Rickettsia prowazekii uses an sn-glycerol-3-phosphate dehydrogenase and a novel dihydroxyacetone phosphate transport system to supply triose phosphate for phospholipid biosynthesis.

Rickettsia prowazekii is an obligate intracellular pathogen that possesses a small genome and a highly refined repertoire of biochemical pathways compared to those of free-living bacteria. Here we describe a novel biochemical pathway that relies on rickettsial transport of host cytosolic dihydroxyacetone phosphate (DHAP) and its subsequent conversion to sn-glycerol-3-phosphate (G3P) for synthesis of phospholipids. This rickettsial pathway compensates for the evolutionary loss of rickettsial glycolysis/gluconeogenesis, the typical endogenous source of G3P. One of the components of this pathway is R. prowazekii open reading frame RP442, which is annotated GpsA, a G3P dehydrogenase (G3PDH). Purified recombinant rickettsial GpsA was shown to specifically catalyze the conversion of DHAP to G3P in vitro. The products of the GpsA assay were monitored spectrophotometrically, and the identity of the reaction product was verified by paper chromatography. In addition, heterologous expression of the R. prowazekii gpsA gene functioned to complement an Escherichia coli gpsA mutant. Furthermore, gpsA mRNA was detected in R. prowazekii purified from hen egg yolk sacs, and G3PDH activity was assayable in R. prowazekii lysed-cell extracts. Together, these data strongly suggested that R. prowazekii encodes and synthesizes a functional GpsA enzyme, yet R. prowazekii is unable to synthesize DHAP as a substrate for the GpsA enzymatic reaction. On the basis of the fact that intracellular organisms often avail themselves of resources in the host cell cytosol via the activity of novel carrier-mediated transport systems, we reasoned that R. prowazekii transports DHAP to supply substrate for GpsA. In support of this hypothesis, we show that purified R. prowazekii transported and incorporated DHAP into phospholipids, thus implicating a role for GpsA in vivo as part of a novel rickettsial G3P acquisition pathway for phospholipid biosynthesis.

Inhibition of the growth of Rickettsia prowazekii in cultured fibroblasts by lymphokines.

The effect of lymphokine treatment of mouse and human fibroblast cell lines on the growth of Rickettsia prowazekii within the fibroblasts was studied. Treatment of mouse L929 cells with concanavalin A- or antigen-induced mouse lymphokines both before and after infection with R. prowazekii led to clearance of the rickettsiae from a substantial proportion of the cells and suppression of rickettsial growth in those cells which remained infected. Similar but less dramatic anti-rickettsial effects were observed in L929 cells treated with mouse lymphokines either only before or after infection with rickettsiae. Mouse lymphokine treatment of L929 cells had similar anti-rickettsial effects on the avirulent E strain and the virulent Breinl strain of R. prowazekii. Addition of cycloheximide or emetine to L929 cells at the same time as the lymphokines markedly suppressed the inhibition of rickettsial growth by the lymphokines. Mouse lymphokine treatment inhibited rickettsial survival and growth in mouse 3T3-A31 cells as well as in mouse L929 cells, but had no effect on rickettsial survival and growth in human foreskin fibroblasts. Conversely, concanavalin A-induced human lymphokines inhibited rickettsial survival and growth in human foreskin fibroblasts but had no effect on rickettsial survival and growth in mouse L929 cells. The rickettsia inhibitory activity in concanavalin A-induced mouse lymphokines was destroyed by heating the lymphokines at 80 degrees C for 10 min or by holding the lymphokines at pH 2 for 24 h but was retained after heating at 56 degrees C for 30 min.

[Cultivation of the clothes lice adapted to feeding on rabbits].

A race of clothes lice adapted to feeding on rabbits is kept at a laboratory longer than 50 years. For this period, more than 850 insect generations undergoing no change in a number of biological tests and morphological indices have been obtained. They have retained a high susceptibility to Provacheck rickettsia infection. All infected lice die, partially with the signs of hemolytic imbibition. Their rickettsial accumulation is as high as 10(5.0) ID50 per insect for albino rats.

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.

Rickettsia prowazekii, ribosomes and slow growth.

Some bacteria, such as Rickettsia prowazekii, grow slowly, not with anticipation of a future feast, but because it is evolutionarily advantageous to do so. This creates apparent paradoxes for understanding their physiology and biochemistry. These rickettsiae have a ribosome concentration higher than expected if these ribosomes support translation at rates comparable to those in Escherichia coli.

Growth of typhus group and spotted fever group rickettsiae in insect cells.

To analyze the host dependency of rickettsial growth, NIAS-AeAl-2 insect cells (AeAl2) derived from mosquito were first used in this study. It was demonstrated that typhus group rickettsiae (TGR) grew well in AeAl2 cells, but spotted fever group rickettsiae (SFGR) failed. To elucidate the inhibitory process of the growth of SFGR in AeAl2 cells, the adherence and invasion were first analyzed. SFGR possessed abilities to adhere to and invade AeAl2 cells as well as TGR in contrast to their inability of the growth in the cells. Morphologically, generation of microvilli could not be observed on AeAl2 cells inoculated with either group of rickettsiae. On the contrary, Vero cells inoculated with rickettsiae generated a great number of microvilli that adhered to rickettsiae and engulfed them into the cells. The roles of rickettsial major outer membrane protein A and B (rOmpA and rOmpB) were later investigated using E. coli expressing either rOmpA or rOmpB on their surface. Bacteria expressing either one of the major outer membrane proteins of rickettsiae as well as bacteria not expressing these proteins showed adherence to and invasion of AeAl2 cells. Thus, it is yet to be elucidated whether these major outer membrane proteins have any roles in these steps.

Obligate intracellular parasites: Rickettsia prowazekii and Chlamydia trachomatis.

Transitions to obligate intracellular parasitism have occurred at numerous times in the evolutionary past. The genome sequences of two obligate intracellular parasites, Rickettsia prowazekii and Chlamydia trachomatis, were published last year. A comparative analysis of these two genomes has revealed examples of reductive convergent evolution, such as a massive loss of genes involved in biosynthetic functions. In addition, both genomes were found to encode transport systems for ATP and ADP, not otherwise found in bacteria. Here, we discuss adaptations to intracellular habitats by comparing the information obtained from the recently published genome sequences of R. prowazekii and C. trachomatis.

Virulence of Rickettsia prowazeki for head lice.

Wild head lice were obtained by combing out adult and instar lice from the uncut hair of school children. Normal body lice were selected from a colony of rabbit-adapted body lice obtained from the United States Department of Agriculture and maintained in the Department of Microbiology for more than 10 yr. Thirty-nine head lice and 60 body lice were fed on a rabbit that had been injected intravenously with a 10% suspension of a yolk sac pool from eggs heavily infected with the Ankara strain of virulent R. prowazeki. Five days after infection, 33 body lice and 16 head lice had survived and were feeding on a volunteer. Between Days 5 and 9, 13 head lice were dead or moribund and all of them were positive by IF for R. prowazeki. The three surviving head lice were also positive. Tests on the 33 body lice showed that 22 were positive for R. prowazeki, including four of the five body lice that survived until Day 15. In summary, head lice can be readily infected with R. prowazeki and disseminate virulent R. prowazeki organisms in their feces. Thus, theoretically, head lice appear to be highly potential as transmitters of R. prowazeki under optimal epidemiologic circumstances.

Interferon-alpha/beta and Rickettsia prowazekii: induction and sensitivity.

Rickettsia prowazekii Madrid E established persistent infections in cultures of growing L-929 cells. Although some L-929 cells died, the cultures survived, remained infected with rickettsiae, and continued to grow. R. prowazekii Madrid E also induced interferon in L-929 cell cultures, and this interferon modulated rickettsial growth. Production of interferon (anti-viral activity) by cultures of R. prowazekii-infected L-929 cells was directly related to the initial rickettsial infection and was blocked by erythromycin. The media collected from R. prowazekii-infected L-929 cells suppressed not only the replication of vesicular stomatitis virus but also the growth of R. prowazekii in fresh L-929 cells. Both anti-viral and anti-rickettsial activities in the media were neutralized by antibodies against murine interferons-alpha and -beta, but not by antibodies against murine interferon-gamma. In addition, a commercial preparation of virus-induced interferons-alpha and -beta also suppressed rickettsial growth in L-929 cells. The combination of treating L-929 cells with this virus-induced interferon and infecting them with R. prowazekii killed some of the L-929 cells.

Properties in culture and persistence in cotton rats of the Rickettsia prowazekii vaccine strain E and its mutants.

Cultural properties and the capacity for persistence were studied in spontaneous erythromycin-resistant (E errSM), in induced erythromycin-resistant (E errI) mutants and in a virulent revertant (E Vir) of the vaccine strain E, as compared with parent vaccine strain E and standard virulent strain Breinl of Rickettsia prowazekii. Cultural properties of the strains were found to differ in passages in chick embryos (CE) and cultures of FL cells. Multiplication indices in CE of mutant E errI were significantly lower than those of other strains (E, E errSM, E Vir, Breinl). The multiplication rate in FL cells was found to be high in strains E errSM, Breinl, E Vir, being much lower in strains E errI and E. The capacity of the virulent revertant E Vir to persist in cotton rat (CR) was higher as compared with that of standard strain Breinl and significantly higher than that of the parent strain E. Low level carrier state of rickettsia was registered in CR infected with the mutant E errI.

Persistence of Rickettsia prowazekii in cotton rat macrophage cultures.

Rickettsia prowazekii is able to multiply and persist for a long time in cotton rat macrophage culture (29-days observation period). Electron microscopic studies showed that the structure of Rickettsiae remained intact at different intervals post-inoculation (p.i.). In the course of persistence Rickettsiae revealed a reduced capacity to infect chick embryos and guinea pigs, however, the infectious agent could be isolated at all stages of persistence of cultured cells such as fibroblasts of the guinea pig embryo, macrophages of intact cotton rats.

Rickettsiae as organisms.

Although most pathogenic rickettsiae are obligate intracellular parasites, it is clear that they are bacteria. As such, form and function in rickettsiae are closely similar to form and function found in their free-living counterparts. This review of rickettsiae as bacteria portrays the broad similarities of rickettsiae and free-living bacteria, as well as the differences which distinquish one group from the other and one rickettsia from another. Growth characteristics and requirements, ecologic influences, special adaptations, antibiotic susceptibilities and host-parasite relationships will be considered in a broad survey of likenesses and differences displayed by rickettsiae pathogenic to man.

Quantitative study on the reproduction of virulent and vaccine Rickettsia prow azeki strains in cells of different origin.

Vaccine and virulent strains of Rickettsia prowazeki differ by the degree of reproduction in McCoy, B, and chick embryo cells but replicate to similar levels in FL cells. As distinct from the virulent Breinl strain, the vaccine E strain rickettsiae permanently lost their capacity for long-term reproduction in McCoy cell cultures but retained their capacity to adsorb on to and penetrate into these cells. Consequently, the reproduction of rickettsiae is limited at later stages of intracellular infection. The E strain of R. prowazeki has been defined as a conditional lethal, host-dependent (hr) mutant.

Multiplication of Rickettsia prowazekii in cotton rat macrophage cultures.

The susceptibility of cotton rat macrophages to Rickettsia (R.) prowazekii, the percentage of the affected cells, and the intensity of damage to individual cells by rickettsiae were found to be much higher than those in guinea pig macrophages infected under similar conditions. At the same time, cotton rat macrophages proved to be more resistant to the effect of rickettsiae than guinea pig macrophages. Some common features of infection in cell culture and in animals have been observed. It is suggested that the outcome of interaction of rickettsiae with macrophages of one or another animal species may be important in generating acute or persistent infection.

Reproduction of vaccine and virulent Rickectsia prow azeki strains in continuous cell lines at different temperatures.

The features of intracellular development of the virulent Breinl strain and 3 vaccine E strains of Rickettsia prowazeki have been followed in continuous FL, McCoy, and B cell cultures at temperatures of 30, 35, 37, 38.5 and 40 degrees C. The virulent Breinl strain multiplied well at these temperatures in McCoy and B cells but in had been gradually lost when cultured at 40 degrees C in FL cells. In contrast to the virulent Breinl strain the vaccine E strains have lost their capacity of long term reproduction at 38.5 degrees C. At 40 degrees C the E strains did not multiply in and had been eliminated from the McCoy and B cells; thus the vaccine E strains revealed a ts-phenotype and, accordingly, it was found to represent a ts-mutant.

Enhancement of the antigenic activity and virulence of the vaccine strain E of Rickettsia prow azeki by passages in cell culture.

Changes in the biological properties of the vaccine strain E of Rickettsia prowazeki occurred upon cultivation of A1 (human amnion) cells infected with this strain. In the course of passages of these cells the antigenic activity and virulence of the rickettsia increased. The changes were observed in 10 out of 22 cell cultures examined: in 6 cultures there was an increase in the antigenic activity and in 4 both in the antigenic activity and in virulence. The time of the occurrence of these changes in the rickettsial populations varied from 12-18 to 53-102 days of passage of the infected cells.

Interaction of Rickettsia prowazekii strains of different virulence with white rat macrophages.

The growth of mildly pathogenic strain E, its virulent revertant EVir, and prototype virulent strain Breinl of Rickettsia prowazekii in peritoneal macrophage cultures of outbread white rats (WR) was evaluated by light microscopy and bioassay in chick embryos (CE). Macrophage cultures infected with strain E were characteristic by limited number of infected cells, poor or moderate accumulation of rickettsiae in individual cells, poor or nil spread of infectious process during first 7 days of infection, and the death of rickettsiae in cultures as determined by the bioassay in CE. Moreover, rickettsiae were not determined in 20.7% of infected macrophage cultures by either microscopic or bioassay methods. In contrast, the growth of virulent strains EVir and Breinl was characteristic by higher proportion of infected cells, considerable accumulation of rickettsiae, and intensive spread of infectious process within 5-7 days post infection (p.i.). However, the intensity of infectious process in macrophage cultures was less expressed with strain EVir than with strain Breinl.

Transcriptional regulation of the gltA and TLC genes in Rickettsia prowazekii growing in a respiration-deficient host cell.

The regulation of the citrate synthase (gltA) and ATP/ADP translocase (tlc) genes of the obligate intracellular bacterium, Rickettsia prowazekii, was analyzed in rickettsia-infected respiration-deficient G14 cells. The level of the gltA mRNAII and the tlc mRNA was much lower in the total RNA isolated from the infected G14 cells grown in 1 g/l glucose (low glucose, GL) medium than in that from infected G14 cells grown in 4.5 g/l glucose (high glucose, GH) medium. However, the level of the gltA mRNAI relative to 16 S rRNA was the same in GL and GH media. An increase in the level of the gltA mRNAII and the tlc mRNA could be observed as early as 2 hrs after shifting from GL to GH medium. We conclude that, under these experimental conditions, the tlc promoter and the gltA promoter P2, but not gltA promoter P1, were transcriptionally regulated.

[Current data on the polymorphism of Rickettsia prowazekii and burneti in cultured cells]. / Novye dannye o polimorfizme rikketsii Provacheka i Berneta pri kul'tivirovanii v kul'ture kletok

In cultivation of Rickettsia prowazeki (strains Breinl and E) in the cell cultures of guinea pig kidneys (GPK) and chick embryo fibroblasts (CEF) ultrastructure of rickettsia of unusual shape (filamentous, irregularpleomorphic and spheroplast-like) were revealed along with rickettsia of the usual shape and size. The polymorphism was less pronounced in the GPK and the CEF cells of Rickettsia burneti (strain M-44). It is supposed that rickettsial polymorphism was not associated with their developmental cycle and served as a morphological expression of the changes in the microorganism under the effect of unfavourable ecological conditions. The appearance of filamentous forms could be associated with disturbed cell division process; changed rigidity of the cell wall could serve as the cause of appearance of pleomorphic rickettsia. In difference from polymorphism, the cycle of rickettsial development is considered to be (in the basis of modern electron microscopic data) as a biological replacement of the vegetative (rod-like, bacillary) forms by those more stable in the external environment, resting (coccoid).

[Characteristics of rickettsial morphology during intracellular development]. / Osobennosti morfologii rikketsii pri ikh vnutrikletochnom razvitii.

The normal anatomy of rickettsiae has been characterized with the use of R. prowazekii, R. conorii and R. akari in continuous cell cultures L-929, Al, FL and in primary chick embryo fibroblast culture. Rickettsiae are short rod-shaped cells with the dense cytoplasm and the regular structure of the cell wall--cytoplasmic membrane complex. The study has shown the absence of polymorphism in rickettsiae growing under permissive conditions, but at the same time these organisms easily develop into pathological forms. Pathological forms can be detected alongside normal rickettsiae in the same cells. The classification of the pathological forms of rickettsiae is presented. In this classification the compensating (reversible) and destructive (irreversible) forms of alterations, as well as hypertrophic and dystrophic processes on the level of the whole rickettsial cell or its organelles, are pointed out.