Opsonized virulent Brucella abortus replicates within nonacidic, endoplasmic reticulum-negative, LAMP-1-positive phagosomes in human monocytes

Cells in the Brucella spp. are intracellular pathogens that survive and replicate within host monocytes. Brucella maintains persistent infections in animals despite the production of high levels of anti-Brucella-specific antibodies. To determine the effect of antibody opsonization on the ability of Brucella to establish itself within monocytes, the intracellular trafficking of virulent Brucella abortus 2308 and attenuated hfq and bacA mutants was followed in the human monocytic cell line THP-1. Early trafficking events of B. abortus 2308-containing phagosomes (BCP) were indistinguishable from those seen for control particles (heat-killed B. abortus 2308, live Escherichia coli HB101, or latex beads). All phagosomes transiently communicated the early-endosomal compartment and rapidly matured into LAMP-1(+), cathepsin D(+), and acidic phagosomes. By 2 h postinfection, however, the number of cathepsin D(+) BCP was significantly lower for live B. abortus 2308-infected cells than for either Brucella mutant strains or control particles. B. abortus 2308 persisted within these cathepsin D(-), LAMP-1(+), and acidic vesicles; however, at the onset of intracellular replication, the numbers of acidic B. abortus 2308 BCP decreased while remaining cathepsin D(-) and LAMP-1(+). In contrast to B. abortus 2308, the isogenic hfq and bacA mutants remained in acidic, LAMP-1(+) phagosomes and failed to initiate intracellular replication. Notably, markers specific for the host endoplasmic reticulum were absent from the BCPs throughout the course of the infection. Thus, opsonized B. abortus in human monocytes survives within phagosomes that remain in the endosomal pathway and replication of virulent B. abortus 2308 within these vesicles corresponds with an increase in intraphagosomal pH.     

Brucella suis-impaired specific recognition of phagosomes by lysosomes due to phagosomal membrane modifications

Brucella species are gram-negative, facultatively intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment in phagocytic and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both types of cells. However, the biochemical mechanisms and microbial factors implicated in Brucella maturation are still completely unknown. We developed two different approaches in an attempt to gain further insight into these mechanisms: (i) a fluorescence microscopy analysis of general intracellular trafficking on whole cells in the presence of Brucella and (ii) a flow cytometry analysis of in vitro reconstitution assays showing the interaction between Brucella suis-containing phagosomes and lysosomes. The fluorescence microscopy results revealed that fusion properties of latex bead-containing phagosomes with lysosomes were not modified in the presence of live Brucella suis in the cells. We concluded that fusion inhibition was restricted to the pathogen phagosome and that the host cell fusion machinery was not altered by the presence of live Brucella in the cell. By in vitro reconstitution experiments, we observed a specific association between killed B. suis-containing phagosomes and lysosomes, which was dependent on exogenously supplied cytosol, energy, and temperature. This association was observed with killed bacteria but not with live bacteria. Hence, this specific recognition inhibition seemed to be restricted to the pathogen phagosomal membrane, as noted in the in vivo experiments.     

Early events and implication of F-actin and annexin I associated structures in the phagocytic uptake of Brucella suis by the J-774A.1 murine cell line and human monocytes

Brucella spp. are facultative, intracellular pathogenic bacteria that cause brucellosis, a zoonosis affecting mammalian species. Brucella entry into myelomonocytic cell lines is highly enhanced by opsonization. Few studies have been undertaken to unravel the first interactions between these bacteria and their host cells. This paper deals with early events following contact of Brucella suis with the J-774A.1 phagocytic cell line and differentiated monocytes. Phagocytic uptake of bacteria was documented under a fluorescence microscope using GFP-expressing B. suis. Unlike entry in the J-774A. 1 cell line, non-opsonized Brucella entered differentiated human monocytes as efficiently as opsonized bacteria. However, following 1 h infections, a mean of only three bacteria were phagocytized and the whole monocyte population was only infected after a 4 h infection. Contact of non-opsonized Brucella with phagocytes did not induce marked structural changes at the cell surface, as revealed by scanning electron microscopy. Contact of Brucella (opsonized or not) elicited transient local recruitment of F-actin, revealed by phalloidin labelling, and of annexin I-associated structures, revealed by immunofluorescence staining. Finally, bacteria appeared to be rapidly internalized in monocytes once they had adhered to the cell surface. A low percentage of infected cells and few adhered and/or internalized bacteria following short-term infections could have resulted either from the fact that there were few sites of entry or the weak bacterial initial interactions with the host-cell membrane or the bacterial receptor.     

Fate of Listeria monocytogenes in murine macrophages: evidence for simultaneous killing and survival of intracellular bacteria.

The intracellular survival of the ubiquitous pathogen Listeria monocytogenes was studied in primary cultures of bone marrow-derived mouse macrophages. Bacteria were able to grow rapidly in these cells, with an apparent multiplication rate of about 40 min. Electron microscopy demonstrated that intracellular bacterial replication was the consequence of simultaneous intracellular killing and replication of bacteria in the same cells. Within the first hour following phagocytosis, most bacteria were destroyed in the phagosomal compartment to which they were confined. This was due to early transfer of hydrolytic enzymes to phagosomes, undoubtedly via phagosome-lysosome (P-L) fusion, as demonstrated by a quantitative analysis after staining for a lysosomal marker, acid phosphatase. One hour after infection, about 14% of the bacteria were free in the cytoplasm, in which they multiplied and induced actin polymerization and spreading to adjacent macrophages, as in epithelial cells. By using the 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine staining procedure, direct evidence is presented that all phagosomes were acidified immediately after phagocytosis, thus indicating that intraphagosomal bacteria were exposed to an acidic environment that might favor vacuolar lysis by listeriolysin O. Intracellular growth in macrophages, therefore, appears to be the result of a competition between the expression of the hydrolytic activity of these cells following P-L fusion and the capacity of L. monocytogenes to escape from the acidified phagosomal compartment before P-L fusion has occurred. The finding that concomitant intracellular killing and survival of L. monocytogenes occurs in the same macrophages might explain the high immunogenicity observed in vivo with live bacteria, as opposed to killed bacteria.     

Ultrastructural Study of the Behavior of Macrophages Toward Parasitic Mycobacteria

Mycobacterium lepraemurium and M. microti (causal agent of vole tuberculosis) were isolated from tissues of experimentally infected mice and used to infect normal mouse peritoneal macrophage cultures. The cellular response to these bacteria up to 4 days after infection was studied quantitatively by electron microscopy. Prelabeling with ferritin was used to facilitate observation of fusion between secondary lysosomes in the cells and phagosomes containing the bacteria. All bacteria were intraphagosomal, and a high proportion of them was morphologically "intact." Nearly all phagosomes containing morphologically damaged (presumed nonviable) bacteria also contained ferritin, having fused with secondary lysosomes. Fusion of lysosomes had also occurred with most phagosomes containing intact M. lepraemurium but was infrequent with phagosomes containing intact M. microti. This tendency of multiplying mycobacteria of the tubercle type to avoid contact with lysosomal contents has already been reported for M. tuberculosis strain H37Rv. The different intracellular circumstances of the parasites may reflect different means of intracellular survival.     

Role of the Brucella suis lipopolysaccharide O antigen in phagosomal genesis and in inhibition of phagosome-lysosome fusion in murine macrophages

Brucella species are gram-negative, facultative intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment inside professional and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both cell types. However, the molecular mechanisms and the microbial factors involved are poorly understood. Smooth lipopolysaccharide (LPS) of Brucella has been reported to be an important virulence factor, although its precise role in pathogenesis is not yet clear. In this study, we show that the LPS O side chain is involved in inhibition of the early fusion between Brucella suis-containing phagosomes and lysosomes in murine macrophages. In contrast, the phagosomes containing rough mutants, which fail to express the O antigen, rapidly fuse with lysosomes. In addition, we show that rough mutants do not enter host cells by using lipid rafts, contrary to smooth strains. Thus, we propose that the LPS O chain might be a major factor that governs the early behavior of bacteria inside macrophages.     

Smooth and rough lipopolysaccharide phenotypes of Brucella induce different intracellular trafficking and cytokine/chemokine release in human monocytes

Virulence of the intracellular pathogen Brucella for humans is mainly associated with its lipopolysaccharide (LPS) phenotype, with smooth LPS phenotypes generally being virulent and rough ones not. The reason for this association is not quite understood. We now demonstrate by flow cytometry, electron microscopy, and ELISA that human peripheral blood monocytes interact both quantitatively and qualitatively different with smooth and rough Brucella organisms in vitro. We confirm that considerably higher numbers of rough than smooth brucellae attach to and enter the monocytes in nonopsonic conditions; but only smooth brucellae replicate in the host cells. We show for the first time that rough brucellae induce higher amounts than smooth brucellae of several CXC (GRO-alpha, IL-8) and CC (MIP-1alpha, MIP-1beta, MCP-1, RANTES) chemokines, as well as pro- (IL-6, TNF-alpha) and anti-inflammatory (IL-10) cytokines released by challenged monocytes. Upon uptake, phagosomes containing rough brucellae develop selective fusion competence to form spacious communal compartments, whereas phagosomes containing smooth brucellae are nonfusiogenic. Collectively, our data suggest that rough brucellae attract and infect monocytes more effectively than smooth brucellae, but only smooth LPS phenotypes establish a specific host cell compartment permitting successful parasitism. These novel findings link the LPS phenotype of Brucella and its virulence for humans at the level of the infected host cells. Whether this is due to a direct effect of the LPS molecules or to upstream bacterial mechanisms remains to be established.     

O-antigen-deficient Francisella tularensis Live Vaccine Strain mutants are ingested via an aberrant form of looping phagocytosis and show altered kinetics of intracellular trafficking in human macrophages

We examined the uptake and intracellular trafficking of F. tularensis Live Vaccine Strain (LVS) and LVS with disruptions of wbtDEF and wbtI genes essential for synthesis of the O antigen of lipopolysaccharide. Unlike parental bacteria, O-antigen-deficient LVS is efficiently killed by serum with intact complement but not by serum lacking terminal complement components. Opsonization of O-antigen-deficient LVS in serum lacking terminal complement components allows efficient uptake of these live bacteria by macrophages. In the presence of complement, whereas parental F. tularensis LVS is internalized within spacious pseudopod loops, mutant LVS is internalized within tightly juxtaposed multiple onion-like layers of pseudopodia. Without complement, both parental and mutant LVSs are internalized within spacious pseudopod loops. Thus, molecules other than O antigen are important in triggering dramatic pseudopod extensions and uptake by spacious pseudopod loops. Following uptake, both parental and mutant LVSs enter compartments that show limited staining for the lysosomal membrane glycoprotein CD63 and little fusion with secondary lysosomes. Subsequently, both parental and mutant LVSs lose their CD63 staining. Whereas the majority of parental LVS escapes into the cytosol by 6 h after uptake, mutant LVS shows a marked lag but does escape by 1 day after uptake. Despite the altered kinetics of phagosome escape, both mutant and parental strains grow to high levels within human macrophages. Thus, the O antigen plays a role in the morphology of uptake in the presence of complement and the kinetics of intracellular growth but is not essential for escape, survival, altered membrane trafficking, or intramacrophage growth.     

Interactions of Neisseria meningitidis with human monocytes

The roles of capsule, pill and Class 5 outer-membrane proteins (Opa and Opc) of Neisseria meningitidis (Nm) in bacterial interactions with human monocytes were investigated using several meningococcal isolates of different serogroups. The presence of either Class I or Class II pili in capsulate strains of several serogroups had no significant effect on adherence to and internalisation by monocytes. Using clonal variants derived from a non-piliated serogroup A strain, C751, it was observed that capsulate bacteria (cap⁺) failed to interact with human monocytes in significant numbers whether or not they expressed outer-membrane proteins. These bacteria were also resistant to phagocytic killing. For capsule-deficient bacteria, expression of the Opc protein or OpaB_c751 correlated with high levels of association, while the expression of OpaD_c751 or OpaA_c751 resulted in comparatively lower levels. Bacteria expressing undetectable levels of Opc or Opa proteins (Opc^-, Opa) failed to interact with monocytes. In phagocytic killing assays, Opc-expressing bacteria (Opc⁺) as well as Opa-expressing bacteria (Opa⁺) were killed more readily than Opc^-, Opa bacteria (30% decrease in viability of Opc⁺ bacteria; 18%, 10% and 8% decrease in viability of OpaB⁺, OpaD⁺ and OpaA⁺ bacteria). A study of intracellular survival showed a gradual decrease in viability of both capsulate and capsule-deficient bacteria. However, proportionately greater numbers of capsule-deficient bacteria were internalized and consequently larger numbers survived over a 4-h period. Prolonged bacterial survival within phagocytic cells may have implications in dissemination of bacteria by carriage within these cells.     

Residual virulence of Brucella abortus in the absence of the cytochrome bc(1)complex in a murine model in vitro and in vivo

To maintain survival in macrophages, Brucella must overcome a hostile phagosomal environment defined as low pH, limited nutrition and low oxygen tension. The specific mechanisms utilized by Brucella to surmount such unfavorable environmental factors in phagosomes are not well understood. In general, to adapt to a change in environmental oxygen tension, bacteria use different terminal oxidases that have different oxygen affinity. To survive in phagosomes where low oxygen tension exists, Brucella, like other bacteria, may require high oxygen affinity terminal oxidases that can accept electrons through a cytochrome bc(1)complex dependent or independent pathway. Using a Brucella abortus cytochrome bc(1)complex deficient mutant, delta fbcF, the requirement for a high oxygen affinity terminal oxidase governed by the cytochrome bc(1)complex dependent pathway was tested. The number of cfu from RAW 264.7 macrophage cells and spleens of BALB/c mice infected with wild-type or the cytochrome bc(1)complex deficient mutant was similar during the course of infection. These results suggest that B. abortus contains no essential terminal oxidase utilized at low oxygen tension in phagosomes requiring the cytochrome bc(1)complex. Alternatively, other branched cytochrome bc(1)complex independent respiratory mechanisms that contain the high oxygen affinity terminal oxidases likely exist to facilitate Brucella survival in phagosomes. This is the first investigation regarding the Brucella respiratory system at the molecular level and the involvement of a respiratory system in Brucella pathogenesis.     

Early events and implication of F-actin and annexin I associated structures in the phagocytic uptake of Brucella suis by the J-774A.1 murine cell line and human monocytes

Brucella spp. are facultative, intracellular pathogenic bacteria that cause brucellosis, a zoonosis affecting mammalian species. Brucella entry into myelomonocytic cell lines is highly enhanced by opsonization. Few studies have been undertaken to unravel the first interactions between these bacteria and their host cells. This paper deals with early events following contact of Brucella suis with the J-774A.1 phagocytic cell line and differentiated monocytes. Phagocytic uptake of bacteria was documented under a fluorescence microscope using GFP-expressing B. suis. Unlike entry in the J-774A.1 cell line, non-opsonized Brucella entered differentiated human monocytes as efficiently as opsonized bacteria. However, following 1 h infections, a mean of only three bacteria were phagocytized and the whole monocyte population was only infected after a 4 h infection. Contact of non-opsonized Brucella with phagocytes did not induce marked structural changes at the cell surface, as revealed by scanning electron microscopy. Contact of Brucella (opsonized or not) elicited transient local recruitment of F-actin, revealed by phalloidin labelling, and of annexin I-associated structures, revealed by immunofluorescence staining. Finally, bacteria appeared to be rapidly internalized in monocytes once they had adhered to the cell surface. A low percentage of infected cells and few adhered and/or internalized bacteria following short-term infections could have resulted either from the fact that there were few sites of entry or the weak bacterial initial interactions with the host-cell membrane or the bacterial receptor.     

Uptake of Aspergillus fumigatus Conidia by phagocytic and nonphagocytic cells in vitro: quantitation using strains expressing green fluorescent protein

Several pathogenic fungal organisms enter eukaryotic cells and manipulate the host cell environment to favor their own growth and survival. Aspergillus fumigatus is a saprophytic fungus that causes invasive lung disease in the immunocompromised host. To determine whether A. fumigatus could enter eukaryotic cells, we studied the uptake of two different GFP-expressing A. fumigatus strains into A549 lung epithelial cells, human umbilical vein endothelial (HUVE) cells, and J774 murine macrophages in vitro. A549 cells internalized 30% of the bound conidia whereas HUVE and J774 cells internalized 50 and 90%, respectively. Conidia within A549 cells remained viable for 6 h; however, 60 to 80% of conidia within J774 cells were killed after only 4 h. Live and heat-killed conidia were internalized to the same extent by A549 cells. After 6 h, almost none of the conidia inside A549 cells had germinated, whereas extracellular conidia had developed germ tubes. Internalization of conidia by A549 cells was a temperature-dependent process and required rearrangement of the underlying host cell cytoskeleton; uptake was inhibited by 75% with 0.5 microM cytochalasin D and by 65% with 5 microM colchicine. Fluorescent labeling of infected A549 cells with rhodamine phalloidin provided visible evidence of cytoskeletal alteration as many of the intracellular conidia were contained in actin-coated phagosomes. These data provide evidence that significant numbers of A. fumigatus conidia can be internalized by nonprofessional phagocytes in vitro and these cells may serve as reservoirs for immune cell evasion and dissemination throughout the host.     

Early Acidification of Phagosomes Containing Brucella suis Is Essential for Intracellular Survival in Murine Macrophages

Brucella suis is a facultative intracellular pathogen of mammals, residing in macrophage vacuoles. In this work, we studied the phagosomal environment of these bacteria in order to better understand the mechanisms allowing survival and multiplication of B. suis. Intraphagosomal pH in murine J774 cells was determined by measuring the fluorescence intensity of opsonized, carboxyfluorescein-rhodamine- and Oregon Green 488-rhodamine-labeled bacteria. Compartments containing live B. suis acidified to a pH of about 4.0 to 4.5 within 60 min. Acidification of B. suis-containing phagosomes in the early phase of infection was abolished by treatment of host cells with 100 nM bafilomycin A(1), a specific inhibitor of vacuolar proton-ATPases. This neutralization at 1 h postinfection resulted in a 2- to 34-fold reduction of opsonized and nonopsonized viable intracellular bacteria at 4 and 6 h postinfection, respectively. Ammonium chloride and monensin, other pH-neutralizing reagents, led to comparable loss of intracellular viability. Addition of ammonium chloride at 7 h after the beginning of infection, however, did not affect intracellular multiplication of B. suis, in contrast to treatment at 1 h postinfection, where bacteria were completely eradicated within 48 h. Thus, we conclude that phagosomes with B. suis acidify rapidly after infection, and that this early acidification is essential for replication of the bacteria within the macrophage.     

Intracellular trafficking of Brucella abortus in J774 macrophages

Brucella abortus is a facultative intracellular bacterium capable of surviving inside professional and nonprofessional phagocytes. The microorganism remains in membrane-bound compartments that in several cell types resemble modified endoplasmic reticulum structures. To monitor the intracellular transport of B. abortus in macrophages, the kinetics of fusion of phagosomes with preformed lysosomes labeled with colloidal gold particles was observed by electron microscopy. The results indicated that phagosomes containing live B. abortus were reluctant to fuse with lysosomes. Furthermore, newly endocytosed material was not incorporated into these phagosomes. These observations indicate that the bacteria strongly affect the normal maturation process of macrophage phagosomes. However, after overnight incubation, a significant percentage of the microorganisms were found in large phagosomes containing gold particles, resembling phagolysosomes. Most of the Brucella bacteria present in phagolysosomes were not morphologically altered, suggesting that they can also resist the harsh conditions prevalent in this compartment. About 50% colocalization of B. abortus with LysoSensor, a weak base that accumulates in acidic compartments, was observed, indicating that the B. abortus bacteria do not prevent phagosome acidification. In contrast to what has been described for HeLa cells, only a minor percentage of the microorganisms were found in compartments labeled with monodansylcadaverine, a marker for autophagosomes, and with DiOC6 (3,3'-dihexyloxacarbocyanine iodide), a marker for the endoplasmic reticulum. These results indicate that B. abortus bacteria alter phagosome maturation in macrophages. However, acidification does occur in these phagosomes, and some of them can eventually mature to phagolysosomes.     

In vitro model of penetration and intracellular growth of Listeria monocytogenes in the human enterocyte-like cell line Caco-2.

Penetration and replication of Listeria monocytogenes within intestinal epithelial cells were studied by infecting the human enterocyte-like cell line Caco-2. Entry was due to directed phagocytosis, as suggested by the inhibiting effect of cytochalasin D on bacterial entry and by electron microscopy showing bacteria inside membrane-limiting vacuoles at the early stage of infection. Only bacteria from pathogenic species (L. monocytogenes and Listeria ivanovii) were able to induce their own phagocytosis by Caco-2 cells, as opposed to Listeria seeligeri, Listeria welshimeri, and Listeria innocua. L. monocytogenes multiplied readily within Caco-2 cells, with an apparent generation time of about 90 min. Listeriolysin O was found to be a major factor promoting intracellular growth of L. monocytogenes. After being internalized at the same rate as that of its hemolytic revertant strain, a nonhemolytic mutant from L. monocytogenes failed to replicate significantly within Caco-2 cells. Electron microscopic study demonstrated that bacteria from the nonhemolytic mutant remained inside phagosomes during cellular infection, whereas hemolytic bacteria from L. monocytogenes were released free within the cytoplasm. This indicates that disruption of vacuole membranes by listeriolysin O-producing strains of L. monocytogenes might be a key mechanism allowing bacteria to escape from phagosomes and to multiply unrestricted within cell cytoplasm.     

Interaction of Legionella micdadei with human monocytes.

We have recently shown that Legionella micdadei is ingested, but not killed, by human neutrophils. Herein we investigate the role of human monocytes in defense against this organism. Serum and monocytes from normal donors having no detectable antibody to L. micdadei were used. Egg-passaged L. micdadei organisms multiplied inside these monocytes with a peak growth of 2 log units within 12 h. No growth occurred when monocytes were omitted or when sonicated monocytes were used. Electron microscopy 18 h after infection revealed these organisms to be intracellular in normal-appearing phagosomes. When the input multiplicity of L. micdadei was greater than 1 CFU per monocyte, no intracellular growth occurred. When egg-passaged Legionella pneumophila organisms were used, intracellular organisms were found in phagosomes studded with ribosomes at the same time period. Peak intracellular growth of L. pneumophilia occurred by 48 h. L. micdadei activated the complement system and was opsonized by C3. However the use of complement-depleted (heat-inactivated) serum as the opsonic source had no effect on the bacterium's ingestion or growth in the monocyte. Thus, L. micdadei multiples in human monocytes. This entry and growth is independent of antibody or complement. The intracellular locations of L. micdadei and L. pneumophila differ, suggesting different mechanisms for the survival of these two organisms in the monocyte.     

Enterococcus faecalis bearing aggregation substance is resistant to killing by human neutrophils despite phagocytosis and neutrophil activation.

Enterococcus faecalis aggregation substance (AS) mediates efficient bacterium-bacterium contact to facilitate plasmid exchange as part of a bacterial sex pheromone system. We have previously determined that AS promotes direct, opsonin-independent binding of E. faecalis to human neutrophils (PMNs) via complement receptor type 3 and other receptors on the PMN surface. We have now examined the functional consequences of this bacterium-host cell interaction. AS-bearing E. faecalis was phagocytosed and internalized by PMNs, as determined by deconvolution fluorescence microscopy. However, these bacteria were not killed by PMNs, and internalized bacteria excluded propidium iodide, indicating intact bacterial membranes. Resistance to killing occurred despite activation of PMNs, as indicated by an increase in both functional and total surface Mac-1 expression, shedding of L-selectin, and an increase in PMN extracellular superoxide and phagosomal oxidant production. Deconvolution fluorescence microscopy also revealed that phagosomes containing AS-bearing bacteria were markedly larger than phagosomes containing opsonized E. faecalis, suggesting that some modification of phagosomal maturation may be involved in AS-induced resistance to killing. PMN phagosomal pH was significantly higher after ingestion of nonopsonized AS-bearing E. faecalis than after that of opsonized bacteria. The novel ability of AS to promote intracellular survival of E. faecalis inside PMNs suggests that AS may be a virulence factor used by strains of E. faecalis.     

Intracellular location of Mycobacterium leprae in macrophages of normal and immune-deficient mice and effect of rifampin

Soon after more than 10(6) Mycobacterium leprae, freshly harvested from armadillo liver or harvested and 60CO irradiated, were inoculated into the hind footpads of either normal or thymectomized and irradiated (T900R) mice, the organisms were found to reside within phagosomes of polymorphonuclear and mononuclear cells. On the other hand, 7 and 8 months after 10(4) freshly harvested M. leprae were inoculated into the footpads of normal or T900R mice and the organisms had multiplied to their maximum in the normal mice, many organisms, largely intact by electron-microscopic criteria, were found to reside free in the cytoplasm of the footpad macrophages, whereas damaged organisms were contained within phagosomes. After 11 months, many intact organisms were found to lie free in the cytoplasm of the macrophages of T900R mice, whereas only damaged intraphagosomal M. leprae cells were observed in the macrophages of normal mice. Finally, a remarkably large proportion of damaged extraphagosomal M. leprae was found in T900R mice administered rifampin for 2 days in a bactericidal dosage. It appears that M. leprae multiplies free in the cytoplasm of the footpad macrophages of infected mice, whereas the M. leprae cells resident within the phagosomes of the macrophages are dead. As the result of treatment with rifampin, the organisms appeared to have been killed in their extraphagosomal location, only afterwards being incorporated into phagosomes. However, the intracellular site in which M. leprae is killed in the course of an effective immune response remains unclear.     

Invasion and intracellular survival of Bordetella bronchiseptica in mouse dendritic cells.

We have studied the interaction between the respiratory pathogen Bordetella bronchiseptica and murine spleen dendritic cells, important antigen-presenting cells that are found in the airway epithelium. Wild-type B. bronchiseptica 5376 attached very efficiently to dendritic cells, whereas the bvg mutant ATCC 10580, wild-type strain BB7865, and its spontaneous delta bvgS mutant BB7866 bound less efficiently. However, all tested B. bronchiseptica strains were able to invade dendritic cells and survive intracellularly for at least 72 h. These results suggest that bvg-independent or bvg-downregulated products are involved in the uptake and intracellular survival. Transmission electron microscopic analysis revealed that bacteria grew and replicated intracellularly and were present in typical phagosomes, which fused with lysosomes during the initial infection period. However, in later infection stages some bacteria seemed to escape into an unfused endocytic compartment, where individual bacteria were tightly surrounded by a membrane. The in vitro interaction of B. bronchiseptica with dendritic cells reported here may be relevant to natural infections caused by this organism that lead to chronicity or an altered immune response.     

Yersinia pseudotuberculosis blocks the phagosomal acidification of B10.A mouse macrophages through the inhibition of vacuolar H(+)-ATPase activity.

Yersinia pseudotuberculosis survived and multiplied in the phagosomes of B10.A mouse peritoneal macrophages. As one of the possible mechanisms for the bacteria's survival in the phagosomes, we demonstrated that live Y. pseudotuberculosis inhibited the phagosomal acidification; pH within phagosomes containing the live Y. pseudotuberculosis remained at about 6.0, whereas pH within phagosomes containing the dead Y. pseudotuberculosis fell to about 4. 5. This ability to inhibit intraphagosomal acidification was also shared by mutants lacking the 42 Md virulence plasmid, indicating that it is chromosomally encoded. The phagosomes containing dead bacteria raised the pH to 6.2 after the treatment of their macrophages with an inhibitor (bafilomycin A1) specific for V-ATPase. Although the amount of V-ATPase in the A and B subunits on the phagosomes was not significantly different between the live and dead bacteria infection, the phagosomes containing live bacteria had a 10-fold smaller V-ATPase activity than those containing the dead bacteria. These results indicated that the inhibition of phagosomal acidification by Y. pseudotuberculosis infection was due to the attenuation of V-ATPase activity, and not due to the exclusion of V-ATPase subunits from the phagosome membrane as found in Mycobacterium avium.