Characterization of rickettsial attachment to host cells by flow cytometry.

The mechanisms of rickettsial attachment have been studied by measuring quantitative changes in rickettsial binding to host cells by flow cytometry after different treatments of the rickettsiae and host cells. Time-dependent binding of Rickettsia conorii to host cells was demonstrated by the increasing intensity of host cell surface fluorescence of rickettsia-host cell combinations when examined with a rickettsia-specific monoclonal antibody. More than 70% of host cells had intensity of fluorescence above the threshold value after 10 min of incubation, owing to rickettsiae bound to the cell surface, and the greatest fluorescence intensity indicative of binding occurred at 20 min. The binding kinetics was rickettsial dose dependent. The binding of rickettsiae to host cells was greatly decreased when host cells or rickettsiae were treated with 1% paraformaldehyde for 30 min or 0.25% trypsin for 5 or 15 min, respectively. Rickettsiae that were heated at 56 degrees C for 15 min lost more than 80% of their ability to attach to host cells. R. rickettsii, an organism closely related to R. conorii, competitively inhibited the attachment of R. conorii (51% inhibition when mixed in equal numbers). These results indicate that the rickettsial binding structures are trypsin and heat sensitive and likely to be surface proteins.     

Entry of Rickettsia tsutsugamushi into polymorphonuclear leukocytes.

Factors involved in the phagocytosis and entry into polymorphonuclear leukocytes (PMNs) of Rickettsia tsutsugamushi were studied by electron microscopy. R. tsutsugamushi propagated in baby hamster kidney cell cultures was incubated with guinea pig peritoneal PMNs in vitro at 35 degrees C. Structurally intact and degenerating rickettsiae were found in phagosomes, but only intact rickettsiae escaped phagosomes and specifically entered the glycogen-rich cytoplasm. The extraphagosomal cytoplasmic rickettsiae were found within 30 min after incubation; continued incubation for 4 h increased the rickettsial entry about fourfold as seen in ultrathin sections. Most rickettsiae in phagosomes were degenerating after 4 h of incubation. When incubated at 25 degrees C, no entry and very few phagocytized rickettsiae were observed. At 40 degrees C, rickettsial entry was greatly reduced, but more rickettsiae were found in phagosomes than at 35 degrees C. Preincubation of rickettsiae at 56 degrees C for 20 min with trypsin or with 2,4-dinitrophenol inhibited entry, but many rickettsiae were in phagosomes. Glutaraldehyde or formaldehyde fixation of rickettsiae and addition of 2-deoxyglucose, iodoacetamide, cytochalasin B, colchicine, or vinblastine inhibited all rickettsial uptake by PMNs. Acid phosphatase cytochemistry of infected PMNs revealed the enzyme activity only in phagosomes with degenerated rickettsiae and not in those with intact rickettsiae. These observations indicated that rickettsiae are passively phagocytized by PMNs, and only those that are intact actively escape from phagosomes, which selectively inhibits lysosomal fusion.     

The nature of the phagosomal membrane: endoplasmic reticulum versus plasmalemma

For decades, the vacuole that surrounds particles engulfed by phagocytosis was believed to originate from the plasma membrane. Conversion of the nascent phagosome into a microbicidal organelle was thought to result from the subsequent, orderly fusion of early endosomes, late endosomes, and ultimately, lysosomes with the original plasma membrane-derived vacuole. This conventional model has been challenged, if not superseded, by a revolutionary model that regards phagosome formation as resulting from the particle sliding into the endoplasmic reticulum via an opening at the base of the phagocytic cup. The merits and implications of these two hypotheses are summarized here and analyzed in light of recent results.     

In vitro model of adhesion and invasion by Bacillus piliformis.

An in vitro model of Bacillus piliformis infection was developed to investigate the mechanisms of adhesion and internalization of this obligate intracellular bacterium. Adhesion and internalization events were examined by electron microscopic evaluation of infected Caco-2 cell monolayers. A few bacteria were identified in apical surface invaginations and in vacuoles subjacent to the apical surface, whereas the majority of bacteria were observed free within the cytoplasm, suggesting that B. piliformis entered epithelial cells via a phagocytic process and rapidly escaped the phagosome. To confirm that host cell phagocytosis was involved in entry of B. piliformis into mammalian cells, Intestine 407 cells were treated with the phagocytic inhibitor cytochalasin D, infected with B. piliformis, and evaluated for bacterial internalization by double-fluorescence labeling. The results showed decreased intracellular bacteria, suggesting that internalization was dependent on host cell microfilament function. To examine the role of B. piliformis in internalization, growth of live and Formalin-killed bacteria was compared. Dead bacteria were not internalized, suggesting that B. piliformis actively participates in internalization. B. piliformis appears to enter host cells by a bacterially directed phagocytic process. The in vitro system described should prove invaluable in further investigations of B. piliformis pathogenic mechanisms.     

Interactions between Rickettsia prowazekii and rabbit polymorphonuclear leukocytes: rickettsiacidal and leukotoxic activities.

Rickettsia prowazekii was assessed for in vitro susceptibility to phagocytosis by rabbit polymorphonuclear leukocytes. [alpha-32P]adenosine triphosphate-labeled rickettsiae were used to determine phagocytosis and adsorption quantitatively. R. prowazekii was less susceptible to phagocytosis than were Escherichia coli and Neisseria gonorrhoeae. Although R. prowazekii was similar to E. coli in susceptibility to superoxide and activated halide, few phagocytized rickettsial cells were inactivated after being ingested by polymorphonuclear leukocytes, and rickettsiae were observed free in polymorphonuclear leukocyte cytoplasm. At low ratios of rickettsiae to polymorphonuclear leukocytes, PMN phagocytosis increased as a linear function of time, but at high ratios (multiplicity of infection, 50) rickettsiae were phagocytized during only the first 10 min of incubation. Polymorphonuclear leukocytes were damaged in the presence of high rickettsial multiplicities such that they released lactate dehydrogenase into the medium and lost the ability to phagocytize both rickettsiae and E. coli. The amount of leukotoxic activity in a given rickettsial sample correlated with the relative hemolytic activity of that sample. The rickettsial leukotoxin was probably not a soluble product, was active in the absence of phagocytosis, and was inhibited by inactivation of the rickettsiae or by incubation at 4 degrees C.     

Experimental infection of mouse peritoneal mesothelium with scrub typhus rickettsiae: an ultrastructural study.

The infection cycle of Rickettsia tsutsugamushi in mouse peritoneal mesothelial cells, observed late in the course of an established infection, intimately involved the host cell plasma membrane. Organisms multiplied in the cytoplasm, moved to the cell periphery, and acquired a host-membrane coat as they budded from the cell surface. Rickettsiae enveloped by this membrane entered other mesothelial cells, apparently by a phagocytic mechanism. Organisms escaped from the phagocytic vacuole as the vacuole membrane and host membrane coat disintegrated. Free rickettsiae replicated by binary fission in the cell cytoplasm. Rickettsial infection of mesothelial cells induced conspicuous cellular hypertrophy with increased numbers of unaltered cytoplasmic organelles.     

Rickettsial interactions with human endothelial cells in vitro: adherence and entry

Rickettsia prowazekii, Madrid E strain, was assessed for its ability to enter endothelial cells derived from the veins of human umbilical cord in vitro. Rickettsial entry increased linearly with multiplicity of infection up to a multiplicity of 500; thereafter, additional rickettsiae adhered, but without a concomitant increase in the number of intracellular rickettsiae. Rickettsial entry required participation both of rickettsiae and endothelial cells; inactivation of rickettsiae with N-ethylmaleimide or Formalin, or of endothelial cells with cytochalasin B or D or NaF greatly reduced rickettsial entry. Because rickettsiae adhered to inactivated endothelial cells, adherence could be examined in the absence of entry. Rickettsial adherence was inhibited by poisons that inhibited rickettsial hemolysis. Calcium ionophore A23187, which did not inhibit endothelial pinocytosis, stimulated rickettsial adherence to endothelial cells, but inhibited rickettsial entry. These results indicated that typhus rickettsiae entered endothelial cells via induced phagocytosis, and that the signal for entry, which was dependent upon rickettsial energy, probably involved formation of a calcium gradient.     

Intracellular and cell-to-cell spread of Listeria monocytogenes involves interaction with F-actin in the enterocytelike cell line Caco-2.

Listeria monocytogenes penetrates and multiplies within professional phagocytes and other cells such as the Caco-2 human enterocytelike cell line. Listeriolysin O, a membrane-damaging cytotoxin accounts for intracellular multiplication through lysis of the membrane-bound phagocytic vacuole. This work demonstrates that once released within the cytosol, L. monocytogenes acquires the capacity to spread intracellularly and infect adjacent cells by interacting with host cell microfilaments. Such evidence was obtained by using drugs which disrupt the cell cytoskeleton. Nocodazole, which blocks polymerization of microtubules, did not affect intracellular spread, whereas cytochalasin D, which blocks polymerization of G-actin, inhibited the intracellular motility of the bacteria. By using fluorescence staining with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin (NBD-phallacidin), transmission electron microscopy, and immunogold labeling, direct evidence was obtained that intracellular bacteria were enveloped with a thick layer of F-actin. Within 2 h after entry, it was demonstrated by confocal microscopy that bacteria were following highly organized routes corresponding to stress fibers. Four hours after entry, some bacteria presented random movements which could be seen by the presence of a large trail of F-actin. Such movements also caused protrusions which deeply penetrated adjacent cells and resulted in the formation of vacuoles limited by a double membrane. After subsequent lysis of these membranes, bacteria released within the cytoplasm were able to multiply and invade new cells. In contrast, an hly::Tn1545 mutant of the wild-type microorganism demonstrated almost no intracellular spread. Only a few bacteria displaying delayed lysis of the phagocytic vacuole behaved like the wild-type strain. Hemolysin-mediated lysis of the phagocytic vacuole and subsequent interaction with host cell microfilaments may represent a major virulence factor allowing tissue colonization during listeriosis.     

Effect of antibody on the rickettsia-host cell interaction

A recent study demonstrated that polyclonal antibodies to Rickettsia conorii and monoclonal antibodies to outer membrane proteins A (OmpA) and B (OmpB) provided effective, Fc-dependent, passive immunity, even in severe combined immunodeficient mice with an established infection. In order to determine the mechanism of protection, mouse endothelial and macrophage-like cell lines were infected with R. conorii that had been exposed to polyclonal antibodies, monoclonal antibodies to OmpA or OmpB, Fab fragments of the polyclonal antibodies, or normal serum or that were left untreated. At 0 h, Fc-dependent antibody enhancement of R. conorii adherence to endothelial and macrophage-like cell lines was inhibited by the presence of normal serum, suggesting Fc receptor-mediated adherence of opsonized rickettsiae. At 3 h, the opsonized rickettsiae had been internalized. After 72 h, inhibited survival of rickettsiae exposed to polyclonal antibodies or monoclonal antibodies to OmpA or OmpB was evident compared with growth of untreated and normal serum-treated and polyclonal Fab antibody-treated R. conorii. Polyclonal antibodies and an anti-OmpB monoclonal antibody inhibited the escape of R. conorii from the phagosome, resulting in intraphagolysosomal rickettsial death. At 48 h of infection, rickettsicidal activity of macrophages by opsonized rickettsiae was inhibited by NG-monomethyl-L-arginine, superoxide dismutase, mannitol, or supplemental L-tryptophan, and endothelial rickettsicidal activity against opsonized rickettsiae was inhibited by NG-monomethyl-L-arginine, superoxide dismutase, catalase, or supplemental L-tryptophan. Thus, Fc-dependent antibodies protected against R. conorii infection of endothelium and macrophages by opsonization that inhibited phagosomal escape and resulted in phagolysosomal killing mediated by nitric oxide, reactive oxygen intermediates, and L-tryptophan starvation.     

Three-dimensional reconstruction of Coxiella burnetii-infected L929 cells by high-voltage electron microscopy.

Previous examination of thin sections of L929 cells heavily infected with the Q fever Priscilla isolate by conventional transmission electron microscopy indicated that the rickettsiae resided within multiple vacuoles. The present study using high-voltage electron microscopy and three-dimensional reconstruction revealed that, in heavily infected cells, the rickettsiae, in fact, reside in one multilobed vacuole. As a result of asymmetric cell division, the multilobed vacuole containing the rickettsiae apparently segregates into one daughter cell, while the companion daughter cell emerges parasite free. This likely explains the appearance of naive uninfected cells in long-term-infected (i.e., ca. 2 years) cell populations that had not been supplemented with uninfected L929 host cells.     

Involvement of lipid rafts in the budding-like exit of Orientia tsutsugamushi

Orientia tsutsugamushi is an intracellular parasite that causes scrub typhus. After entering the cytoplasm by induced phagocytosis, O. tsutsugamushi escapes from the primary phagosome into the host cytosol, where it replicates slowly. Subsequently, it is released from the host cells by a process resembling viral budding with a remaining bacterial aggregate near the nucleus. Lipid rafts have been implicated in the life cycle of a wide variety of pathogenic microorganisms. We have observed that proteins of O. tsutsugamushi were co-fractionated with the lipid rafts over a sucrose density gradient, suggesting the possible involvement of lipid rafts during the intracellular life cycle of O. tsutsugamushi. The entry of O. tsutsugamushi into the host cells was shown to be independent on lipid rafts as judged by the inability of lipid raft-disrupting agents to inhibit bacterial entry and no co-localization of bacterial proteins with caveolin. To our interest, a 47-kDa protein (HtrA) was observed to be co-localized with caveolin at the cell membrane at 72 h after infection, when bacterial particles move to the cell membrane and initiate the exit into the extracellular environment. Our results suggest that O. tsutsugamushi involves lipid rafts of the host cells in the budding-like process to exit from host cells.     

Interferon-gamma and tumor necrosis factor-alpha exert their antirickettsial effect via induction of synthesis of nitric oxide.

How the host defenses control rickettsiae in the cytosol of nonphagocytic host cells, where they are not exposed to antibodies or phagocytes, has posed a difficult question. Rickettsia conorii infection of a mouse fibroblast cell line was inhibited in a dose-dependent manner by nitrogen oxide synthesized by eukaryotic host cells stimulated by interferon-gamma or tumor necrosis factor-alpha. L-arginine was the source of the nitric oxide as demonstrated by competitive inhibition by NG-monomethyl-L-arginine. Nitric oxide synthesis required host cell protein synthesis and had an approximately 48-hour lag phase following cytokine stimulation. At low doses of interferon-gamma and tumor necrosis factor-alpha, which had no detectable response as single agents, dramatic synergistic nitric oxide synthesis and antirickettsial effects were observed.     

Acquisition of the vacuolar ATPase proton pump and phagosome acidification are essential for escape of Francisella tularensis into the macrophage cytosol

The Francisella tularensis-containing phagosome (FCP) matures to a late-endosome-like phagosome prior to bacterial escape into the cytosols of macrophages, where bacterial proliferation occurs. Our data show that within the first 15 min after infection of primary human monocyte-derived macrophages (hMDMs), approximately 90% of the FCPs acquire the proton vacuolar ATPase (vATPase) pump and the lysomotropic dye LysoTracker, which concentrates in acidic compartments, similar to phagosomes harboring the Listeria monocytogenes control. The acquired proton vATPase pump and lysomotropic dye are gradually lost by 30 to 60 min postinfection, which coincides with bacterial escape into the cytosols of hMDMs. Colocalization of phagosomes harboring the iglD mutant with the vATPase pump and the LysoTracker dye was also transient, and the loss of colocalization was faster than that observed for the wild-type strain, which is consistent with the faster escape of the iglD mutant into the macrophage cytosol. In contrast, colocalization of both makers with phagosomes harboring the iglC mutant was persistent, which is consistent with fusion to the lysosomes and failure of the iglC mutant to escape into the macrophage cytosol. We have utilized a fluorescence microscopy-based phagosome integrity assay for differential labeling of vacuolar versus cytosolic bacteria, using antibacterial antibodies loaded into the cytosols of live hMDMs. We show that specific inhibition of the proton vATPase pump by bafilomycin A1 (BFA) blocks rapid bacterial escape into the cytosols of hMDMs, but 30% to 50% of the bacteria escape into the cytosol by 6 to 12 h after BFA treatment. The effect of BFA on the blocking of bacterial escape into the cytosol is completely reversible, as the bacteria escape after removal of BFA. We also show that the limited fusion of the FCP to lysosomes is not due to failure to recruit the late-endosomal fusion regulator Rab7. Therefore, within few minutes of its biogenesis, the FCP transiently acquires the proton vATPase pump to acidify the phagosome, and this transient acidification is essential for subsequent bacterial escape into the macrophage cytosol.     

von Willebrand factor release and thrombomodulin and tissue factor expression in Rickettsia conorii-infected endothelial cells.

Mediterranean spotted fever, a tick-borne rickettsiosis caused by Rickettsia conorii, may lead to small-vessel or deep-vein thrombosis. In order to evaluate the role of endothelial cell alteration in this lesion, we infected human endothelial cells derived from umbilical veins with R. conorii. We report the induction of two previously unreported prothrombotic mechanisms in rickettsial disease: (i) a progressive decline in thrombomodulin antigen and (ii) early expression of tissue factor, and, as described for R. rickettsii infection, later release of von Willebrand factor from Weibel-Palade bodies. Thrombomodulin expression in infected endothelial cells, measured by the thrombin-dependent activation of protein C or flow cytometric analysis, decreased steadily between 4 and 24 h after inoculation with rickettsiae. R. conorii infection induced tissue factor expression, measured by clotting assay and flow cytometric analysis, which was detectable 2 h postinoculation, reached its maximum 4 h postinoculation, and progressively decreased thereafter. Infection resulted in a relatively late release of von Willebrand factor antigen into the culture medium. A double-label immunofluorescence assay for the simultaneous evaluation of von Willebrand factor and R. conorii showed that the depletion of cytoplasmic von Willebrand factor stored in Weibel-Palade bodies was due to a direct effect of the intracellular R. conorii. These disturbances of endothelial function observed with R. conorii-infected cells may provide a paradigm for the elucidation of thrombotic pathobiology with Mediterranean spotted fever.     

Phagocytosis of chrysotile fibers by pleural mesothelial cells in culture.

Pleural mesothelial cells (PMC) from the parietal pleura of rats were incubated in culture with UICC A chrysotile fibers. The sequence of events in phagocytosis was studied by electron microscopy: phases of attachment sequestration, and degranulation of lysosomal content into the phagocytic vacuole were observed. This demonstrates that PMC can engage in phagocytosis of chrysotile fibers.     

Electron Microscopy of the Phagocytosis of Capsulated Bacillus anthracis

The phagocytosis of capsulated vegetative cells of Bacillus anthracis in the mouse spleen was studied by thin sectioning techniques of electron microscopy. Mice were injected with autoclaved suspensions of capsulated and noncapsulated vegetative cells via the tail vein. The animals were killed 5, 10, and 30 min and 4 hr postinjection, with the central portion of the spleen being removed and procssed for electron microscopy. Fixation was with 2% KMnO₄ for 2 hr. Results of this study indicated that phagocytosis of autoclaved, capsulated anthrax bacilli was through the normal phagocytic process. The vesicular membrane or phagosome membrane was still present around these killed, capsulated organisms 4 hr postinjection. It is concluded that such cells possess no toxic principle to account for the supposed destruction of the phagosome membrane previously reported.     

Localization of electron-dense tracers during entry of Rickettsia tsutsugamushi into polymorphonuclear leukocytes.

The invasion of Rickettsia tsutsugamushi, Gilliam strain, into guinea pig polymorphonuclear leukocytes (PMNs) and the localization and distribution of tracers were followed during the process by electron microscopy. The seven tracers used were: cationized ferritin, ferritin, thorium dioxide (ThO2), carbon particles, latex spheres, paraffin oil, and Escherichia coli. These markers were added to the incubation medium containing the PMNs before or simultaneously with R. tsutsugamushi-infected BHK-21 cells. Both morphologically intact and degenerating rickettsiae were present in the phagosomes in PMNs, but only the viable-appearing rickettsiae were free in the cytoplasm. The intact rickettsiae were singly and selectively phagocytized in tightly enclosed phagosomal membranes which usually excluded the tracers, except when ThO2 or ferritin was used. When ThO2, which labels the plasma membrane of PMNs, was used. ThO2-labeled phagosomal membranes enclosing rickettsiae were observed and short membrane fragments still labeled with this tracer were found in the vicinity of rickettsiae in the cytoplasmic matrix of PMNs. When ferritin or ThO2 was used as a tracer, some of the phagosomes contained rickettsiae still enclosed in an envelope of BHK-21 cytoplasm and cell membrane. Phagolysosomes preloaded with electron-dense markers fused with subsequently formed phagosomes containing degenerated rickettsiae but not with those containing intact rickettsiae. These results support our interpretation that viable rickettsial entry into PMNs is by selective phagocytosis and escape from these phagosomes.     

Cross-reactive lymphocyte responses and protective immunity against other spotted fever group rickettsiae in mice immunized with Rickettsia conorii.

Lymphocyte proliferation in response to antigens on spotted fever group rickettsiae was used as a method to investigate the group-specific protective immunity to rechallenge characteristic of this group of rickettsiae at the T-cell receptor level. Spleen cells from Rickettsia conorii-immune C3H/HeJ mice proliferated in response to R. rickettsii Sheila Smith, R. sibirica 246, R. australis, and all tested strains of R. conorii (Casablanca, Moroccan, and Malish). Spleen cells from these mice, however, responded poorly or not at all to antigens prepared from the Kaplan or Hartford strain of R. akari. Proliferation of immune T cells maintained as in vitro cell lines showed a similar pattern of reactivity to these antigens; however, response to R. akari was consistently demonstrable. Spleen cells from C3H/HeJ mice immunized with R. akari responded to R. akari and R. conorii antigens as well as antigens from the other spotted fever group rickettsiae. Lymphocytes obtained from lymph nodes draining foot pads infected with R. conorii or R. akari demonstrated cross-reactivity similar to that found with immune spleen cells. If immunization was accomplished with R. conorii antigen emulsified in Freund complete adjuvant, the resulting lymph node cells were able to respond to R. akari antigens. These data suggest that infection with R. conorii induces a population of T lymphocytes that recognize an antigen(s) that also is found on other spotted fever rickettsiae and that may be responsible for cross-protective immunity. This antigen probably is not a major antigen on R. akari.     

Selective enrichment of NADPH oxidase activity in phagosomes from guinea pig polymorphonuclear leukocytes

Recent studies have demonstrated that the activated NADPH oxidase, the enzyme responsible for the stimulation of O₂ consumption with O ₂ ^– formation during phagocytosis, is located in the plasma membrane of leukocytes. The present work deals with whether the activation induced by phagocytosis involves the enzyme of the entire membrane or only that of the portion of the membrane that interacts with the phagocytosable particle and forms the phagosome. The results presented show that the activity of the NADPH oxidase of phagosomal membrane, isolated by centrifugation of homogenates on discontinuous sucrose gradients, is increased 12.6-fold with respect that of homogenate. In contrast, the activities of 5-nucleotidase and of acidp-nitrophenyl phosphatase, enzyme markers of the plasma membrane not activated during phagocytosis and uniformly distributed on the entire membrane, are increased only about threefold with respect to that of homogenate. These results indicate that during phagocytosis the activation of NADPH oxidase is a segmentary response that involves only the enzyme that forms the phagocytic vacuole. This fact is relevant for the function of toxic intermediates of oxygen reduction that are discharged in direct contact with the engulfed agent.