The success of Mycobacterium species as pathogens depends on their ability to maintain an infection inside the phagocytic vacuole of the macrophage. Although the bacteria are reported to modulate maturation of their intracellular vacuoles, the nature of such modifications is unknown. In this study, vacuoles formed around Mycobacterium avium failed to acidify below pH 6.3 to 6.5. Immunoelectron microscopy of infected macrophages and immunoblotting of isolated phagosomes showed that Mycobacterium vacuoles acquire the lysosomal membrane protein LAMP-1, but not the vesicular proton-adenosine triphosphatase (ATPase) responsible for phagosomal acidification. This suggests either a selective inhibition of fusion with proton-ATPase-containing vesicles or a rapid removal of the complex from Mycobacterium phagosomes.
Abstract. Physical fixation by rapid freezing followed by freeze-fracture and deep-etching has provided the means for potentially seeing the three-dimensional arrangement in the native state of particles on mitochondrial inner membranes. We have used these techniques to study the tubular cristae of Paramecium in the hope of determining the arrangement of Ft complexes, their abundance, and location in the membranes. We also sought information regarding other respiratory complexes in these membranes. Our results, supported by stereo pairs, show that F, complexes are arranged as a double row of particles spaced at 12 nm along each row as a zipper following the full length of the outer curve of the helically shaped tubular cristae. There are an average of 1,500 highly ordered Ft complexes per micrometer squared of 50-nm tubular cristae surface. The F~ complexes definitely lie outside the membranes in their native state. Other particle subsets, also nonrandomly arrayed, were seen. One such population located along the inner helical curve consisted of large 13-nm-wide particles that were spaced at 30 nm center-to-center. Such particles, because of their large size and relative abundance when compared to F, units, resemble complex I of the respiratory complexes. Any models attempting to understand the coupling of respiratory complexes with FoF, ATPase in Paramecium must take into account a relatively high degree of order and potential immobility of at least some of these integral membrane complexes.M ITOCHONDRIAL cristae are known to contain the electron transport complexes as well as the FoFt ATPase complexes involved in oxidative phosphorylation. In addition, the ADP-ATP translocase, a transhydrogenase, and several less abundant integral membrane proteins reside in these membranes (6). Over 25 years ago the presence of 9-nm projections extending from these membranes into the mitochondrial matrix was discovered in beef heart muscle by Fermindez-Mor~in (9) who used the negative-staining technique. In the ensuing years, similar globular units have been shown to be present in a wide variety of organisms across all living kingdoms of eukaryotic life (5, 26-28, 31, 35, 38). Thus it is likely that all mitochondrial cristae bear these projections. The role of the 9-nm projections was established by reconstitution studies which confinned that the F~ head groups represented the ATPase complexes (34).At the same time some work was also done on estimating the relative numbers of FoF~ complexes per unit membrane area, e.g., Smith (38) reported that insect flight muscle has •4,000 complexes per micrometer squared of cristae membrane, but these reports were based on use of the negative staining technique where projections can best be seen when the membranes are viewed in profile. More recent estimates of the Ft concentrations are based on biochemical and enzymatic methods. For example, Schwerzmann et al. (42) calculated that rat liver mitochondria have 2,568 FoF~ complexes per micrometer squared of inner membrane.Since the discove...
ABSTRACT. The temporal changes in the size and pH of digestive vacuoles (DV) in Paramecium caudatum were reevaluated. Cells were pulsed briefly with polystyrene latex spheres or heat‐killed yeast stained with three sulfonphthalein indicator dyes. Within 5 min of formation the intravacuolar pH declined from ∼7 to 3. With the exception of a transient and early increase in vacuolar size, vacuole condensation occurred rapidly and paralleled the acidification so that vacuoles reached their lowest pH and minimal size simultaneously. Neutralization and expansion of vacuole size began when vacuoles were GT8 min old. No labeled vacuoles were defecated prior to 21 min after formation but almost all DV were defecated within 1 h so that the digestive cycle of individual vacuoles ranged from 21 to 60 min. Based on these size and pH changes, the presence of acid phosphatase activity, and membrane morphology, digestive vacuoles can be grouped into four stages of digestion. The DV‐I are GT6 min old and undergo rapid condensation and acidification. The DV‐II are between 4 to 10 min old and are the most condensed and acidic vacuoles. The DV‐III range in age from 8 to ∼20 min and include the expanding or expanded vacuoles that result from lysosomes fusing with DV‐II. The DV‐IV are GD21 min old, and since digestion is presumably completed, they can be defecated. The rise in intravacuolar pH that accompanies vacuole expansion suggests that lysosomes play a role in vacuole neutralization in addition to their degradative functions. The acidification and condensation processes in DV‐I appear to be unrelated to lysosomal function, as no acid phosphaiase activity has been detected at this stage, but may be related to phagosomal functions important in killing food organisms, denaturing proteins prior to digestion, and preparing vacuole membrane for fusion with lysosomes.
Although acidification of phagocytic vacuoles has received a broadened interest with the development of pH-sensitive fluorescent probes to follow the pH changes of vacuoles and acidic vesicles in living cells, the mechanism responsible for the acidification of such vacuoles still remains in doubt. In previous studies of the digestive vacuole system in the ciliate Paramecium caudatum we observed and described a unique population of apparently nonlysosomal vesicles that quickly fused with the newly released vacuole before the vacuole became acid and before lysosomes fused with the vacuole. In this paper we report the following: (a) these vesicles, named acidosomes, are devoid of acid phosphatase; (b) these vesicles accumulate neutral red as well as acridine orange, two observations that demonstrate their acid content; (c) cytochalasin B given 15 s after exposure of the cells to indicator dye-stained yeast will inhibit the acidification of yeast-containing vacuoles; and that (d) we observed using electron microscopy, that fusion of acidosomes with the vacuole is inhibited by cytochalasin B. We conclude that the mechanism for acidification of phagocytic vacuoles in Paramecium resides, at least partially if not entirely, in the acidosomes.Recent papers that have used pH-sensitive fluorescent probes (1) to follow the time course of the pH changes in phagosomes (2-5) and endosomes ( l, 4, 6-8) of various cells suggest that the pH of a vesicle or vacuole that derives from the cell surface often becomes acid before such an organelle fuses with lysosomes. Thus the mechanism for the initial acidification of these organelles would not come from the lysosomes, though some lysosome membranes, such as those of rat liver cells, have now been shown to have a proton pump (9, 10), nor would the acidification in these cases result from the digestive processes that would typically follow lysosome fusion. In Paramecium it has long been known that the pH of the digestive vacuole falls quickly following the release of the vacuole from the oral region ( 1 l, 12). We recently reported a detailed study of the time-course of the pH changes in the vacuoles of Paramecium caudatum grown in axenic medium and showed that the vacuolar pH falls from 7.0 to 3.0 within 5 min (13). By 8 min, the pH begins to rise rapidly to neutrality. The initial acidification parallels the early rapid vacuole condensation (13), while the rise in pH at 8 min corresponds to the approximate time when vacuoles first fuse with lysosomes (14) and acquire acid phosphatase activity (15).We have also described a population of rather large vesicles (up to l ~zm in diameter), which we termed phagosomal fusion vesicles (PFVs) ~ (16, 17). These PFVs were observed to bind to the forming digestive vacuole membrane, but they do not fuse with the vacuole until the vacuole has been released from the oral region of the cell and has moved a distance of some 30 um to the posterior end of the cell. By 15 to 30 s these PFVs have fused with the vacuole. Because these PFVs fuse w...
Strains ofListeria monocytogenes serotype 4b account for a large fraction of sporadic listeriosis cases, as well as all major food-borne epidemics attributed to this pathogen. We have identified a set of three monoclonal antibodies which showed a high degree of specificity for strains of L. monocytogenes serotype 4b. Two of these antibodies (c74.33 and c74.180, isotypes immunoglobulin M [IgM] and IgG3, respectively) recognized all serotype 4b strains, whereas antibody c74.22 (isotype IgGl) failed to recognize certain epidemic-associated strains. The corresponding antigens were located on the surface of the bacteria and were expressed following bacterial growth in different media and over a wide range of temperatures (4, 22, and 37°C). Heating L. monocytogenes cells at 80, 90, or 100°C abolished reactivity for c74.22 but not for c74.33 MAb. These MAbs were negative for all of the non-Listeria strains tested, including representatives of several gram-negative and gram-positive species. The surface antigen recognized by c74.22 appeared to be associated with the ability of the bacteria to enter (invade) mammalian cells in culture.
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