Analysis of antigenic characteristics of Rickettsia tsutsugamushi Boryong strain and antigenic heterogeneity of Rickettsia tsutsugamushi using monoclonal antibodies.

Twenty-four monoclonal antibodies were produced by immunizing BALB/c mice with Rickettsia tsutsugamushi Boryong strain and used for the analysis of antigenic characteristics of R.tsutsugamushi Boryong strain and antigenic heterogeneity of R.tsutsugamushi by indirect immunofluorescent(IF) test. R. tsutsugamushi Kato, Karp, Gilliam, TA686, TA716, TA763, TC586, TH1817, and Boryong were used for the analysis of antigenic heterogeneity of R.tsutsugamushi. Five monoclonal antibodies were reactive with 27-kDa protein, four monoclonal antibodies were reactive with 47-kDa protein, and eight monoclonal antibodies were reactive with 56-kDa protein of R.tsutsugamushi Boryong strain. The reactive protein of seven monoclonal antibodies could not be identified by immunoblotting method. All monoclonal antibodies to 27-kDa protein and three monoclonal antibodies to 47-kDa protein, and five monoclonal antibodies to 56-kDa protein were reactive with three to eight strains among nine strains of R. tsutsugamushi tested. One monoclonal antibody reactive to 47-kDa protein(KI18) and two monoclonal antibodies reactive to 56-kDa protein(KI36, and KI37) reacted with all the strains of R. tsutsugamushi tested. Strain-specific monoclonal antibody(KI58) could be found among antibodies which were reactive with 56-kDa protein. There was no strain which showed same reactivity pattern to these 24 monoclonal antibodies among nine strains. From this results, it could be concluded that Boryong strain is antigenically different from other strains of R.tsutsugamushi and antigenic heterogeneity of R.tsutsugamushi is due to the antigenic diversity of several proteins of R. tsutsugamushi including 56-kDa protein.     

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.     

Deficiency of peptidoglycan and lipopolysaccharide components in Rickettsia tsutsugamushi.

Analyses of chemical composition in whole cells of Rickettsia tsutsugamushi were performed and compared with those of the other rickettsiae and gram-negative bacteria. The results indicated that R. tsutsugamushi does not contain detectable amounts of 3-deoxy-D-mannooctulosonic acid, heptose, muramic acid, or glucosamine (less than 2, less than 2, less than 3, and less than 3 nmol/mg, respectively). The microorganism was found to contain four kinds of fatty acids (16:0, 18:0, 18:1, and 18:2), but not hydroxy fatty acids. Furthermore, in analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver or Coomassie blue staining, lipopolysaccharide bands were not detected in preparations treated with proteinase K. It is concluded that R. tsutsugamushi has little or no peptidoglycan or lipopolysaccharide.     

Metabolism of Rickettsia tsutsugamushi and Rickettsia rickettsi in Irradiated Host Cells

The metabolism of Rickettsia tsutsugamushi (Gilliam strain) multiplying in irradiated L cells was investigated by methods involving the use of (14)C-labeled substrates and cycloheximide, an inhibitor of eukaryotic metabolism. Cycloheximide-resistant amino acid and adenine incorporations were appreciably higher in infected than in uninfected cultures during the period from 3 to 5 or 6 days postinoculation. The metabolism of R. rickettsi was similarly studied in primary duck embryo cells, which are more susceptible to infection with this rickettsia than are L cells. A difference in cycloheximide-resistant activity between infected and uninfected cultures was also noted, but was small. This finding is attributed to the more limited growth of R. rickettsi.     

Comparative ultrastructural study on the cell envelopes of Rickettsia prowazekii, Rickettsia rickettsii, and Rickettsia tsutsugamushi.

Rickettsia tsutsugamushi differs from other rickettsiae in its cell envelope organization. The differences were made evident through a comparative study of the outer envelope of R. tsutsugamushi, R. prowazekii, and R. rickettsii by electron microscopy.     

Growth characteristics and proteins of plaque-purified strains of Rickettsia tsutsugamushi.

Six plaque-purified strains of Rickettsia tsutsugamushi (Karp, Gilliam, Kato, JC472B, TA716, and TA763) that fall into three categories of virulence for mice were compared by several parameters. Five of the six strains formed plaques of identical size in mouse cells, but each of three strains tested (representing three mouse virulence types) had a different doubling time in mouse cell cultures. Neither of these properties correlated strictly with virulence in mice, although the avirulent TA716 strain replicated much more slowly than the more virulent Karp and Gilliam strains. R. tsutsugamushi strain heterogeneity was also manifested at the polypeptide level by migration rates in sodium dodecyl sulfate-polyacrylamide gels of three of the major scrub typhus antigens (Sta110, Sta56, and Sta47), with those of Sta110 differing most widely. As expected, immunoblotting with polyclonal mouse sera showed substantial cross-reactivity among the major antigens of the six strains. Similar tests with Karp-induced monoclonal antibodies (MAb) demonstrated that some epitopes on Sta110 and Sta56 were shared by fewer than the six strains, but they identified no epitope unique to Karp. In contrast to the ready demonstration of antigenic heterogeneity in Sta110 and Sta56, four of the five Sta47-specific MAb reacted well with Sta47 from each of the six strains; the remaining MAb bound Sta47 from Karp and the Karp-like JC472B strain more strongly than Sta47 from the other four strains. The MAb also were useful in indicating the possible occurrence of Sta47 as dimers and trimers, the presence of Sta110 (as well as Sta56 and Sta47) in the rickettsial membrane, and the apparent interaction of the putative heat shock protein Sta58 with Sta47 or Sta47-Sta56 complexes.     

Identification and partial characterization of Rickettsia tsutsugamushi major protein immunogens.

Strains of Rickettsia tsutsugamushi so far examined have either three or four quantitatively predominant proteins, which apparently are surface proteins and which range in size between 50 and 63 kilodaltons. These polypeptides also were the major immunogens detected by polyacrylamide gel electrophoresis of extracted rickettsial proteins which had been precipitated by hyperimmune rabbit sera. The major proteins from different rickettsial strains share some epitopes, as evidenced by the lack of strain specificity of the rabbit sera in the immunoprecipitation tests. However, similar experiments with a limited number of monoclonal antibodies showed that strain-specific determinants also are associated with at least the 58/60-kilodalton polypeptide. A lack of strain-specific epitopes on the rickettsial surface was indicated by our inability to detect binding of heterologous antisera to the rickettsial surface by immunoferritin labeling. Because the three major proteins of the Karp and Gilliam strains are accessible to antibody in unextracted organisms, it is possible that the exteriorly exposed epitopes of these three polypeptides are strain specific and that their common determinants are normally buried in the membrane or otherwise inaccessible. Attempts to absorb out specific antibody with intact rickettsiae gave equivocal results; however, when immune complexes formed before rickettsial extraction were examined by electrophoresis, antibody appeared to have bound strain specifically with at least the 60-kilodalton protein.     

Effect of immune serum on infectivity of Rickettsia tsutsugamushi.

Hyperimmune antirickettsial serum was shown to prevent the attachment/penetration stage of Rickettsia tsutsugamushi infection of suspended chicken cells. The extent of the inhibition depended on the serum concentration but not on the presence of complement. The neutralizing activity was reduced by prior adsorption of immune serum with staphylococcal protein A or with intact rickettsiae but was not affected by adsorption with target cells. In the neutralization tests, there was no cross-reactivity between the Karp and Gilliam strains of R. tsutsugamushi. Incubation of rickettsiae with immune serum did not alter their capacity to metabolize glutamate nor grossly damage the permeability barrier function of their cytoplasmic membranes. Although the assay method had the capacity to detect some aggregated infectious organisms, none were found in immune serum-treated suspensions. It was concluded that immune serum may inhibit rickettsial infection by blocking a surface component(s) whose function is necessary for attachment to and/or penetration of target cells.     

Characterization of factors determining Rickettsia tsutsugamushi pathogenicity for mice.

Pathogenicity of Rickettsia tsutsugamushi for laboratory mice is known to be influenced by at least three factors: (i) route of inoculation, (ii) antigenic strain, and (iii) natural resistance of the host. By using Karp, Gilliam, and Kato strains of R. tsutsugamushi, we examined the effect of these three pathogenicity factors on the kinetics of infection and the development of immunity in BALB/cDub and C3H/HeDub mice. The appearance of rickettsemia in the pathogenic infections generally preceded infections of reduced pathogenicity by 1 to 2 days in both magnitude and time of onset. Mice infected by the subcutaneous route with normally pathogenic rickettsiae, i.e., Gilliam-infected C3H/HeDub mice and Karp-infected BALB/cDub mice, consistently maintained a detectable rickettsemia over a 1-year period. Rickettsiae were recovered from the spleens of 95% (19 of 20) of these mice 52 weeks postinfection. In contrast, mice with infections of reduced pathogenicity, i.e., BALB/cDub mice infected by intraperitoneal and subcutaneous inoculation with Gilliam, did not have detectable rickettsemia from week 20 through week 52 postinfection except for a single mouse on week 44 postinfection. Rickettsiae were detected in the spleens of only 40% (8 of 20) of these mice after 1 year. In both Gilliam-infected mouse strains, protection against heterologous challenge with Karp or Kato rickettsial strains was incomplete up to 7 days postimmunization. Infections of reduced pathogenicity did not result from an enhanced systemic immune response by the host. The onset of the humoral response was not different for the pathogenic and reduced-pathogenicity infections. Pathogenicity differences seemed to result from the more rapid growth of the rickettsiae in the pathogenic infections.     

Role of T-lymphocytes in production of antibody to antigens of Rickettsia tsutsugamushi and other Rickettsia species.

The requirement of thymus-dependent lymphocytes for antibody production to Rickettsia tsutsugamushi, Rickettsia akari, Rickettsia conorii, and Rickettsia typhi was investigated by comparing antibody production in athymic (nu/nu) or thymus-bearing BALB/c mice. Athymic BALB/c mice produced antibody after infection with R. akari, R. conorii, and R. typhi as measured by indirect fluorescent antibody titration or radioimmunoassay. Antibody production in these mice was a great or greater than in the thymus-bearing mice and demonstrated similar kinetics. In contrast, athymic BALB/c mice infected either intraperitoneally or subcutaneously with the Gilliam strain of R. tsutsugamushi failed to produce demonstrable antibody. The requirement of thymus-dependent lymphocytes for antibody production to R. tsutsugamushi was further suggested by the demonstration of antibody production after transfer of immune thymus-dependent lymphocytes to athymic mice and the demonstration of R. tsutsugamushi-specific T helper cells in immune thymus-bearing mice. The antibody produced in athymic mice after infection with R. akari, R. conorii, and R. typhi was predominantly immunoglobulin M, based on isotype-specific radioimmunoassays and sucrose gradient fractionation. Furthermore, the antibody produced by athymic mice in response to R. akari infection reacted with a carbohydrate-containing outer membrane component.     

Purification and partial characterization of a type-specific antigen of Rickettsia tsutsugamushi.

A type-specific antigen (54- to 56-kilodalton polypeptide) in the envelope of Rickettsia tsutsugamushi was purified from each of three prototype strains (Gilliam, Karp, and Kato) by a combination of mild anionic detergent treatment, gel filtration, and reverse-phase high-performance liquid chromatography. The purified antigens from the three strains were shown to have similar amino acid compositions: primarily aspartic acid, glutamic acid, and glycine, with lesser amounts of cysteine, methionine, and tyrosine. The N-terminal amino acid sequences of the antigens were 74.3% homologous among the three strains.     

Establishment and characterization of a T-cell line specific for Rickettsia tsutsugamushi.

To study the immunological protective system against rickettsial infection, a T-cell line specific for Rickettsia tsutsugamushi antigen was established by long-term culture of splenocytes from mice immunized with live Gilliam strain R. tsutsugamushi and then propagated in the presence of homologous rickettsial antigen and syngeneic filler cells. The characteristics of the T-cell line and its capacity to induce antirickettsial protection in vivo were studied. Flow cytometric analysis demonstrated that the T-cell line showed the phenotype Thy-1.2+ L3T4+ Lyt-2-, suggestive of helper T cells. In a lymphocyte proliferation assay, this cell line showed a specific response to Gilliam antigen, partial cross-reactivity to Karp antigen, but no response to Kato antigen. The proliferative response of this T-cell line was filler cell dependent, and genetic restriction was observed between the T-cell line and filler cells. The T-cell line produced gamma interferon, one of the macrophage-activating factors, in cultures with specific antigen and was able to adoptively mediate antirickettsial protection in vivo. The data presented here suggest that antigen-specific helper T cells play an important role in protection against rickettsial infection.     

Production of gamma interferon in mice immune to Rickettsia tsutsugamushi.

C3H/He mice immunized by subcutaneous infection with Rickettsia tsutsugamushi Gilliam were examined for the production of immune interferon after intravenous administration of irradiated strain Gilliam antigen, in supernatants of immune lymphocytes stimulated with specific antigen, and after a secondary challenge with viable rickettsiae. Mice administered various doses of irradiated whole-organism antigen 28 days after immunization showed circulating levels of interferon which peaked 4 h after inoculation and were antigen dose dependent. The interferon produced was pH 2 sensitive and stable at 56 degrees C for 1 h and was neutralized by antiserum directed against immune, but not against alpha/beta, interferon. The production of another lymphokine, macrophage migration inhibition factor, paralleled that of interferon. The interferon produced by cultures of spleen cells obtained from immune animals was antigen specific and dose dependent. Peak levels were obtained 48 to 72 h after the addition of antigen. The interferon produced by spleen cell cultures after stimulation with Gilliam antigen was characterized as immune interferon by the same physical and antigenic criteria used for serum interferon. Interferon was produced in vitro by the Thy-1.2+ lymphocyte and required the presence of a spleen-adherent cell population. Immune mice produced high circulating levels of immune interferon after intraperitoneal challenge with viable rickettsiae, which suggested a possible role for interferon in the resistance of immune mice to rechallenge with R. tsutsugamushi.     

Antigenic and genetic relatedness of eight Rickettsia tsutsugamushi antigens

The genetic and antigenic relatedness of eight antigens in three strains of Rickettsia tsutsugamushi has been studied by using recombinant organisms expressing epitopes of the 150-, 110-, 72-, 58-, 56-, 49-, 47-, and 20-kilodalton (kDa) polypeptide antigens of the Karp strain. Southern blot analysis of Karp, Kato, and Gilliam strain genomic DNA by using probes specific for each antigen class indicated that while strong homology exists between each of the corresponding antigen genes in these three strains, some restriction fragment length polymorphism exists. Antibodies affinity purified against each recombinant antigen class reacted with a comparably sized polypeptide in the Karp, Kato, and Gilliam strains in Western blots (immunoblots). Against more recent human isolates of R. tsutsugamushi, the affinity-purified antibodies against the 58-kDa recombinant antigen (anti-58-kDa) reacted with all nine isolates, anti-56-kDa reacted with eight of nine isolates, anti-47-kDa reacted with eight of nine isolates, anti-72-kDa reacted with eight of nine isolates, and anti-110-kDa reacted with four of nine isolates. Additional analysis indicated that the 110-kDa antigen may contain strain-specific epitopes similar to those previously reported for the 56-kDa polypeptide. Evidently, the strain heterogeneity among scrub typhus rickettsiae is a result of multiple components that exhibit variability in a background of strong homology.     

Detection of Rickettsia tsutsugamushi in Experimentally infected mice by PCR.

We developed a rapid procedure for the detection of Rickettsia tsutsugamushi DNA by the PCR technique. The primer pair used for the PCR was designed from the DNA sequence of the gene encoding a 120-kDa antigen, which was proven to be group specific by immunoblot analysis with mouse hyperimmune sera against various rickettsial strains. This PCR method was able to detect up to 10 ag of plasmid DNA (pKT12). Specific PCR products were obtained with DNAs from R. tsutsugamushi Kato, Karp, Gilliam, TA716, TA1817, and Boryong, but not with DNAs from other rickettsiae, such as R. prowazekii, R. typhi, R. akari, and strain TT118. In a study with experimentally infected mice, the PCR method could detect rickettsial DNA from 2 days after inoculation (DAI), whereas serum antibody against R. tsutsugamushi could be detected from 6 to 8 DAI by an immunofluorescence test. Although clinical manifestations subsided after 14 DAI, rickettsial DNA in blood samples could be detected by PCR for up to 64 DAI. These results suggest that this PCR method can be applied to the early diagnosis of scrub typhus and can also be used to detect the residual rickettsiae after clinical symptoms subside.     

Murine T-cell response to native and recombinant protein antigens of Rickettsia tsutsugamushi.

A polyclonal T-cell line with TH1 characteristics was used to assess the murine cellular immune response to native and recombinant Rickettsia tsutsugamushi antigens. Proliferation of this T-cell line was observed in response to numerous native antigen fractions, which indicates that the murine T-helper-cell response is directed at multiple scrub typhus antigens with no apparent antigenic immunodominance. Subsequent analysis of recombinant R. tsutsugamushi antigens made it possible to identify a 47-kDa scrub typhus antigen (Sta47) that was stimulatory for the polyclonal T-cell line. Recombinant clones encoding 56-, 58-, and 110-kDa antigens (Sta56, Sta58, and Sta110, respectively) were unable to induce proliferation of this T-cell line. DNA sequence analysis of the cloned rickettsial insert encoding the Sta47 protein revealed the presence of four open reading frames potentially encoding proteins of 47, 30, 18, and 13 kDa. Analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated and eluted fractions of lysates from the recombinant HB101(pRTS47B4.3) demonstrated that the fractions containing the 47-kDa protein as well as those containing proteins less than 18 kDa were stimulatory. Selected synthetic amphipathic peptides derived from the Sta47 antigen sequence identified a 20-amino-acid peptide that gave a 10-fold increase in T-cell proliferation over a control malarial peptide of similar length. Recognition of the 47-kDa antigen by a T-cell line with TH1 characteristics implicates this protein as one of potential importance in protection studies and future vaccine development.     

Comparative susceptibility to mouse interferons of Rickettsia tsutsugamushi strains with different virulence in mice and of Rickettsia rickettsii.

Three strains of Rickettsia tsutsugamushi (Karp, Gilliam, and TA716, representing three virulence types in mice) were examined for their sensitivity to the inhibitory effects of recombinant gamma interferon (IFN-gamma) and purified IFN-alpha/beta in two cultured mouse fibroblast cell lines. The susceptibilities of another species, Rickettsia rickettsii, and of encephalomyocarditis virus (EMCV) were also tested for comparative purposes. IFN-gamma inhibited rickettsial replication in only one of the six combinations of R. tsutsugamushi strains and mouse cells (strain Gilliam and the BALB/c mouse-derived cell line). In contrast, R. rickettsii and EMCV replication were markedly inhibited in both cell types, but to a greater extent in the BALB/c line than in the C3H cells. IFN-alpha/beta (300 to 450 U/ml) was uniformly ineffective in three of the combinations of R. tsutsugamushi strains and mouse cells (Gilliam in C3H cells and Karp in both C3H and BALB/c cells); in the remaining sets, IFN-alpha/beta-mediated inhibition of rickettsial replication was variable and in no case was it very pronounced. The tests with R. rickettsii in both cell types also indicated slight, variable sensitivity to IFN-alpha/beta. EMCV, on the other hand, was very susceptible to IFN-alpha/beta, confirming the potency of the preparation used; as with IFN-gamma, virus replication was inhibited to a greater degree in the BALB/c cell line than in the C3H cultures. These results are discussed in terms of their relationship to the virulence properties of the R. tsutsugamushi strains in BALB/c and C3H mice and to the known IFN-sensitivities of the more widely studied Rickettsia prowazekii.     

Genomic comparison of virulent Rickettsia rickettsii Sheila Smith and avirulent Rickettsia rickettsii Iowa

Rickettsia rickettsii is an obligate intracellular pathogen that is the causative agent of Rocky Mountain spotted fever. To identify genes involved in the virulence of R. rickettsii, the genome of an avirulent strain, R. rickettsii Iowa, was sequenced and compared to the genome of the virulent strain R. rickettsii Sheila Smith. R. rickettsii Iowa is avirulent in a guinea pig model of infection and displays altered plaque morphology with decreased lysis of infected host cells. Comparison of the two genomes revealed that R. rickettsii Iowa and R. rickettsii Sheila Smith share a high degree of sequence identity. A whole-genome alignment comparing R. rickettsii Iowa to R. rickettsii Sheila Smith revealed a total of 143 deletions for the two strains. A subsequent single-nucleotide polymorphism (SNP) analysis comparing Iowa to Sheila Smith revealed 492 SNPs for the two genomes. One of the deletions in R. rickettsii Iowa truncates rompA, encoding a major surface antigen (rickettsial outer membrane protein A [rOmpA]) and member of the autotransporter family, 660 bp from the start of translation. Immunoblotting and immunofluorescence confirmed the absence of rOmpA from R. rickettsii Iowa. In addition, R. rickettsii Iowa is defective in the processing of rOmpB, an autotransporter and also a major surface antigen of spotted fever group rickettsiae. Disruption of rompA and the defect in rOmpB processing are most likely factors that contribute to the avirulence of R. rickettsii Iowa. Genomic differences between the two strains do not significantly alter gene expression as analysis of microarrays revealed only four differences in gene expression between R. rickettsii Iowa and R. rickettsii strain R. Although R. rickettsii Iowa does not cause apparent disease, infection of guinea pigs with this strain confers protection against subsequent challenge with the virulent strain R. rickettsii Sheila Smith.