates the sustained production of low levels of reactive oxygen species, causing active and maintained alkalinization of the phagosomal lumen. DCs lacking NOX2 show enhanced phagosomal acidification and increased antigen degradation, resulting in impaired crosspresentation. Therefore, NOX2 plays a critical role in conferring DCs the ability to function as specialized phagocytes adapted to process antigens rather than kill pathogens.

Invasion and survival strategies of Anaplasma phagocytophilum.

Anaplasma phagocytophilum is an aetiological agent of human granulocytic ehrlichiosis, an emerging tick-borne zoonosis in the United States and Europe. This obligate intracellular bacterium is unique in that it colonizes polymorphonuclear leucocytes (neutrophils). Neutrophils are key players in innate immunity. These short-lived phagocytes ingest invading microorganisms and destroy them by various means, which include fusing the bacteria-containing phagosome with acidic lysosomes as well as directing toxic oxidative and proteolytic compounds into the phagosomal lumen. Its tropism for neutrophils indicates that A. phagocytophilum uses strategies for evading and/or neutralizing these microbicidal activities. This review focuses on some of the mechanisms that A. phagocytophilum uses for neutrophil adhesion, surviving within the hostile intracellular environment of its host neutrophil and for effectively disseminating to naïve host cells.

Natural resistance to intracellular infections: natural resistance-associated macrophage protein 1 (Nramp1) functions as a pH-dependent manganese transporter at the phagosomal membrane.

Mutations at the natural resistance-associated macrophage protein 1 (Nramp1) locus cause susceptibility to infection with antigenically unrelated intracellular pathogens. Nramp1 codes for an integral membrane protein expressed in the lysosomal compartment of macrophages, and is recruited to the membrane of phagosomes soon after the completion of phagocytosis. To define whether Nramp1 functions as a transporter at the phagosomal membrane, a divalent cation-sensitive fluorescent probe was designed and covalently attached to a porous particle. The resulting conjugate, zymosan-FF6, was ingested by macrophages and its fluorescence emission was recorded in situ after phagocytosis, using digital imaging. Quenching of the probe by Mn(2+) was used to monitor the flux of divalent cations across the phagosomal membrane in peritoneal macrophages obtained from Nramp1-expressing (+/+) and Nramp1-deficient (-/-) macrophages. Phagosomes from Nramp1(+/+) mice extrude Mn(2+) faster than their Nramp(-/-) counterparts. The difference in the rate of transport is eliminated when acidification of the phagosomal lumen is dissipated, suggesting that divalent metal transport through Nramp1 is H(+) dependent. These studies suggest that Nramp1 contributes to defense against infection by extrusion of divalent cations from the phagosomal space. Such cations are likely essential for microbial function and their removal from the phagosomal microenvironment impairs pathogenesis, resulting in enhanced bacteriostasis or bactericidal activity.

Characterization of a two-component system, devR-devS, of Mycobacterium tuberculosis

By subtractive hybridization, we isolated genes, differentially expressed in virulent strain (dev), that are expressed at higher levels in the virulent Mycobacterium tuberculosis H37Rv strain in comparison to its avirulent counterpart, H37Ra, and consequently may be associated with the virulence phenotype of M. tuberculosis. A two-component system, devR-devS, was identified by DNA sequencing of a dev clone. DevR, the predicted gene product of devR, is a response regulator (RR) in the NarL/ UhpA subfamily of two-component systems. The devS gene product displayed homology with histidine protein kinases (HPKs) including UhpB, NarX and NarQ. The devR-devS locus is preceded by gene Rv3134c that encodes a putative alanine–aline- rich protein. This locus was conserved in M. tuberculosis and M. bovis BCG but not in other mycobacteria. A devR -lacZ transcription fusion demonstrated β-galactosidase activity in M. smegmatis and in M. tuberculosis. The devR and devS genes were cotranscribed and the levels of their transcripts were lower in two isolates of the avirulent H37Ra strain in comparison to the virulent H37Rv strain of M. tuberculosis. The level of DevR protein was also lower in one of the H37Ra strains in comparison to the H37Rv strain. However, in a third isolate of H37Ra, RNA and protein expression was equivalent to that in the H37Rv strain. Electron microscopic immunogold analysis of M. tuberculosis grown in laboratory medium and within human monocytes revealed specific labelling for DevR protein within the bacteria and the phagosomal lumen of infected monocytes. These findings collectively suggest a potential role for devR-devS in the regulation of genetic programmes unique to the tubercle bacillus.

Regulation of Phagosomal Acidification: DIFFERENTIAL TARGETING OF Na+/H+EXCHANGERS, Na+/K+-ATPases, AND VACUOLAR-TYPE H+-ATPases

Vacuolar-type (V) ATPases are thought to be the main determinant of phagosomal acidification. In phagosomes containing mycobacteria, which ostensibly impair the delivery of V-ATPases to the phagosomal membrane, the pH would be expected to be near neutral. This prediction was tested by microfluorescence ratio imaging using macrophages from mice susceptible to mycobacterial infection. Although less acidic than their counterparts containing dead bacteria, phagosomes containing live Mycobacteria bovis were nearly 1 pH unit more acidic than the cytosol, suggesting the existence of alternate H+ transport mechanisms. We therefore investigated whether Na+/H+ exchange (NHE) contributes to phagosomal acidification. Immunoblotting, reverse transcriptase-polymerase chain reaction, and pharmacological studies indicated that NHE1 is the predominant isoform of the exchanger in macrophages. Fractionation revealed that NHE1 is incorporated into the phagosomal membrane, and measurements of pH indicated that it is functional in this location. Nevertheless, acidification of the lumen of phagosomes containing either latex beads or live M. bovis was insensitive to (3-methylsulfonyl-4-piperidinobenzoyl)-guanidine methanesulfonate, a potent inhibitor of NHE1. This may have been due to the absence of an appropriate lumen to cytosol Na+ gradient, because the phagosomal membrane was found to be devoid of Na+/K+ pumps. Unexpectedly, the acidification of M. bovis phagosomes was fully reversed by specific inhibitors of the vacuolar H+-ATPase, suggesting that ATPases are present only transiently or in reduced quantities in the phagosomal membrane. Alternatively, acid equivalents accumulated in endosomes by V-ATPases may be delivered to the mycobacterial phagosome by carrier vesicles devoid of ATPases.

Subcellular distribution of docking/fusion proteins in neutrophils, secretory cells with multiple exocytic compartments.

Neutrophils contain at least four distinct types of secretory organelles, which undergo exocytosis during infection and inflammation. The signaling pathways leading to secretion of individual granules and their kinetics of exocytosis vary greatly, causing temporal and regional differences in docking and fusion with the plasma membrane. As a step toward understanding the processes underlying differential granular secretion in neutrophils, we assessed the presence and distribution of a number of proteins reported to be involved in vesicular docking and/or fusion in other systems. Specific Abs were used for immunoblotting of cells fractionated by density gradients and free-flow electrophoresis, and for localization by confocal immunofluorescence and electron microscopy. Syntaxin 1, VAMP (vesicle-associated membrane protein)-1, synaptosome-associated protein-25 (SNAP-25), synaptophysin, and cellubrevin were not detectable in human neutrophils. In contrast, syntaxin 4, VAMP-2, and the 39-kDa isoform of secretory carrier membrane protein (SCAMP) were present. SCAMP was found mainly in secondary and tertiary granules and in a fraction containing secretory vesicles, but was virtually absent from the primary (lysosomal) granules. This profile is consistent with the proposed "post-Golgi" distribution of SCAMP. VAMP-2 was largely absent from primary and secondary granules, but concentrated in tertiary granules and secretory vesicles. This pattern of distribution parallels the increasing sensitivity of these exocytic compartments to intracellular free calcium. Accordingly, ionomycin induced translocation of VAMP-2 toward the plasma membrane. Syntaxin 4 was found almost exclusively in the plasma membrane, and it accumulated in lamellipodia of migrating cells. This regional accumulation may contribute to localized secretion into the phagosomal lumen.

Determinants of the phagosomal pH in macrophages. In situ assessment of vacuolar H(+)-ATPase activity, counterion conductance, and H+ “leak”.

We studied the factors that determine the intraphagosomal pH (pHp) in elicited murine peritoneal macrophages. pHp was measured in situ by recording the fluorescence of covalently fluoresceinated Staphylococcus aureus ingested by the macrophages. Following spontaneous acidification of the phagosomes, passive (leak) H+ permeability was determined measuring the rate of change of pHp upon complete inhibition of the H+ pump with bafilomycin A1. A significant, but comparatively low passive H+ permeability was detected. The existence of a passive H+ leak implies that continuous energy expenditure is required for the maintenance of an acidic pHp. In combination with ionophores, bafilomycin was also used to estimate the counterion permeability. The counterion conductance was found to be severalfold higher than the H+ leak. Ion substitution experiments in electropermeabilized cells and the inhibitory effects of quinine and 5-nitro-2-(3-phenylpropylamino)benzoic acid suggest that both monovalent anions and cations permeate the phagosomal membrane. The activity of the H+ pump was measured at various pHp levels. In the steady state, the rate of H+ pumping was considerably lower than counterion permeation. These findings suggest that the phagosomal membrane potential is insignificant. Consistent with this notion, increasing phagosomal conductance with ionophores failed to accelerate the rate of H+ pumping. Thus, the transmembrane delta pH is the predominant component of the proton-motive force across the phagosomal membrane in the steady state. The rate of H+ pumping was found to decrease steeply as the phagosomal lumen became acidified. Therefore, the pH sensitivity of the H+ pump, which possibly reflects a kinetic or allosteric effect, is the primary determinant of pHp.