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Acyl carrier proteins (ACPs) are small helical proteins found in all kingdoms of life, primarily involved in fatty acid and polyketide biosynthesis. In eukaryotes, ACPs are part of the fatty acid synthase (FAS) complex, where they act as flexible tethers for the growing lipid chain, enabling access to the distinct active sites in FAS. In the type II synthesis systems found in bacteria and plastids, these proteins exist as monomers and perform various processes, from being a donor for synthesis of various products such as endotoxins, to supplying acyl chains for lipid A and lipoic acid FAS (quorum sensing), but also as signaling molecules, in bioluminescence and activation of toxins. The essential and diverse nature of their functions makes ACP an attractive target for antimicrobial drug discovery. Here, we report the structure, dynamics and evolution of ACPs from three human pathogens: Borrelia burgdorferi, Brucella melitensis and Rickettsia prowazekii, which could facilitate the discovery of new inhibitors of ACP function in pathogenic bacteria., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

AbstractFlea-borne (murine) typhus is a global rickettsiosis caused by Rickettsia typhi . Although flea-borne typhus is no longer nationally notifiable, cases are reported for surveillance purposes in a few U.S. states. The infection is typically self-limiting, but may be severe or life-threatening in some patients. We performed a retrospective review of confirmed or probable cases of fatal flea-borne typhus reported to the Texas Department of State Health Services during 1985-2015. When available, medical charts were also examined. Eleven cases of fatal flea-borne typhus were identified. The median patient age was 62 years (range, 36-84 years) and 8 (73%) were male. Patients presented most commonly with fever (100%), nausea and vomiting (55%), and rash (55%). Respiratory (55%) and neurologic (45%) manifestations were also identified frequently. Laboratory abnormalities included thrombocytopenia (82%) and elevated hepatic transaminases (63%). Flea or animal contact before illness onset was frequently reported (55%). The median time from hospitalization to administration of a tetracycline-class drug was 4 days (range, 0-5 days). The median time from symptom onset to death was 14 days (range, 1-34 days). Flea-borne typhus can be a life-threatening disease if not treated in a timely manner with appropriate tetracycline-class antibiotics. Flea-borne typhus should be considered in febrile patients with animal or flea exposure and respiratory or neurologic symptoms of unknown etiology.

In August 2012, laboratory tests confirmed a mixed outbreak of epidemic typhus fever and trench fever in a male youth rehabilitation center in western Rwanda. Seventy-six suspected cases and 118 controls were enrolled into an unmatched case-control study to identify risk factors for symptomatic illness during the outbreak. A suspected case was fever or history of fever, from April 2012, in a resident of the rehabilitation center. In total, 199 suspected cases from a population of 1,910 male youth (attack rate = 10.4%) with seven deaths (case fatality rate = 3.5%) were reported. After multivariate analysis, history of seeing lice in clothing (adjusted odds ratio [aOR] = 2.6, 95% confidence interval [CI] = 1.1-5.8), delayed (≥ 2 days) washing of clothing (aOR = 4.0, 95% CI = 1.6-9.6), and delayed (≥ 1 month) washing of beddings (aOR = 4.6, 95% CI = 2.0-11) were associated with illness, whereas having stayed in the rehabilitation camp for ≥ 6 months was protective (aOR = 0.20, 95% CI = 0.10-0.40). Stronger surveillance and improvements in hygiene could prevent future outbreaks., (© The American Society of Tropical Medicine and Hygiene.)

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.

Methylation of rickettsial OmpB (outer membrane protein B) has been implicated in bacterial virulence. Rickettsial methyltransferases RP789 and RP027-028 are the first biochemically characterized methyltransferases to catalyze methylation of outer membrane protein (OMP). Methylation in OMP remains poorly understood. Using semiquantitative integrated liquid chromatography-tandem mass spectroscopy, we characterize methylation of (i) recombinantly expressed fragments of Rickettsia typhi OmpB exposed in vitro to trimethyltransferases of Rickettsia prowazekii RP027-028 and of R. typhi RT0101 and to monomethyltransferases of R. prowazekii RP789 and of R. typhi RT0776, and (ii) native OmpBs purified from R. typhi and R. prowazekii strains Breinl, RP22, and Madrid E. We found that in vitro trimethylation occurs at relatively specific locations in OmpB with consensus motifs, KX(G/A/V/I)N and KT(I/L/F), whereas monomethylation is pervasive throughout OmpB. Native OmpB from virulent R. typhi contains mono- and trimethyllysines at locations well correlated with methylation in recombinant OmpB catalyzed by methyltransferases in vitro. Native OmpBs from highly virulent R. prowazekii strains Breinl and RP22 contain multiple clusters of trimethyllysine in contrast to a single cluster in OmpB from mildly virulent R. typhi. Furthermore, OmpB from the avirulent strain Madrid E contains mostly monomethyllysine and no trimethyllysine. The native OmpB from Madrid E was minimally trimethylated by RT0101 or RP027-028, consistent with a processive mechanism of trimethylation. This study provides the first in-depth characterization of methylation of an OMP at the molecular level and may lead to uncovering the link between OmpB methylation and rickettsial virulence.

Several of the infectious diseases associated with human lice are life-threatening, including epidemic typhus, relapsing fever, and trench fever, which are caused by Rickettsia prowazekii, Borrelia recurrentis, and Bartonella quintana, respectively. Although these diseases have been known for several centuries, they remain a major public health concern in populations living in poor-hygiene conditions because of war, social disruption, severe poverty, or gaps in public health management. Poor-hygiene conditions favour a higher prevalence of body lice, which are the main vectors for these diseases. Trench fever has been reported in both developing and developed countries in populations living in poor conditions, such as homeless individuals. In contrast, outbreaks of epidemic typhus and epidemic relapsing fever have occurred in jails and refugee camps in developing countries. However, reports of a significantly high seroprevalence for epidemic typhus and epidemic relapsing fever in the homeless populations of developed countries suggest that these populations remain at high risk for outbreaks of these diseases. Additionally, experimental laboratory studies have demonstrated that the body louse can transmit other emerging or re-emerging pathogens, such as Acinetobacter baumannii and Yersinia pestis. Therefore, a strict survey of louse-borne diseases and the implementation of efficient delousing strategies in these populations should be public health priorities., (© 2012 The Authors. Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.)

Background: Rickettsia prowazekii is the etiological agent of epidemic typhus and is an obligate intracellular bacterium that grows as a parasite freely within the cytoplasm of a eukaryotic host cell. Previous studies have shown that rOmpA and rOmpB which belong to the family of rickettsial cell surface antigens are involved in vitro in the adhesion of Rickettsiae to epithelial cells. Recently, two putative rickettsial adhesins have been identified using high resolution 2D-PAGE coupled with mass spectrometry. In this study, we further characterize and describe the adhesin Adr2 from R. prowazekii., Methodology/principal Findings: Using an overlay assay coupled with mass spectrometry two adhesins, Adr1 (RP827) and Adr2 (RP828), were identified from the R. prowazekii proteome Recombinant R. prowazekii Adr2 was expressed through fusion with Dsbc in Escherichia coli, purified and concentrated, thus allowing production of specific monoclonal antibodies, as confirmed by western blot assays. Finally, inhibition of rickettsiae-induced cytotoxicity with monoclonal anti-Adr2 antibody has showed a greatest impact on bacterial cell entry at 8 h post-infection (ca50%) and then decreased progressively to attempt 18% of inhibition at day 7. These, correlated to the inhibition of rickettsiae-induced cytotoxicity with monoclonal anti-rOmpB antibody. Thus, Adr2 is sufficient to mediate R. prowazekii entry into the cell at early stage of mammalian cell infection., Conclusions: Our results suggest that R. prowazekii Adr2 could be the main actor promoting the entry of rickettsiae into the host cells. The present study opens the framework for future investigations for better understanding of the Adr2 -mediated mechanisms involved in adhesion/invasion or intracellular survival of R. prowazekii., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Rickettsia prowazekii, the agent of epidemic typhus, is an obligate intracellular bacterium that is transmitted to human beings by the body louse. Several strains that differ considerably in virulence are recognized, but the genetic basis for these variations has remained unknown since the initial description of the avirulent vaccine strain nearly 70 yr ago. We use a recently developed murine model of epidemic typhus and transcriptomic, proteomic, and genetic techniques to identify the factors associated with virulence. We identified four phenotypes of R. prowazekii that differed in virulence, associated with the up-regulation of antiapoptotic genes or the interferon I pathway in the host cells. Transcriptional and proteomic analyses of R. prowazekii surface protein expression and protein methylation varied with virulence. By sequencing a virulent strain and using comparative genomics, we found hotspots of mutations in homopolymeric tracts of poly(A) and poly(T) in eight genes in an avirulent strain that split and inactivated these genes. These included recO, putative methyltransferase, and exported protein. Passage of the avirulent Madrid E strain in cells or in experimental animals was associated with a cascade of gene reactivations, beginning with recO, that restored the virulent phenotype. An area of genomic plasticity appears to determine virulence in R. prowazekii and represents an example of adaptive mutation for this pathogen.

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligately intracytoplasmic bacterium, a lifestyle that imposes significant barriers to genetic manipulation. The key to understanding how this unique bacterium evades host immunity is the mutagenesis of selected genes hypothesized to be involved in virulence. The R. prowazekii pld gene, encoding a protein with phospholipase D activity, has been associated with phagosomal escape. To demonstrate the feasibility of site-directed knockout mutagenesis of rickettsial genes and to generate a nonrevertible vaccine strain, we utilized homologous recombination to generate a pld mutant of the virulent R. prowazekii strain Madrid Evir. Using linear DNA for transformation, a double-crossover event resulted in the replacement of the rickettsial wild-type gene with a partially deleted pld gene. Linear DNA was used to prevent potentially revertible single-crossover events resulting in plasmid insertion. Southern blot and PCR analyses were used to confirm the presence of the desired mutation and to demonstrate clonality. While no phenotypic differences were observed between the mutant and wild-type strains when grown in tissue culture, the pld mutant exhibited attenuated virulence in the guinea pig model. In addition, animals immunized with the mutant strain were protected against subsequent challenge with the virulent Breinl strain, suggesting that this transformant could serve as a nonrevertible, attenuated vaccine strain. This study demonstrates the feasibility of generating site-directed rickettsial gene mutants, providing a new tool for understanding rickettsial biology and furthering advances in the prevention of epidemic typhus.

Rickettsia prowazekii, the etiologic agent of epidemic typhus, infects vascular endothelium, leading to vasculitis and tissue infiltration of leukocytes. Murine and human endothelial cells (ECs) were infected with R. prowazekii, including the virulent Breinl strain and the attenuated Madrid E strain. The transendothelial migration (TM) of murine and human peripheral blood mononuclear cells (PBMCs) across ECs infected with Breinl organisms was significantly increased compared with that for uninfected ECs or for ECs infected with attenuated organisms, demonstrating that increased TM was related to R. prowazekii virulence. Increased TM was associated with a specific inflammatory pattern. Indeed, only Breinl organisms induced the expression of transcripts for inflammatory cytokines and chemokines by ECs. Murine PBMCs that had transmigrated across ECs infected with Breinl organisms overexpressed inflammatory cytokines and chemokines as well as tissue factor, whereas interleukin-10 expression was down-regulated. The impact of R. prowazekii infection on the TM of PBMCs may play a prominent role in the development of lesions in epidemic typhus.

Comparative genomics of Rickettsia and Orientia has revealed an exciting interplay between reductive evolutionary forces acting on metabolic genes in all species and proliferation of mobile genetic elements in some species. These contradictory evolutionary forces highlight the influence of chance, adaptation and host-cell exploitation during the evolution of intracellular bacteria.

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.

Rudolph Weigl gained a high estimation for his spectacular invention of the first efficient vaccine against typhus fever. Although born a German native speaker, he changed into a great Pole with Polish education and upbringing in a patriotic atmosphere. His scientific career developed in the Universities of Lvov, Cracow and Poznan. For him, academic employment was a constant exploration of practical truths and a response to human needs, and this remained a deep obligation during his lifetime. His vaccine saved millions, particularly during World War II. Despite many nominations, he was never awarded the Nobel Prize.

Rickettsia prowazekii, an obligate intracellular Gram-negative bacterium, is the etiologic agent of epidemic typhus. We analyzed the proteome of the virulent Breinl strain of R. prowazekii purified from infected egg yolk sacs. Total proteins from purified R. prowazekii Breinl strain were reduced by dithiothreitol, alkylated by iodoacetic acid and digested with trypsin followed by analysis with an integrated two-dimensional liquid chromatography and mass spectrometry system (2D-LC/MS/MS). A comparison was made using previously analyzed proteome of the Madrid E strain and current analysis of the Breinl strain. For Breinl 251 proteins were identified, representing 30% of the total protein-encoding genes, using a shotgun 2D-LC/MS/MS proteomic approach. This result is identical to that of Madrid E strain. Among the identified proteins, 33 from Breinl and 37 from Madrid E have an unknown function. A methyltransferase, RP028/RP027, whose gene is mutated in the avirulent Madrid E strain but not in the virulent Breinl strain, was only detectable in the Breinl strain, consistent with the genetic mutation in Madrid E. This result suggests the possible relationship between this gene product and the virulence of the strains.

Rickettsia prowazekii Madrid E (E) strain is an effective vaccine, but can revert to virulent status when passaged in animals. The aim of this study is to identify the reverse mutation that may determine the virulence of R. prowazekii by comparing the genetic structures of E strain and its virulent revertant Evir strain. We determined that the gene (Rp028/Rp027) encoding the methyltransferase was mutated by frameshift in avirulent E strain but not in virulent revertant Evir strain and wild type virulent Breinl strain. We conclude that the mutation in the E strain gene reverts to wild type in the virulent revertant Evir strain. Whether the mutation plays an essential role in the attenuation of E strain needs to be further investigated.

Rickettsia prowazekii is an obligate intracellular gram-negative bacterium. Comparative proteomics study of a virulent strain (Breinl) versus an avirulent strain (Madrid E) was performed using an integrated liquid chromatography and mass spectrometer. About 30% of predicted proteins were detected and identified. Among the detected proteins, more than 30 proteins were of unknown function in both strains. Although several proteins were detected in only one strain, the overall distribution of detected proteins in different COGs (clusters of orthologs groups) was very similar between the two strains. Functional analysis of differentially expressed proteins, either qualitatively or quantitatively, may lead to the discovery of pathogenesis-related factors.

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.

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.

Members of the genus Rickettsia possess the ability to invade host cells and promptly escape from phagosomal vacuoles into the host cell cytosol, thereby avoiding destruction within the endosomal pathway. The mechanism underlying rickettsial phagosomal escape remains unknown, although the genomic sequences of several rickettsial species have allowed for the identification of four genes with potential membranolytic activities (tlyA, tlyC, pat1, and pld). This study was undertaken to determine which of the selected genes of Rickettsia prowazekii mediate the escape process. Quantitative ultrastructural analyses indicated that the period of active phagosomal escape was between 30 and 50 min postinfection. Reverse transcriptase PCR analyses determined that tlyC and pld were transcribed during the period of active phagosomal escape but that tlyA and pat1 were not. The functionality of both tlyC and pld was determined by complementation studies of Salmonella, which replicates within endosomes. Complementation of Salmonella organisms with either tlyC or pld resulted in the escape of transformants from endosomal vacuoles into the host cell cytosol demonstrated by quantitative ultrastructural analyses. These data suggest a role for tlyC and pld in the process of phagosomal escape by R. prowazekii.

The outer membrane protein B (OmpB) of the typhus group rickettsiae is an immunodominant antigen and has been shown to provide protection against typhus in animal models. Consequently, OmpB is currently being considered as a potential rickettsiae vaccine candidate to be used in humans. The OmpB from virulent strains are heavily methylated while the attenuated strains are hypomethylated. Western blot analysis of partially digested OmpB revealed that one of the reactive fragments was located at the N-terminus (fragment A, aa 33-272). Recently, we have over expressed, purified, and chemically methylated the recombinant fragment A from Rickettsia prowazekii (Ap). The methylated Ap was thoroughly characterized by LC/MS/MS on the ProteomeX workstation. The protein sequence of Ap with and without methylation was 87.7% and 100% identified, respectively. This high sequence coverage enabled us to determine the sites and extent of methylation on the lysine residues in Ap. All the lysine residues except the C-terminus lysine were either mono-, di- or tri-methylated. In addition, carbamylation on the N-terminus glycine was identified using a combination of denovo sequencing (DeNovoX) and the pattern recognition (SALSA) program with accurate mass measurement. We demonstrated that the use of peptide identification (SEQUEST) in combination with SALSA and denovo sequencing provided a useful means to characterize the sequence and posttranslational modifications of given proteins.

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.

Rickettsia prowazekii, the causative agent of epidemic typhus, has been responsible for millions of human deaths. Madrid E is an attenuated strain of R. prowazekii, while Breinl is a virulent strain. The genomic DNA sequence of Madrid E has recently been published. To study the genomic variations between Madrid E (reference) and Breinl (test) DNAs, cohybridization experiments were performed on a DNA microarray containing all 834 protein-coding genes of Madrid E. Of the 834 genes assessed, 24 genes showed 1.5- to 2.0-fold increases in hybridization signals in Breinl DNA compared to Madrid E DNA, indicating the presence of genomic variations in approximately 3% of the total genes. Eighteen of these 24 genes are predicted to be involved in different functions. Southern blot analysis of five genes, virB4, ftsK, rfbE, lpxA, and rpoH, suggested the presence of an additional paralog(s) in Breinl, which might be related to the observed increase in hybridization signals. Studies by real-time reverse transcription-PCR revealed an increase in expression of the above-mentioned five genes and five other genes. In addition to the elevated hybridization signals of 24 genes observed in the Breinl strain, one gene (rp084) showed only 1/10 the hybridization signal of Madrid E. Further analysis of this gene by PCR and sequencing revealed a large deletion flanking the whole rp084 gene and part of the rp083 gene in the virulent Breinl strain. The results of this first rickettsial DNA microarray may provide some important information for the elucidation of pathogenic mechanisms of R. prowazekii.

Rickettsia prowazekii is transmitted to humans by the body louse. A new experimental model of body louse infection with R. prowazekii is reported here. Eight hundred human lice were infected by feeding on a rabbit that was made bacteremic by injecting 2x106 plaque-forming units of R. prowazekii. The bacterium invaded the stomach cells and was released in feces, in which it was detected 5 days after infection. At day 6 after infection, as a result of the cell burst and the spread of erythrocytes in the hemolymph, the louse became bright red and died within 4 h. The life span of infected lice was shortened by 20-23 days, compared with that of uninfected control lice. Infected lice did not transmit R. prowazekii to their progeny. Through cell culture, rickettsiae were cultivated from fecal samples up to 10 days after their emission. The administration of doxycycline to the rabbit during louse feeding did not cure lice from R. prowazekii infection.

Using polymerase chain reaction and sequencing, we investigated the prevalence of Rickettsia prowazekii, Bartonella quintana, and Borrelia recurrentis in 841 body lice collected from various countries. We detected R. prowazekii in body lice from Burundi in 1997 and in lice from Burundi and Rwanda in 2001; B. quintana infections of body lice were widespread. We did not detect B. recurrentis in any lice.

A growing number of pathogens are being found to possess specialized secretion systems which they use in various ways to subvert host defenses. One class, called type IV, are defined as having homology to the conjugal transfer systems of naturally occurring plasmids. It has been proposed that pathogens with type IV secretion systems have acquired and adapted the conjugal transfer systems of plasmids and now use them to export toxins. Several well-characterized intracellular pathogens, including Legionella pneumophila, Coxiella burnetii, Brucella abortus, and Rickettsia prowazekii, contain type IV systems which are known or suspected to be of critical importance in their ability to cause disease. Specifically, these systems are believed to be the key factors determining intracellular fate, and thus the ability to replicate and cause disease.

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.

Within the alpha subdivision of proteobacteria, the arthropod-borne human pathogens Rickettsia prowazekii and Bartonella henselae provide examples of bacteria with obligate and facultative intracellular life styles, respectively. The complete genome sequence of R. prowazekii has been published, whereas the sequencing of the B. henselae genome is in its final stage. Here, we provide a brief overview of a comparative analysis of both genomes based on the delineated metabolic properties. The relative proportion of genes devoted to basic information processes is similar in the two genomes. In contrast, a full set of genes encoding proteins involved in the biosynthesis of amino acids and nucleotides is present in B. henselae, while the majority of these genes is absent from R. prowazekii. This suggests that B. henselae has a better potential for growth in the free-living mode, whereas R. prowazekii is more specialised to growth in an intracellular environment. Functional genomics will provide the potential to further resolve the genetic basis for successful human infections by these important parasites.

The analysis of the results of prolonged observations on the prophylactic immunization of employees working with R. prowazekii is presented. The necessity of the differentiated approach to the determination of the immunization schedule and the choice of vaccine is shown. The presence of specific antibodies (Ab) and the level of their titers have been found to be related to the degree of anti-infectious protection. The following characteristics indicate the presence of profound immunological transformation in vaccinees: complement-fixing Ab in titers 1:10 and more and/or immunofluorescent Ab in titers not below 1:180, Ab to protein in the hemagglutination test in titers not below 1:1000. These specific Ab and the level of their titers can be registered after the second injection of live combined typhus vaccine E and the third injection of chemical typhus vaccine. Cases of laboratory infection and their relationship to the character of immunization and the intensity of contacts with R. prowazekii virulent strains are discussed. Attention is drawn to the strict observance of professional safety rules.

We describe here the complete genome sequence (1,111,523 base pairs) of the obligate intracellular parasite Rickettsia prowazekii, the causative agent of epidemic typhus. This genome contains 834 protein-coding genes. The functional profiles of these genes show similarities to those of mitochondrial genes: no genes required for anaerobic glycolysis are found in either R. prowazekii or mitochondrial genomes, but a complete set of genes encoding components of the tricarboxylic acid cycle and the respiratory-chain complex is found in R. prowazekii. In effect, ATP production in Rickettsia is the same as that in mitochondria. Many genes involved in the biosynthesis and regulation of biosynthesis of amino acids and nucleosides in free-living bacteria are absent from R. prowazekii and mitochondria. Such genes seem to have been replaced by homologues in the nuclear (host) genome. The R. prowazekii genome contains the highest proportion of non-coding DNA (24%) detected so far in a microbial genome. Such non-coding sequences may be degraded remnants of 'neutralized' genes that await elimination from the genome. Phylogenetic analyses indicate that R. prowazekii is more closely related to mitochondria than is any other microbe studied so far.

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.

Rickettsiae are bacterial obligate intracellular parasites ranging from harmless endosymbionts to the etiologic agents of some of the most devastating diseases known to mankind. Rickettsiae are primarily associated with arthropod vectors in which they may exist commensally and, in most cases, only accidentally infect humans. These fascinating microbes are the prototypical obligate intracellular parasites. Other than being extremely fastidious in their growth requirements, however, rickettsiae are typical gram-negative bacteria. Only a few intracellular parasites multiply within the cytoplasm of eukaryotic cells. In this environment, rickettsiae are provided with a rich source of biosynthetic precursors not normally encountered by free-living bacteria and have evolved a number of unique mechanisms to transport such metabolites as nucleotides and nucleotide sugars. The physiologic basis for their obligate parasitism, however, has remained elusive for > 90 years. Other than the obvious property of replicating inside eukaryotic cells, the molecular mechanisms of cellular damage are ill defined. The typhus-group rickettsiae multiply within host cells to great numbers without profound damage until lysis occurs. In contrast, the spotted fever-group rickettsiae spread rapidly from cell to cell by an actin-based motility. This property, in itself, is not sufficient to cause cell death, because avirulent spotted fever-group rickettsiae also spread by actin-based movement but do not cause lysis of the host cell. Despite the obvious limitations imposed by their obligate intracellular lifestyle and the current lack of methods for genetic manipulation, there are enough interesting biological properties of rickettsiae to offer an attractive area for research.

A 29.5 kda outer membrane protein (OmpB) of R. prowazekii virulent Breinl strain is known to differ from its counterpart in attenuated Madrid E strain, while OmpB of this latter one and of its virulent variant EVir coincide in mobility. The infectivity of these strains for macrophages was previously shown to be different as well, and to correlate with their virulence. Previously cloned R. prowazekii Breinl strain, 1.644-bp insert from lambda gtll recombinant expressing as OmpB in inducer-independent fashion was sequenced and used as a query to search for similarity in non-redundant GenBank/EMBL/DDBJ Data Base aided by BLASTX mail server. Extensive homology of inferred 282-aa sequence to peptidyl-propyl cis/trans isomerase C (PPIase C) of E. coli belonging to parvulin family of rotamases (foldases), and related proteins such as Campylobacter jejuni cell binding factor 2 (Cbf2), B. subtilis PrsA, and Lactococcus lactis PrtM has been revealed.

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.

Rickettsia prowazekii invades nucleated cells through phagocytosis and subsequently proliferates in the cytoplasm of the host cell. Hemolysis and a phospholipase A2 (PLA2) activity at neutral pHs have previously been reported; even though the phagosomal environment is most likely acidic. We here show that R. prowazekii and R. typhi also lyse erythrocytes at mildly acidic pHs, compatible with an early phagosomal compartment. For R. prowazekii, hemolysis at an acidic pH but not a neutral pH is enhanced by Ca2+, raising the possibility that more than one membranolytic factor may be produced by the rickettsiae. The rickettsiae alone display PLA2 activity, implying that the enzyme is of bacterial rather than erythrocyte or host cell origin. Moreover, the PLA2 activity requires divalent cations (Ca2+ or Mg2+), and, as with many extracellular PLA2s from other species, it has a preference for acidic over neutral phospholipids. The pH dependence of PLA2 is similar to that of the hemolysis without Ca2+, but in the presence of the hemolysis buffers (which contain Mg2+), there is no calcium-induced enhancement at acidic pHs. Thus, these rickettsiae are endowed with a membranolytic activity that could contribute to the escape of the bacteria from early phagosomal compartments, and it is likely that multiple toxins may be used for membrane lysis.

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)

Five strains of Rickettsia prowazekii different in origin, biological and genetic properties were compared in protein and LPS patterns by the polyacrylamide gel electrophoresis and in antigenic properties by immunoblotting with specific sera of infected white mice. Three virulent strains Breinl, G and Katsinjan had identical protein patterns and differed from isogenic pair of strains E and EVir in the electrophoretic properties of 29-30 kDa proteins. Silver-strained LPS patterns were different in five compared strains. Strain G and strain Katsinjan had the longest O-chaines of LPS. Polyclonal mouse antisera contained specific antibodies which mainly directed against LPS and 25-60 kDa proteins. Strains E and EVir were identical in all performed immunoblotting reactions and separated from three virulent strains. Out of virulent strains, whole cell antigen of strain Katsinjan and LPS antigen of strain G had different reactions in comparison with correspondent antigen of the standard strain Breinl.

Macrophage-like RAW264.7 cells are killed by the combination of gamma interferon (IFN-gamma) treatment and infection with Rickettsia prowazekii. The roles of tumor necrosis factor alpha (TNF-alpha), the nitric oxide synthase pathway, and lipopolysaccharide (LPS) in this killing were investigated. R. prowazekii, both the Breinl and Madrid E strains, induced RAW264.7 cells to produce TNF-alpha. However, dead rickettsiae (which cannot kill the IFN-gamma-treated RAW264.7 cells) induced the production of as much TNF-alpha as viable rickettsiae. Inhibition of the production of TNF-alpha (by the addition of actinomycin D or emetine during the rickettsial infection) or neutralization of TNF-alpha (by the addition of polyclonal rabbit anti-mouse TNF-alpha serum both during the IFN-gamma treatment and during the rickettsial infection) did not inhibit the killing of the RAW264.7 cells. Addition of polymyxin B (which inhibits many effects of LPS) during the IFN-gamma treatment did not inhibit the ability of IFN-gamma to prepare the RAW264.7 cells to be killed by R. prowazekii. Suppression of nitrite production by addition of the nitric oxide synthase inhibitor aminoguanidine both during the IFN-gamma treatment and during the rickettsial infection also did not inhibit the killing of the RAW264.7 cells. R. prowazekii-mediated killing of the RAW264.7 cells was dramatically suppressed in cultures treated with IFN-gamma plus LPS compared with that in cultures treated with IFN-gamma alone, and inhibition of nitric oxide synthase restored the rickettsia-induced killing of the RAW264.7 cells in cultures treated with IFN-gamma plus LPS. These data indicate that (i) TNF-alpha, LPS, and the nitric oxide synthase pathway are not required in order for IFN-gamma to prepare RAW264.7 cells to be killed by R. prowazekii; (ii) neither TNF-alpha nor the nitric oxide synthase pathway is responsible for the killing of the IFN-gamma-treated RAW264.7 cells by R. prowazekii; and (iii) in cultures treated with IFN-gamma plus LPS and then incubated with rickettsiae, a nitric oxide synthase pathway-dependent mechanism inhibits the killing of the RAW264.7 cells.

Endothelial cells (EC) synthesize platelet-activating factor (PAF) when activated by agents such as ATP or thrombin, and PAF production occurs as a consequence of endothelial phospholipase A activity. Because interactions between Rickettsia prowazekii and a variety of host cells result in the expression of phospholipase A activity, we assessed the relative abilities of uninfected and rickettsia-infected EC to synthesize PAF. Endothelial cells were infected with rickettsiae and examined at 24-h intervals for rickettsial multiplication, EC viability, and PAF synthesis. By 24 h postinfection, 80% of the EC were infected with an average of 10.6 rickettsiae per cell; by 72 h, the rickettsiae were too numerous to count and the numbers of viable EC began to decrease. Both rickettsia-infected and sham-treated EC synthesized PAF when stimulated with either thrombin or ATP, but rickettsia-infected EC synthesized about three times as much PAF in response to cell activation as did their uninfected counterparts. Additionally, unlike their uninfected counterparts, rickettsia-infected EC synthesized significant amounts of PAF in the absence of cell activation; rickettsia-infected EC synthesized as much PAF in the absence of activation as did uninfected EC in response to ATP. In each case, essentially all of the newly synthesized PAF remained with the cell pellet. Finally, EC incubated with high numbers of rickettsiae (1,000 rickettsiae per EC) for 30 min synthesized more PAF when activated with ATP than did their sham-treated activated counterparts.

The protein antigens of two distinct lines of genetically related strains, namely the nonpathogenic strain E and its virulent revertant EVir and of the standard virulent strain Breinl were compared in SDS-PAGE and immunoblot assay using typhus patient sera and immune rabbit sera. No differences in the polypeptide pattern as detected in SDS-PAGE were found between strain E and EVir; the Breinl strain differed in a 30 kD protein. The high immunogenicity of the protein antigens of E, EVir and Breinl strains was demonstrated by immunoblot assay with human sera, which did not show any differences between the strains studied. Immunoblot analysis with immune rabbit sera to the strain E, EVir, and Breinl showed differences in immunological response to the 70 kD and 60 kD polypeptides of low virulent strain E and those of virulent strains EVir and Breinl.

The major M(r) 100 kD protein (protein I) from the standard virulent R. prowazekii strain Breinl, from the nonvirulent strain E and its virulent revertant EVir were isolated by chromatography and characterized. Purified protein I from the three strains of different virulence and origin had the same physico-chemical and antigenic properties, protected guinea pigs against infection with the virulent strain Breinl and induced the production of antibodies, which neutralized the toxic and haemolytic activities of R. prowazekii. The amino acid composition of protein I as determined for the three above mentioned strains was similar. Modified residues of Lys, Asn, and/or Gln were found in protein I. Protein I from virulent strains Breinl and EVir differed from that of nonpathogenic strain E by the quantity of N epsilon-Me-Lys and N epsilon-Me3-Lys, but all had the same total amount of Lys and its derivatives. It may be suggested that a difference may exist in the processing of the protein I of nonpathogenic strain E and of virulent strains of R. prowazekii.

Electron microscopy has provided valuable insights into the study of rickettsiae as intracellular parasites from several important perspectives. This tool has allowed researchers to delineate the fine structural features of these organisms and to show that they truly resemble free-living bacteria. Furthermore, it has been shown that there are subtle, but distinct differences in the outer envelope structure of some members of the genus Rickettsia that may explain reported differences in tinctorial properties and in their sensitivity to certain antibiotics. With Coxiella burnetii, electron microscopy has helped significantly in the characterization of the pleomorphic nature of the organism including formation of terminal bodies that resemble endospores of gram-positive bacteria. Electron microsxopy has also helped to define the relationship of the rickettsiae to their host cells. For example, ultrastructural analysis can reveal whether organisms exist free within the cytoplasm or nucleus (members of the genus Rickettsia), or whether they are bound by a phagosomal or phagolysosomal membrane (Ehrlichia and Coxiella). Finally, although all rickettsiae eventually destroy their host cell, it has been shown through transmission electron microscopy that this destruction might be mediated by different mechanisms that are specific for different rickettsial species.

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.

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.