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On the nature of obligate intracellular symbiosis of rickettsiae--Rickettsia prowazekii cells import mitochondrial porin.

Mitochondrial porin was identified in Rickettsia prowazekii by Western blot analysis of whole cells and membrane fractions with monoclonal antibody against porin VDAC 1 of animal mitochondria. Using the BLAST server, no protein sequences homologous to mitochondrial porin were found among the rickettsial genomes. Rickettsiae also do not contain their own porin. The protein imported by rickettsiae is weakly extracted by nonionic detergents and, like porin in mitochondria, is insensitive to proteinase K in whole cells. Immunocytochemical analysis showed that it localizes to the outer membrane of the bacterial cells. These data support an earlier suggestion about import by rickettsiae of indispensable proteins from cytoplasm of the host cell as a molecular basis of obligate intracellular parasitism. They are also consistent with the hypothesis invoking a transfer of genes specifying surface proteins from the last common ancestor of rickettsiae and mitochondria to the host genome, and preservation by rickettsiae of the primitive ability to import these proteins.

Active Transport, Cell Nucleus↗

Structural properties of lipopolysaccharides from Rickettsia typhi and Rickettsia prowazekii and their chemical similarity to the lipopolysaccharide from Proteus vulgaris OX19 used in the Weil-Felix test.

The lipopolysaccharides (LPSs) isolated from typhus group (TG) rickettsiae Rickettsia typhi and Rickettsia prowazekii were characterized by chemical analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining. LPSs from two species of TG rickettsiae contained glucose, 3-deoxy-D-manno-octulosonic acid, glucosamine, quinovosamine, phosphate, and fatty acids (beta-hydroxylmyristic acid and heneicosanoic acid) but not heptose. The O-polysaccharides of these LPSs were composed of glucose, glucosamine, quinovosamine, and phosphorylated hexosamine. Resolution of these LPSs by their apparent molecular masses by SDS-PAGE showed that they have a common ladder-like pattern. Based on the results of chemical composition and SDS-PAGE pattern, we suggest that these LPSs act as group-specific antigens. Furthermore, glucosamine, quinovosamine, and phosphorylated hexosamine were also found in the O-polysaccharide of the LPS from Proteus vulgaris OX19 used in the Weil-Felix test, suggesting that they may represent the antigens common to LPSs from TG rickettsiae and P. vulgaris OX19.

Antigens, Bacterial↗

Evaluation of the pathogenic potential of Rickettsia canada and Rickettsia prowazekii organisms in dogs.

An unusual pattern of seroreactivity to antigens of rickettsial organisms (Rickettsia rickettsii, R rhipicephali, R montana, and R bellii), particularly to R bellii antigen, was detected in 3 dogs during a 2-month period. Thus, studies were initiated to clarify the pathogenic potential of the more distantly related rickettsial organisms (R canada and R prowazekii) in dogs. Because R bellii are nonpathogenic rickettsiae that share numerous common properties with spotted fever-group and typhus-group rickettsiae, and because closely related pathogenic relatives of R bellii have not been identified, we examined the pathogenic potential of these typhus-group rickettsiae by testing stored serum samples, by attempting rickettsial isolation from febrile dogs, and by experimentally inoculating dogs with R canada and R prowazekii. Evaluation of results of a serosurvey of acute and convalescent serum samples from 80 dogs in which Rocky Mountain spotted fever had been considered as a differential diagnosis, but seroconversion to R rickettsii had not been documented, identified 1 dog with a fourfold increase in antibody titer to R rhipicephali and 3 dogs with fourfold increases in antibody titer to 1 or more antigens of typhus-group rickettsial organisms. A study of 15 dogs that were febrile during summer months failed to identify serologic or tissue culture evidence of typhus-group rickettsial infection or typhus-group rickettsemia, but did result in isolation of R rickettsii and Ehrlichia canis, respectively, from 1 dog each. In our final study, after experimentally inoculating 6 dogs with R canada and R prowazekii, all dogs seroconverted to the respective rickettsiae, but rickettsemia or clinical and hematologic evidence of disease was not observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Antibodies to Orientia tsutsugamushi, Rickettsia typhi and spotted fever group rickettsiae among febrile patients in rural areas of Malaysia.

A serosurvey was conducted in 1995-97 among 1596 febrile patients from 8 health centres in Malaysia for antibodies against Orientia tsutsugamushi (OT), Rickettsia typhi (RT) and TT118 spotted fever group rickettsiae (SFGR) by using an indirect immunoperoxidase assay. A total of 51.4% patients had antibody against at least 1 of those rickettsiae. Antibody to SFGR was most prevalent (42.5%), followed by RT (28.1%) and OT (24.9%). The seroprevalences of antibodies to SFGR, RT or OT alone were 12.4, 3.6 and 4.3%, respectively. Antibodies against more than 1 species of rickettsiae were presence in 31.1% of the patients, suggesting the possibility of co-infection, previous exposures or serological cross-reactivities. Seroprevalence of the various rickettsiae varied according to locality, with SFGR antibodies being the most prevalent in most areas. There was no significant association of prevalence of rickettsial antibody with gender. The seroprevalence of OT, SFGR and RT increased with patient age but an increase of antibody titre with age was not significant. Those working in the agricultural sectors had significantly higher seroprevalence of OT, SFGR and RT than those not related with agricultural activities. Scrub typhus remains a public health problem with an estimated annual attack rate of 18.5%. Tick typhus and murine typhus as shown in this serosurvey appear much more widespread than scrub typhus in this country.

Adolescent↗

The rickettsial outer-membrane protein A and B genes of Rickettsia australis, the most divergent rickettsia of the spotted fever group.

The genes for rickettsial outer-membrane protein A (rOmpA), a distinguishing feature of spotted fever group (SFG) rickettsiae, and rOmpB, a genus-specific protein, were identified and sequenced in Rickettsia australis. The amino acid sequences of domains I, III and IV of the R. australis rOmpA share close homology with those of rOmpA of other SFG rickettsiae, but the repeat region (domain II) is dramatically different from that of other known SFG rOmpA. R. australis rOmpB is more similar to rOmpB of other SFG rickettsiae than to that of typhus group rickettsiae.

Amino Acid Substitution↗

Progress in rickettsial genome analysis from pioneering of Rickettsia prowazekii to the recent Rickettsia typhi.

Three rickettsial genomes have been sequenced and annotated. Rickettsia prowazekii and R. typhi have similar gene order and content. The few differences between R. prowazekii and R. typhi include a 12-kb insertion in R. prowazekii, a large inversion close to the origin of replication in R. typhi, and loss of the complete cytochrome c oxidase system by R. typhi. R. prowazekii, R. typhi, and R. conorii have 13, 24, and 560 unique genes, respectively, and share 775 genes, most likely their essential genes. The small genomes contain many pseudogenes and much noncoding DNA, reflecting the process of genome decay. R. typhi contains the largest number of pseudogenes (41), and R. conorii the fewest, in accordance with its larger number of genes and smaller proportion of noncoding DNA. Conversely, typhus rickettsiae contain fewer repetitive sequences. These genomes portray the key themes of rickettsial intracellular survival: lack of enzymes for sugar metabolism, lipid biosynthesis, nucleotide synthesis, and amino acid metabolism, suggesting that rickettsiae depend on the host for nutrition and building blocks; enzymes for the complete TCA cycle and several copies of ATP/ADP translocase genes, suggesting independent synthesis of ATP and acquisition of host ATP; and type IV secretion system. All rickettsiae share two outer membrane proteins (OmpB and Sca 4) and LPS biosynthesis machinery. RickA, unique to spotted fever rickettsiae, plays a role in induction of actin polymerization in R. conorii, but not in R. prowazekii or R. typhi. The genome of R. typhi contains four potentially membranolytic genes (tlyA, tlyC, pldA, and pat-1) and five autotransporter genes, sca 1, sca 2, sca 3, ompA, and ompB. The presence of six 50-amino acid repeat units in Sca 2 suggests function as an adhesin. The high laboratory passage of the sequenced strains raises the issue of the occurrence of laboratory mutations in genes not required for growth in cell culture or eggs. Resequencing revealed that eight annotated pseudogenes of E strain are actually intact genes. Comparative genomics of virulent and avirulent strains of rickettsial species may reveal their virulence factors.

Genome, Bacterial↗

Rickettsial antibody prevalence in southern Israel: IgG antibodies to Coxiella burnetii, Rickettsia typhi, and spotted fever group rickettsiae among urban- and rural-dwelling and Bedouin women.

A retrospective serological survey was carried out using sera obtained from women at childbirth in the southern desert region of Israel to determine exposure experience to three rickettsial agents: Coxiella burnetii, Rickettsia typhi, and spotted fever group rickettsiae. Using the indirect fluorescent antibody method for determining IgG antibodies, it was found that about 40% of all sera examined demonstrated antibodies to one or more rickettsiae. Bedouin women appeared to be at greater risk of having antibodies to C. burnetii and spotted fever group rickettsiae than did Jewish residents of Beersheba, agricultural settlements, and development towns. The residents of development towns appeared to be at lower risk of developing antibodies to spotted fever group rickettsiae than did other populations sampled. Possible reasons for these differences are discussed.

Antibodies, Bacterial↗

Isolation of a spotted fever group Rickettsia, Rickettsia peacockii, in a Rocky Mountain wood tick, Dermacentor andersoni, cell line.

An embryonic cell line (DAE100) of the Rocky Mountain wood tick, Dermacentor andersoni, was observed by microscopy to be chronically infected with a rickettsialike organism. The organism was identified as a spotted fever group (SFG) rickettsia by PCR amplification and sequencing of portions of the 16S rRNA, citrate synthase, Rickettsia genus-specific 17-kDa antigen, and SFG-specific 190-kDa outer membrane protein A (rOmpA) genes. Sequence analysis of a partial rompA gene PCR fragment and indirect fluorescent antibody data for rOmpA and rOmpB indicated that this rickettsia was a strain (DaE100R) of Rickettsia peacockii, an SFG species presumed to be avirulent for both ticks and mammals. R. peacockii was successfully maintained in a continuous culture of DAE100 cells without apparent adverse effects on the host cells. Establishing cell lines from embryonic tissues of ticks offers an alternative technique for isolation of rickettsiae that are transovarially transmitted.

Animals↗

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.

Animals↗

Rickettsia felis: a new species of pathogenic rickettsia isolated from cat fleas.

A flea-borne rickettsia, previously referred to as ELB, has been implicated as a cause of human illness. Using sequence data obtained from a fragment of the citrate synthase gene, we compared ELB, Rickettsia australis, R. rickettsii, and R. akari with the louse-borne R. prowazekii. We tallied 24 base pair differences between ELB and R. prowazekii and 25 between R. rickettsii and R. prowazekii; there were 30 base pair differences between R. australis and R. prowazekii and 29 between R. akari and R. prowazekii. We observed 32 differences between Rickettsia typhi and ELB. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses of ELB, with typing sera against R. typhi indicate that ELB surface antigens are more closely related to the flea-borne R. typhi than to the mite-borne R. akari. On the basis of the results of citrate synthase gene sequence comparisons, as well as previous comparisons with 16S rRNA and 17-kDa-protein gene segments, we found that ELB is sufficiently genetically distinct from other rickettsiae to be designated a new species, Rickettsia felis.

Animals↗

Rickettsia rickettsii and Rickettsia montana from Ixodid ticks in Connecticut.

Dermacentor variabilis, infected with spotted fever group rickettsiae, parasitized 8 of 70 raccoons captured in Newtown, Connecticut. The spotted fever agent, Rickettsia rickettsii, was isolated and identified from 4 adult D. variabilis and from 1 nymphal Ixodes texanus removed from raccoons. This verifies the presence of this etiologic agent in ticks in an area where 6 people had clinical signs and symptoms of Rocky Mountain spotted fever (RMSF) and antibodies to R. rickettsii. These are the first isolations of R. rickettsii from D. variabilis in southern New England and the first identified rickettsiae from I. texanus. No rickettsiae were isolated from Ixodes muris or I. cookei. Rickettsia montana was recovered in Vero cell culture from a D. variabilis collected in East Haddam, Connecticut where RMSF is not known to be prevalent.

Animals↗

Development of quantitative real-time PCR assays to detect Rickettsia typhi and Rickettsia felis, the causative agents of murine typhus and flea-borne spotted fever.

Rickettsia typhi and Rickettsia felis are the etiologic agents of murine typhus and flea-borne spotted fever, respectively. We have constructed two quantitative real-time polymerase chain reaction (qPCR) assays to detect these pathogenic rickettsiae. The qPCR assays were developed utilizing unique sequences of the R. typhi and R. felis outer membrane protein B genes (ompB) to design the specific primers and molecular beacon probes. The assays were found to be species-specific and did not yield false-positive reactions with nucleic acid from other rickettsiae, orientiae, neorickettsiae or unrelated bacteria. In addition, the assays were sensitive enough to detect three target sequence copies per reaction and were capable of detecting R. typhi and R. felis nucleic acid in the cat flea, Ctenocephalides felis. These results demonstrate that two sensitive and specific qPCR assays have been successfully developed to detect and enumerate R. typhi and R. felis.

Animals↗

Prevalence of antibodies to Coxiella burnetti, Rickettsia conorii, and Rickettsia typhi in seven African countries.

The prevalences of antibodies reactive with Coxiella burnetti, Rickettsia conorii, and Rickettsia typhi were determined by indirect fluorescent antibody testing of sera from seven African countries. The seroprevalences of antibodies reactive with C. burnetti and R. conorii in countries from North Africa were similar to those reported from southern Europe. In countries of sub-Saharan Africa, the seroprevalence of antibodies reactive with C. burnetti varied greatly; the generally higher figures for West Africa, where stock breeding is prominent, suggested that domestic animals might be the main reservoirs of infection. The prevalence of antibodies to the recently described species Rickettsia africae was higher than that reported for spotted fever-group rickettsiae from elsewhere in the world and paralleled the distribution of Amblyomma species. Western blot results suggested that the antibodies detected were more likely to be reactive with R. africae than with R. conorii, the main vector of which (Rhipicephalus species) rarely feeds on humans. The seroprevalences of antibodies reactive with R. typhi were higher in coastal regions, where Rattus norvegicus--the natural host of the vector Xenopsylla--is more prevalent, than in inland areas.

Africa↗

Differentiation of Rickettsia japonica by restriction endonuclease fragment length polymorphism using products of polymerase chain reaction amplification with Rickettsia rickettsii 190-kilodalton surface antigen gene primers.

Restriction fragment length polymorphism of polymerase chain reaction (PCR) amplification products differentiated Rickettsia japonica, a causative agent of Oriental spotted fever, from other spotted fever group (SFG) rickettsiae. Primer pair Rr190. 70p and Rr190. 602n of R. rickettsii 190-kDa antigen gene sequence primed genomic DNAs obtained from R. japonica, type strain YH and strains NT, NK, YKI, and TKN. The products were cleaved by PstI but not by AfaI restriction endonuclease. The PstI digestion pattern of PCR-products amplified from all strains of R. japonica was identical and easily differentiated from that of other SFG rickettsiae. The present study demonstrated a genotypic difference between R. japonica and other pathogenic SFG rickettsiae.

Animals↗

Experimentally infected human body lice (pediculus humanus humanus) as vectors of Rickettsia rickettsii and Rickettsia conorii in a rabbit model.

The human body louse, the natural vector of Rickettsia prowazekii, is able to experimentally transmit the normally flea-borne rickettsia R. typhi, suggesting that the relationships between the body louse and rickettsiae are not specific. We used our experimental infection model to test the ability of body lice to transmit two prevalent tick-borne rickettsiae. Each of two rabbits was made bacteremic by injecting intravenously 2 x 10(6) plaque-forming units of either R. rickettsii or R. conorii. Four hundred body lice were infected by feeding on the bacteremic rabbit and were compared with 400 uninfected lice. Each louse group was fed once a day on a separate seronegative rabbit. The survival of infected lice was not different from that of uninfected controls. Lice remained infected for their lifespan, excreted R. rickettsii and R. conorii in their feces, but did not transmit the infection to their progeny. The nurse rabbit of uninfected lice remained asymptomatic and seronegative. Those rabbits used to feed infected lice developed bacteremia and seroconverted. Although the body louse is not a known vector of spotted fevers, it was able in our study to acquire, maintain, and transmit both R. rickettsii and R. conorii.

Animals↗

Metabolic Activity of the Trench Fever Rickettsia, Rickettsia quintana.

A study of the metabolic activity of Rickettsia quintana was carried out by conventional Warburg and radioisotope techniques with intact cells harvested while growing in the fluid counterpart of the medium of Vinson and Fuller. Like other rickettsiae, R. quintana did not utilize glucose, but did metabolize glutamate and glutamine. Unlike typhus rickettsiae, R. quintana did not require a diluent high in K(+) for metabolic activity, and it utilized glutamine more efficiently than glutamate. In typical experiments, this microorganism produced 1.6 to 2.0 mumoles of CO(2) from glutamine per mg of rickettsial protein per hr at 37 C, while consuming 1.5 to 1.7 mumoles of O(2). R. quintana also utilized, in descending order, succinate, alpha-ketoglutarate, glutamate, pyruvate, and citrate; the first-named substrate was utilized more rapidly than glutamine. R. quintana, like typhus rickettsiae, has a glutamate-oxaloacetate transaminase because aspartate was isolated, by means of thin-layer chromatography, as one of the end products of the utilization of glutamine. When the microorganisms were incubated with glutamine-(14)C and unlabeled intermediates of the citric acid cycle, labeled dicarboxylic acids of the cycle were recovered. Labeled tricarboxylic acids, however, were not recovered, possibly because of cellular impermeability to the corresponding unlabeled intermediates. In the case of cis-aconitate, it was shown that this substrate interfered with the uptake of glutamine. These observations are believed to provide convincing evidence that glutamine is utilized through the citric acid cycle and that R. quintana, with the differences noted, resembles other rickettsiae.

Journal Article↗

Mapping of monoclonal antibody binding sites on CNBr fragments of the S-layer protein antigens of Rickettsia typhi and Rickettsia prowazekii.

The 120 kDa surface protein antigens (SPAs) of typhus rickettsiae lie external to the outer membrane in regular arrays and chemically resemble the S-layer proteins of other bacteria. These proteins elicit protective immune responses against the rickettsiae. In order to study the immunochemistry of these proteins, purified SPAs from Rickettsia typhi and Rickettsia prowazekii were fragmented with CNBr. The fragments were separated by SDS-PAGE and were recovered on PVDF membrane following electroblotting. The origin of eight major fragments from R. prowazekii and seven major fragments from R. typhi was determined by automated N-terminal amino acid sequencing and by comparison with the DNA sequence encoding R. prowazekii SPA. The cleavage patterns and protein sequences of the two proteins differed significantly. CNBr fragments corresponding to the C-terminus (amino acid 1372-1612 of the deduced sequence from encoding gene spaP) were not present in both SPAs. This suggests that the corresponding C-terminal region was not synthesized or was removed during SPA translocation to the cell surface. Modified amino acids were detected in each protein. Eighteen monoclonal antibodies selected for varied reactivity with both native and denatured SPA proteins could be classified into eight different types based on western blot analysis of the CNBr fragments. Six of the monoclonal antibody types reacted predominantly with a single region of the SPAs. Two types of antibodies bound to several CNBr fragments which contained both limited sequence similarity and modified amino acids either of which might account for the multisite binding of these antibodies.

Amino Acid Sequence↗

Characterization of the gene encoding the protective paracrystalline-surface-layer protein of Rickettsia prowazekii: presence of a truncated identical homolog in Rickettsia typhi.

The DNA sequence of the gene encoding the protective surface protein antigen (SPA) of Rickettsia prowazekii has been determined. The open reading frame of 4836 nucleotides with promoter and ribosome-binding site is present on a 10.1-kilobase EcoRI fragment. The encoded carboxyl terminus of the 169-kDa protein contains a potential transmembrane region and hydrophilic regions with many lysine and arginine residues potentially accessible to proteolytic cleavage. Because the rickettsia-derived SPA has an estimated molecular mass of only 120 kDa and does not contain several predicted large carboxyl-region CNBr fragments, the SPA product appears to be processed by the rickettsiae. Eight other CNBr fragments were identical in sequence to those predicted from the encoded gene. A complementary 8.7-kilobase EcoRI fragment of Rickettsia typhi DNA was cloned. This fragment lacked a 1433-base-pair region that included the promoter, ribosome-binding site, and the initial 1162 base pairs of the open reading frame encoding the R. prowazekii SPA but had a 3674-base-pair region identical with the remainder of the R. prowazekii SPA gene sequence.

Amino Acid Sequence↗