A 29.5 kDa heat-modifiable major outer membrane protein of Rickettsia prowazekii, putative virulence factor, is a peptidyl-prolyl cis/trans isomerase

Allelic genes from three Rickettsia prowazekii strains encoding parvulin-like protein (Plp), a heat-modifiable 29.5 kDa major outer membrane protein, were earlier cloned into expression vector pQE 30. In this work, recombinant proteins were overproduced in E. coli, purified, and found to exhibit an expected peptidyl-prolyl cis/trans isomerase activity of a parvulin type in vitro with oligopeptide substrates. Native polypeptide of prototype virulent Breinl strain is known to differ by SDS-PAGE mobility from those of both vaccine Madrid E and virulent EVir isolates. Being different in electrophoretic behavior, heat-unmodified forms of the three strains were shown to migrate apart from lipopolysaccharides. A EVir Plp gene was sequenced, and deduced protein sequence was found to be identical to previously published Breinl and Madrid E. Present data indicate that unknown post-translational modification(s) in rickettsiae are responsible for both interstrain difference and heat-modifiability of Plp.     

Nucleotide sequence of the gene and features of the major outer membrane protein of a virulent Rickettsia prowazekii strain.

We have determined the nucleotide sequence of the gene for a major outer membrane protein (MOMP) of apparent molecular weight 29.5 kD of the virulent Breinl strain of Rickettsia prowazekii. The gene contains an open reading frame (ORF) that encodes a 282-amino-acid polypeptide with a calculated molecular mass of 31549 daltons. A signal-like peptide sequence is found at the deduced N terminus. A heterologous 29.5-kD antigen expressed in Escherichia coli was shown to be secreted into the periplasm. A database search for similar protein sequences revealed considerable homology of the polypeptide with the E. coli peptidyl-prolyl cis/trans isomerase and related proteins of the parvulin family. The genes for MOMP of the virulent Breinl and EVir strains and the vaccine Madrid E strain were amplified using specific primers and cloned into expression vector pQE-30. We found that the polypeptides encoded by the recombinant DNAs do not differ in SDS-PAGE mobility, while the native MOMP of the Breinl strain is known to be different from the corresponding proteins of the Madrid E and EVir strains. Furthermore, no differences within the ORF for the 29.5-kD proteins of the three strains were found by restriction endonuclease analysis of polymerase chain reaction (PCR) products. A possible role of parvulin-like protein (Plp) in the virulence of epidemic typhus agent and the nature of interstrain differences are discussed. Near the plp gene on the opposite strand, an origin of the gene that codes for the SecA subunit of a preprotein translocase was found.     

The rickettsial genome studied by DNA restriction analysis

The restriction analysis of 6 Rickettsia prowazekii strains with the use of 8 restrictases (Cfr13I, EcoRI, HindIII, MSpI, MvaI, PstI, XhoI, BamHI) has been carried out. In the presence of considerable homology in the restriction pictures of DNA in these strains some differences in 1-2 fragments within the range of 8,000-20,000 nucleotide pairs have been established. The strains under study have been divided into two groups according to the character of differences in their restrictograms: the group of virulent typing strain Breinl (Breinl, G. Anan'ev) and the group of strain E with low pathogenicity (E, EVir, Katsinian). Differences in the restrictograms of DNA do not correlate with the virulence of R. prowazekii strains and the areas of their isolation.     

Cloning and expression in Escherichia coli of three Rickettsia prowazekii genes, coding outer membrane proteins]

Rickettsia prowazekii (virulent Breinl strain) random genomic DNA fragments were cloned in the lambda gt11 expression vector by using non-palindromic adaptors. Several immunoreactive clones were selected after screening 20,000 individual recombinant plaques with human convalescent serum. Some recombinants synthesized the complete 60 K protein, and others synthesized beta-galactosidase fusion polypeptides containing epitopes of 134 K protein of the R. prowazekii outer membrane. The amplified genomic library was screened with monospecific antibodies directed against abundant 31 K and 29.5 K outer membrane proteins. Several recombinant clones expressing full or part of 29.5 K polypeptide, and none expressing 31 K polypeptide were revealed. The serum of a patient convalescing from epidemic typhus did not react in western blot with recombinant 29.5 K protein.     

Comparative evaluation of vaccinal and virulent strains of Rickettsia prowazekii by the plaque method

The comparative evaluation of R. prowazekii vaccine and virulent strains has revealed that strain Breinl possesses greater capacity for plaque formation than strain E. The possibility of using the plaque assay for differentiation of substrains 288 and 281 of strain E has been established.     

Comparison of in vitro susceptibilities of Rickettsia prowazekii, R. rickettsii, R. sibirica and R. tsutsugamushi to antimicrobial agents

The in vitro activities of 16 antimicrobial agents against Rickettsia prowazekii (Breinl strain), R. rickettsii (Bitterroot strain), R. sibirica (ATCC No. VR151) and R. tsutsugamushi (Gilliam, Karp, Kato, Shimokoshi, Kawasaki and Kuroki strains) were determined by the cell culture method. Tetracycline, demethylchlortetracycline, doxycycline, minocycline, chloramphenicol, kitasamycin and rifampicin were generally effective (MIC, 0.005-0.78 micrograms/ml) to all strains tested. Quinolones such as norfloxacin, ciprofloxacin and ofloxacin were moderately active, but they were less active against R. tsutsugamushi than other rickettsial species. Penicillins and cephems showed low activity against most of the strains tested, but high concentrations of benzylpenicillin (MIC, 25-50 micrograms/ml) inhibited R. prowazekii, R. rickettsii and R. sibirica. These findings may be applicable for differentiation of species of genus Rickettsia.     

Rickettsial metK-encoded methionine adenosyltransferase expression in an Escherichia coli metK deletion strain

The obligate intracellular bacterium Rickettsia prowazekii has recently been shown to transport the essential metabolite S-adenosylmethionine (SAM). The existence of such a transporter would suggest that the metK gene, coding for the enzyme that synthesizes SAM, is unnecessary for rickettsial growth. Genome sequencing has revealed that this is the case for the metK genes of the spotted fever group and the Madrid E strain of R. prowazekii, which contain recognizable inactivating mutations. However, several strains of the typhus group rickettsiae possess metK genes lacking obvious mutations. In order to determine if these genes code for a product that retains MAT function, an Escherichia coli metK deletion mutant was constructed in which individual rickettsial metK genes were tested for the ability to complement the methionine adenosyltransferase deficiency. Both the R. prowazekii Breinl and R. typhi Wilmington metK genes complemented at a level comparable to that of an E. coli metK control, demonstrating that the typhus group rickettsiae have the capability of synthesizing as well as transporting SAM. However, the appearance of mutations that affect the function of the metK gene products (a stop codon in the Madrid E strain and a 6-bp deletion in the Breinl strain) provides experimental support for the hypothesis that these typhus group genes, like the more degenerate spotted fever group orthologs, are in the process of gene degradation.     

Determination of genome size and restriction pattern polymorphism of Rickettsia prowazekii and Rickettsia typhi by pulsed field gel electrophoresis

Abstract Pulsed field gel electrophoresis (PFGE) of Sma I, Mlu I and Sal I digested DNA was used to estimate genome size and perform restriction fragment length polymorphism analysis for Rickettsia prowazekii and Rickettsia typhi . We concluded that the genome of R. prowazekii and R. typhi consisted of a single chromosomal DNA. The total length of DNA of R. prowazekii was 1,106±54 kb and of R. typhi was 1,133±44kb. It was possibleto differentiate two strains of R. prowazekii , Breinl and EVir, by PFGE analysis after Sal I digestion. Restriction fragment length polymorphism analysis did not reveal intraspecies differences between three human isolates and one Xenopsilla cheopis isolate of R. typhi .     

The surface antigens of Rickettsia prowazekii studied with monoclonal antibodies]

R. prowazekii antigens have been tested with the use of monoclonal antibodies (McAb) to different epitopes of the microorganism. As revealed in these tests, McAb B4/4 and A-3/D, active against species-specific thermolabile antigen, interact with protein having a molecular weight of 90-120 KD. McAb C5/2, active against thermostable group antigen common with that of Rickettsia typhi, interact with LPS-like antigen having a molecular weight of 30 KD. Ultrastructural immunochemical studies have revealed that both R. prowazekii antigens are located on surface structures of rickettsiae, such as the microcapsule and cell wall.     

Methods of isolation and polypeptide composition of membrane fractions of Rickettsia prowazekii

The methods of cell lysis by lysozyme in tris-EDTA-sucrose with the consequent disruption of spheroplasts by the osmotic shock were used to obtain the total membranes from the intact or temperature-inactivated Rickettsia prowazekii. Detergents solubilization methods were used for analysis of outer membrane proteins. Sarcosyl insoluble material is shown to contain the main 134, 31, 29.5 and 25 Kd proteins, the minor 78, 60, 42, 17 Kd proteins, while the mixture of both membranes possess a more complex composition. Treatment of total membranes by the 2% octylglycoside results in elimination of the 31 Kd polypeptide. Inactivated Rickettsia can be used for isolation of the outer layer proteins diminishing the risk of working with this pathogenic microorganism.     

Genotype characterization of the bacterium expressing the male-killing trait in the ladybird beetle Adalia bipunctata with specific rickettsial molecular tools.

The male-killing ladybird beetle (LB) bacterium (AB bacterium) was analyzed with specific rickettsial molecular biology tools in the LB Adalia bipunctata strains. Eight phenotype-positive LB strains showing mortality of male embryos were amplified with rickettsial genus-specific primers from the gene for citrate synthase (CS) and the gene for a 17-kDa protein and spotted fever group-specific primers from the gene for the 120-kDa outer membrane protein (ompB). The specificity of amplification was confirmed by Southern hybridization and the absence of the above-listed gene products in three phenotype-negative LB strains. Restriction polymorphism patterns of three examined amplicons from the CS gene, 17-kDa-protein gene, and ompB gene were identical among the eight phenotype-positive LB strains and were unique among all known rickettsiae of the spotted fever and typhus groups. Amplified fragments of the CS genes of the AB bacterium, Rickettsia prowazekii Breinl, Rickettsia typhi Wilmington, Rickettsia canada 2678, and Rickettsia conorii 7 (Malish) were sequenced. The greatest differences among the above-listed rickettsial and AB bacterium CS gene sequences were between bp 1078 and 1110. Numerical analysis based on CS gene fragment sequences shows the close relationships of the AB bacterium to the genus Rickettsia. Expanding of knowledge about rickettsial arthropod vectors and participation of rickettsiae in the cytoplasmic maternal inheritance of arthropods is discussed.     

S-adenosylmethionine transport in Rickettsia prowazekii

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate, intracellular, parasitic bacterium that grows within the cytoplasm of eucaryotic host cells. Rickettsiae exploit this intracellular environment by using transport systems for the compounds available in the host cell's cytoplasm. Analysis of the R. prowazekii Madrid E genome sequence revealed the presence of a mutation in the rickettsial metK gene, the gene encoding the enzyme responsible for the synthesis of S-adenosylmethionine (AdoMet). Since AdoMet is required for rickettsial processes, the apparent inability of this strain to synthesize AdoMet suggested the presence of a rickettsial AdoMet transporter. We have confirmed the presence of an AdoMet transporter in the rickettsiae which, to our knowledge, is the first bacterial AdoMet transporter identified. The influx of AdoMet into rickettsiae was a saturable process with a K(T) of 2.3 micro M. Transport was inhibited by S-adenosylethionine and S-adenosylhomocysteine but not by sinfungin or methionine. Transport was also inhibited by 2,4-dinitrophenol, suggesting an energy-linked transport mechanism, and by N-ethylmaleimide. AdoMet transporters with similar properties were also identified in the Breinl strain of R. prowazekii and in Rickettsia typhi. By screening Escherichia coli clone banks for AdoMet transport, the R. prowazekii gene coding for a transporter, RP076 (sam), was identified. AdoMet transport in E. coli containing the R. prowazekii sam gene exhibited kinetics similar to that seen in rickettsiae. The existence of a rickettsial transporter for AdoMet raises intriguing questions concerning the evolutionary relationship between the synthesis and transport of this essential metabolite.     

A murine model of infection with Rickettsia prowazekii: implications for pathogenesis of epidemic typhus

Epidemic typhus remains a major disease threat, furthermore, its etiologic agent, Rickettsia prowazekii, is classified as a bioterrorism agent. We describe here a murine model of epidemic typhus that reproduced some features of the human disease. When BALB/c mice were inoculated intravenously with R. prowazekii (Breinl strain), they survived but did not clear R. prowazekii infection. Immunohistological analysis of tissues and quantitative PCR showed that R. prowazekii was present in blood, liver, lungs and brain 1 day after infection and persisted for at least 9 days. Importantly, infected mice developed interstitial pneumonia, with consolidation of the alveoli, hemorrhages in lungs, multifocal granulomas in liver, and hemorrhages in brain, as seen in humans. Circulating antibodies directed against R. prowazekii were detected at day 4 post-infection and steadily increased for up to 21 days, demonstrating that R. prowazekii lesions were independent of humoral immune response. R. prowazekii-induced lesions were associated with inflammatory response, as demonstrated by elevated levels of inflammatory cytokines including interferon-gamma, tumor necrosis factor and the CC chemokine RANTES in the lesions. We concluded that the BALB/c mouse strain provides a useful model for studying the pathogenic mechanisms of epidemic typhus and its control by the immune system.     

The sensitivity of a number of cell cultures to different strains of Rickettsia prowazekii

The sensitivity of some cell cultures to different R. prowazekii strains (strain E with low pathogenicity, virulent strain Breinl, strains ERifRI and EVir) has been studied with a view to the selection of an adequate culture for growing these strains and the study of their biological properties. Experiments on titration in cells have revealed that 6- to 7-day primary and secondary irradiated quail fibroblasts and human amnion cells FL show the maximum sensitivity to all strains under study, comparable to that of chick embryos. The sensitivity of 6- to 7-day primary and secondary irradiated chick fibroblasts is faintly pronounced, and 24-hour chick and quail fibroblasts are still less sensitive. Cells FL have shown high sensitivity to strain E and mutant ERifRI in prolonged subculturing for 140 and 63 days (the term of observation) respectively after a single inoculation.     

Inactivation of SAM-Methyltransferase is the Mechanism of Attenuation of a Historic Louse Borne Typhus Vaccine Strain

Louse borne typhus (also called epidemic typhus) was one of man''s major scourges, and epidemics of the disease can be reignited when social, economic, or political systems are disrupted. The fear of a bioterrorist attack using the etiologic agent of typhus, Rickettsia prowazekii, was a reality. An attenuated typhus vaccine, R. prowazekii Madrid E strain, was observed to revert to virulence as demonstrated by isolation of the virulent revertant Evir strain from animals which were inoculated with Madrid E strain. The mechanism of the mutation in R. prowazekii that affects the virulence of the vaccine was not known. We sequenced the genome of the virulent revertant Evir strain and compared its genome sequence with the genome sequences of its parental strain, Madrid E. We found that only a single nucleotide in the entire genome was different between the vaccine strain Madrid E and its virulent revertant strain Evir. The mutation is a single nucleotide insertion in the methyltransferase gene (also known as PR028) in the vaccine strain that inactivated the gene. We also confirmed that the vaccine strain E did not cause fever in guinea pigs and the virulent revertant strain Evir caused fever in guinea pigs. We concluded that a single nucleotide insertion in the methyltransferase gene of R. prowazekii attenuated the R. prowazekii vaccine strain E. This suggested that an irreversible insertion or deletion mutation in the methyl transferase gene of R. prowazekii is required for Madrid E to be considered a safe vaccine.     

Seroimmunologic monitoring of microorganisms of Rickettsia and Bartonella species microorganisms in the Moscow region

Serological study of 788 blood sera, taken from residents of the Moscow region was conducted using antigens of microorganisms of the genera Rickettsia and Bartonella. The first group under examination consisted of 355 patients with diagnosed diseases of nonreckettsial nature. The second group includes 433 healthy adults working at a meat processing and packing factory. The main method used for sera survey was the indirect immunofluorescence test. In the sera taken from the first group of subjects specific antibodies to R. prowazekii, R. typhi, B. quintana, B. henselae antigens were detected in 2.3%, 5.1%, 4.0% and 2.9% of serum samples respectively. In the serum samples taken from the second group the proportion of antibodies to R. prowazekii, R. typhi, B. quintana, B. henselae antigens was different: 0.5%, 3.3%, 1.7% and 4.0% respectively. In total, specific antibodies to R. typhi and B. henselae prevailed over specific antibodies to R. prowazekii and B. quintana twofold.     

Proteome analysis of Madrid E strain of Rickettsia prowazekii

Rickettsia prowazekii, an obligate intracellular Gram-negative bacterium, is the etiologic agent of epidemic typhus. The threat of typhus as a biological weapon lies in its stability in the dried louse feces and in its infection by inhalation of an aerosol. Consequently, it is listed as a select agent and warrants more research to understand its pathogenesis. Although the genomic DNA sequence of strain Madrid E has been completed, the actual expression of the individual protein has not been investigated. In order to provide a global view of the expressed protein profile, the whole cell lysate of purified rickettsia (Madrid E strain) was reduced, alkylated, and digested with trypsin. The total digest was characterized by a two-dimensional liquid chromatography mass spectrometry system and analyzed with a modified version of the ProteomeX workstation. A total of 252 proteins out of 834 predicted protein-coding genes were identified, 238 proteins were identified by the detection of at least two unique peptides. Only 14 proteins were identified by the detection of one unique peptide in all three separate analyses. Among the 238 proteins identified by multiple unique peptides, 230 proteins were found in at least two of three separate analyses. The reproducible and convenient methodology and the information described here have provided a foundation for future proteome study of various R. prowazekii strains with different virulence.     

Use of the immunofluorescence reaction for evaluating the immunological transformation in those inoculated with a chemical typhus vaccine

The results of the serological examination of persons immunized with chemical typhus vaccine (CTV) are presented. The examination was carried out by means of the complement fixation test (CFT), the passive hemagglutination test (PHAT), the toxin neutralization test (TNT) and the immunofluorescence test (IFT). The acetone-fixed live culture of Rickettsia prowazekii, strain Breinl, served as antigen in IFT. If persons immunized with CTV showed positive titers in CFT, TNT and PHAT, the results of IFT were highly correlated with the CFT titers. In 6-12 months after immunization with CTV the titers of CFT, TNT and PHAT became negative, while the IFT titers remained positive for several subsequent years.     

Characterization of the Rickettsia prowazekii pepA gene encoding leucine aminopeptidase.

The pepA gene, encoding a protein with leucine aminopeptidase activity, was isolated from Rickettsia prowazekii, an obligate intracellular parasitic bacterium. Nucleotide sequence analysis revealed an open reading frame of 1,502 bp that would encode a protein of 499 amino acids with a calculated molecular weight of 53,892, a size comparable to that of the protein produced in Escherichia coli minicells containing the rickettsial gene. Also, heat-stable leucine aminopeptidase activity was demonstrable in an E. coli peptidase-deficient strain containing R. prowazekii pepA. Comparison of the amino acid sequence of the R. prowazekii PepA with the characterized leucine aminopeptidases from E. coli, Arabidopsis thaliana, and bovine eye lens revealed that 39.8, 34.9, and 34.0% of the residues were identical, respectively. Residues proposed to be part of the active site or involved in the binding of metal ions in the bovine metalloenzyme were all conserved in R. prowazekii PepA. However, despite the structural and enzymatic similarity to E. coli PepA, the R. prowazekii protein was unable to complement the cer site-specific, PepA-dependent recombination system found in E. coli that resolves ColE1-type plasmid multimers into their monomeric forms.