- Email: [email protected]
Rickettsia prowazekii and Bartonella henselae: Differences in the intracellular life styles revisited
IJ M
Int. J. Med. Microbial. 290, 135-141 (2000) © Urban & Fischer Verlag http://www.urbanfischer.de/jaurnals/ijmm
Rickettsia prowazekii and Bartonella henselae: Differences in the intracellular life styles revisited Siv G. E. Andersson 1, Christoph Dehio 2 1 2
Department of Molecular Evolution, Evolutionary Biology Center, Uppsala University, S-75236 Uppsala, Sweden Department of Molecular Microbiology, Biozentrum of the University of Basel, CH-4056 Basel, Switzerland
Received March 9, 2000 . Accepted March 12,2000
Abstract 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. Key words: Genome sequence - infection - pathogenesis - intracellular life style - alpha subdivision of proteobacteria - Rickettsia - Bartonella
Introduction Several bacterial pathogens have evolved the ability to 'invade' mammalian cells and to survive and replicate within the nutrient-rich but often hostile intracellular habitat. While facultative intracellular parasites have retained their ability to live outside the host, obligate intracellular pathogens can replicate only within the host cell. While the dichotomy - obligate vs. facultative - primarily mirrors our ability to axenically culture
pathogens, these terms might also reflect the different levels of adaptation of the pathogen to life within the host cell. Assuming that intracellular bacterial parasites have free-living ancestors, it is important to place discussions about their pathogenic and metabolic life styles in an evolutionary perspective. Transition to intracellular growth environments is typically associated with minimisation of cell volume, massive loss of genetic information, extensive genomic rearrangements and enhanced fixation rates for mutations (Andersson
Corresponding author: Siv G. E. Andersson, Department of Molecular Evolution, Evolutionary Biology Center, Uppsala University, S-75236 Uppsala, Sweden, Phone: +46-18-4714379, Fax: +46-18-5577 23, E-mail: [email protected] 1438-4221100/290/2-135 $ 12.00/0
136
Siv G. E. Andersson, Christoph Dehio
and Kurland, 1995, 1998). This raises intriguing questions about the processes and mechanisms by which waves of intracellular parasites sweep across human populations. Thea subdivision of proteobacteria comprises a large number of bacteria that live in close association with eukaryotic host cells, many of which are intracellular pathogens. The rickettsiae as a broad group have traditionally comprised obligate intracellular pathogens of the genera Rickettsia, Coxiella and Ehrlichia. The Ehrlichia and Coxiella species reside within parasitophorous vacuoles and acidic phagolysosomes, respectively, whereas the Rickettsia species enter the host cell cytoplasm. The latter is the only group of obligate intra-cytoplasmic pathogens. The Rhizobiaceae group comprises several facultative intracellular bacteria including the genera Bartonella and Brucella, which once internalised will remain within a phagosome. As representatives of an obligate and a facultative intracellular life style, the human pathogens Rickettsia prowazekii and Bartonella henselae have been chosen for genome sequencing projects, respectively. Here, we discuss implications of the genome sequence information obtained on the metabolic capabilities of these pathogens.
The obligate intracellular parasite Rickettsia prowazekii The genus Rickettsia includes small pathogenic bacteria that grow strictly within the cytoplasm or the nuclei of eukaryotic cells. The majority of species are human pathogens that are spread from one host to another by arthropod vectors such as ticks, mites and insects. The genus Rickettsia is currently subdivided into the typhus group (TG) and the spotted fever group (SFG) (Raoult and Roux, 1997). The TG has only two members, namely Rickettsia prowazekii and Rickettsia typhi, which are pathogenic to humans. Currently 13 pathogenic SFG species have been described, and additionally 20 species have been identified but not yet been correlated with any human disease (NCB! Taxonomy Database, http://www.ncbi.nlm.nih.gov/). R. prowazekii is the causative agent of epidemic typhus, which throughout history has caused more deaths than all the wars combined. The disease is transmitted among humans by the body louse, Pediculus humanus corporis, and is fatal in 10 to 30 % of the patients. The bacteria can persist for life and are often activated under stressful conditions (recrudescent typhus). This disease, also referred to as Brill-Zinsser disease, can initiate an Qutbreak of epidemic typhus if louse infestations are widespread in the population (Raoult and Raux, 1997). R. typhi is the causative
agent of murine typhus, which is a milder form of typhus in humans. Rocky Mountain spotted fever is one of the most severe rickettsial diseases and is induced by transfer of Rickettsia rickettsii from a tick bite (Raoult and Roux, 1997). Rickettsia conorii causes a disease as severe as Rocky Mountain spotted fever, whereas the other pathogenic species cause similar, but milder, diseases (Raoult and Roux, 1997). The pathology of rickettsial infections results from host-cell destruction from within. To survive and reproduce, the organism must enter the eukaryotic host cell, grow within the host cell, exit, and re-establish this cycle in another host cell. Although Rickettsia species can enter various nucleated host cells in vitro, the primary target in vivo is the endothelium. Rickettsia enters cultured endothelial cells by an actin-dependent process (Walker, 1984). Once inside the cell, the bacteria lyse the phagosomal membrane in order to get access to the cytoplasm. Some species, i. e. R. rickettsii (Heinzen et aI., 1999) and R. conorii (Gouin et aI., 1999), are able to polymerise actin into 'comet tail' structures and move within the cytosol of infected cells, analogous to the paradigms of actin-based motility by Listeria or Shigella (Gouin et aI., 1999). This process may facilitate cell-to-cell spread from the initial site of cell invasion, which may occur without any further extracellular stage. Indeed, endothelial cell culture plaques induced by R. rickettsii have been reported, which is indicative of cell-to-cell spreading in association with a cytopathic effect (Walker et aI., 1982). However, infection of endothelial cells has also been shown to exert ananti-apoptotic effect which is essential for host cell survival, thus modulating the host cell's apoptotic response to its own advantage by potentially allowing the host cell to remain as a site of infection (Clifton et aI., 1998). Further studies are necessary to provide a better understanding of the fascinating aspects of endothelial interaction exhibited by these ancient human pathogens.
The facultative intracellular parasite Bartonella henselae The genus Bartonella comprises small, curved, often piliated (Fig.1A) or flagellated (Fig.1B) bacteria, which have been cultured in recent years on axenic media from the blood of many mammalian hosts. This resulted in a dramatic expansion of the genus Bartonella from one described species in 1993 to currently 13 species, among which at least 6 have been associated with human diseases (Dehio and Sander, 1999). A hemotropic life style together with transmission by blood-sucking arthropods appear to provide a unique
Intracellular parasites: Rickettsia and Bartonella
B
Fig. 1. Surface structures of Bartonella. Transmission electron micrographs of piliated B. henselae (A) and flagellated B. clarridgeiae (8). Scale bars correspond to 1 ~lm.
parasitic strategy for all Bartonella spp. However, each species appears to be highly adapted to one or a few mammalian hosts, cau~ing a hemotropic infection characterised by the ability to parasitise erythrocytes intracellularly. In contrast, incidental infection of nonreservoir hosts does not lead to erythrocyte invasion but may cause various clinical manifestations (Dehio and Sander, 1999). This may be best illustrated for the world-wide distribu.ted pathogen B. henselae. Cats are the natural reservoir for the cat flee-borne B. henselae. Infected cats develop a typically asymptomatic intraerythrocytic bacteremia (Kordick and Breitschwert, 1995). In contrast, incidental human infection via a cat scratch or cat bite may result in various clinical manifestations . The clinical outcome is mainly determined by the immunological status of the iQfected individual. Cat scratch disease (CSD), a persistent, necrotising inflammation of the lymph nodes, and endocarditis, are mainly observed among immunocompetent individuals. Immunocompromised patients develop severe clinical manifestations, such as bacillary angiomatosis (BA), bacillary peliosis (BP) and relapsing bacteremia. BA and BP are particularly remarkable as they represent vasoproliferative lesions resulting from a process of pathogen-stimulated angiogenesis (Anderson and Neuman, 1997). Similar to B. henselae, rat-adapted B. elizabethae, cat-adapted B. clarridgeiae and mouse-adapted B. grahamii appear to infect humans only incidentally (Dehio and Sander, 1999). In contrast, humans are the only known reservoir for B. bacilliformis and B. quintana. B. bacilliformis appears in endemic regions of South America as the agent of Carrion's disease transmitted by the sand fly Lutzomyia verrucarum (GarciaCaceres and Garcia, 1991). In the acute phase of this biphasic disease a -hemolytic anemia with fever (referred to as Oroya fever) occurs, during which almost every erythrocyte becomes invaded. When untreated,
137
Oroya fever comes close to having the highest death rate of all infectious diseases (up to 85 %) (Reynafarje and Ramos, 1961). After resolution, the second chronic phase (referred to as Verruga Peruana) is characterized by hemangiomatous eruptions of the skin as a result of endothelial cell proliferation. B. quintana became known during world war I as the agent of trench fever, which stroke more than one million soldiers at both front lines in many European countries. The disease, also known as 5-day fever, was recognised due to the multiple relapses of fever often associated with leg and back pain. B. quintana bacteremia may also have an asymptomatic course (Bass et aI., 1997), reflecting the characteristic hemotropic infection strategy of Bartonella spp. in the reservoir host. Interestingly, B. quintana infection of immunocompromised patients leads to the same spectrum of vasoproliferative disorders as caused by B. henselae (BA and BP), underlining the unique tropism of Bartonella towards endothelial cells and their capacity to trigger vascular proliferation. In contrast to erythrocyte invasion, which is restricted to the reservoir host, vascular proliferation in an immunocompromised situation appears to occur in both the reservoir as well as the incidental host. The remarkable tropism of Bartonella for erythrocytes and endothelial cells has attracted considerable attention of both clinicians and researchers. Erythrocyte invasion by B. henselae was studied in vitro (Mehock et aI., 1998) and the resulting bacteremia has been investigated in vivo in experimentally infected cats (Regnery et aI., 1996). A detailed description of the in vivo course of erythrocyte parasitism by the closely related species B. tribocorum (Heller et aI., 1998) performed in a novel rat infection model revealed (i) a synchronous onset of bacteremia marked by singular bacteria found in close association with circulating erythrocytes, (ii) intra erythrocytic replication
Fig. 2. Intracellular colonisation of erythrocytes by B. tribocorum. (A) Erythrocytes are visualised by differential interference contrast, bacteria are detected by fluorescence detection based on constitutive expression of green fluorescent protein. (8) Transmission electron micrograph of an infected erythrocyte providing evidence for a vacuolar membrane surrounding the intracellular bacterium. The scale bars correspond to 2 f!m.
138
Siv G. E. Andersson, Christoph Dehio
of bacteria (Fig.2) paralleled by a constant rate of clearance of infected erythrocytes, (iii) the appearance of periodic, overlapping erythrocyte re-infection waves and (iv) cessation of the bacteremia within three months of infection (R. Schulein, A. Seubert, C. Gille, C. Lanz, Y. Hansmann, and C. Dehio, submitted). Several Bartonella species were shown to colonise and invade endothelial cells in vivo (Dehio, 1999). In vitro infection models demonstrated that bacterial invasion can occur by a phagocytosis-like process involving the cellular actin cytoskeleton. A novel bacterial uptake mechanism could be described for B. henselae, which triggers the formation, engulfment and uptake of a large bacterial aggregate, the so-called invasome, by vascular endothelial cells (Fig. 3, Dehio et al., 1997). In both uptake processes, intracellular bacteria remain entrapped within a vacuolar membrane. The specific colonisation of endothelial cells by epiand intracellular bacteria can trigger the activation and proliferation of this cell type. Both processes are likely to be associated indirectly or directly with the formation of the unique vasoproliferative lesion caused by Bartonella infections in immunocompromised patients. Bacteria or outer membrane preparations of B. henselae activate endothelial cells by up-regulating the surface expression of adhesion molecules, which in turn cause enhanced rolling"and adhesion of polymorphonuclear neutrophils (PMNs) (0. Fuhrmann, M. Arvand, M. Krull, S. Hippenstiel, J. Seybold, C. Dehio, and N. Suttorp, submitted). This proinflammatory phenotype of vascular endothelial cells likely contributes to the mixed infiltrations of PMNs and monocytes typically seen in BA lesions, which account for inflammation and may indirectly contribute to the formation and maintenance of the vasoproliferative BA lesions (Dehio, 1999). Complete bacteria, outer membrane preparations as well as culture supernatants also contain an angiogenic activity that directly stimulates endothelial cell proliferation (Conley et al., 1994, Maeno et al.,
1999, M. Rottgen, M. Roger and C. Dehio, unpublished results; Dehio and Sander, 1999) as well as in vitro angiogenesis (M. Rottgen and C. Dehio, unpublished results). The molecular nature of this angiogenic factor, as well as the factors involved in the process of endothelial and erythrocyte invasion remain intriguing problems to be solved.
Metabolic exploitation A myriad of metabolic and virulence genes have to be expressed to allow survival in the hostile intracellular environment. Elucidation of the complex and dynamic patterns of gene expression and protein interaction during the infectious cycle requires detailed knowledge about the putative functions of the complete set of genes in the organisms being studied. Recently, genome sequencing projects on two intracellular bacteria of the ex subdivision of proteobacteria have allowed systematic characterisation of gene functions and expression profiles in these organisms. The 1.1 Mb genome sequence of R. prowazekii has already been published (Andersson et al., 1998) and the 2.0 Mb genome sequence of B. henselae is near to be completed (c. Alsmark, B. Canback, C. G. Kurland, S. G. E. Andersson, unpublished data). The eukaryotic host cell is an extremely rich growth environment as compared to many other ecological niches that harbour bacteria. Therefore it is not surprising to find that many bacteria of different phylogenetic affiliations have 'learnt' to exploit the intracellular growth environment. However, the full spectrum of microbe-host interactions is broad, as reflected in different genomic footprints for different intracellular parasites. For example, the metabolic profiles of Rickettsia and Bartonella differ in a manner that directly reflects their different degrees of host-cell dependence. Thus, the obligate parasite R. prowazekii has discard-
Fig. 3. The invasome-mediated -invasion of endothelial cells by B. hense/ae. Transmission electron micrographs illustrate the sequential steps of (A) aggregation, (B) engulfment and (C) internalisation of B. henselae into endothelial cells. The scale bars correspond to 211m. Reproduced with permission from Dehio etal.,1997.
Intracellular parasites: Rickettsia and Bartonella
ed most genes allocated to biosynthetic functions and is dependent upon its host cell for supply of small molecules such as amino acids and nucleoside monophosphates (Andersson et aI., 1998). In contrast, the facultative parasite B. henselae has a full complement of genes required for the biosynthesis of these basic building blocks (c. Alsmark, B. Canback, C. G. Kurland, S. G. E. Andersson, unpublished results). That is to say, Bartonella has retained the capacity for growth in the free-living mode, whereas Rickettsia seems to be irreversibly specialised to living in the intracellular environment. Chlamydia represents another group of obligate intracellular parasites that are highly adapted to the intracellular milieu (Stephens et a!', 1998). Like Rickettsia, the relative proportion of genes allocated to biosynthetic functions is very low in Chlamydia. For example, both Rickettsia and Chlamydia contain genes required for the interconversion of nucleoside monophosphates into all of the required nucleotides and deoxynucleotides, but lack genes involved in de novo purine and pyrimidine biosynthesis (Zomorodipour and Andersson, 1999). Since the two parasites do not share a common intracellular ancestor, the loss of biosynthetic genes is more likely to result from reductive, convergent evolution. A key transport protein in obligate intracellular parasites is the ATP/ADP translocase that enables the parasite to utilise cytoplasmic ATP as a source of energy. R. prowazekii has as many as five genes coding for ATP/ADP translocases (Andersson et aI., 1998), whereas C. trachomatis has only two (Stephens et aI., 1998). ATP/ADP transport exchange proteins have so far only been observed in Rickettsia~ Chlamydia, mitochondria and chloroplasts (Andersson, 1998). Phylogenetic analyses suggest that the ATP/ADP translocases in R. prowazekii and C. trachomatis are of a similar type, but characteristically different from their functional homologs in mitochondria. No genes coding for ATP/ADP translocases have been found in the genome of B. henselae, suggesting that Bartonella has not yet evolved transport systems for utilising the compounds available in the cytosol to the same extent as Rickettsia (c. Alsmark, B. Canback, C. G. Kurland, S. G. E. Andersson, unpublished results).
Inactivation, degradation and elimination It seems reasonable to assume that the genome of a freeliving ancestor of Rickettsia and Bartonella had a metabolic repertoire similar to that of modern free-living bacteria. If so, as much as 80 % of the original gene complement may have been lost during the evolution of the obligate intracellular lineages in this subdivision. Indeed, genome sequence data suggests that genes are
139
constantly being inactivated and discarded from the Rickettsia genomes. For example, a detailed comparative analysis of the metK gene, which codes for Sadenosylmethionine synthetase has revealed the presence of deleterious mutations in most of the Rickettsia species analysed (Andersson and Andersson, 1999a,b). It has been speculated that the degradation of the metK gene may be due to the invention of a transport system for S-adenosylmethionine (AdoMet) (Andersson and Andersson, 1999a). The novel import system and the ancestral biosynthetic gene may have worked hand-inhand for a while, until the transport system for AdoMet was efficient enough to compensate for the internal biosynthesis of AdoMet. If speciation occurred during the time both systems were operating, the time at which the first inactivation mutation was fixed in the population may have been determined solely by chance. A large number of pseudogenes have since been identified in the Rickettsia genomes, one of these is located downstream of the metK gene. The ancestral gene sequences of the two neighboring genes could be reconstructed by the removal of potential insertion and deletion mutations (Andersson and Andersson, 1999a). These reconstructed, ancestral gene sequences displayed the characteristic variations in G + C content values at the three codon positions, confirming that they have once been actively transcribed and translated genes. A closer inspection of the disruptive mutations has shown that there is a strong mutational bias towards deletions in Rickettsia (Andersson and Andersson, 1999a). This means that the inactivated gene sequences will deteriorate in a gradual manner until they are no longer recognisable as genes. It has been speculated that a large fraction of the noncoding DNA in the R. prowazekii genome may represent remnants of ancestral, inactivated genes in the very latest stages of degradation (Andersson et aI., 1998). Indeed, some of the sequences classified as noneading DNA in R. prowazekii display strong sequence similarities to fragments of genes or pseudogenes in other Rickettsia species (J. O. Andersson and S. G. E. Andersson, unpublished). Because of their obligate intracellular life style, there may be few possibilities for genetic exchange between different individuals in the rickettsial populations. Small populations with low recombination frequencies are expected to be particularly sensitive to the influence of the so-called Muliers ratchet, which will result in higher fixation rates for mutations (Felsenstein, 1974; Andersson and Kurland, 1995). Indeed, synonymous , substitution rates for a wide range of genes from the TG and the SFG are approximately similar, suggesting that these rates directly reflect the rate at which mutations are being produced. This suggests that selection may not be as effective in small, obligate intracellular
140
Siv G. E. Andersson, Christoph Dehio
populations as in large free-living bacterial populations. Phylogenetic reconstructions provide some indications of ancient gene transfer events into the R. prowazekii genome. For example, the lysyl amino acyltRNA synthetase and the valyl aminoacyl-tRNA synthetases are most similar to their homologs in archaea (Andersson et al., 1998). However, the nucleotide composition patterns of modern Rickettsia genes are extremely homogenous, providing no or only few indications of recent horizontal gene transfer events (M. Remm and S. G. E. Andersson, unpublished results). Taken together, it can be concluded that intracellular parasites are metabolically and genetically very divergent from bacteria with a free-living life style. This is in part explained by a high concentration of nutrients in the cytoplasm, which enables high rates of gene efflux. The isolated environment inside the eukaryotic cell provides protection from competitors on the one hand, but eliminates possibilities for genetic exchange on the other. Furthermore, intracellular bacteria have to go through a series of bottlenecks during the transmission from one host to the other, which may lead to high fixation rates for deleterious mutations. Each of these three factors is likely to contribute to the overall degradation and loss of genetic information. This leaves us with a paradox, namely that the intracellular growth environment that initially seemed so rich and attractive for bacterial growth may in the long-term induce stagnation and degradation (Andersson and Kurland, 1998).
Conclusions and perspectives The obligate intracellular life style and the pathogenicity of many Rickettsia species have made it very difficult to study the biology of these bacteria. The first genetic manipulation of a rickettsial gene was recently performed by the successful transformation of a rifampin resistance gene into the genome of R. prowazekii (Rachek et al., 1998). This represents the first step in the development of a genetic system for Rickettsia, which, together with the availability of the complete genome sequence of R. prowazekii (Andersson et al., 1998), will radically change our possibilities to study and understand the molecular biology of this group of obligate intracellular parasites. However, while experimental studies on obligate intracellular parasites such as Rickettsia are still challenging, we have genetics (Dehio and Meyer, 1997; Dehio et al., 1998) as well as suitable in vitro and animal infection models available for Bartonella. In combination with the genome sequence, this will enable us to perform modern types of functional genomic studies. These will include tran~~riptome analysis and saturational mutagenesis approaches to aid in the systematic
exploration of the molecular basis for facultative intracellular parasitism. Acknowledgements.The authors acknowledge the members of their laboratories for providing unpublished results. We thank Bozena Pichler-Brand and Christa Lanz for their help in electron microscopy and confocal laser scanning microscopy. We are grateful to Dr. Terry Kwok for critically reading of the manuscript. The genome sequencing projects are supported by grants to S. G. E. Andersson from the Swedish Foundation for Strategic Research, the National Science Research Council and the Knut and Alice Wallenberg Foundation.
References Anderson, B., Neuman, M. A.: Bartonella spp. as emerging human pathogens. Clin. Microbio!. Rev. 10, 203-219 (1997). Andersson,]. 0., Andersson, S. G. E.: Genome degradation is an ongoing process in Rickettsia. Mo!. Bio!. Evo!. 16, 1178-1191 (1999a). Andersson, J. 0., Andersson, S. G. E.: Insights into the evolutionary process of genome degradation. Curro Opin. Genet. Dev. 9, 664-671 (1999b). Andersson, S. G. E.: Bioenergetics of the obligate intracellular parasite Rickettsia prowazekii. Biochim. Biophys. Acta 1365, 105-111 (1998). Andersson, S. G. E., Kurland, C. G.: Genomic evolution drives the evolution of the translation system. Biochem. Cell Bio!. 73, 775-787 (1995). Andersson, S. G. E., Kurland C. G.: Reductive evolution of resident genomes. Trends Microbio!' 6,263-278 (1998). Andersson, S. G. E., Zomorodipour, A., Andersson, J. 0., Sicheritz-Ponten, T., Alsmark, U. C. M., Podowski, R. M., Naslund, A. K., Eriksson, A.-S., Winkler, H. H., Kurland C. G.: The genome sequence of Rickettsia prowazekii and the origin of mitochondria, Nature 396, 133-140 (1998). Bass, J. W., Vincent, J. M., Person, D. A.: The expanding spectrum of Bartonella infections: I. Bartonellosis and trench fever. Pediatr. Infect. Dis. J. 16, 2-10 (1997). Clifton, D. R., Goss, R. A., Sahni, S. K., Antwerp, D. v., Baggs, R.B., Marder, V.]., Silverman, D.]., Spron, L.A.: NF-xB-dependent inhibition of apoptosis is essential for host cell survival during Rickettsia rickettsii infection. Proc. Nat!' Acad. Sci. USA 95, 4646-4651 (1998). Conley, T., Slater, L., Hamilton, K.: Rochalimaea spp. stimulate human endothelial cell proliferation and migration in vitro.J.Lab. Clin. Med.124, 521-528 (1994). Dehio, c.: Interactions of Bartonella henselae with vascular endothelial cells. Curro Opin. Microbio!. 2, 78-82 (1999). Dehio, c., Meyer, M.: Maintenance of broad-host-range incompatibility group P and group Q plasmids and transposition of Tn5 in Bartonella henselae following conjugal plasmid transfer from Escherichia coli. ]. Bacterio!' 179, 538-540 (1997). Dehio, c., Meyer, M., Berger,]., Schwarz, H., Lanz, c.: Interaction of Bartonella henselae with endothelial cells results in bacterial aggregation on the cell surface and the subse-
Intracellular parasites: Rickettsia and Bartonella quent engulfment and internalisation of the bacterial aggregate by a unique structure, the invasome. J. Cell Sci. 110,2141-2154 (1997) . Dehio, C, Sander, A.: Bartonella as emerging pathogens. Trends Microbiol. 7,226-228 (1999). Dehio, M., Knorre, A., Lanz, C, Dehio C: Construction of versatile high-level expression vectors for Bartonella henselae and the use of green fluorescent protein as a new expression marker. Gene 215, 223-229 (1998). Felsenstein, J.: The evolutionary advantage of recombination. Genetics 78,157-159 (1974). Garcia-Caceres, U., Garcia, F. U.: Bartonellosis: An immunodepressive disease and the life of Daniel Alcides Carrion. Am.J. Clin. Pathol. 95 , S58-S66 (1991). Gouin, E., Gantelet, H., Egile, C, Lasa, I., Ohayon, H., Villiers, V., Gounon, P., Sansonetti, P. J., Cossart, P.: A comparative study of the actin-based motilitites of the pathogenic bacteria Listeria monocytogenes, Shigella {lexneri and Rickettsia conorii, J. Cell Sci. 112, 1697-1708 (1999). Heinzen, R. A., Grieshaber, S. S., Van Kirk, L. S., Devin, C J.: Dynamics of actin-based movement by Rickettsia rickettsii in vero cells. Infect. Immun. 67, 4201-4207 (1999). Heller, R., Riegel, P., Hansmann, Y., Delacour, G., Bermond, D., Dehio, c., Lamarque, F., Monteil, H., Chomel, B., Piemont, Y.: Bartonella tribocorum sp. nov., a new Bartonella species isolated from the blood of wild rats. Int. J. Syst. Bacteriol. 48, 1333-1339 (1998). Kordick, D. L., Breitschwerdt, E. B.: Intraerythrocytic presence of Bartonella henselae. J. Clin. Microbiol. 33, 16551656 (1995). Maeno, N., Oda, H., Yoshiie, K., Wahid, M. R., Fujimura, T., Matayoshi, S.: Live Bartonella henselae enhances endothe-
141
lial cell proliferation without direct contact. Microb. Pathog. 27, 419-427 (1999). Mehock, J. R., Greene, C E., Gherardini, F. C, Hahn, T. W., Krause, D. C: Bartonella henselae invasion of feline erythrocytes in vitro. Infect. Immun. 66, 3462-3466 (1998). Rachek, L.I., Tucker, A. M., Winkler, H. H., Wood, D.O.: Transformation of Rickettsia prowazekii to rifampin resistance.J. Bacterial. 180,2118-2124 (1998). Raoult, D., Raux, V.: Rickettsioses as paradigms of new or emerging infectious diseases. Clin. Microbial. Rev. 10, 694-719 (1997). Regnery, R. L., Rooney, J. A. , Johnson, A. M., Nesby, S. L., Manzewitsch, P., Beaver, K., Olson, J. G.: Experimentally induced Bartonella henselae infections followed by challenge exposure and antimicrobial therapy in cats. Am.J.Vet. Res. 57, 1714-1719 (1996). Reynafarje, C, Ramos, J.: The hemolytic anemia of human bartonellosis, Blood 17,562-578 (1961). Stephens, R. S., Kalman, S., Lammel, C, Fan, J., Marathe, R., et al.: Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis. Science 282, 754-759 (1998). Walker, T. S. : Rickettsial interactions with human endothelial cells in vitro: adherence and entry. Infect. Immun. 44, 205-210 (1984). Walker, D. H., Firth, W. T., Edgell, CJ.: Human endothelial cell culture plaques induced by Rickettsia rickettsii. Infect. Immun. 37, 301-306 (1982). Zomorodipour, A., Andersson, S. G. E.: Obligate intracellular parasites: Rickettsia prowazekii and Chlamydia trachomatis. FEBS Lett. 452,11-15 (1999).
Int. J. Med. Microbial. 290, 135-141 (2000) © Urban & Fischer Verlag http://www.urbanfischer.de/jaurnals/ijmm
Rickettsia prowazekii and Bartonella henselae: Differences in the intracellular life styles revisited Siv G. E. Andersson 1, Christoph Dehio 2 1 2
Department of Molecular Evolution, Evolutionary Biology Center, Uppsala University, S-75236 Uppsala, Sweden Department of Molecular Microbiology, Biozentrum of the University of Basel, CH-4056 Basel, Switzerland
Received March 9, 2000 . Accepted March 12,2000
Abstract 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. Key words: Genome sequence - infection - pathogenesis - intracellular life style - alpha subdivision of proteobacteria - Rickettsia - Bartonella
Introduction Several bacterial pathogens have evolved the ability to 'invade' mammalian cells and to survive and replicate within the nutrient-rich but often hostile intracellular habitat. While facultative intracellular parasites have retained their ability to live outside the host, obligate intracellular pathogens can replicate only within the host cell. While the dichotomy - obligate vs. facultative - primarily mirrors our ability to axenically culture
pathogens, these terms might also reflect the different levels of adaptation of the pathogen to life within the host cell. Assuming that intracellular bacterial parasites have free-living ancestors, it is important to place discussions about their pathogenic and metabolic life styles in an evolutionary perspective. Transition to intracellular growth environments is typically associated with minimisation of cell volume, massive loss of genetic information, extensive genomic rearrangements and enhanced fixation rates for mutations (Andersson
Corresponding author: Siv G. E. Andersson, Department of Molecular Evolution, Evolutionary Biology Center, Uppsala University, S-75236 Uppsala, Sweden, Phone: +46-18-4714379, Fax: +46-18-5577 23, E-mail: [email protected] 1438-4221100/290/2-135 $ 12.00/0
136
Siv G. E. Andersson, Christoph Dehio
and Kurland, 1995, 1998). This raises intriguing questions about the processes and mechanisms by which waves of intracellular parasites sweep across human populations. Thea subdivision of proteobacteria comprises a large number of bacteria that live in close association with eukaryotic host cells, many of which are intracellular pathogens. The rickettsiae as a broad group have traditionally comprised obligate intracellular pathogens of the genera Rickettsia, Coxiella and Ehrlichia. The Ehrlichia and Coxiella species reside within parasitophorous vacuoles and acidic phagolysosomes, respectively, whereas the Rickettsia species enter the host cell cytoplasm. The latter is the only group of obligate intra-cytoplasmic pathogens. The Rhizobiaceae group comprises several facultative intracellular bacteria including the genera Bartonella and Brucella, which once internalised will remain within a phagosome. As representatives of an obligate and a facultative intracellular life style, the human pathogens Rickettsia prowazekii and Bartonella henselae have been chosen for genome sequencing projects, respectively. Here, we discuss implications of the genome sequence information obtained on the metabolic capabilities of these pathogens.
The obligate intracellular parasite Rickettsia prowazekii The genus Rickettsia includes small pathogenic bacteria that grow strictly within the cytoplasm or the nuclei of eukaryotic cells. The majority of species are human pathogens that are spread from one host to another by arthropod vectors such as ticks, mites and insects. The genus Rickettsia is currently subdivided into the typhus group (TG) and the spotted fever group (SFG) (Raoult and Roux, 1997). The TG has only two members, namely Rickettsia prowazekii and Rickettsia typhi, which are pathogenic to humans. Currently 13 pathogenic SFG species have been described, and additionally 20 species have been identified but not yet been correlated with any human disease (NCB! Taxonomy Database, http://www.ncbi.nlm.nih.gov/). R. prowazekii is the causative agent of epidemic typhus, which throughout history has caused more deaths than all the wars combined. The disease is transmitted among humans by the body louse, Pediculus humanus corporis, and is fatal in 10 to 30 % of the patients. The bacteria can persist for life and are often activated under stressful conditions (recrudescent typhus). This disease, also referred to as Brill-Zinsser disease, can initiate an Qutbreak of epidemic typhus if louse infestations are widespread in the population (Raoult and Raux, 1997). R. typhi is the causative
agent of murine typhus, which is a milder form of typhus in humans. Rocky Mountain spotted fever is one of the most severe rickettsial diseases and is induced by transfer of Rickettsia rickettsii from a tick bite (Raoult and Roux, 1997). Rickettsia conorii causes a disease as severe as Rocky Mountain spotted fever, whereas the other pathogenic species cause similar, but milder, diseases (Raoult and Roux, 1997). The pathology of rickettsial infections results from host-cell destruction from within. To survive and reproduce, the organism must enter the eukaryotic host cell, grow within the host cell, exit, and re-establish this cycle in another host cell. Although Rickettsia species can enter various nucleated host cells in vitro, the primary target in vivo is the endothelium. Rickettsia enters cultured endothelial cells by an actin-dependent process (Walker, 1984). Once inside the cell, the bacteria lyse the phagosomal membrane in order to get access to the cytoplasm. Some species, i. e. R. rickettsii (Heinzen et aI., 1999) and R. conorii (Gouin et aI., 1999), are able to polymerise actin into 'comet tail' structures and move within the cytosol of infected cells, analogous to the paradigms of actin-based motility by Listeria or Shigella (Gouin et aI., 1999). This process may facilitate cell-to-cell spread from the initial site of cell invasion, which may occur without any further extracellular stage. Indeed, endothelial cell culture plaques induced by R. rickettsii have been reported, which is indicative of cell-to-cell spreading in association with a cytopathic effect (Walker et aI., 1982). However, infection of endothelial cells has also been shown to exert ananti-apoptotic effect which is essential for host cell survival, thus modulating the host cell's apoptotic response to its own advantage by potentially allowing the host cell to remain as a site of infection (Clifton et aI., 1998). Further studies are necessary to provide a better understanding of the fascinating aspects of endothelial interaction exhibited by these ancient human pathogens.
The facultative intracellular parasite Bartonella henselae The genus Bartonella comprises small, curved, often piliated (Fig.1A) or flagellated (Fig.1B) bacteria, which have been cultured in recent years on axenic media from the blood of many mammalian hosts. This resulted in a dramatic expansion of the genus Bartonella from one described species in 1993 to currently 13 species, among which at least 6 have been associated with human diseases (Dehio and Sander, 1999). A hemotropic life style together with transmission by blood-sucking arthropods appear to provide a unique
Intracellular parasites: Rickettsia and Bartonella
B
Fig. 1. Surface structures of Bartonella. Transmission electron micrographs of piliated B. henselae (A) and flagellated B. clarridgeiae (8). Scale bars correspond to 1 ~lm.
parasitic strategy for all Bartonella spp. However, each species appears to be highly adapted to one or a few mammalian hosts, cau~ing a hemotropic infection characterised by the ability to parasitise erythrocytes intracellularly. In contrast, incidental infection of nonreservoir hosts does not lead to erythrocyte invasion but may cause various clinical manifestations (Dehio and Sander, 1999). This may be best illustrated for the world-wide distribu.ted pathogen B. henselae. Cats are the natural reservoir for the cat flee-borne B. henselae. Infected cats develop a typically asymptomatic intraerythrocytic bacteremia (Kordick and Breitschwert, 1995). In contrast, incidental human infection via a cat scratch or cat bite may result in various clinical manifestations . The clinical outcome is mainly determined by the immunological status of the iQfected individual. Cat scratch disease (CSD), a persistent, necrotising inflammation of the lymph nodes, and endocarditis, are mainly observed among immunocompetent individuals. Immunocompromised patients develop severe clinical manifestations, such as bacillary angiomatosis (BA), bacillary peliosis (BP) and relapsing bacteremia. BA and BP are particularly remarkable as they represent vasoproliferative lesions resulting from a process of pathogen-stimulated angiogenesis (Anderson and Neuman, 1997). Similar to B. henselae, rat-adapted B. elizabethae, cat-adapted B. clarridgeiae and mouse-adapted B. grahamii appear to infect humans only incidentally (Dehio and Sander, 1999). In contrast, humans are the only known reservoir for B. bacilliformis and B. quintana. B. bacilliformis appears in endemic regions of South America as the agent of Carrion's disease transmitted by the sand fly Lutzomyia verrucarum (GarciaCaceres and Garcia, 1991). In the acute phase of this biphasic disease a -hemolytic anemia with fever (referred to as Oroya fever) occurs, during which almost every erythrocyte becomes invaded. When untreated,
137
Oroya fever comes close to having the highest death rate of all infectious diseases (up to 85 %) (Reynafarje and Ramos, 1961). After resolution, the second chronic phase (referred to as Verruga Peruana) is characterized by hemangiomatous eruptions of the skin as a result of endothelial cell proliferation. B. quintana became known during world war I as the agent of trench fever, which stroke more than one million soldiers at both front lines in many European countries. The disease, also known as 5-day fever, was recognised due to the multiple relapses of fever often associated with leg and back pain. B. quintana bacteremia may also have an asymptomatic course (Bass et aI., 1997), reflecting the characteristic hemotropic infection strategy of Bartonella spp. in the reservoir host. Interestingly, B. quintana infection of immunocompromised patients leads to the same spectrum of vasoproliferative disorders as caused by B. henselae (BA and BP), underlining the unique tropism of Bartonella towards endothelial cells and their capacity to trigger vascular proliferation. In contrast to erythrocyte invasion, which is restricted to the reservoir host, vascular proliferation in an immunocompromised situation appears to occur in both the reservoir as well as the incidental host. The remarkable tropism of Bartonella for erythrocytes and endothelial cells has attracted considerable attention of both clinicians and researchers. Erythrocyte invasion by B. henselae was studied in vitro (Mehock et aI., 1998) and the resulting bacteremia has been investigated in vivo in experimentally infected cats (Regnery et aI., 1996). A detailed description of the in vivo course of erythrocyte parasitism by the closely related species B. tribocorum (Heller et aI., 1998) performed in a novel rat infection model revealed (i) a synchronous onset of bacteremia marked by singular bacteria found in close association with circulating erythrocytes, (ii) intra erythrocytic replication
Fig. 2. Intracellular colonisation of erythrocytes by B. tribocorum. (A) Erythrocytes are visualised by differential interference contrast, bacteria are detected by fluorescence detection based on constitutive expression of green fluorescent protein. (8) Transmission electron micrograph of an infected erythrocyte providing evidence for a vacuolar membrane surrounding the intracellular bacterium. The scale bars correspond to 2 f!m.
138
Siv G. E. Andersson, Christoph Dehio
of bacteria (Fig.2) paralleled by a constant rate of clearance of infected erythrocytes, (iii) the appearance of periodic, overlapping erythrocyte re-infection waves and (iv) cessation of the bacteremia within three months of infection (R. Schulein, A. Seubert, C. Gille, C. Lanz, Y. Hansmann, and C. Dehio, submitted). Several Bartonella species were shown to colonise and invade endothelial cells in vivo (Dehio, 1999). In vitro infection models demonstrated that bacterial invasion can occur by a phagocytosis-like process involving the cellular actin cytoskeleton. A novel bacterial uptake mechanism could be described for B. henselae, which triggers the formation, engulfment and uptake of a large bacterial aggregate, the so-called invasome, by vascular endothelial cells (Fig. 3, Dehio et al., 1997). In both uptake processes, intracellular bacteria remain entrapped within a vacuolar membrane. The specific colonisation of endothelial cells by epiand intracellular bacteria can trigger the activation and proliferation of this cell type. Both processes are likely to be associated indirectly or directly with the formation of the unique vasoproliferative lesion caused by Bartonella infections in immunocompromised patients. Bacteria or outer membrane preparations of B. henselae activate endothelial cells by up-regulating the surface expression of adhesion molecules, which in turn cause enhanced rolling"and adhesion of polymorphonuclear neutrophils (PMNs) (0. Fuhrmann, M. Arvand, M. Krull, S. Hippenstiel, J. Seybold, C. Dehio, and N. Suttorp, submitted). This proinflammatory phenotype of vascular endothelial cells likely contributes to the mixed infiltrations of PMNs and monocytes typically seen in BA lesions, which account for inflammation and may indirectly contribute to the formation and maintenance of the vasoproliferative BA lesions (Dehio, 1999). Complete bacteria, outer membrane preparations as well as culture supernatants also contain an angiogenic activity that directly stimulates endothelial cell proliferation (Conley et al., 1994, Maeno et al.,
1999, M. Rottgen, M. Roger and C. Dehio, unpublished results; Dehio and Sander, 1999) as well as in vitro angiogenesis (M. Rottgen and C. Dehio, unpublished results). The molecular nature of this angiogenic factor, as well as the factors involved in the process of endothelial and erythrocyte invasion remain intriguing problems to be solved.
Metabolic exploitation A myriad of metabolic and virulence genes have to be expressed to allow survival in the hostile intracellular environment. Elucidation of the complex and dynamic patterns of gene expression and protein interaction during the infectious cycle requires detailed knowledge about the putative functions of the complete set of genes in the organisms being studied. Recently, genome sequencing projects on two intracellular bacteria of the ex subdivision of proteobacteria have allowed systematic characterisation of gene functions and expression profiles in these organisms. The 1.1 Mb genome sequence of R. prowazekii has already been published (Andersson et al., 1998) and the 2.0 Mb genome sequence of B. henselae is near to be completed (c. Alsmark, B. Canback, C. G. Kurland, S. G. E. Andersson, unpublished data). The eukaryotic host cell is an extremely rich growth environment as compared to many other ecological niches that harbour bacteria. Therefore it is not surprising to find that many bacteria of different phylogenetic affiliations have 'learnt' to exploit the intracellular growth environment. However, the full spectrum of microbe-host interactions is broad, as reflected in different genomic footprints for different intracellular parasites. For example, the metabolic profiles of Rickettsia and Bartonella differ in a manner that directly reflects their different degrees of host-cell dependence. Thus, the obligate parasite R. prowazekii has discard-
Fig. 3. The invasome-mediated -invasion of endothelial cells by B. hense/ae. Transmission electron micrographs illustrate the sequential steps of (A) aggregation, (B) engulfment and (C) internalisation of B. henselae into endothelial cells. The scale bars correspond to 211m. Reproduced with permission from Dehio etal.,1997.
Intracellular parasites: Rickettsia and Bartonella
ed most genes allocated to biosynthetic functions and is dependent upon its host cell for supply of small molecules such as amino acids and nucleoside monophosphates (Andersson et aI., 1998). In contrast, the facultative parasite B. henselae has a full complement of genes required for the biosynthesis of these basic building blocks (c. Alsmark, B. Canback, C. G. Kurland, S. G. E. Andersson, unpublished results). That is to say, Bartonella has retained the capacity for growth in the free-living mode, whereas Rickettsia seems to be irreversibly specialised to living in the intracellular environment. Chlamydia represents another group of obligate intracellular parasites that are highly adapted to the intracellular milieu (Stephens et a!', 1998). Like Rickettsia, the relative proportion of genes allocated to biosynthetic functions is very low in Chlamydia. For example, both Rickettsia and Chlamydia contain genes required for the interconversion of nucleoside monophosphates into all of the required nucleotides and deoxynucleotides, but lack genes involved in de novo purine and pyrimidine biosynthesis (Zomorodipour and Andersson, 1999). Since the two parasites do not share a common intracellular ancestor, the loss of biosynthetic genes is more likely to result from reductive, convergent evolution. A key transport protein in obligate intracellular parasites is the ATP/ADP translocase that enables the parasite to utilise cytoplasmic ATP as a source of energy. R. prowazekii has as many as five genes coding for ATP/ADP translocases (Andersson et aI., 1998), whereas C. trachomatis has only two (Stephens et aI., 1998). ATP/ADP transport exchange proteins have so far only been observed in Rickettsia~ Chlamydia, mitochondria and chloroplasts (Andersson, 1998). Phylogenetic analyses suggest that the ATP/ADP translocases in R. prowazekii and C. trachomatis are of a similar type, but characteristically different from their functional homologs in mitochondria. No genes coding for ATP/ADP translocases have been found in the genome of B. henselae, suggesting that Bartonella has not yet evolved transport systems for utilising the compounds available in the cytosol to the same extent as Rickettsia (c. Alsmark, B. Canback, C. G. Kurland, S. G. E. Andersson, unpublished results).
Inactivation, degradation and elimination It seems reasonable to assume that the genome of a freeliving ancestor of Rickettsia and Bartonella had a metabolic repertoire similar to that of modern free-living bacteria. If so, as much as 80 % of the original gene complement may have been lost during the evolution of the obligate intracellular lineages in this subdivision. Indeed, genome sequence data suggests that genes are
139
constantly being inactivated and discarded from the Rickettsia genomes. For example, a detailed comparative analysis of the metK gene, which codes for Sadenosylmethionine synthetase has revealed the presence of deleterious mutations in most of the Rickettsia species analysed (Andersson and Andersson, 1999a,b). It has been speculated that the degradation of the metK gene may be due to the invention of a transport system for S-adenosylmethionine (AdoMet) (Andersson and Andersson, 1999a). The novel import system and the ancestral biosynthetic gene may have worked hand-inhand for a while, until the transport system for AdoMet was efficient enough to compensate for the internal biosynthesis of AdoMet. If speciation occurred during the time both systems were operating, the time at which the first inactivation mutation was fixed in the population may have been determined solely by chance. A large number of pseudogenes have since been identified in the Rickettsia genomes, one of these is located downstream of the metK gene. The ancestral gene sequences of the two neighboring genes could be reconstructed by the removal of potential insertion and deletion mutations (Andersson and Andersson, 1999a). These reconstructed, ancestral gene sequences displayed the characteristic variations in G + C content values at the three codon positions, confirming that they have once been actively transcribed and translated genes. A closer inspection of the disruptive mutations has shown that there is a strong mutational bias towards deletions in Rickettsia (Andersson and Andersson, 1999a). This means that the inactivated gene sequences will deteriorate in a gradual manner until they are no longer recognisable as genes. It has been speculated that a large fraction of the noncoding DNA in the R. prowazekii genome may represent remnants of ancestral, inactivated genes in the very latest stages of degradation (Andersson et aI., 1998). Indeed, some of the sequences classified as noneading DNA in R. prowazekii display strong sequence similarities to fragments of genes or pseudogenes in other Rickettsia species (J. O. Andersson and S. G. E. Andersson, unpublished). Because of their obligate intracellular life style, there may be few possibilities for genetic exchange between different individuals in the rickettsial populations. Small populations with low recombination frequencies are expected to be particularly sensitive to the influence of the so-called Muliers ratchet, which will result in higher fixation rates for mutations (Felsenstein, 1974; Andersson and Kurland, 1995). Indeed, synonymous , substitution rates for a wide range of genes from the TG and the SFG are approximately similar, suggesting that these rates directly reflect the rate at which mutations are being produced. This suggests that selection may not be as effective in small, obligate intracellular
140
Siv G. E. Andersson, Christoph Dehio
populations as in large free-living bacterial populations. Phylogenetic reconstructions provide some indications of ancient gene transfer events into the R. prowazekii genome. For example, the lysyl amino acyltRNA synthetase and the valyl aminoacyl-tRNA synthetases are most similar to their homologs in archaea (Andersson et al., 1998). However, the nucleotide composition patterns of modern Rickettsia genes are extremely homogenous, providing no or only few indications of recent horizontal gene transfer events (M. Remm and S. G. E. Andersson, unpublished results). Taken together, it can be concluded that intracellular parasites are metabolically and genetically very divergent from bacteria with a free-living life style. This is in part explained by a high concentration of nutrients in the cytoplasm, which enables high rates of gene efflux. The isolated environment inside the eukaryotic cell provides protection from competitors on the one hand, but eliminates possibilities for genetic exchange on the other. Furthermore, intracellular bacteria have to go through a series of bottlenecks during the transmission from one host to the other, which may lead to high fixation rates for deleterious mutations. Each of these three factors is likely to contribute to the overall degradation and loss of genetic information. This leaves us with a paradox, namely that the intracellular growth environment that initially seemed so rich and attractive for bacterial growth may in the long-term induce stagnation and degradation (Andersson and Kurland, 1998).
Conclusions and perspectives The obligate intracellular life style and the pathogenicity of many Rickettsia species have made it very difficult to study the biology of these bacteria. The first genetic manipulation of a rickettsial gene was recently performed by the successful transformation of a rifampin resistance gene into the genome of R. prowazekii (Rachek et al., 1998). This represents the first step in the development of a genetic system for Rickettsia, which, together with the availability of the complete genome sequence of R. prowazekii (Andersson et al., 1998), will radically change our possibilities to study and understand the molecular biology of this group of obligate intracellular parasites. However, while experimental studies on obligate intracellular parasites such as Rickettsia are still challenging, we have genetics (Dehio and Meyer, 1997; Dehio et al., 1998) as well as suitable in vitro and animal infection models available for Bartonella. In combination with the genome sequence, this will enable us to perform modern types of functional genomic studies. These will include tran~~riptome analysis and saturational mutagenesis approaches to aid in the systematic
exploration of the molecular basis for facultative intracellular parasitism. Acknowledgements.The authors acknowledge the members of their laboratories for providing unpublished results. We thank Bozena Pichler-Brand and Christa Lanz for their help in electron microscopy and confocal laser scanning microscopy. We are grateful to Dr. Terry Kwok for critically reading of the manuscript. The genome sequencing projects are supported by grants to S. G. E. Andersson from the Swedish Foundation for Strategic Research, the National Science Research Council and the Knut and Alice Wallenberg Foundation.
References Anderson, B., Neuman, M. A.: Bartonella spp. as emerging human pathogens. Clin. Microbio!. Rev. 10, 203-219 (1997). Andersson,]. 0., Andersson, S. G. E.: Genome degradation is an ongoing process in Rickettsia. Mo!. Bio!. Evo!. 16, 1178-1191 (1999a). Andersson, J. 0., Andersson, S. G. E.: Insights into the evolutionary process of genome degradation. Curro Opin. Genet. Dev. 9, 664-671 (1999b). Andersson, S. G. E.: Bioenergetics of the obligate intracellular parasite Rickettsia prowazekii. Biochim. Biophys. Acta 1365, 105-111 (1998). Andersson, S. G. E., Kurland, C. G.: Genomic evolution drives the evolution of the translation system. Biochem. Cell Bio!. 73, 775-787 (1995). Andersson, S. G. E., Kurland C. G.: Reductive evolution of resident genomes. Trends Microbio!' 6,263-278 (1998). Andersson, S. G. E., Zomorodipour, A., Andersson, J. 0., Sicheritz-Ponten, T., Alsmark, U. C. M., Podowski, R. M., Naslund, A. K., Eriksson, A.-S., Winkler, H. H., Kurland C. G.: The genome sequence of Rickettsia prowazekii and the origin of mitochondria, Nature 396, 133-140 (1998). Bass, J. W., Vincent, J. M., Person, D. A.: The expanding spectrum of Bartonella infections: I. Bartonellosis and trench fever. Pediatr. Infect. Dis. J. 16, 2-10 (1997). Clifton, D. R., Goss, R. A., Sahni, S. K., Antwerp, D. v., Baggs, R.B., Marder, V.]., Silverman, D.]., Spron, L.A.: NF-xB-dependent inhibition of apoptosis is essential for host cell survival during Rickettsia rickettsii infection. Proc. Nat!' Acad. Sci. USA 95, 4646-4651 (1998). Conley, T., Slater, L., Hamilton, K.: Rochalimaea spp. stimulate human endothelial cell proliferation and migration in vitro.J.Lab. Clin. Med.124, 521-528 (1994). Dehio, c.: Interactions of Bartonella henselae with vascular endothelial cells. Curro Opin. Microbio!. 2, 78-82 (1999). Dehio, c., Meyer, M.: Maintenance of broad-host-range incompatibility group P and group Q plasmids and transposition of Tn5 in Bartonella henselae following conjugal plasmid transfer from Escherichia coli. ]. Bacterio!' 179, 538-540 (1997). Dehio, c., Meyer, M., Berger,]., Schwarz, H., Lanz, c.: Interaction of Bartonella henselae with endothelial cells results in bacterial aggregation on the cell surface and the subse-
Intracellular parasites: Rickettsia and Bartonella quent engulfment and internalisation of the bacterial aggregate by a unique structure, the invasome. J. Cell Sci. 110,2141-2154 (1997) . Dehio, C, Sander, A.: Bartonella as emerging pathogens. Trends Microbiol. 7,226-228 (1999). Dehio, M., Knorre, A., Lanz, C, Dehio C: Construction of versatile high-level expression vectors for Bartonella henselae and the use of green fluorescent protein as a new expression marker. Gene 215, 223-229 (1998). Felsenstein, J.: The evolutionary advantage of recombination. Genetics 78,157-159 (1974). Garcia-Caceres, U., Garcia, F. U.: Bartonellosis: An immunodepressive disease and the life of Daniel Alcides Carrion. Am.J. Clin. Pathol. 95 , S58-S66 (1991). Gouin, E., Gantelet, H., Egile, C, Lasa, I., Ohayon, H., Villiers, V., Gounon, P., Sansonetti, P. J., Cossart, P.: A comparative study of the actin-based motilitites of the pathogenic bacteria Listeria monocytogenes, Shigella {lexneri and Rickettsia conorii, J. Cell Sci. 112, 1697-1708 (1999). Heinzen, R. A., Grieshaber, S. S., Van Kirk, L. S., Devin, C J.: Dynamics of actin-based movement by Rickettsia rickettsii in vero cells. Infect. Immun. 67, 4201-4207 (1999). Heller, R., Riegel, P., Hansmann, Y., Delacour, G., Bermond, D., Dehio, c., Lamarque, F., Monteil, H., Chomel, B., Piemont, Y.: Bartonella tribocorum sp. nov., a new Bartonella species isolated from the blood of wild rats. Int. J. Syst. Bacteriol. 48, 1333-1339 (1998). Kordick, D. L., Breitschwerdt, E. B.: Intraerythrocytic presence of Bartonella henselae. J. Clin. Microbiol. 33, 16551656 (1995). Maeno, N., Oda, H., Yoshiie, K., Wahid, M. R., Fujimura, T., Matayoshi, S.: Live Bartonella henselae enhances endothe-
141
lial cell proliferation without direct contact. Microb. Pathog. 27, 419-427 (1999). Mehock, J. R., Greene, C E., Gherardini, F. C, Hahn, T. W., Krause, D. C: Bartonella henselae invasion of feline erythrocytes in vitro. Infect. Immun. 66, 3462-3466 (1998). Rachek, L.I., Tucker, A. M., Winkler, H. H., Wood, D.O.: Transformation of Rickettsia prowazekii to rifampin resistance.J. Bacterial. 180,2118-2124 (1998). Raoult, D., Raux, V.: Rickettsioses as paradigms of new or emerging infectious diseases. Clin. Microbial. Rev. 10, 694-719 (1997). Regnery, R. L., Rooney, J. A. , Johnson, A. M., Nesby, S. L., Manzewitsch, P., Beaver, K., Olson, J. G.: Experimentally induced Bartonella henselae infections followed by challenge exposure and antimicrobial therapy in cats. Am.J.Vet. Res. 57, 1714-1719 (1996). Reynafarje, C, Ramos, J.: The hemolytic anemia of human bartonellosis, Blood 17,562-578 (1961). Stephens, R. S., Kalman, S., Lammel, C, Fan, J., Marathe, R., et al.: Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis. Science 282, 754-759 (1998). Walker, T. S. : Rickettsial interactions with human endothelial cells in vitro: adherence and entry. Infect. Immun. 44, 205-210 (1984). Walker, D. H., Firth, W. T., Edgell, CJ.: Human endothelial cell culture plaques induced by Rickettsia rickettsii. Infect. Immun. 37, 301-306 (1982). Zomorodipour, A., Andersson, S. G. E.: Obligate intracellular parasites: Rickettsia prowazekii and Chlamydia trachomatis. FEBS Lett. 452,11-15 (1999).