- Email: [email protected]
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Wang, J. et al. (1993)5. Infect. Dis. 167,1320-1329 Blakebrough, I.S. et al. (1982) J. Infect. Dis. 146, 626-637 Salih, M.A.M. et al. (1990) 1. Clin. Micro&o/. 28,1711-1719 Caugant, D.A. et al. (1986) J. Gen. Microbial. 132,641-652 Caugant, D.A. et al. (1987) 1. Bacterial. 169,2781-2792 Caugant, D.A. et al. (1994) 1. Clin. Microbial. 32,323-330 Achtman, M. (1990) Rev. Med. Microbial. 1,29-38 Achtman, M. et al. (1992) J. Infect. Dis. 165,53-68 Moore, P.S. and Broome, C.V. (1994) Sci. Am. 271,38-47 Cartwright, K. (1995) in Meningococcal Disease (Cartwright, K., ed.), pp. 115-146, John Wiley & Sons Griffiss, J.M. (1995) in Meningococcal Disease (Cartwright, K., ed.), pp. 35-70, John Wiley & Sons Greenwood, B.M. et al. (1984) Lancet i, 1339-1342 Rouse, B.T. and Horohov, D.W. (1986) Rev. Infect. Dis. 8, 850-873 Reilly, S. and Gaunt, P.N. (1991) Lancet 338, 1143-1144 Cartwright, K.A.V. et al. (1991) Lancet 338,554-557 Young, L.S. et al. (1972) N. Engl. 1. Med. 287,5-9 Hubert, B. et al. (1992) J. Infect. Dis. 166,542-545 Moore, P.S. et al. (1990) J. Am. Med. Assoc. 264,1271-1275 Filice, G.A. et al. (1984) Am. J. Public Health 74,253-254 Griffiss, J.M., Brandt, B.L. and Jarvis, G.A. (1987) in Eoohtion ofMeningococca1 Disease (Vol. 2) (Vedros, N.A., ed.),
pp. 99-l 19, CRC Press Robertson, B.D. and Meyer, T.F. (1992) Trends Genet. 8,422-427 Achtman, M. et al. (1988) 1. Exp. Med. 168,507-525 Achtman, M. et al. (1991) J. Infect. Dis. 164,375-382 Suker, J. et al. (1994) Mol. Microbial. 12,253-265 Maiden, M.C.J. et al. (1991) Mol. Microbial. 5,727-736 Moore, P.S. et al. (1988) 1. Am. Med. Assoc.260,2686-2689 46 Moore, P.S. et al. (1989) Lancet ii, 260-263 47 Jones, D.M. and Sutcliffe,E.M. (1990) 1. Infect. 21,21-25 48 Riou, J.Y. et al. (1991) Eur. J. Clin. Microbial. Infect. Dis. 10, 405-409 49 Haimanot, R.T. et al. (1990) Stand. 1. Infect. Dis. 22, 171-174 50 Guibourdenche, M. et al. (1994) Eur. I. Clin. Microbial. Infect. Dis. 13,174-177 51 Morelli, G. et al. (1994) Mol. Microbial. 11, 175-187 52 McGuinness, B.T., Lambden, P.R. and Heckels, J.E. (1993) Mol. Microbial. 7,505-514 53 Rosenqvist, E. et al. (1993) Microb. Patbog. 15,197-205 54 McGuinness, B.T. et al. (1991) Lancet 337,514-517 55 Atwood, KC., Schneider, L.K. and Ryan, F.J. (1951) Proc. Nat1 Acad. Sci. USA 37,146-155 56 Olyhoek, A.J.M. et al. (1991) Microb. Pathog. 11,249-257 57 Aho, E.L. et al. (1991) Mol. Microbial. 5,1429-1437 40 41 42 43 44 45
Regulation of extracellular toxin production in CZostridium perfrilzgelzs Julian I. Rood and Michael Lyristis Until recently, little was known about In the past few years, there he aerotolerant anaerobe has been a dramatic expansion the regulation of toxin production in Clostridium perfringens Clostridium perfringens. Three regulatory of genetic studies on C. peris the primary causative fringens toxins. The genes engenes that control the production of agent of gas gangrene, or coding the CItoxin2”, 8 toxin7e9, 8 toxin (perfringolysin 0) have now been clostridial myonecrosis, and E toxinlo, t toxin”, K toxin12, identified. Two of these genes make up a also causes food poisoning p toxin13, enterotoxin14-17, sialitwo-component sensor-kinase-responseand several human and animal dase18 and hyaluronidase19 have regulator system that also controls the enterotoxaemic diseases. It is a all been cloned and sequenced production of other extracellular toxins. fast-growing putrefactive and and their location on the C. persaccharolytic bacterium that ].I. Rood* and M. Lyristis are in the Dept of fringens genome determined20. produces many different extraMicrobiology, Mona& University, Clayton 3168, Genetic modification of the gene cellular toxins and enzymes, Australia. *tel: +61 3 905 4825, fax: +61 3 905 encoding a toxin, plc (or CPU), several of which have been 481 I, e-mail: [email protected] has been used to produce a implicated in disease. The most modified toxin for vaccine studsignificant of the extracellular _. ies21, and chromosomal mutations in both plc and the toxins are a toxin (phospholipase C), 0 toxin gene encoding 8 toxin, pfoA, have been constructed and (perfringolysin 0) and K toxin (collagenase), which are used in virulence studiesz2J3. These studies have clearly believed to be important in gas gangrene, and p toxin shown that a toxin is an essential component in the and E toxin, which are involved in different enteropathogenesis of C.-perfringens-mediated gas gangrene. toxaemic syndromes. Clinical isolates of C. perfringens The precise role of these toxins in disease has not can be divided into five toxin types (A-E) based on been elucidated. The a toxin has phospholipase C, their ability to produce ~1, p, E and t toxins’.
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sphingomyelinase, haemolytic and lethal activities. Its mode of action may involve either cytolysis an&or more-complex tissue reactions, such as activation of the arachidonic-acid cascadez4. The 8 toxin is a thiolactivated oxygen-labile cytolysin that is both haemolytic and lethal. Both toxins may act by destroying host inflammatory cells and decreasing the effectiveness of the host inflammatory response, thereby enhancing the ability of C. perfringens cells to invade the tissueszs. Evidence for genes that regulate a toxin production Although type A strains appear to produce more a toxin than do strains of other toxin types+, until recently, very little was known about the regulation of a toxin production in C. perfringens. Unfortunately, our knowledge of how a toxin expression is regulated is still very incomplete and considerable research is needed before this process is understood clearly. Early studies on the cloning and sequence analysis of the plc gene yielded little information about its regulation2,4-6. Other studies carried out in Escberichia coli showed that deletion of the region upstream of the plc promoter leads to a tenfold increase in a toxin production3,27. This increase is associated with elevated levels of plc-specific mRNA, indicating that the upstream region is involved in negatively regulating a toxin production in cis at the transcriptional leve12’. Although this region contains a potential gene4, orf2, which is transcribed in the opposite direction to the plc gene, the evidence suggests that it is unlikely that the putative orf2-gene product is involved in the regulation of the plc gene28. Deletion analysis indicates that the 77 bp immediately upstream of the plc gene is primarily responsible for the repression of a toxin production (Fig. la). This region contains three adjacent AT-rich regions just upstream of the pk promoter and has anomalous behaviour on low-temperature agarose gels. It was concluded that the AT-rich regions result in an intrinsic DNA curvature of the region upstream of the pk gene, and that the repression of transcription is due to the effects of DNA bending2’. However, the functional role in C. perfringens of this region of DNA curvature remains to be determined. Comparison of the regions upstream of plc from C. perfringens type A and C strains, which produce different levels of a toxin, reveals that they have similar upstream sequences and that they result in similar levels of cxtoxin when expressed in E. coli28. However, the high-level a-toxin-producing type A strain produces much more plc-specific mRNA than does the type C strain, which produces only low levels of a toxin. These results suggest that other factors are involved in the regulation of the plc gene in these strains. Gelretardation studies have shown that extracts from both type A and C strains retard the migration of a DNA fragment containing the AT-rich region, the promoter and the start of the pk gene. In addition, the lower level of p/c-gene expression in the type C strain may be related to the absence of a protein that specifically binds to the plc gene ‘*. These data suggest that regu-
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1
(W
omoc
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virS
Fig. 1.The gene regions involved in regulation of extracellular toxin production in Clostridium perfringens. The direction of transcription is indicated by the arrows. (a) The p/c locus includes the AT-rich region (AT) and the p/c promoter (P). The figure also shows the c1 toxin structural gene, p/c, and orf2. (b) Shows the 8 toxin gene region, including the structural gene pfoA and the upstream activator pfof?. (c) Shows the global-regulatory locus of the virR-vi& system, including orfIOC, the function of which is unknown. lation of a toxin production may involve both a protein that is present in both type A and C strains and that binds to the plc promoter region, and a protein that binds to the plc gene, but is only present in type A strains. The latter protein, which we postulate to be the product of a gene that we have designated plR, is presumably an activator that is responsible for the higher level of a toxin production in type A strains. However, as neither of these putative proteins has been identified or characterized, nor have their structural genes been cloned, their role in the regulation of a toxin production is speculative. Role of PfoR in regulating 8 toxin production The pfoA gene was one of the first toxin genes from C. perfringens to be cloned and sequenced7,s. The pfoA gene encodes the 52.5 kDa 8 toxin, which is expressed in E. coli and secreted into the periplasm. Subsequently, the pfoA gene was cloned as part of a larger restriction fragment’ and sequence analysis has revealed that there is a second gene, pfoR, located 591 bp upstream of pfoA and transcribed from the same DNA strand (Fig. lb). Genetic studies using E. coli have shown that deletion of all or part of the pfoR gene leads to decreased levels of expression of 0 toxin. Complementation studies suggest that the product of this gene is significantly more active when the pfoR gene is present on the same plasmid and in the same orientation as the pfoA gene, suggesting that pfoR encodes a cisacting regulatory protein, PfoR, that behaves as a positive activator of 8 toxin production. PfoR contains sequence motifs commonly found in regulatory proteins, including a helix-turn-helix mot@. Deletion of the pfoR gene does not completely eliminate the production of 8 toxin from pfoA in E. coli. This result suggests either that the PfoR protein is not essential for 8 toxin production or that, in E. coli, some expression
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bally regulate toxin production in C. perfringens 32,33. Recent work in two independent laboratories has confirmed this hypothesis, and has led to the identification of a twocomponent signal transduction system that regulates the production of 8 toxin and several other extracellular toxins in C. perfringens34,3s. This system consists of two genes, virR and virS, that appear to form , virR virS an operon (Fig. lc). The virS gene encodes a putative protein with Phosphorylation \ JVirR motifs similar to those in the cytoplasmic carboxy-terminal domains of sensor histidine kinases3j. In particular, VirS contains the histidine residue that is the site of autophosphorylation in other sensor kinases, as well as other motifs that have been proposed to be involved in kinase activity and nucleotide binding29-31. The amino-terminal domain of VirS appears to contain six or seven domains, transmembrane suggesting that this portion of the molecule is imbedded in the cell membrane, and acts as the receptor Nan Prl 8 Toxin a Toxin K Toxin or sensor of the biochemical signal that leads to the autophosphorylFig. 2. Model for the regulation of extracellular toxin and enzyme production in Clostridiur; ation of the cytoplasmic carboxyperfringens. The figure shows the structural genes for CItoxin (p/c), 9 toxin (pfoA), Ktoxin (co/A) terminal VirS domain35. The virR sialidase/neuraminidase (nan) and protease (pit), together with the regulatory genes pfoR, virl and vi6 The putative p/c-specific regulatory gene is indicated as p/c/?. Based on the mode gene encodes a putative protein described previously35. similar to the amino-terminal domain of bacterial response regulators34J5. VirR contains aspartate residues that are conserved in response regulators and of the pfoA gene results from vector-encoded or E.-co/ithat probably act as the site of phosphorylation by specific promoters. The construction and analysis of the VirS protein. chromosomal pfoR mutants in C. perfringens is reGenetic studies in C. perfringens have shown that quired to determine the precise role of the PfoR protein. the virR and v&S genes regulate more than one extracellular toxin34,35. Tn92 6 insertion mutagenesis led to Global-regulatory role of VirR and VlrS the isolation of a chromosomal vi& mutant that proThe expression of many bacterial genes is controlled duces no detectable 6 toxin and also has reduced levels by the products of genes that encode a two-component of a toxin, protease and sialidase3s. This mutation signal transduction system. Such systems generally could be complemented by recombinant plasmids consist of two genes, which encode a sensor histidine carrying the virS gene, providing direct confirmation kinase and a cytoplasmic response regulator. The senthat the pleiotropic effects are due to the insertion of sor kinase usually contains at least one transmemTn916 into the virS gene. We have found that mulbrane domain and is localized at least partly in the cell tiple copies of the virR gene can partially override the membrane. Conformational changes induced by the effects of a mutation in vir& presumably either by crossdetection of either low-molecular-mass compounds in talk from another sensor kinase or by less-efficient the membrane or membrane perturbations induced VirR-mediated autophosphorylation. Chromosomal by the growth phase lead to the autophosphorylation mutations in the virR gene have been isolated both by of the cytoplasmic domain of the sensor kinase. The chemical mutagenesis and by homologous recombiactivated sensor kinase then phosphorylates and actination with an insertionally inactivated virR gene34. vates the response regulator. The activated regulator These mutants do not produce any detectable 8 toxin then either activates or represses the transcription of and produce reduced levels of K toxin. These pleiotropic the target genes29-31. effects can be complemented in truns by providing the Early genetic studies of C. perfringens resulted in virR gene on a recombinant plasmid. Northern blots the isolation of mutants with an altered ability to proshowed that regulation of the pfoA gene is mediated duce more than one toxin or extracellular enzyme, at the transcriptional leveP4. suggesting that there may be a gene or genes that gloEnvironmental or growth stimulus
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Based on our studies, we proposed a model for the regulation of extracellular toxin production in C. perenfringens 35. In this model (Fig. 2), an unknown vironmental or growth-stage stimulus is detected by the transmembrane domain of the VirS protein. The presence of six or seven transmembrane segments in this protein suggests that signal transduction may involve the transport of a solute across the cell membrane. This results in an alteration in the conformation of VirS, enabling it to autophosphorylate the His255 residue. Subsequently, we propose that the cytoplasmic domain of the phosphorylated VirS protein phosphorylates the Asp73 residue of the cytoplasmic VirR protein. Activated VirR then either activates the transcription of pfoA directly, or, more likely, activates the transcription of pfoR, which, in turn, leads to increased transcription of pfoA. The presence of an activated VirR protein must be essential for the expression of pfoA because mutation of either virR or virS completely eliminates 8 toxin production34,3s. In contrast, phosphorylated VirR protein only partially activates genes involved in the production of a toxin, K toxin, sialidase and protease. These differences may reflect a requirement for additional regulatory proteins to control the expression of toxins other than 0 toxin, as has already been suggested for the plc gene, or may reflect differences in the VirR-binding sites. Other workers reported that a C. perfringens strain 13 plc mutant derived from a single crossover event produces increased amounts of 8 toxin, and implied that changes in a toxin production may act via the VirR-VirS system to regulate the production of other toxinsz3. However, two independent strain-13-derived plc mutants constructed in this laboratory by allelic exchange do not show the same effects on the production of 8 toxin’“. Future perspectives The discovery of the VirR-VirS signal transduction system and the results of studies on the regulation of plc lead to many more questions than can be answered with the data available. Despite the rapid advances of the past few years, it is clear that we currently have only a very cursory view of how extracellular toxin production is regulated in C. perfringens. Important questions need to be asked. What are the environmental or growth-phase signals that activate the VirRVirS cascade? What are the precise sites at which VirR binds, and how does binding at these sites activate transcription? Does VirR activate the expression of pfoA directly or by activating pfoR? What is the precise role of the PfoR protein, and what is the mechanism by which it activates transcription of pfoA? What is the molecular basis for the different effects on the regulation of 8 toxin production and the production of other extracellular toxins? What other genes are regulated by this two-component system? What other genes are involved in regulating the production of other toxins, such as a toxin? Additional basic studies are also needed to understand this regulatory network. For example, it will not be possible to understand the mechanism of action of
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the VirR or PfoR proteins until the promoters of the pfoA and pfoR genes have been clearly identified. It is also essential that genetic studies of these regulatory genes, and their target sites, are carried out, where possible, in C. perfringens and not in E. coli. Fortunately, the genetic tools that are now available in C. perfringens are considerable: appropriate vectors for shuttling recombinant plasmids between C. perfringens and E. coli are available36J7. Methods have been developed for the isolation of defined chromosomal mutants either by Tn916 mutagenesis3”, by the insertion of suicide plasmids using homologous recombination’“,‘“, or by allelic exchange2’. Therefore, further genetic studies on this regulatory system are feasible and exciting new data should result from several laboratories over the next few years. All the studies carried out to date, as well as those suggested here, involve the analysis of toxin production in culture media. Although it has been shown in vivo that the VirR-VirS system affects virulence”s, it is critical that researchers are cognizant of the fact that different regulatory mechanisms may operate when the cells are growing in mammalian tissues. Acknowledgements The research in this laboratory was supported by grants from the Australian National Health and Medical ResearchCouncil and CSL
Ltd. M.L. was the recipient of an Australian Postgraduate Research Award (Industry). References 1 Rood, J.I. and Cole, S.T. (1991) Microbio!. Rev. 55,621-648 2 Titball, R.W. et ul. (1989) Infect. lmmun. 57,367-376 3 Okabe, A., Shim& T. and Hayashi, H. (1989) Biochem. Biophys. Res. Commun. 160,33-39 4 Saint-Joanis, B., Garnier, T. and Cole, S.T. (1989) Mol. Gen. Genet. 219,453-460 5 Leslie, D. et al. (1989) Mol. Micro&o/. 3, 383-392 6 Tso, J.Y. and Siebel, C. (1989) Infect. Immun. 57,468-476 7 Tweten, R.K. (1988) Infect. fmmun. 56,3228-3234 8 Tweten, R.K. (1988) Infect. lmmun. 56,3235-3240 9 Shim& T. et al. (1991) Infect. Immun. 59, 137-142 10 Hunter, S.E.C. et a/. (1992) Infect. lmmun. 60, 102-l 10 11 PereIIe, S. et al. (1993) Infect. Immun. 61,5147-5156 12 Matsushita, 0. et ul. (1994) J. Bacterial. 176, 149-156 13 Hunter, S.E.C. et al. (1993) Infect. Immun. 61,3958-3965 14 Hanna, P.C., Wnek, A.P. and McClane, B.A. (1989) 1. Bacterial. 171,6815-6820 15 Iwanejko, L.A., Routledge, M.N. and Stewart, G.S.A.B. (1989) J. Gen. Micro&o/. 135, 903-909 16 Van Damme-Jongsten, M., Werners, K. and Notermans, S. (1989) Antonie van LeeuwenhoekJ. Microbial. 56, 181-190 17 Czeczulin, J.R., Hanna, P.C. and McClane, B.A. (1993) Infect. Immun. 61, 3429-3439 18 Roggentin, I’. et al. (1988) FEBS Lett. 238,31-34 19 Canard, B. et al. (1994) Mol. Gen. Genet. 243,215-224 20 Canard, B., Saint-Joanis, B. and Cole, S.T. (1992) Mol. Microblol. 6, 1421-1429 21 Williamson, E.D. and Titball, R.W. (1993) Vaccine 11, 1253-1258 22 Awad, MM. et al. (1995) Mol. Microbial. 15, 191-202 23 Ninomiya, M. et al. (1994) Infect. lmmun. 62,5032-5039 24 Titball, R.W. (1993) Microbial. Rev. 57, 347-366 25 Bryant, A.E. et al. (1993) FEMS lmmunol. Med. Microbial. 7, 321-336 26 McDonel, J.L. (1980) Phurmucol. Ther. 10,617-635 27 Toyonaga, T. et al. (1992) Microbial. lmmunol. 36,603-613 28 Katayama, S-I. et al. (1993) Infect. lmmun. 61,457-463
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29 Gross, R. (1993) EMS Microbial. Rev. 104,301-326 30 Alex, L.A. and Simon, M.I. (1994) TrendsGenet.10,133-138 31 Bourret, R.B., Borkovich, K.A. and Simon, M.I. (1991) Annx Rev. Biochem. 60,401-441 32 Rood, J.I. and Wilkinson, R.G. (1975) 1. Bacterial.123,419-427
33 34 35 36
Imagawa, T. etal. (1981) &ken]. 24,13-21 Shim& T. et al. (1994) J. Bacterial. 176, 1616-1623 Lyristis, M. et al. (1994) Mol. Microbial.12, 761-777 Sloan, J. et al. (1992) Plasmid 27,207-219 37 Bannam, T.L. and Rood, J.1. (1993) Plusmid229,233-235
Rickettsiaprowazekii, ribosomes and slow growth Herbert H. Winkler
R
ickettsia prowazekii, the etio-
logical agent of louse-borne typhus, is a bacterium that can live only within the cytoplasm of a eukaryotic cel11e3.These rickettsiae are cousins of those that cause other arthropod-borne typhus and spotted fevers. Epidemic typhus is second only to malaria as an affliction of humankind throughout recorded history; however, little epidemic typhus is seen today. This is largely because of better public health and hygiene, and to some degree because it is socially and politically incorrect for any ‘nice’ country to have body lice infesting The rickettsiae, its population. members of the present-day alphapurple eubacteria, are an essential evolutionary model since they are the only known human bacterial pathogens that are obligate intracytoplasmic (free in the cytoplasm) parasites. This article considers the slow growth of R. prowazekii and the concentration of stable RNA in this, and other, slowly growing organisms. Evolutionary advantages of slow growth
With a generation time of about lOh, R. prowazekii still grows almost twice as fast as Mycobacterium tuberculosis. But we are not looking for the champion of slow growth. Even the familiar Escherichia coli, when conditions are very poor, can have a generation time of many hours - much longer than the 20min that aficionados of speed and exponential explosiveness enthuse about. The crucial difference
Some bacteria, such as Rickettsia prowuzekii, grow slowly, not with anticipation of a future feast, but because it is evolutionarily advantageous to do so. This creates apparent paradoxes for understanding their physiology and biochemistry. These rickettsiae have a ribosome concentration higher than expected if these ribosomes support translation at rates comparable to those in Escherichia coli. H.H. Winkler is in the Laboratory of Molecular Biology, University of South Alabama College of Medicine, Mobile, AL 36688, USA. tel: +1 334 460 6108, fax: +I 334 460 7269, e-mail: [email protected]
is that R. prowazekii (and M. tuberculosis) grows slowly without the opportunity ever to grow fast; this is not the common bacteriological paradigm of feast and famine. The pathogenicity of rickettsial disease is not caused by the elaboration of any special toxins, but is caused by the growth of the rickettsiae and the response of the host cell to its increased occupation. In epidemic typhus, a single R. prowazekii organism enters the cytoplasm of the host cell and grows by binary fission until there are many hundreds of rickettsiae in the cell. As a result of this rickettsial burden, the cell bursts, releasing the rickettsiae, which in turn will infect other cells. This intracytoplasmic growth results in the destruction of the endothelial cells lining the capillaries of the host. 0
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When an individual succumbs to epidemic typhus, the millions of rickettsiae growing within the cytoplasm of the host are doomed to lose their habitat and die because these rickettsiae have no route to another host. Transmission of rickettsial diseases is always by an arthropod vector (lice, fleas, mites and ticks). The vector for epidemic typhus, the human body louse, will feed only on living humans. Thus, the survival of these rickettsiae depends on a louse obtaining at least one rickettsia in its bloodmeal on the infected host so that the rickettsia can grow within the cells of the louse and be transmitted eventually to another human host. Clearly, the evolutionary success of R. prowazekii as a species depends on the success of rickettsial transmission; a common theme in parasitology, but one that is rarely voiced in bacteriology. The success of transmission is, in turn, related to the longevity and vigor of the infected person, and the concentration of rickettsiae in the blood. To obtain the highest level of rickettsiae in the blood and at the same time to preserve the life of the host for as long as possible, every cell that bursts must release the maximum number of rickettsiae. A high concentration of intracytoplasmic rickettsiae is hypothesized to be necessary for the escape of the rickettsiae from the host cell because the phospholipase A activity of the entire intracytoplasmic rickettsial mass is needed to overcome the ability of the host cell to repair the damage to its plasma membrane4.
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Wang, J. et al. (1993)5. Infect. Dis. 167,1320-1329 Blakebrough, I.S. et al. (1982) J. Infect. Dis. 146, 626-637 Salih, M.A.M. et al. (1990) 1. Clin. Micro&o/. 28,1711-1719 Caugant, D.A. et al. (1986) J. Gen. Microbial. 132,641-652 Caugant, D.A. et al. (1987) 1. Bacterial. 169,2781-2792 Caugant, D.A. et al. (1994) 1. Clin. Microbial. 32,323-330 Achtman, M. (1990) Rev. Med. Microbial. 1,29-38 Achtman, M. et al. (1992) J. Infect. Dis. 165,53-68 Moore, P.S. and Broome, C.V. (1994) Sci. Am. 271,38-47 Cartwright, K. (1995) in Meningococcal Disease (Cartwright, K., ed.), pp. 115-146, John Wiley & Sons Griffiss, J.M. (1995) in Meningococcal Disease (Cartwright, K., ed.), pp. 35-70, John Wiley & Sons Greenwood, B.M. et al. (1984) Lancet i, 1339-1342 Rouse, B.T. and Horohov, D.W. (1986) Rev. Infect. Dis. 8, 850-873 Reilly, S. and Gaunt, P.N. (1991) Lancet 338, 1143-1144 Cartwright, K.A.V. et al. (1991) Lancet 338,554-557 Young, L.S. et al. (1972) N. Engl. 1. Med. 287,5-9 Hubert, B. et al. (1992) J. Infect. Dis. 166,542-545 Moore, P.S. et al. (1990) J. Am. Med. Assoc. 264,1271-1275 Filice, G.A. et al. (1984) Am. J. Public Health 74,253-254 Griffiss, J.M., Brandt, B.L. and Jarvis, G.A. (1987) in Eoohtion ofMeningococca1 Disease (Vol. 2) (Vedros, N.A., ed.),
pp. 99-l 19, CRC Press Robertson, B.D. and Meyer, T.F. (1992) Trends Genet. 8,422-427 Achtman, M. et al. (1988) 1. Exp. Med. 168,507-525 Achtman, M. et al. (1991) J. Infect. Dis. 164,375-382 Suker, J. et al. (1994) Mol. Microbial. 12,253-265 Maiden, M.C.J. et al. (1991) Mol. Microbial. 5,727-736 Moore, P.S. et al. (1988) 1. Am. Med. Assoc.260,2686-2689 46 Moore, P.S. et al. (1989) Lancet ii, 260-263 47 Jones, D.M. and Sutcliffe,E.M. (1990) 1. Infect. 21,21-25 48 Riou, J.Y. et al. (1991) Eur. J. Clin. Microbial. Infect. Dis. 10, 405-409 49 Haimanot, R.T. et al. (1990) Stand. 1. Infect. Dis. 22, 171-174 50 Guibourdenche, M. et al. (1994) Eur. I. Clin. Microbial. Infect. Dis. 13,174-177 51 Morelli, G. et al. (1994) Mol. Microbial. 11, 175-187 52 McGuinness, B.T., Lambden, P.R. and Heckels, J.E. (1993) Mol. Microbial. 7,505-514 53 Rosenqvist, E. et al. (1993) Microb. Patbog. 15,197-205 54 McGuinness, B.T. et al. (1991) Lancet 337,514-517 55 Atwood, KC., Schneider, L.K. and Ryan, F.J. (1951) Proc. Nat1 Acad. Sci. USA 37,146-155 56 Olyhoek, A.J.M. et al. (1991) Microb. Pathog. 11,249-257 57 Aho, E.L. et al. (1991) Mol. Microbial. 5,1429-1437 40 41 42 43 44 45
Regulation of extracellular toxin production in CZostridium perfrilzgelzs Julian I. Rood and Michael Lyristis Until recently, little was known about In the past few years, there he aerotolerant anaerobe has been a dramatic expansion the regulation of toxin production in Clostridium perfringens Clostridium perfringens. Three regulatory of genetic studies on C. peris the primary causative fringens toxins. The genes engenes that control the production of agent of gas gangrene, or coding the CItoxin2”, 8 toxin7e9, 8 toxin (perfringolysin 0) have now been clostridial myonecrosis, and E toxinlo, t toxin”, K toxin12, identified. Two of these genes make up a also causes food poisoning p toxin13, enterotoxin14-17, sialitwo-component sensor-kinase-responseand several human and animal dase18 and hyaluronidase19 have regulator system that also controls the enterotoxaemic diseases. It is a all been cloned and sequenced production of other extracellular toxins. fast-growing putrefactive and and their location on the C. persaccharolytic bacterium that ].I. Rood* and M. Lyristis are in the Dept of fringens genome determined20. produces many different extraMicrobiology, Mona& University, Clayton 3168, Genetic modification of the gene cellular toxins and enzymes, Australia. *tel: +61 3 905 4825, fax: +61 3 905 encoding a toxin, plc (or CPU), several of which have been 481 I, e-mail: [email protected] has been used to produce a implicated in disease. The most modified toxin for vaccine studsignificant of the extracellular _. ies21, and chromosomal mutations in both plc and the toxins are a toxin (phospholipase C), 0 toxin gene encoding 8 toxin, pfoA, have been constructed and (perfringolysin 0) and K toxin (collagenase), which are used in virulence studiesz2J3. These studies have clearly believed to be important in gas gangrene, and p toxin shown that a toxin is an essential component in the and E toxin, which are involved in different enteropathogenesis of C.-perfringens-mediated gas gangrene. toxaemic syndromes. Clinical isolates of C. perfringens The precise role of these toxins in disease has not can be divided into five toxin types (A-E) based on been elucidated. The a toxin has phospholipase C, their ability to produce ~1, p, E and t toxins’.
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sphingomyelinase, haemolytic and lethal activities. Its mode of action may involve either cytolysis an&or more-complex tissue reactions, such as activation of the arachidonic-acid cascadez4. The 8 toxin is a thiolactivated oxygen-labile cytolysin that is both haemolytic and lethal. Both toxins may act by destroying host inflammatory cells and decreasing the effectiveness of the host inflammatory response, thereby enhancing the ability of C. perfringens cells to invade the tissueszs. Evidence for genes that regulate a toxin production Although type A strains appear to produce more a toxin than do strains of other toxin types+, until recently, very little was known about the regulation of a toxin production in C. perfringens. Unfortunately, our knowledge of how a toxin expression is regulated is still very incomplete and considerable research is needed before this process is understood clearly. Early studies on the cloning and sequence analysis of the plc gene yielded little information about its regulation2,4-6. Other studies carried out in Escberichia coli showed that deletion of the region upstream of the plc promoter leads to a tenfold increase in a toxin production3,27. This increase is associated with elevated levels of plc-specific mRNA, indicating that the upstream region is involved in negatively regulating a toxin production in cis at the transcriptional leve12’. Although this region contains a potential gene4, orf2, which is transcribed in the opposite direction to the plc gene, the evidence suggests that it is unlikely that the putative orf2-gene product is involved in the regulation of the plc gene28. Deletion analysis indicates that the 77 bp immediately upstream of the plc gene is primarily responsible for the repression of a toxin production (Fig. la). This region contains three adjacent AT-rich regions just upstream of the pk promoter and has anomalous behaviour on low-temperature agarose gels. It was concluded that the AT-rich regions result in an intrinsic DNA curvature of the region upstream of the pk gene, and that the repression of transcription is due to the effects of DNA bending2’. However, the functional role in C. perfringens of this region of DNA curvature remains to be determined. Comparison of the regions upstream of plc from C. perfringens type A and C strains, which produce different levels of a toxin, reveals that they have similar upstream sequences and that they result in similar levels of cxtoxin when expressed in E. coli28. However, the high-level a-toxin-producing type A strain produces much more plc-specific mRNA than does the type C strain, which produces only low levels of a toxin. These results suggest that other factors are involved in the regulation of the plc gene in these strains. Gelretardation studies have shown that extracts from both type A and C strains retard the migration of a DNA fragment containing the AT-rich region, the promoter and the start of the pk gene. In addition, the lower level of p/c-gene expression in the type C strain may be related to the absence of a protein that specifically binds to the plc gene ‘*. These data suggest that regu-
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1
(W
omoc
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Fig. 1.The gene regions involved in regulation of extracellular toxin production in Clostridium perfringens. The direction of transcription is indicated by the arrows. (a) The p/c locus includes the AT-rich region (AT) and the p/c promoter (P). The figure also shows the c1 toxin structural gene, p/c, and orf2. (b) Shows the 8 toxin gene region, including the structural gene pfoA and the upstream activator pfof?. (c) Shows the global-regulatory locus of the virR-vi& system, including orfIOC, the function of which is unknown. lation of a toxin production may involve both a protein that is present in both type A and C strains and that binds to the plc promoter region, and a protein that binds to the plc gene, but is only present in type A strains. The latter protein, which we postulate to be the product of a gene that we have designated plR, is presumably an activator that is responsible for the higher level of a toxin production in type A strains. However, as neither of these putative proteins has been identified or characterized, nor have their structural genes been cloned, their role in the regulation of a toxin production is speculative. Role of PfoR in regulating 8 toxin production The pfoA gene was one of the first toxin genes from C. perfringens to be cloned and sequenced7,s. The pfoA gene encodes the 52.5 kDa 8 toxin, which is expressed in E. coli and secreted into the periplasm. Subsequently, the pfoA gene was cloned as part of a larger restriction fragment’ and sequence analysis has revealed that there is a second gene, pfoR, located 591 bp upstream of pfoA and transcribed from the same DNA strand (Fig. lb). Genetic studies using E. coli have shown that deletion of all or part of the pfoR gene leads to decreased levels of expression of 0 toxin. Complementation studies suggest that the product of this gene is significantly more active when the pfoR gene is present on the same plasmid and in the same orientation as the pfoA gene, suggesting that pfoR encodes a cisacting regulatory protein, PfoR, that behaves as a positive activator of 8 toxin production. PfoR contains sequence motifs commonly found in regulatory proteins, including a helix-turn-helix mot@. Deletion of the pfoR gene does not completely eliminate the production of 8 toxin from pfoA in E. coli. This result suggests either that the PfoR protein is not essential for 8 toxin production or that, in E. coli, some expression
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bally regulate toxin production in C. perfringens 32,33. Recent work in two independent laboratories has confirmed this hypothesis, and has led to the identification of a twocomponent signal transduction system that regulates the production of 8 toxin and several other extracellular toxins in C. perfringens34,3s. This system consists of two genes, virR and virS, that appear to form , virR virS an operon (Fig. lc). The virS gene encodes a putative protein with Phosphorylation \ JVirR motifs similar to those in the cytoplasmic carboxy-terminal domains of sensor histidine kinases3j. In particular, VirS contains the histidine residue that is the site of autophosphorylation in other sensor kinases, as well as other motifs that have been proposed to be involved in kinase activity and nucleotide binding29-31. The amino-terminal domain of VirS appears to contain six or seven domains, transmembrane suggesting that this portion of the molecule is imbedded in the cell membrane, and acts as the receptor Nan Prl 8 Toxin a Toxin K Toxin or sensor of the biochemical signal that leads to the autophosphorylFig. 2. Model for the regulation of extracellular toxin and enzyme production in Clostridiur; ation of the cytoplasmic carboxyperfringens. The figure shows the structural genes for CItoxin (p/c), 9 toxin (pfoA), Ktoxin (co/A) terminal VirS domain35. The virR sialidase/neuraminidase (nan) and protease (pit), together with the regulatory genes pfoR, virl and vi6 The putative p/c-specific regulatory gene is indicated as p/c/?. Based on the mode gene encodes a putative protein described previously35. similar to the amino-terminal domain of bacterial response regulators34J5. VirR contains aspartate residues that are conserved in response regulators and of the pfoA gene results from vector-encoded or E.-co/ithat probably act as the site of phosphorylation by specific promoters. The construction and analysis of the VirS protein. chromosomal pfoR mutants in C. perfringens is reGenetic studies in C. perfringens have shown that quired to determine the precise role of the PfoR protein. the virR and v&S genes regulate more than one extracellular toxin34,35. Tn92 6 insertion mutagenesis led to Global-regulatory role of VirR and VlrS the isolation of a chromosomal vi& mutant that proThe expression of many bacterial genes is controlled duces no detectable 6 toxin and also has reduced levels by the products of genes that encode a two-component of a toxin, protease and sialidase3s. This mutation signal transduction system. Such systems generally could be complemented by recombinant plasmids consist of two genes, which encode a sensor histidine carrying the virS gene, providing direct confirmation kinase and a cytoplasmic response regulator. The senthat the pleiotropic effects are due to the insertion of sor kinase usually contains at least one transmemTn916 into the virS gene. We have found that mulbrane domain and is localized at least partly in the cell tiple copies of the virR gene can partially override the membrane. Conformational changes induced by the effects of a mutation in vir& presumably either by crossdetection of either low-molecular-mass compounds in talk from another sensor kinase or by less-efficient the membrane or membrane perturbations induced VirR-mediated autophosphorylation. Chromosomal by the growth phase lead to the autophosphorylation mutations in the virR gene have been isolated both by of the cytoplasmic domain of the sensor kinase. The chemical mutagenesis and by homologous recombiactivated sensor kinase then phosphorylates and actination with an insertionally inactivated virR gene34. vates the response regulator. The activated regulator These mutants do not produce any detectable 8 toxin then either activates or represses the transcription of and produce reduced levels of K toxin. These pleiotropic the target genes29-31. effects can be complemented in truns by providing the Early genetic studies of C. perfringens resulted in virR gene on a recombinant plasmid. Northern blots the isolation of mutants with an altered ability to proshowed that regulation of the pfoA gene is mediated duce more than one toxin or extracellular enzyme, at the transcriptional leveP4. suggesting that there may be a gene or genes that gloEnvironmental or growth stimulus
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Based on our studies, we proposed a model for the regulation of extracellular toxin production in C. perenfringens 35. In this model (Fig. 2), an unknown vironmental or growth-stage stimulus is detected by the transmembrane domain of the VirS protein. The presence of six or seven transmembrane segments in this protein suggests that signal transduction may involve the transport of a solute across the cell membrane. This results in an alteration in the conformation of VirS, enabling it to autophosphorylate the His255 residue. Subsequently, we propose that the cytoplasmic domain of the phosphorylated VirS protein phosphorylates the Asp73 residue of the cytoplasmic VirR protein. Activated VirR then either activates the transcription of pfoA directly, or, more likely, activates the transcription of pfoR, which, in turn, leads to increased transcription of pfoA. The presence of an activated VirR protein must be essential for the expression of pfoA because mutation of either virR or virS completely eliminates 8 toxin production34,3s. In contrast, phosphorylated VirR protein only partially activates genes involved in the production of a toxin, K toxin, sialidase and protease. These differences may reflect a requirement for additional regulatory proteins to control the expression of toxins other than 0 toxin, as has already been suggested for the plc gene, or may reflect differences in the VirR-binding sites. Other workers reported that a C. perfringens strain 13 plc mutant derived from a single crossover event produces increased amounts of 8 toxin, and implied that changes in a toxin production may act via the VirR-VirS system to regulate the production of other toxinsz3. However, two independent strain-13-derived plc mutants constructed in this laboratory by allelic exchange do not show the same effects on the production of 8 toxin’“. Future perspectives The discovery of the VirR-VirS signal transduction system and the results of studies on the regulation of plc lead to many more questions than can be answered with the data available. Despite the rapid advances of the past few years, it is clear that we currently have only a very cursory view of how extracellular toxin production is regulated in C. perfringens. Important questions need to be asked. What are the environmental or growth-phase signals that activate the VirRVirS cascade? What are the precise sites at which VirR binds, and how does binding at these sites activate transcription? Does VirR activate the expression of pfoA directly or by activating pfoR? What is the precise role of the PfoR protein, and what is the mechanism by which it activates transcription of pfoA? What is the molecular basis for the different effects on the regulation of 8 toxin production and the production of other extracellular toxins? What other genes are regulated by this two-component system? What other genes are involved in regulating the production of other toxins, such as a toxin? Additional basic studies are also needed to understand this regulatory network. For example, it will not be possible to understand the mechanism of action of
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the VirR or PfoR proteins until the promoters of the pfoA and pfoR genes have been clearly identified. It is also essential that genetic studies of these regulatory genes, and their target sites, are carried out, where possible, in C. perfringens and not in E. coli. Fortunately, the genetic tools that are now available in C. perfringens are considerable: appropriate vectors for shuttling recombinant plasmids between C. perfringens and E. coli are available36J7. Methods have been developed for the isolation of defined chromosomal mutants either by Tn916 mutagenesis3”, by the insertion of suicide plasmids using homologous recombination’“,‘“, or by allelic exchange2’. Therefore, further genetic studies on this regulatory system are feasible and exciting new data should result from several laboratories over the next few years. All the studies carried out to date, as well as those suggested here, involve the analysis of toxin production in culture media. Although it has been shown in vivo that the VirR-VirS system affects virulence”s, it is critical that researchers are cognizant of the fact that different regulatory mechanisms may operate when the cells are growing in mammalian tissues. Acknowledgements The research in this laboratory was supported by grants from the Australian National Health and Medical ResearchCouncil and CSL
Ltd. M.L. was the recipient of an Australian Postgraduate Research Award (Industry). References 1 Rood, J.I. and Cole, S.T. (1991) Microbio!. Rev. 55,621-648 2 Titball, R.W. et ul. (1989) Infect. lmmun. 57,367-376 3 Okabe, A., Shim& T. and Hayashi, H. (1989) Biochem. Biophys. Res. Commun. 160,33-39 4 Saint-Joanis, B., Garnier, T. and Cole, S.T. (1989) Mol. Gen. Genet. 219,453-460 5 Leslie, D. et al. (1989) Mol. Micro&o/. 3, 383-392 6 Tso, J.Y. and Siebel, C. (1989) Infect. Immun. 57,468-476 7 Tweten, R.K. (1988) Infect. fmmun. 56,3228-3234 8 Tweten, R.K. (1988) Infect. lmmun. 56,3235-3240 9 Shim& T. et al. (1991) Infect. Immun. 59, 137-142 10 Hunter, S.E.C. et a/. (1992) Infect. lmmun. 60, 102-l 10 11 PereIIe, S. et al. (1993) Infect. Immun. 61,5147-5156 12 Matsushita, 0. et ul. (1994) J. Bacterial. 176, 149-156 13 Hunter, S.E.C. et al. (1993) Infect. Immun. 61,3958-3965 14 Hanna, P.C., Wnek, A.P. and McClane, B.A. (1989) 1. Bacterial. 171,6815-6820 15 Iwanejko, L.A., Routledge, M.N. and Stewart, G.S.A.B. (1989) J. Gen. Micro&o/. 135, 903-909 16 Van Damme-Jongsten, M., Werners, K. and Notermans, S. (1989) Antonie van LeeuwenhoekJ. Microbial. 56, 181-190 17 Czeczulin, J.R., Hanna, P.C. and McClane, B.A. (1993) Infect. Immun. 61, 3429-3439 18 Roggentin, I’. et al. (1988) FEBS Lett. 238,31-34 19 Canard, B. et al. (1994) Mol. Gen. Genet. 243,215-224 20 Canard, B., Saint-Joanis, B. and Cole, S.T. (1992) Mol. Microblol. 6, 1421-1429 21 Williamson, E.D. and Titball, R.W. (1993) Vaccine 11, 1253-1258 22 Awad, MM. et al. (1995) Mol. Microbial. 15, 191-202 23 Ninomiya, M. et al. (1994) Infect. lmmun. 62,5032-5039 24 Titball, R.W. (1993) Microbial. Rev. 57, 347-366 25 Bryant, A.E. et al. (1993) FEMS lmmunol. Med. Microbial. 7, 321-336 26 McDonel, J.L. (1980) Phurmucol. Ther. 10,617-635 27 Toyonaga, T. et al. (1992) Microbial. lmmunol. 36,603-613 28 Katayama, S-I. et al. (1993) Infect. lmmun. 61,457-463
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29 Gross, R. (1993) EMS Microbial. Rev. 104,301-326 30 Alex, L.A. and Simon, M.I. (1994) TrendsGenet.10,133-138 31 Bourret, R.B., Borkovich, K.A. and Simon, M.I. (1991) Annx Rev. Biochem. 60,401-441 32 Rood, J.I. and Wilkinson, R.G. (1975) 1. Bacterial.123,419-427
33 34 35 36
Imagawa, T. etal. (1981) &ken]. 24,13-21 Shim& T. et al. (1994) J. Bacterial. 176, 1616-1623 Lyristis, M. et al. (1994) Mol. Microbial.12, 761-777 Sloan, J. et al. (1992) Plasmid 27,207-219 37 Bannam, T.L. and Rood, J.1. (1993) Plusmid229,233-235
Rickettsiaprowazekii, ribosomes and slow growth Herbert H. Winkler
R
ickettsia prowazekii, the etio-
logical agent of louse-borne typhus, is a bacterium that can live only within the cytoplasm of a eukaryotic cel11e3.These rickettsiae are cousins of those that cause other arthropod-borne typhus and spotted fevers. Epidemic typhus is second only to malaria as an affliction of humankind throughout recorded history; however, little epidemic typhus is seen today. This is largely because of better public health and hygiene, and to some degree because it is socially and politically incorrect for any ‘nice’ country to have body lice infesting The rickettsiae, its population. members of the present-day alphapurple eubacteria, are an essential evolutionary model since they are the only known human bacterial pathogens that are obligate intracytoplasmic (free in the cytoplasm) parasites. This article considers the slow growth of R. prowazekii and the concentration of stable RNA in this, and other, slowly growing organisms. Evolutionary advantages of slow growth
With a generation time of about lOh, R. prowazekii still grows almost twice as fast as Mycobacterium tuberculosis. But we are not looking for the champion of slow growth. Even the familiar Escherichia coli, when conditions are very poor, can have a generation time of many hours - much longer than the 20min that aficionados of speed and exponential explosiveness enthuse about. The crucial difference
Some bacteria, such as Rickettsia prowuzekii, grow slowly, not with anticipation of a future feast, but because it is evolutionarily advantageous to do so. This creates apparent paradoxes for understanding their physiology and biochemistry. These rickettsiae have a ribosome concentration higher than expected if these ribosomes support translation at rates comparable to those in Escherichia coli. H.H. Winkler is in the Laboratory of Molecular Biology, University of South Alabama College of Medicine, Mobile, AL 36688, USA. tel: +1 334 460 6108, fax: +I 334 460 7269, e-mail: [email protected]
is that R. prowazekii (and M. tuberculosis) grows slowly without the opportunity ever to grow fast; this is not the common bacteriological paradigm of feast and famine. The pathogenicity of rickettsial disease is not caused by the elaboration of any special toxins, but is caused by the growth of the rickettsiae and the response of the host cell to its increased occupation. In epidemic typhus, a single R. prowazekii organism enters the cytoplasm of the host cell and grows by binary fission until there are many hundreds of rickettsiae in the cell. As a result of this rickettsial burden, the cell bursts, releasing the rickettsiae, which in turn will infect other cells. This intracytoplasmic growth results in the destruction of the endothelial cells lining the capillaries of the host. 0
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When an individual succumbs to epidemic typhus, the millions of rickettsiae growing within the cytoplasm of the host are doomed to lose their habitat and die because these rickettsiae have no route to another host. Transmission of rickettsial diseases is always by an arthropod vector (lice, fleas, mites and ticks). The vector for epidemic typhus, the human body louse, will feed only on living humans. Thus, the survival of these rickettsiae depends on a louse obtaining at least one rickettsia in its bloodmeal on the infected host so that the rickettsia can grow within the cells of the louse and be transmitted eventually to another human host. Clearly, the evolutionary success of R. prowazekii as a species depends on the success of rickettsial transmission; a common theme in parasitology, but one that is rarely voiced in bacteriology. The success of transmission is, in turn, related to the longevity and vigor of the infected person, and the concentration of rickettsiae in the blood. To obtain the highest level of rickettsiae in the blood and at the same time to preserve the life of the host for as long as possible, every cell that bursts must release the maximum number of rickettsiae. A high concentration of intracytoplasmic rickettsiae is hypothesized to be necessary for the escape of the rickettsiae from the host cell because the phospholipase A activity of the entire intracytoplasmic rickettsial mass is needed to overcome the ability of the host cell to repair the damage to its plasma membrane4.
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