Complete genome sequence of Rickettsia typhi and comparison with sequences of other rickettsiae

Rickettsia typhi, the causative agent of murine typhus, is an obligate intracellular bacterium with a life cycle involving both vertebrate and invertebrate hosts. Here we present the complete genome sequence of R. typhi (1,111,496 bp) and compare it to the two published rickettsial genome sequences: R. prowazekii and R. conorii. We identified 877 genes in R. typhi encoding 3 rRNAs, 33 tRNAs, 3 noncoding RNAs, and 838 proteins, 3 of which are frameshifts. In addition, we discovered more than 40 pseudogenes, including the entire cytochrome c oxidase system. The three rickettsial genomes share 775 genes: 23 are found only in R. prowazekii and R. typhi, 15 are found only in R. conorii and R. typhi, and 24 are unique to R. typhi. Although most of the genes are colinear, there is a 35-kb inversion in gene order, which is close to the replication terminus, in R. typhi, compared to R. prowazekii and R. conorii. In addition, we found a 124-kb R. typhi-specific inversion, starting 19 kb from the origin of replication, compared to R. prowazekii and R. conorii. Inversions in this region are also seen in the unpublished genome sequences of R. sibirica and R. rickettsii, indicating that this region is a hot spot for rearrangements. Genome comparisons also revealed a 12-kb insertion in the R. prowazekii genome, relative to R. typhi and R. conorii, which appears to have occurred after the typhus (R. prowazekii and R. typhi) and spotted fever (R. conorii) groups diverged. The three-way comparison allowed further in silico analysis of the SpoT split genes, leading us to propose that the stringent response system is still functional in these rickettsiae.     

Molecular phylogeny and rearrangement of rRNA genes in Rickettsia species.

It has previously been observed that Rickettsia prowazekii has an unusual arrangement of the rRNA genes. In this species, the three rRNA genes, 16S (rrs), 23S (rrl), and 5S (rrf), are not linked in the typical arrangements for bacteria. Rather, the 16S rRNA gene has been separated from the 23S and 5S rRNA gene cluster, and the 23S rRNA gene is preceded by a gene which codes for methionyl-tRNAf(Met) formyltransferase (fmt). In this study, we screened the genus Rickettsia for the fmt-rrl motif in order to examine the phylogenetic depth of this unusual rRNA gene organization. A rearranged operon structure was observed in Rickettsia conorii, Rickettsia parkeri, Rickettsia sibirica, Rickettsia rickettsii, Rickettsia amblyomii, Rickettsia montana, Rickettsia rhipicephali, Rickettsia australis, Rickettsia akari, Rickettsia felis, Rickettsia canada, and Rickettsia typhi. There is also evidence for a divided operon in Rickettsia belli, but in this species, the fmt gene could not be identified upstream of the 23S rRNA gene. In order to place the rearrangement event in the evolutionary history of the Rickettsia, phylogenetic analyses were performed based on the fmt-rrl spacer regions and the 23S rRNA genes. Based on these phylogenies, we suggest that the genomic rearrangement of the rRNA genes preceded the divergence of the typhus group and the spotted fever group Rickettsia. The unique organization of the 23S rRNA genes provides a simple diagnostic tool for identification of Rickettsia species.     

Sequence analysis of the gene encoding a spotted fever group-specific intracytoplasmic protein PS120 of Rickettsia japonica

The 3,438-nucleotide (nt) sequence containing a 3,054-nt open reading frame of the gene (rps120) encoding an antigenic, intracytoplasmic, spotted fever group-specific and heat-stable 120-kilodalton protein (PS120) of Rickettsia japonica was determined. The nt and deduced 1,018 amino-acid (aa) sequences were compared to those of R. conorii since only those of this species had been determined among SFG rickettsiae. The homologies of these sequences between R. japonica and R. conorii were considerably high at 97 and 95%, respectively. These high homologies were comparable to those of beta-peptides encoded by the ompB genes among SFG rickettsiae. It was also found that the genome of R. prowazekii contained a nt sequence with 68% homology to that of the rps120 gene of R. japonica.     

Protein candidates for the serodiagnosis of rickettsioses

The laboratory diagnosis of rickettsioses is based on serology (reference method), cell culture and/or molecular tools. However, the main drawback of serology is its incapacity to provide identification of Rickettsiae at the level of species. The aim of this study was to propose the versatile protein markers able to discriminate the patients with murine typhus from those with Mediterranean spotted fever. We have cloned and expressed 20 proteins of Rickettsia prowazekii and Rickettsia rickettsii, respectively, using the GATEWAY approach. These recombinant proteins were screened by ELISA with sera of infected patients with Rickettsia typhi and Rickettsia conorii, respectively. We identified several potential markers which allowed infection due to R.?typhi to be discriminated from those due to R.?conorii. However, the values of test-operating parameters were not sufficient for its 'routine' clinical use. Our diagnostic test requires further optimization for be applied as a point-of-care strategy in the management of patients with suspected cases of rickettsiosis.     

Characterization of the ftsZ gene from Ehrlichia chaffeensis,Anaplasma phagocytophilum,and Rickettsia rickettsii,and use as a differential PCR target

Degenerate primers corresponding to highly conserved regions of previously characterized ftsZ genes were used to PCR amplify a portion of the ftsZ gene from the genomic DNA of Ehrlichia chaffeensis (ftsZ(Ech)), Anaplasma phagocytophilum (ftsZ(Ap)), and Rickettsia rickettsii (ftsZ(Rr)). Genome walking was then used to amplify the 5' and 3' termini of the genes. The DNA sequences of the resulting amplification products yielded open reading frames coding for proteins with molecular masses of 42.0, 45.7, and 48.3 kDa for A. phagocytophilum, E. chaffeensis, and R. rickettsii, respectively. These homologs are 20 to 70 amino acids longer than the FtsZ proteins characterized in bacteria such as Escherichia coli and Bacillus subtilis, but do not possess the large extended carboxyl-termini found in the FtsZ proteins of Bartonella, Rhizobium, and Agrobacterium species. The functional domains important for FtsZ activity are conserved within the ehrlichial and rickettsial FtsZ protein sequences. The R. rickettsii FtsZ sequence is highly homologous to the FtsZ protein previously described for Rickettsia prowazekii (89% identity), and identical to the FtsZ protein of Rickettsia conorii. The percent identity observed between the A. phagocytophilum and E. chaffeensis FtsZ proteins is only 79% and is particularly low in the carboxyl-terminal region (15.8% identity). Primers were designed to PCR amplify a portion of the variable carboxyl-terminal region of the ftsZ gene, and used to differentiate each agent based on the size of the amplicons: A. phagocytophilum, 278 bp; E. chaffeensis, 341 bp; and Rickettsia spp., 425 bp.     

Genotypic identification and phylogenetic analysis of the spotted fever group rickettsiae by pulsed-field gel electrophoresis.

Using pulsed-field gel electrophoresis, we studied the chromosomes of spotted fever group rickettsiae. We digested the DNA of 16 species currently known to belong to this group with SmaI, EagI, and BssHII. The genome size of 13 rickettsiae was between 1,200 and 1,300 kb. "Rickettsia massiliae" and "R. helvetica" genome sizes were 1,370 and 1,397 kb, respectively, and that of R. bellii was 1,660 kb. It was possible to obtain distinctive patterns for each species, but in R. conorii, 10 isolates exhibited the same profiles, showing that pulsed-field gel electrophoresis is a good interspecies identification tool. We achieved a phylogenetic analysis of these bacteria by using the Dice coefficient and UPGMA and Package Philip programming. We established a dendrogram of the genetic relationships between the different species showing the existence of a cluster in the spotted fever group rickettsiae including R. conorii, R. rickettsii, R. parkeri, R. sibirica, "R. africae," "R. slovaca," Thai tick typhus rickettsia, and Israeli tick typhus rickettsia. We located three genes previously cloned and sequenced (genes encoding the R. rickettsii surface proteins of 120 and 190 kDa and the R. prowazekii citrate synthase gene), using Southern hybridization. The genes encoding citrate synthase and the surface protein of 190 kDa were usually located on the same band, and it is hypothesized that they are relatively close on the chromosome.     

Ku70, a component of DNA-dependent protein kinase, is a mammalian receptor for Rickettsia conorii

Rickettsia conorii, a strictly intracellular and category C priority bacterial pathogen (NIAID), invades different mammalian cells. Although some signaling events involved in bacterial entry have been documented, the bacterial and host proteins mediating entry were not known. We report the identification of the Ku70 subunit of DNA-dependent protein kinase (DNA-PK) as a receptor involved in R. conorii internalization. Ku70 is recruited to R. conorii entry sites, and inhibition of Ku70 expression impairs R. conorii internalization. Bacterial invasion is dependent on the presence of cholesterol-enriched microdomains containing Ku70. R. conorii infection stimulates the ubiquitination of Ku70. In addition, the ubiquitin ligase c-Cbl is recruited to R. conorii entry foci, and downregulation of endogenous c-Cbl blocks bacterial invasion and Ku70 ubiquitination. An affinity chromatography approach identified the rickettsial protein rOmpB as a ligand for Ku70. This is the first report of a receptor-ligand interaction involved in the internalization of any rickettsial species.     

Proteome analysis of Rickettsia conorii by two-dimensional gel electrophoresis coupled with mass spectrometry

The availability of genome sequence offers the opportunity to further expand our knowledge about proteins expressed by Rickettsia conorii, strictly intracellular bacterium responsible for Mediterranean spotted fever. Using two-dimensional polyacrylamide gel electrophoresis combined with MALDI-TOF mass spectrometry, we established the first reference map of R. conorii proteome. This approach also allowed identification of GroEL as the major antigen recognized by rabbit serum and sera of infected patients. Altogether, this work opens the way to characterize the proteome of R. conorii, to compare protein profiles of different isolates or of bacteria maintained under different experimental conditions and to identify immunogenic proteins as potential vaccine targets.     

Transmission electron microscopy as a tool for exploring bacterial proteins: model of RickA in Rickettsia conorii

Rickettsia conorii, the etiologic agent of Mediterranean spotted fever, belongs to the spotted fever group of Rickettsia. It is an obligate intracellular bacterium that grows within the cytoplasm of its eukaryotic host cells. It is motile in the cytoplasm of infected cells and RickA is reported as critical protein in this aspect. However, the subcellular localization of RickA remains uncertain. We describe a simple method allowing RickA protein to be localized by immunofluorescence assay (IFA) and transmission electron microscopy (TEM). By using IFA we showed the global expression of surface protein RickA in R. conorii organisms. The TEM results showed that RickA is widely expressed over the entire bacterial surface of R. conorii.     

The insertion of palindromic repeats in the evolution of proteins

The current theory of protein evolution is that all contemporary proteins are derived from an ancestral subset. However, each new sequenced genome exhibits many genes with no detectable homologues in other species, leading to the paradoxical picture of a universal ancestor with more genes than any of its progeny. Standard explanations indicate that fast evolving genes might disappear into the 'twilight zone' of sequence similarity. Regardless of the size of the original ancestral subset, its origin and the potential mechanisms of its subsequent enlargement are rarely addressed. Sequencing of Rickettsia conorii genome recently led to the discovery of three families of repeat-mobile elements frequently inserted into the middle of protein coding genes. Although not yet identified in other species of bacteria, this discovery has provided the first clear evidence for the de novo creation of long protein segments (up to 50 amino acid residues) by repeat insertion. Based on previous results and theories on the coding potential of palindromic elements, we speculate that their insertion and mobility might have played a significant role in the early stages of protein evolution.     

Widespread use of real-time PCR for rickettsial diagnosis

We report 2?years of experience with rickettsial molecular diagnosis using real-time PCR at the French National Reference Center. All Rickettsia genomes available were compared to discover specific sequences to design new sets of primers and probes. The specificity was verified in silico and against a panel of 30 rickettsial species. Sensitivity was determined using 10-fold serial dilutions. Finally, primers and probes that were both specific and sensitive were routinely used for the diagnosis of rickettsial infections from clinical specimens. We retained sets of primers and probes to detect spotted fever group Rickettsia, typhus group Rickettsia,Rickettsia conorii,Rickettsia slovaca,Rickettsia africae and Rickettsia australis; 643 clinical samples were screened for the presence of Rickettsia DNA. Overall, 45 positive samples were detected, including 15 Rickettsia africae, nine R.?conorii, five Rickettsia sibirica mongolitimonae, four R.?slovaca, two R.?australis, four Rickettsia massiliae, one Rickettsia honei, one Rickettsia typhi and eight Rickettsia sp. Positive samples were detected mainly from cutaneous biopsies and swabs (31/45). Widespread use of real-time PCR is inexpensive and reduces delay in the diagnosis of rickettsial infections. These real-time PCR assays could be implemented easily in laboratories that have molecular facilities and may be added to existing molecular tools as a point-of-care strategy.     

Rickettsia conorii infection stimulates the expression of ISG15 and ISG15 protease UBP43 in human microvascular endothelial cells

Rickettsia conorii, an obligate intracellular bacterium and the causative agent of Mediterranean spotted fever, preferentially infects microvascular endothelial cells of the mammalian hosts leading to onset of innate immune responses, characterized by the activation of intracellular signaling mechanisms, release of pro-inflammatory cytokines and chemokines, and killing of intracellular rickettsiae. Our recent studies have shown that interferon (IFN)-β, a cytokine traditionally considered to be involved in antiviral immunity, plays an important role in the autocrine/paracrine regulation of host defense mechanisms and control of R. conorii growth in the host endothelial cells. Here, we show that R. conorii infection induces the expression of ISG15 (an interferon-stimulated gene coding a protein of 17kD) and UBP43 (an ISG15-specific protease) at the levels of mRNA and protein and report the evidence of ISGylation of as yet unidentified target proteins in cultured human microvascular endothelium. Infection-induced expression of ISG15 and UBP43 requires intracellular replication of rickettsiae and production of IFN-β, because treatment with tetracycline and presence of an antibody capable of neutralizing IFN-β activity resulted in near complete attenuation of both responses. Inhibition of R. conorii-induced ISG15 by RNA interference results in significant increase in the extent of rickettsial replication, whereas UBP43 knockdown yields a reciprocal inhibitory effect. In tandem, these results demonstrate the stimulation of interferon-β-mediated innate immune mechanisms capable of perturbing the growth and replication of pathogenic rickettsiae and provide first evidence for ISG15-mediated post-translational modification of host cellular proteins during infection with an intracellular bacterium.     

Birth and death of orphan genes in Rickettsia

The origin and evolution of the thousands of species-specific genes with unknown functions, the so-called orphan genes, has been a mystery. Here, we have studied the rates and patterns of orphan sequence evolution, using the Rickettsia as our reference system. Of the Rickettsia conorii orphans examined in this study, 80% were found to be short gene fragments or fusions of short segments from neighboring genes. We reconstructed the putative sequences of the full-length genes from which the short orphan fragments are thought to have originated. One of the genes thus reconstructed displays weak similarity to the ankyrin-repeat protein family, an identification that is strongly supported by comparative molecular modeling. Studies of the patterns of gene fragmentation underscore the importance of short repeated sequences as targets for recombination events that result in sequence loss and the formation of short, transient open reading frames. Our analysis demonstrates that gene sequences present in the common ancestor can be inferred even in cases when no full-length open reading frame is present in any of the contemporary species. Such reconstructions support the identification of lost protein functions and hint at important lifestyle changes.     

Development of recombinant OmpA and OmpB proteins as diagnostic antigens for rickettsial disease

In this study the diagnostic potential of Rickettsia conorii recombinant antigens was analyzed. For this, site-specific PCR primers were used to clone the OmpA and OmpB genes of R. conorii into pMAL-c2X plasmids. Six fragments of OmpA and four of OmpB were expressed as fusion proteins with maltose-binding protein in Escherichia coli. OmpA_1350-1784, OmpB_801-1269, and OmpB_1227-1634 regions from truncated proteins were selected as diagnostic candidate antigens by ELISA using control sera. ELISA results of three antigens were compared to the results obtained by using a commercial ELISA kit which contained whole OmpA and OmpB antigens from R. conorii . For this analysis, 40 serum samples taken from febrile patients and uninfected controls were tested. Of the 20 R. conorii test results which were positive with the commercial kit, 18 were shown to be positive by ELISA using OmpA_1350-1784 (a sensitivity of 90%). The specificity of the ELISA was 100%; all of the 20 samples shown to be negative using the commercial kit were also negative in our assay. The sensitivities of the ELISA using the OmpB_801-1269 and OmpB_1227-1634 were 90% and 95%, respectively. The specificities of the OmpB_801-1269 and the OmpB_1227-1634 were 100% and 95%, respectively. These results suggest that specific regions of OmpA and OmpB effectively detect antibodies against R. conorii , and the truncated recombinant antigens could be used for development of diagnostic tools for rickettsial disease.     

Emerging rickettsioses

Ticks are known to carry and transmit a number of microbial agents that cause diseases in humans and animals. Among these are members of the order Rickettsiales (alpha-proteobacteria), which include the genera Rickettsia and Ehrlichia. The most common and well-known Rickettsial human disease in Europe is Mediterranean Spotted Fever (MSF), caused by Rickettsia conorii. In recent years, a number of new Rickettsia species have been discovered in Europe, some of which have been shown to be pathogenic to humans. These discoveries have been facilitated by use of sequence-based molecular identification techniques. In Italy, it is generally believed that R. conorii is the only Rickettsia species present, and clinical tests for MSF rely on antigens raised against this bacterium. We are currently undertaking a molecular screening study of Rickettsiales-bacteria in ticks from various regions of Italy, to check for the potential presence of species from this order recently discovered in other parts of Europe. So far, we have identified a number of additional species in ticks collected from northern, central and southern regions. These include the known pathogens R. helvetica and R. slovaca as well as two species which may or may not be of medical relevance: R. monacensis and R. sp. IRS4. As a part of this survey, we have identified a novel alphaproteobacterium from the medically important tick Ixodes ricinus. This bacterium, tentatively named IricES1, has the unusual property of existing within the mitochondria, as well as the cytoplasm, of ovarian cells. To our knowledge, this is the only known example of a bacterium that is able to enter the mitochondria of animals. Our recently published electron microscopic data indicates that the bacterium enters mitochondria between the inner and outer membranes, and then proceeds to consume the inner mitochondrial matrix. We will present further data on this bacterium, including: 1) its phylogenetic position based on various molecular sequences, 2) its localization within the tick based on in situ hybridization; 3) its distribution among tick populations in Europe; 4) preliminary data on attempts at culturing this bacterium in a variety of cell types. Possible interactions between the bacterium and its host will be discussed. Ticks are known to carry and transmit a number of microbial agents that cause diseases in humans and animals. Among these are members of the order Rickettsiales (alpha-proteobacteria), which include the genera Rickettsia and Ehrlichia. The most common and well-known Rickettsial human disease in Europe is Mediterranean Spotted Fever (MSF), caused by Rickettsia conorii. In recent years, a number of new Rickettsia species have been discovered in Europe, some of which have been shown to be pathogenic to humans. These discoveries have been facilitated by use of sequence-based molecular identification techniques. In Italy, it is generally believed that R. conorii is the only Rickettsia species present, and clinical tests for MSF rely on antigens raised against this bacterium. We are currently undertaking a molecular screening study of Rickettsiales-bacteria in ticks from various regions of Italy, to check for the potential presence of species from this order recently discovered in other parts of Europe. So far, we have identified a number of additional species in ticks collected from northern, central and southern regions. These include the known pathogens R. helvetica and R. slovaca as well as two species which may or may not be of medical relevance: R. monacensis and R. sp. IRS4. As a part of this survey, we have identified a novel alphaproteobacterium from the medically important tick Ixodes ricinus. This bacterium, tentatively named IricES1, has the unusual property of existing within the mitochondria, as well as the cytoplasm, of ovarian cells. To our knowledge, this is the only known example of a bacterium that is able to enter the mitochondria of animals. Our recently published electron microscopic data indicates that the bacterium enters mitochondria between the inner and outer membranes, and then proceeds to consume the inner mitochondrial matrix. We will present further data on this bacterium, including: 1) its phylogenetic position based on various molecular sequences, 2) its localization within the tick based on in situ hybridization; 3) its distribution among tick populations in Europe; 4) preliminary data on attempts at culturing this bacterium in a variety of cell types. Possible interactions between the bacterium and its host will be discussed.     

Comparison of the virulence plasmid genomes of two strains of Shigella which lost the ability to bind Congo red

As an enteric pathogen and Gram negative bacte-rium, Shigella possesses high infectivity and leads to serious illness. Since its discovery in 1898 by Shiga, Shigella species have been studied widely. These studies have elucidated the Shigella pathogenicit…     

Expression of an epitope-tagged virulence protein in Rickettsia parkeri using transposon insertion

Despite recent advances in our ability to genetically manipulate Rickettsia, little has been done to employ genetic tools to study the expression and localization of Rickettsia virulence proteins. Using a mariner-based Himar1 transposition system, we expressed an epitope-tagged variant of the actin polymerizing protein RickA under the control of its native promoter in Rickettsia parkeri, allowing the detection of RickA using commercially-available antibodies. Native RickA and epitope-tagged RickA exhibited similar levels of expression and were specifically localized to bacteria. To further facilitate protein expression in Rickettsia, we also developed a plasmid for Rickettsia insertion and expression (pRIE), containing a variant Himar1 transposon with enhanced flexibility for gene insertion, and used it to generate R. parkeri strains expressing diverse fluorescent proteins. Expression of epitope-tagged proteins in Rickettsia will expand our ability to assess the regulation and function of important virulence factors.