rOmpA is a critical protein for the adhesion ofRickettsia rickettsiito host cells

rOmpA and rOmpB are immunodominant, surface-exposed proteins ofRickettsia rickettsii.Prior evidence suggests that adhesion ofR. rickettsiito the host cell is mediated by a rickettsial protein. Five monoclonal antibodies to rOmpA, five to rOmpB, and one to the rickettsial lipopolysaccharide (LPS) were tested for inhibition of rickettsial attachment. All the monoclonal antibodies to rOmpA inhibited adhesion of rickettsiae to the L-929 cells with some inhibition rates as high as 90%. In contrast, monoclonal antibodies to rOmpB and LPS did not block attachment. When Fab fragments of monoclonal antibodies against rOmpA and rOmpB were used, similar results were observed as for the intact monoclonals, non-adhesion and adhesion, respectively. Purified rOmpA showed a competitive inhibitive effect on the attachment ofR. rickettsiito host cells. Trypsin completely digested rOmpA but not rOmpB from the surface of intactR. rickettsii, resulting in loss of the ability of the rickettsiae to attach to the host cell. rOmpA appears to play an important role in the initial adhesion ofR. rickettsiito the host cell.     

Gamma interferon as a crucial host defense against Rickettsia conorii in vivo.

Gamma interferon (IFN-gamma) plays an important role as a host defense in rickettsial infection. Swiss Webster mice, which are resistant to Rickettsia conorii (Malish 7 strain) infection, were treated with a monoclonal antibody against mouse IFN-gamma. When the antibody-treated mice were inoculated with 12 50% tissue culture infective doses of R. conorii, the mortality was 47% and the morbidity was 100%. None of the control mice, which received the same dose of R. conorii, died or became ill. The enumeration of rickettsiae in organs by direct immunofluorescence in paraffin sections demonstrated higher quantities of rickettsiae in the spleen had liver of IFN-gamma-depleted mice as compared with those of the infected controls. The kinetic analysis of IFN-gamma levels in sera showed depletion in the treated mice. These results indicate that IFN-gamma plays an important role as a host defense in the early stage of rickettsial infection. Survival of some mice despite continued treatment with antibody to IFN-gamma suggests that other immune mechanisms may also be important.     

Intracellular movements of Rickettsia conorii adn R. typhi based on actin polymerization

Human vascular endothelial, Vero and human embryonic lung cells infected with rickettsiae for 24 h or 48 h were labelled for polymerized actin with NBD-phallacidin. Between 20 and 68% of the intracellular Rickettsia conorii had an actin tail of between 0.33 and 15 microns, with the longest tails being observed in Vero cells. In the case of R. typhi less than 1% of the organisms had actin tails and these were considerably shorter than those of R. conorii. These findings provide new information concerning the different cytopathic effects observed with the two rickettsial species.     

Mechanisms of intracellular killing of Rickettsia conorii in infected human endothelial cells, hepatocytes, and macrophages

The mechanism of killing of obligately intracellular Rickettsia conorii within human target cells, mainly endothelium and, to a lesser extent, macrophages and hepatocytes, has not been determined. It has been a controversial issue as to whether or not human cells produce nitric oxide. AKN-1 cells (human hepatocytes) stimulated by gamma interferon, tumor necrosis factor alpha, interleukin 1beta, and RANTES (regulated by activation, normal T-cell-expressed and -secreted chemokine) killed intracellular rickettsiae by a nitric oxide-dependent mechanism. Human umbilical vein endothelial cells (HUVECs), when stimulated with the same concentrations of cytokines and RANTES, differed in their capacity to kill rickettsiae by a nitric oxide-dependent mechanism and in the quantity of nitric oxide synthesized. Hydrogen peroxide-dependent intracellular killing of R. conorii was demonstrated in HUVECs, THP-1 cells (human macrophages), and human peripheral blood monocytes activated with the cytokines. Rickettsial killing in the human macrophage cell line was also mediated by a limitation of the availability of tryptophan in association with the expression of the tryptophan-degrading enzyme indoleamine-2,3-dioxygenase. The rates of survival of all of the cell types investigated under the conditions of activation and infection in these experiments indicated that death of the host cells was not the explanation for the control of rickettsial infection. This finding represents the first demonstration that activated human hepatocytes and, in some cases, endothelium can kill intracellular pathogens via nitric oxide and that RANTES plays a role in immunity to rickettsiae. Human cells are capable of controlling rickettsial infections intracellularly, the most relevant location in these infections, by one or a combination of three mechanisms involving nitric oxide synthesis, hydrogen peroxide production, and tryptophan degradation.     

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.     

Analysis of T-cell-dependent and -independent antigens of Rickettsia conorii with monoclonal antibodies.

Four monoclonal antibodies from euthymic mice and two monoclonal antibodies from athymic mice were directed against antigens of Rickettsia conorii, as shown by both indirect immunofluorescence and an enzyme immunoassay. There was extensive cross-reactivity with other spotted fever group rickettsiae. Euthymic monoclonal antibodies 3-2 and 9-2 (immunoglobulin G2a [IgG2a]) and 27-10 (IgG1) distinctly outlined the acetone-fixed rickettsial surface, as determined by indirect immunofluorescence; only monoclonal antibody 3-2 reacted with the intact rickettsial surface, as determined by colloidal gold-protein A negative-stain electron microscopy. Athymic monoclonal antibodies 32-2 and 35-3 (IgM) and euthymic monoclonal antibody 31-15 (IgG3) all demonstrated an irregular, extrarickettsial morphology, as determined by immunofluorescence, and ultrastructural cell wall blebs that were readily shed from the rickettsial surface. Monoclonal antibody 3-2, the only antibody to confer protection in lethally challenged mice, reacted with a high-molecular-weight protein in Western immunoblots. Monoclonal antibodies 31-15, 32-2, and 35-3 reacted with a "ladder" of proteinase K-resistant, lipopolysaccharidelike antigens. None of the monoclonal antibodies stabilized the ultrastructural rickettsial slime layer, but both athymic and euthymic polyclonal antibodies to R. conorii did. This is, to the best of our knowledge, the first report of the production of monoclonal antibodies to R. conorii and their use for antigenic analysis.     

Interaction of rickettsiae with haemocytes of Alveonasus lahorensis ticks

The development of different rickettsial species (Rickettsia sibirica, R. conorii, R. acari and coxiella burnetii) in haemocytes of Alveonasus lahorensis ticks was compared by viral observations and time-lapse cine-micrography. The interaction of different rickettsial species with the haemocytes in vitro had specific characteristic features and reflected the pattern of interaction of the rickettsiae with tick cells in vivo.     

Protective monoclonal antibodies recognize heat-labile epitopes on surface proteins of spotted fever group rickettsiae.

Thirty-eight monoclonal antibodies that have not been reported previously were developed from mice immunized with Rickettsia rickettsii, R. conorii, and R. sibirica. Western immunoblotting showed that these monoclonal antibodies are directed against heat-sensitive epitopes which are located on two major surface polypeptides with molecular sizes ranging from 115 to 150 kilodaltons. The detection of the two bands did not depend on the presence of 2-mercaptoethanol. Both bands were destroyed by treatment with proteinase K. Monoclonal antibodies examined by immunofluorescence assay reacted with epitopes that are species specific, group reactive, or shared among a smaller subset of species of spotted fever group rickettsiae. Nine of the monoclonal antibodies were evaluated for their ability to neutralize rickettsial infection and thus protect animals against disease caused by homologous species of rickettsiae. Treatment of rickettsiae with monoclonal antibodies F3-12, F3-14, and F3-36 completely protected guinea pigs against illness caused by the homologous organism R. rickettsii. Monoclonal antibodies F9-5G11 and F15-5B12, derived from mice immunized with R. sibirica, conferred partial protection by delaying the onset and shortening the duration of fever in guinea pigs inoculated with R. sibirica. Monoclonal antibodies F2-15, F2-31, F2-53, and F3-12 protected mice from a lethal infection with R. conorii. Heat-labile epitopes of spotted fever group rickettsial surface proteins are important candidate antigens for development of vaccines to confer protective immunity.     

Directional actin polymerization associated with spotted fever group Rickettsia infection of Vero cells.

Members of the spotted fever group (SFG) of rickettsiae spread rapidly from cell to cell by an unknown mechanism(s). Staining of Rickettsia rickettsii-infected Vero cells with rhodamine phalloidin demonstrated unique actin filaments associated with one pole of intracellular rickettsiae. F-actin tails greater than 70 microns in length were seen extending from rickettsiae. Treatment of infected cells with chloramphenicol eliminated rickettsia-associated F-actin tails, suggesting that de novo protein synthesis of one or more rickettsial proteins is required for tail formation. Rickettsiae were coated with F-actin as early as 15 min postinfection, and tail formation was detected by 30 min. A survey of virulent and avirulent species within the SFG rickettsiae demonstrated that all formed actin tails. Typhus group rickettsiae, which do not spread directly from cell to cell, lacked F-actin tails entirely or exhibited only very short tails. Transmission electron microscopy demonstrated fibrillar material in close association with R. rickettsii but not Rickettsia prowazekii. Biochemical evidence that actin polymerization plays a role in movement was provided by showing that transit of R. rickettsii from infected cells into the cell culture medium was inhibited by treatment of host cells with cytochalasin D. These data suggest that the cell-to-cell transmission of SFG rickettsiae may be aided by induction of actin polymerization in a fashion similar to that described for Shigella flexneri and Listeria monocytogenes.     

Serological approach to the occurrence of rickettsioses in the Central African Republic

A serosurvey for evidence of human rickettsial infections was carried out in the Republic of Central Africa on 144 sera by indirect immunofluorescence (IIF) and microagglutination tests (MA). There was no serological evidence of epidemic typhus and only two sera were positive for murine typhus. Approximately 15% of the surveyed population was serologically positive by MA for R. conorii antibodies. However, 48% of this population had spotted fever group antibodies as detected by IIF but were negative in MA for R. conorii, R. rickettsii and R. akari antibodies. These sera with high titers in IIF and negative in MA lead us to believe that in Central Africa there are rickettsiae pathogenic for man that are related to the Spotted Fever group and are yet to be identified.     

Inhibition of Rickettsia conorii growth by recombinant tumor necrosis factor alpha: enhancement of inhibition by gamma interferon.

Purified human recombinant tumor necrosis factor alpha (rTNF-alpha) inhibited the growth of Rickettsia conorii (Casablanca strain) in HEp-2 cell culture. The effect was observed when the cells were pretreated with rTNF-alpha or when rTNF-alpha was added after adsorption of the rickettsiae. The inhibitory effect of rTNF-alpha on rickettsial growth was enhanced by gamma interferon. Cycloheximide had no effect on inhibition of the rickettsial yield, suggesting that de novo protein synthesis is not required for the inhibitory effect of rTNF-alpha. The addition of tryptophan partially abolished the inhibitory effect of rTNF-alpha and rTNF-alpha plus gamma interferon.     

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.     

Rickettsia conorii entry into Vero cells.

The entry of rickettsiae into eukaryotic cells is mediated by an induced phagocytosis, but rickettsiae have never been observed in a closed phagocytic vacuole. In this study, Rickettsia conorii entry into Vero cells was observed by transmission electron microscopy during a period of 3 to 20 min after bacterium-cell contact. The entry occurred within 3 min after bacterium-cell contact, and R. conorii was observed in the process of engulfment, within a phagocytic vacuole, or free in the cytosol. Escape from the phagosome is a very rapid step since phagosome lysis was only occasionally observed. By 12 min, 90% of bacteria were internalized and half were free in the cytosol. This report confirms that rickettsiae penetrate nonphagocytic cells by induced phagocytosis and is the first demonstration of rickettsiae within a complete phagocytic vacuole.     

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.     

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.     

Distribution of immunogenic epitopes on the two major immunodominant proteins (rOmpA and rOmpB) of Rickettsia conorii among the other rickettsiae of the spotted fever group.

Forty-four monoclonal antibodies were raised against strain Seven, the type strain of Rickettsia conorii. Of these 44 monoclonal antibodies, 13, 27, and 4 were demonstrated to be directed against the 116-kDa protein (rOmpA), the 124-kDa protein (rOmpB), and lipopolysaccharide-like antigen, respectively. The antiprotein monoclonal antibodies were found to be directed against 29 distinct epitopes, which were located on the two major immunodominant proteins discussed above. Further analysis showed that strain-specific epitopes were located on the rOmpA protein and species- and subgroup-specific epitopes were located on the rOmpB protein. R. conorii Manuel, Indian tick typhus rickettsia, and Kenya tick typhus rickettsia also possessed all 29 epitopes, whereas the other rickettsiae of the spotted fever group (SFG) expressed between 3 and 25 epitopes, with the exception of Rickettsia helvetica, R. akari, and R. australis which did not possess any epitopes. Additional analyses by Western immunoblotting confirmed that the epitopes shared among the SFG rickettsiae were located on the same two high-molecular-mass proteins as on R. conorii. However, although epitopes on the R. conorii rOmpB protein were expressed on the rOmpB proteins of most other SFG rickettsiae, some were found on the rOmpA proteins of R. aeschlimannii, R. rickettsii, and R. rhipicephali. Both proteins possessing the common epitopes were found to have different sizes in the SFG rickettsial species. The different distributions of common epitopes in the SFG rickettsiae were also used to build a taxonomic dendrogram, which demonstrated that all the R. conorii strains formed a relatively independent cluster within the SFG rickettsiae and was generally consistent with previously proposed taxonomies.     

Rickettsia prowazekii requires host cell serine and glycine for growth.

The growth requirement of Rickettsia prowazekii for the amino acids serine and glycine was assessed in both wild-type cell lines and a mutant cell line. X-irradiated L929 cells supported the growth of R. prowazekii when the cells were incubated in Eagle minimal essential medium supplemented with serum. In contrast, in this medium, X-irradiated Vero cells did not support the growth of rickettsiae unless cycloheximide, serine, or glycine was added. Other nonessential amino acids, additional glucose, and potential products of host cell metabolism of serine and glycine were nonstimulatory. The concentration of serine or glycine required to support rickettsial growth had no effect on the doubling time of uninfected, unirradiated Vero cells. A comparison of intracellular amino acid pools indicated that the serine and glycine concentrations in mock-infected Vero cells were approximately 31 and 14% of the respective concentrations in mock-infected L929 cells. The pools of both amino acids in Vero cells increased markedly upon treatment of the cells with cycloheximide. Interconversion of serine and glycine catalyzed by serine hydroxymethyltransferase was detected in cell-free extracts of purified rickettsiae. However, this enzymatic activity did not permit rickettsial growth in a glycine-requiring clone (772-56d) of the Chinese hamster ovary cell CHO-K1 in the absence of glycine supplementation. These data indicate that R. prowazekii depends on the host cell for serine or glycine.     

Differential interaction of dendritic cells with Rickettsia conorii: impact on host susceptibility to murine spotted fever rickettsiosis

Spotted fever group rickettsioses are emerging and reemerging infectious diseases, some of which are life-threatening. In order to understand how dendritic cells (DCs) contribute to the host resistance or susceptibility to rickettsial diseases, we first characterized the in vitro interaction of rickettsiae with bone marrow-derived DCs (BMDCs) from resistant C57BL/6 (B6) and susceptible C3H/HeN (C3H) mice. In contrast to the exclusively cytosolic localization within endothelial cells, rickettsiae efficiently entered and localized in both phagosomes and cytosol of BMDCs from both mouse strains. Rickettsia conorii-infected BMDCs from resistant mice harbored higher bacterial loads compared to C3H mice. R. conorii infection induced maturation of BMDCs from both mouse strains as judged by upregulated expression of classical major histocompatibility complex (MHC) and costimulatory molecules. Compared to C3H counterparts, B6 BMDCs exhibited higher expression levels of MHC class II and higher interleukin-12 (IL-12) p40 production upon rickettsial infection and were more potent in priming na?ve CD4(+) T cells to produce gamma interferon. In vitro DC infection and T-cell priming studies suggested a delayed CD4(+) T-cell activation and suppressed Th1/Th2 cell development in C3H mice. The suppressive CD4(+) T-cell responses seen in C3H mice were associated with a high frequency of Foxp3(+) T regulatory cells promoted by syngeneic R. conorii-infected BMDCs in the presence of IL-2. These data suggest that rickettsiae can target DCs to stimulate a protective type 1 response in resistant hosts but suppressive adaptive immunity in susceptible hosts.     

Genetic variation in Australian spotted fever group rickettsiae.

Rickettsiae were isolated by cell culture of buffy coat blood from six patients with spotted fever from southeastern Australia and Flinders Island in Bass Strait. The isolates were genetically compared with two previous Rickettsia australis patient isolates. The genus-specific 17-kDA genes from the isolates were compared after DNA amplification and restriction fragment analysis of the amplified DNA. This comparison revealed that mainland rickettsial isolates from southeastern Australia were identical to two previous isolates of R. australis from northeastern Australia. Rickettsial isolates from Flinders Island were distinct from the mainland isolates. The 16S rRNA gene sequences from the isolates were determined and compared. The Flinders Island rickettsial agent was most closely related (0.3% structural divergence) to Rickettsia rickettsii, Rickettsia conorii, and Rickettsia slovaca. The Flinders Island rickettsial agent was 1.3 and 2.1% structurally divergent from R. australis and Rickettsia akari, respectively. The 16S rRNA gene sequence from the Flinders Island agent shows that this rickettsia is more closely related to the rickettsial spotted fever group than is R. australis. We conclude that there are two populations of spotted fever group rickettsiae in Australia and propose that the genetically distinct causative organism of Flinders Island spotted fever be designated Rickettsia honei. The extent of distribution and animal host reservoirs remain to be elucidated.