Rickettsia prowazekii methionine aminopeptidase as a promising target for the development of antibacterial agents.

Methionine aminopeptidase (MetAP) is a class of ubiquitous enzymes essential for the survival of numerous bacterial species. These enzymes are responsible for the cleavage of N-terminal formyl-methionine initiators from nascent proteins to initiate post-translational modifications that are often essential to proper protein function. Thus, inhibition of MetAP activity has been implicated as a novel antibacterial target. We tested this idea in the present study by targeting the MetAP enzyme in the obligate intracellular pathogen Rickettsia prowazekii. We first identified potent RpMetAP inhibitory species by employing an in vitro enzymatic activity assay. The molecular docking program AutoDock was then utilized to compare published crystal structures of inhibited MetAP species to docked poses of RpMetAP. Based on these in silico and in vitro screens, a subset of 17 compounds was tested for inhibition of R. prowazekii growth in a pulmonary vascular endothelial cell (EC) culture infection model system. All compounds were tested over concentration ranges that were determined to be non-toxic to the ECs and 8 of the 17 compounds displayed substantial inhibition of R. prowazekii growth. These data highlight the therapeutic potential for inhibiting RpMetAP as a novel antimicrobial strategy and set the stage for future studies in pre-clinical animal models of infection.

Pathogenic rickettsiae as bioterrorism agents.

Because of their unique biological characteristics, such as environmental stability, small size, aerosol transmission, persistence in infected hosts, low infectious dose, and high associated morbidity and mortality, Rickettsia prowazekii and Coxiella burnetii have been weaponized. These biological attributes would make the pathogenic rickettsiae desirable bioterrorism agents. However, production of highly purified, virulent, weapon-quality rickettsiae is a daunting task that requires expertise and elaborate, state-of-the art laboratory procedures to retain rickettsial survival and virulence. Another drawback to developing rickettsial pathogens as biological weapons is their lack of direct transmission from host to host and the availability of very effective therapeutic countermeasures against these obligate intracellular bacteria.

Transposon mutagenesis of the obligate intracellular pathogen Rickettsia prowazekii.

Genetic analysis of Rickettsia prowazekii has been hindered by the lack of selectable markers and efficient mechanisms for generating rickettsial gene knockouts. We have addressed these problems by adapting a gene that codes for rifampin resistance for expression in R. prowazekii and by incorporating this selection into a transposon mutagenesis system suitable for generating rickettsial gene knockouts. The arr-2 gene codes for an enzyme that ADP-ribosylates rifampin, thereby destroying its antibacterial activity. Based on the published sequence, this gene was synthesized by PCR with overlapping primers that contained rickettsial codon usage base changes. This R. prowazekii-adapted arr-2 gene (Rparr-2) was placed downstream of the strong rickettsial rpsL promoter (rpsL(P)), and the entire construct was inserted into the Epicentre EZ::TN transposome system. A purified transposon containing rpsL(P)-Rparr-2 was combined with transposase, and the resulting DNA-protein complex (transposome) was electroporated into competent rickettsiae. Following selection with rifampin, rickettsiae with transposon insertions in the genome were identified by PCR and Southern blotting and the insertion sites were determined by rescue cloning and inverse PCR. Multiple insertions into widely spaced areas of the R. prowazekii genome were identified. Three insertions were identified within gene coding sequences. Transposomes provide a mechanism for generating random insertional mutations in R. prowazekii, thereby identifying nonessential rickettsial genes.

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.

Rickettsial phospholipase A2 as a pathogenic mechanism in a model of cell injury by typhus and spotted fever group rickettsiae.

Phospholipase A2 activity by typhus group rickettsiae causes hemolysis in vitro. Rickettsial phospholipase A2 has been proposed to mediate entry into the host cell, escape from the phagosome, and cause injury to host cells by both typhus and spotted fever group rickettsiae. In a rickettsial contact-associated cytotoxicity model, the interaction of Rickettsia prowazekii or R. conorii with Vero cells caused temperature-dependent release of 51Cr from the cells. Treatment of rickettsiae, but not the cells, with a phospholipase A2 inhibitor (bromophenacyl bromide) or with antibody to king cobra venom inhibited cell injury. Rickettsial treatment with bromophenacyl bromide inhibited the release of free fatty acids from the host cell. Neither the inhibitor nor antivenom impaired rickettsial active transport of L-lysine. Thus, host cell injury was mediated by a rickettsial phospholipase A2-dependent mechanism.

Failure of azithromycin in treatment of Brill-Zinsser disease.

Two patients suffering from Brill-Zinsser disease were treated with azithromycin, which did not prove effective. Rickettsia prowazekii, the agent causing Brill-Zinsser disease, cannot be treated with azithromycin. Both patients had epidemiological features consistent with and a clinical course typical of the disease. The diagnosis of Brill-Zinsser disease was serologically confirmed.

In vitro activities of telithromycin (HMR 3647) against Rickettsia rickettsii, Rickettsia conorii, Rickettsia africae, Rickettsia typhi, Rickettsia prowazekii, Coxiella burnetii, Bartonella henselae, Bartonella quintana, Bartonella bacilliformis, and Ehrlichia chaffeensis.

In vitro activities of telithromycin compared to those of erythromycin against Rickettsia spp., Bartonella spp., Coxiella burnetii, and Ehrlichia chaffeensis were determined. Telithromycin was more active than erythromycin against Rickettsia, Bartonella, and Coxiella burnetii, with MICs of 0.5 microg/ml, 0.003 to 0.015 microg/ml, and 1 microg/ml, respectively, but was inactive against Ehrlichia chaffeensis.

Rickettsia prowazekii transports UMP and GMP, but not CMP, as building blocks for RNA synthesis.

Rickettsia prowazekii, the etiological agent of epidemic typhus, is an obligate intracellular bacterium and is apparently unable to synthesize ribonucleotides de novo. Here, we show that as an alternative, isolated, purified R. prowazekii organisms transported exogenous uridyl- and guanylribonucleotides and incorporated these labeled precursors into their RNA in a rifampin-sensitive manner. Transport systems for nucleotides, which we have shown previously and show here are present in rickettsiae, have never been reported in free-living bacteria, and the usual nucleobase and nucleoside transport systems are absent in rickettsiae. There was a clear preference for the monophosphate form of ribonucleotides as the transported substrate. In contrast, rickettsiae did not transport cytidylribonucleotides. The source of rickettsial CTP appears to be the transport of UMP followed by its phosphorylation and the amination of intrarickettsial UTP to CTP by CTP synthetase. A complete schema of nucleotide metabolism in rickettsiae is presented that is based on a combination of biochemical, physiological, and genetic information.

Transformation of Rickettsia prowazekii to rifampin resistance.

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular parasitic bacterium that grows directly within the cytoplasm of the eucaryotic host cell. The absence of techniques for genetic manipulation hampers the study of this organism's unique biology and pathogenic mechanisms. To establish the feasibility of genetic manipulation in this organism, we identified a specific mutation in the rickettsial rpoB gene that confers resistance to rifampin and used it to demonstrate allelic exchange in R. prowazekii. Comparison of the rpoB sequences from the rifampin-sensitive (Rifs) Madrid E strain and a rifampin-resistant (Rifr) mutant identified a single point mutation that results in an arginine-to-lysine change at position 546 of the R. prowazekii RNA polymerase beta subunit. A plasmid containing this mutation and two additional silent mutations created in codons flanking the Lys-546 codon was introduced into the Rifs Madrid E strain of R. prowazekii by electroporation, and in the presence of rifampin, resistant rickettsiae were selected. Transformation, via homologous recombination, was demonstrated by DNA sequencing of PCR products containing the three mutations in the Rifr region of rickettsial rpoB. This is the first successful demonstration of genetic transformation of Rickettsia prowazekii and represents the initial step in the establishment of a genetic system in this obligate intracellular pathogen.

[The use of a method of estimating the count of viable Rickettsia prowazekii for the screening of efficacious antibiotics]. / Ispol'zovanie metoda otsenki kolichestva zhiznesposobnykh Rickettsia prowazekii dlia skrininga éffektivnykh antibiotikov.

The possibility of the evaluation of antirickettsial activity of chemopreparations, both in vitro (by the inhibition of hemolytic activity) and in vivo (under the conditions of experimental pulmonary rickettsiosis in mice), with the use of the rapid method for the determination of the number of metabolically active rickettsiae is shown. The proposed approach may be used in the complex of mutually complementary methods for the screening of antirickettsial preparations.

Cytokine sensitivity and methylation of lysine in Rickettsia prowazekii EVir and interferon-resistant R. prowazekii strains.

Modified Rickettsia prowazekii strains have been derived from the avirulent Madrid E strain by passage in the lungs of white mice (strain EVir) or by selection for resistance to gamma interferon (IFN-gamma) (strains 427-19 and 87-17) or alpha/beta interferon (IFN-alpha/beta) (strains 83-2P, 60P, 103-2P, and 110-1P). Compared with the Madrid E strain, strain EVir has increased virulence (N. M. Balayeva and V. N. Nikolskaya, J. Hyg. Epidemiol. Microbiol. Immunol. 17:11-20, 1973) and a different lysine methylation profile in its surface protein antigen (A. V. Rodionov, M. E. Eremeeva, and N. M. Balayeva, Acta Virol. 35:557-565, 1991). The other six strains differ from the Madrid E strain in their resistance to IFN and their ability to grow well in untreated macrophagelike RAW264.7 cells. In the present study, to determine which properties are shared by these strains, we examined R. prowazekii EVir for the following: (i) the sensitivity of its growth in L929 cells to the cytokines IFN-alpha/beta, IFN-gamma, tumor necrosis factor alpha (TNF-alpha), and IFN-gamma plus TNF-alpha; (ii) the ability to grow in untreated RAW264.7 cells; and (iii) the ability to induce interferon in L929 cell cultures; we also evaluated strains 83-2P and 87-17 for lysine methylation. Multiplication of strain EVir in growing L929 cells was not markedly inhibited by either IFN-alpha/beta or IFN-gamma. In X-irradiated L929 cells, growth of strain EVir was slightly inhibited (11%) by TNF-alpha alone, somewhat inhibited (38%) by IFN-gamma alone, and markedly inhibited (87%) by IFN-gamma plus TNF-alpha. Nitrite production was induced in X-irradiated, strain EVir-infected L929 cell cultures treated with TNF-alpha alone or IFN-gamma alone; however, more nitrite was produced in infected cultures treated with IFN-gamma plus TNF-alpha. Nitrite production, the dramatic inhibitory effect of IFN-gamma plus TNF-alpha, and the modest inhibitory effect of IFN-gamma on the growth of strain EVir in X-irradiated L929 cells were all alleviated by the addition of the nitric oxide synthase inhibitor NG-methyl-L-arginine. Strain EVir grew very well in untreated macrophagelike RAW264.7 cells and appeared defective in the ability to induce IFN in L929 cell cultures. All strains grown in L929 cells in the presence of radiolabeled lysine had similar percentages of their radioactivity as methylated lysines.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of the peptidoglycan of Rickettsia prowazekii.

In the present study, peptidoglycan from Rickettsia prowazekii, an obligate intracellular bacterium, was purified. The rickettsial peptidoglycan is like that of gram-negative bacteria; that is, it is sodium dodecyl sulfate insoluble, lysozyme sensitive, and composed of glutamic acid, alanine, and diaminopimelic acid in a molar ratio of 1.0:2.3:1.0. The small amount of lysine found in the peptidoglycan preparation suggests that a peptidoglycan-linked lipoprotein(s) may be present in the rickettsiae. D-Cycloserine, a D-alanine analog which inhibits the biosynthesis of bacterial cell walls, prevented rickettsial growth in mouse L929 cells at a high concentration and altered the morphology of the rickettsiae at a low concentration. These effects were prevented by the addition of D-alanine. This suggests that R. prowazekii contains D-alanine in the peptidoglycan and has D-Ala-D-Ala ligase and alanine racemase activities.

[Comprehensive evaluation of the effectiveness of antibiotics in experimental typhus infection]. / Kompleksnaia otsenka éffektivnosti antibiotikov pri éksperimental'noi sypnotifoznoi infektsii.

The informative indices of antibiotic efficacy in the treatment of typhus were determined in experiments on guinea pigs and used for the design of an integral index of a drug prophylactic activity against Rickettsia prowazekii. A procedure for a comprehensive estimation of an antibiotic prophylactic efficacy in experimental typhus by the whole complex of clinical, immunological and rickettsiological properties is described.

Synthesis of DNA, rRNA, and protein by Rickettsia prowazekii growing in untreated or gamma interferon-treated mouse L929 cells.

The syntheses of DNA, rRNA, and protein by Rickettsia prowazekii growing in mouse fibroblastic L929 cells were measured at various times after the addition of gamma interferon (IFN-gamma) to correlate the inhibition of a site of macromolecular synthesis with the established IFN-gamma-induced inhibition of rickettsial growth. A method was developed to measure the syntheses of DNA, rRNA, and protein by R. prowazekii during a 2-h pulse-labeling period while the rickettsiae were growing within cultured host cells that had intact macromolecular synthesis. This method involved incubation of the rickettsia-infected cells with a radioactive precursor (H3 32PO4 or Tran35S-label), purification of the rickettsiae, purification of rickettsial nucleic acids, and analysis of rickettsial nucleic acids and proteins by electrophoresis and autoradiography. A key feature of the method involved the use of calculated specific activities from a densitometric analysis of gels and autoradiograms, a procedure that made the data independent of rickettsial recovery. Rickettsial DNA and rRNA syntheses were both inhibited 12 h after the addition of IFN-gamma to infected cultures, whereas the synthesis of rickettsial proteins was not inhibited at this time. In contrast, at 20 h after the addition of IFN-gamma, rickettsial DNA, rRNA, and protein syntheses were all inhibited.

Comparison of properties of virulent, avirulent, and interferon-resistant Rickettsia prowazekii strains.

Several properties of virulent, avirulent, and interferon-resistant Rickettsia prowazekii strains were compared. All of the interferon-resistant rickettsial strains (which were derived from the avirulent Madrid E strain) resembled the virulent Breinl strain in that they grew well in untreated mouse macrophagelike RAW264.7 cells. In contrast, the avirulent Madrid E strain grew poorly in untreated RAW264.7 cells. Pretreatment of interferon-resistant rickettsiae or R. prowazekii Breinl with antirickettsial serum or immunoglobulin G suppressed the ability of the rickettsiae to grow in untreated RAW264.7 cells. Interferon-resistant R. prowazekii strains, like the Madrid E and Breinl strains, rapidly killed a substantial proportion of RAW264.7 cells that had been treated with gamma interferon or very high concentrations of alpha/beta interferon. Untreated infected RAW264.7 cells and interferon-treated mock-infected RAW264.7 cells were not killed during the same period. In cultures of RAW264.7 cells treated with either alpha/beta interferon (120 to 1,200 U/ml) or a subsaturating concentration of gamma interferon (0.5 U/ml), R. prowazekii Breinl organisms killed a higher percentage of the cells than did comparable numbers of R. prowazekii Madrid E organisms or interferon-resistant rickettsiae. Although R. prowazekii Breinl (like R. prowazekii Madrid E) was quite sensitive to gamma interferon in mouse L929 cells, the Breinl strain was resistant to murine alpha/beta interferon compared with the Madrid E strain and the two strains selected for resistance to murine gamma interferon. One of the interferon-resistant strains (strain 60P, which was selected for resistance to murine alpha/beta interferon) differed from the other R. prowazekii strains in that it induced little or no detectable interferon in L929 cell cultures.

Selection of alpha/beta interferon- and gamma interferon-resistant rickettsiae by passage of Rickettsia prowazekii in L929 cells.

The ability of endogenously produced alpha/beta interferon (IFN-alpha/beta) to inhibit rickettsial growth in infected L929 cell cultures was evaluated by comparing the growth of Rickettsia prowazekii Madrid E in untreated cultures and cultures treated with anti-mouse IFN (alpha + beta) serum. The endogenously produced IFN was neutralized, and rickettsial growth was enhanced in the antiserum-treated cultures. This inhibitory effect of endogenously produced IFN-alpha/beta was used to select rickettsiae resistant to IFN-alpha/beta. Rickettsiae were screened for resistance to IFN-alpha/beta after being cultured in untreated L929 cells for several weeks to several months. Two isolates derived from R. prowazekii Madrid E and two isolates derived from plaque-purified R. prowazekii Madrid E were plaque-purified twice, grown in embryonated hen eggs, and evaluated for resistance to IFN-alpha/beta and IFN-gamma. Compared with the parental rickettsial strain, all four isolates were significantly resistant to IFN-alpha/beta and IFN-gamma. In addition, they were as resistant or more resistant to IFN-gamma when compared with two previously described IFN-gamma resistant isolates that were selected in IFN-gamma-treated L929 cells. One of the two isolates from IFN-gamma-treated L929 cells was also resistant to IFN-alpha/beta; the other isolate was similar to the parental Madrid E strain in sensitivity to IFN-alpha/beta.

[The effect of antibiotics and immunostimulants on rickettsial persistence in experimental typhus infection]. / Vliianie antibiotikov i immunostimuliatorov na persistirovanie rikketsii pri éksperimental'noi sypnotifoznoi infektsii.

A model of experimental typhus infection has shown that not only specific protection factors but also nonspecific ones are very important to remove Rickettsia prowazekii from macroorganisms. A complex application of antibiotics and drugs increasing immunobiological reactivity of the organism (prodigiosan, methyl uracil) noticeably decreases the rickettsia persistence level in the organism of experimental animals, the indices of specific and nonspecific protection being increased.