Adaptation of Borrelia burgdorferi in the tick and the mammalian host

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1 FEMS Microbiology Reviews 27 (2003) 493^504 Adaptation of Borrelia burgdorferi in the tick and the mammalian host b Juan Anguita a, Michael N. Hedrick a, Erol Fikrig b; a Department of Biology, University of North Carolina at Charlotte, Charlotte, NC, USA Section of Rheumatology, Department of Internal Medicine, Yale University School of Medicine, P.O. Box , S525A 300 Cedar Street, New Haven, CT , USA Abstract Received 4 November 2002; received in revised form 10 February 2003; accepted 12 March 2003 First published online 10 April 2003 Borrelia burgdorferi, the causative agent of Lyme disease, shows a great ability to adapt to different environments, including the arthropod vector, and the mammalian host. The success of these microorganisms to survive in nature and complete their enzootic cycle depends on the regulation of genes that are essential to their survival in the different environments. This review describes the current knowledge of gene expression by B. burgdorferi in the tick and the mammalian host. The functions of the differentially regulated gene products as well as the factors that influence their expression are discussed. A thorough understanding of the changes in gene expression and the function of the differentially expressed antigens during the life cycle of the spirochete will allow a better control of this prevalent infection and the design of new, second generation vaccines to prevent infection with the spirochete. ß 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Lyme disease; Gene expression; Vaccine; Borrelia burgdorferi Contents 1. Introduction Lyme disease pathogenesis Changes in gene expression during the B. burgdorferi life cycle Gene expression in the tick Gene expression in the mammalian host Strategies to survive mammalian immune responses Recombination as a means of immune escape Erps and complement inhibition Signals that trigger gene expression changes and recombination Environmental factors and cell density Immune pressure Host factors Molecular mechanisms of gene regulation Concluding remarks Acknowledgements References * Corresponding author. Tel.: +1 (203) address: erol. krig@yale.edu (E. Fikrig) / 03 / $22.00 ß 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi: /s (03)

2 494 J. Anguita et al. / FEMS Microbiology Reviews 27 (2003) 493^ Introduction Borrelia burgdorferi, the causative agent of Lyme disease, is an example of a microorganism that has adapted through evolution to survive in di erent environments. The ability of spirochetes to survive in two divergent surroundings, the tick and the vertebrate host, is primarily based on changes in B. burgdorferi gene expression that are hallmarks during infection of both the arthropod and the mammal. How the spirochete manages to induce the changes necessary to survive in these settings, the nature of those changes, and the function of the genes di erentially regulated are the subjects of intensive investigation. Despite the knowledge that we have on particular genes that are either up- or downregulated during the transition from the vector to the mammalian host, and to some extent during persistent infection of the mammal, little is known about the function of the vast majority of the proteins encoded by these genes, and the signals that trigger these changes. The limitations inherent to the study of the spirochetal biology, namely the imperfect genetic manipulation systems in pathogenic isolates, and the complex interactions between ticks, the mammalian hosts, and the responses triggered by these contacts, have prevented a thorough understanding of the survival mechanisms of B. burgdorferi and the design of e ective therapeutics and second generation vaccines targeting antigens expressed in the mammalian host. Recently, signi cant advances have been made to better understand the biology of the spirochete, and the development of new tools has allowed a dramatic increase in our knowledge of the regulation of gene expression and protein function, and their relationship with the transmission of B. burgdorferi between the arthropod and the mammal. 2. Lyme disease pathogenesis Lyme disease is caused by spirochetes belonging to the B. burgdorferi sensu lato group of microorganisms. These include species that cause the disease in North America (B. burgdorferi sensu stricto) and Europe (Borrelia garinii, Borrelia afzelii and B. burgdorferi sensu stricto). The life cycle of the bacterium involves the arthropod vector and the mammalian host. Ticks of the Ixodes genus are the main vectors for B. burgdorferi transmission, both in the United States and Western Europe [1^3]. The cycle of transmission begins when uninfected ticks feed on animals that carry the spirochete. These are usually small mammals, including white-footed mice (Peromyscus leucopus), but can also include birds and other animals, although their role in the maintenance of the microorganism in nature has not been completely elucidated [4,5]. Once the tick acquires the spirochete, the arthropod remains infected during the molting period. Therefore, when the next tick stage is ready to feed they can promptly transmit B. burgdorferi to the next mammalian host. The lack of vertical transmission in the mammalian host is probably a determinant factor that contributes to the maintenance of an obligate enzootic life cycle of the microorganism [1]. Ticks may play a major role for the distribution of the disease. In the United States, ticks feed mainly on small rodents in the northeast and also on lizards in the southeast. Lizards are not competent carriers for the spirochete [1], which could explain the low incidence of the disease in this part of the country. Lyme disease is endemic in areas of the United States, Europe and some countries in Asia. In the United States, these areas include the northeast, upper midwest and northern regions of the Paci c coast. The prevalence of the Ixodes vector and the percentage of ticks that are infected with the spirochete help determine the distribution of the disease. Lyme disease usually begins with a skin rash (erythema migrans) that can be accompanied by u-like symptoms [6]. Even without treatment, the rash generally resolves within 3^4 weeks. However, the spirochete can be detected in the blood and if the patient is not treated, the disease can evolve into secondary and tertiary complications [6^ 19]. Secondary disease may be manifested by disseminated skin rashes, carditis, aseptic meningitis, or acute arthritis [7,16,20^22]. Cardiac involvement occurs approximately in 8% of untreated patients and is characterized by palpitations and atrioventricular conduction abnormalities that usually resolve within 6 weeks. Around 10% of the untreated infected individuals develop neurologic complications that include meningitis, meningoencephalitis, nerve palsies and radiculitis [23]. Arthritis can be mono- or oligo-articular, and occurs in 10^20% of the untreated patients. The most frequently a ected are large joints, particularly the knees. Arthritic attacks last several months and can recur over periods of years [16]. Unusual late manifestations of disease include chronic antibiotic-resistant Lyme arthritis, which primarily occurs in the USA, and acrodermatitis chronica atrophicans, a purplish cutaneous lesion that is more common in Europe. Among the di erent animal models of Lyme borreliosis, the murine model is especially useful for the study of acute arthritis induced by B. burgdorferi. Mice are persistently infected with the spirochete and consistently develop in- ammation of the joints with a temporal and histological pattern that partially resembles human disease. The in- ammation peaks at 2^4 weeks of infection, and resolves over a period of months [24,25]. It is characterized by a neutrophilic in ltrate that may be accompanied by edema, thickening of the tendon sheath and, in severe cases, cartilage destruction and bone resorption [24,25]. The murine model does not represent, however, a good system to study other arthritic processes elicited by infection with the spirochete, namely, chronic antibiotic-resistant Lyme arthritis that occurs in a small percentage of patients and that may have autoimmune etiology [26^28]. Several factors in uence the pathogenesis of Lyme dis-

3 J. Anguita et al. / FEMS Microbiology Reviews 27 (2003) 493^ ease. The number of B. burgdorferi in the a ected organs [29], spirochetal virulence [30^32], and the humoral and cellular responses arising during infection [33^40], a ect the severity of the symptoms found in the murine model. The murine model has also provided proof that antibodies play an essential role in the control of infection and resolution of disease, and mice with genetic de ciencies that lead to impaired antibody production maintain a high spirochetal burden throughout the infection period and do not resolve the in ammatory symptoms associated with the disease [41^52]. In SCID mice, the control of infection is achieved by the transfer of infected mouse sera [52], presensitized splenocytes or partially by B cells, but not T cells [53], which underscores the role for B cellmediated responses to the spirochete to control infection. The study of the biology of B. burgdorferi improved dramatically with the development of media that support its growth, a milestone that has not occurred yet for another medically important spirochete, Treponema pallidum, the causative agent of syphilis. The design of Barbour^ Stoenner^Kelly (BSK) II and a variation, BSK H, media allowed researchers to study gene and protein expression in di erent environmental conditions [54,55]. Changes that occur in vitro re ect the e ect of physio-chemical factors on the spirochete and established the basis for further studies in vivo both in the arthropod and the mammalian host [56^61]. The combination of both analyses and the correlation of gene expression with infectivity and/or pathogenicity set a starting point to elucidate the complex biology of the spirochete and the function of some of the genes that are di erentially regulated during the life cycle of the bacterium. These studies have bene ted enormously from the publication of the whole genome sequence of B. burgdorferi [62]. This important achievement was a turning point in spirochetal research, and allowed the concentration of the e orts of investigators in gene regulation and protein function of the numerous di erentially regulated genes during the life cycle of the microorganism. Table 1 Some of the B. burgdorferi genes di erentially expressed in the tick and the mammalian host 3. Changes in gene expression during the B. burgdorferi life cycle It is well known that B. burgdorferi alters its gene expression during its life cycle. Intuitively, these changes are due to the di erent environmental conditions that dictate enzymatic activities that need to be performed. Alternatively, or complementarily, these changes in gene expression may be related to immune escape strategies. Although little is known about the exact function of a vast majority of the di erentially expressed genes, it seems obvious that proteins that bind tick or mammalian proteins are di erentially expressed in the vector and the reservoir host, respectively (see Table 1). These include OspA, DbpA, Bbk32and the gene family that encodes OspE/F-related proteins (Erps), among others Gene expression in the tick The tick environment is exposed to changes related to temperature and the blood meal. Among the proteins expressed by B. burgdorferi in the tick, OspA is the object of investigation because it is one of the best examples of an antigen with a de nitive spatial pattern of expression, is the object of the only FDA approved vaccine available for human use although recently retired from the market due to poor sales, and its function has been recently revealed. OspA serves as an anchor for the spirochete to the tick midgut [63] in such a way that antibodies that prevent its binding function also interfere with e cient colonization of the bacteria entering the arthropod from the mammalian host [64]. This function suggests an important role of the lipoprotein in the colonization of the midgut of the invertebrate host. OspA binds speci cally to a protein component or components in the tick midgut but very weakly to the salivary gland of the vector, implying that the ligand serves as a directional anchor once they enter the arthropod. Similarly, the downregulation of ospa expression during blood meal may allow the spirochete to Gene Function Regulation Tick ospa anchor to the tick midgut expressed in unfed ticks; downregulated during blood meal by unknown signals ospc target of the spirochete to the salivary glands regulated by temperature and ph Mammalian host dbpa (bba24) decorin-binding protein expressed in the mammalian host; upregulated by a decrease in ph bbk32 bronectin-binding protein induced upon engorgement of the tick bbk50 unknown function same kinetics as bbk32 ospe/f complement inhibition expressed in the mammalian host Bba64 P35, of unknown function regulated by ph, temperature and cell density vls immune escape; highly variable recombination induced by unknown factors; induced by in ammatory signals mlp unknown function; multicopy lipoprotein genes induced by body or nearly body temperature and some unknown mammalian factor(s) rev unknown function induced upon feeding of ticks The function, when known and their mode of regulation are also given.

4 496 J. Anguita et al. / FEMS Microbiology Reviews 27 (2003) 493^504 Fig. 1. A model depicting OspA and OspC expression during the life cycle of B. burgdorferi. OspA is expressed in the tick midgut, serving as an anchor protein. Its downregulation allows the spirochetes to translocate to the salivary gland from where they can be transmitted to the mammalian host. OspA expression can also be detected in some patients during late infection. OspC is upregulated when the tick start its blood meal and probably allows the spirochete to move through the hemocele of the arthropod. Its expression in the salivary gland and the mammal is minimal and vanishes over time. escape this environment and continue to the salivary gland, from where they can readily pass to the mammalian host during tick feeding. Importantly, OspA shows a great degree of self-aggregation which could potentially further help the spirochete to colonize the tick midgut [63]. This phenomenon would also explain the high levels of clumping found in spirochetes that are grown in vitro, which express high levels of the lipoprotein. The downregulation of OspA does not explain, however, the tropism of the spirochete for the salivary gland during tick feeding. Experiments have revealed the heterogeneity of spirochetes during the period that covers tick feeding and spirochetal migration from the gut to the salivary glands and ultimately to the mammalian host. These events are more complex than envisioned previously. The working hypothesis stated that once the tick starts feeding, unknown signals would induce the downregulation of ospa (permitting therefore the tick to detach from the tissue [63,64]), and the upregulation of ospc (involved in migration [65]) (Fig. 1). However, once the feeding process starts, several populations are found in the midgut, including spirochetes that express only OspA, OspC, both or neither [66]. These ndings indicate that cross-regulation of both genes is not likely to occur, since spirochetes expressing both ospa and ospc can be found during the blood meal. They also support the notion of OspA acting as a gut-anchor protein, since very few spirochetes were found in the salivary gland expressing the protein. These may represent escapees from the gut that keep expressing the lipoprotein, or spirochetes that upregulate the gene once they are in the salivary gland. Surprisingly, these results also show minimal expression of ospc, suggesting that the function of the protein is neither related to the migration of the bacterium to the salivary gland nor to the infection of the mammalian host [66]. Nevertheless, a burst of bacterial expression of ospc in the gut implies that the protein may facilitate or be necessary for the initial movement of the spirochetes across the hemocele [66] and consequently, antibodies that target the lipoprotein block the translocation of the microorganism from the gut to the salivary gland [65] Gene expression in the mammalian host After transmission to the mammalian host, the spirochetes remain in the skin for several days before they colonize di erent tissues, including the joints and the heart in the mouse, where they can induce in ammatory responses. Several genes have been reported to be upregulated during the part of the life of the bacterium that occurs in the mammal with variations in their time frame of expression. Other proteins with clear function in the mammalian host, such as the bronectin-binding protein (Bbk32 [67]) are upregulated during tick engorgement and retain their expression during infection of the mice [68,69]. Its function seems to be important for the preservation of

5 J. Anguita et al. / FEMS Microbiology Reviews 27 (2003) 493^ infectivity both in the vector and the vertebrate host, since antibodies that target Bbk32and Bbk50 (an antigen of unknown function with similar expression kinetics [70]) have protective capacity when mice are passively immunized [68]. OspC is one of the genes that was thought to be expressed readily in the mammalian host, although a careful examination of spirochetes as they enter the host revealed that its expression is limited to early infection (Fig. 1), decreasing over time [71], which agrees with a fading antibody response over time [72,73]. The analysis of other genes that are expressed during infection of the mammalian host shortly after transmission from the tick also indicated that the time frame required for the upregulation of these genes is longer than 3 days, the time period analyzed by Hodzic et al. recently [74]. These authors analyzed the expression of dbpa until 3 days post-infection by tick challenge showing that the mrna was absent, although the spirochetes were readily present in the skin of the mice [74]. As an interesting suggestion from these results, we can speculate that the upregulation of these genes may coincide in time with the initiation of the spirochetal dissemination in the host, and that dissemination may depend on this expression. OspA is generally not expressed in the murine host, even when the mice are infected with culture-grown spirochetes that express high levels of the protein [71] suggesting that it is positively regulated by a tick factor, negatively regulated by a murine component or both. In humans, anti- OspA antibody production has been reported in a small number of patients, suggestive of expression of the protein at later stages of the disease [75] (Fig. 1). Its role in infection is not clear, but a hypothesis proposes that crossreaction of certain epitopes of the protein with portions of LFA-1 are able to break tolerance to the self-antigen and provoke immune responses against the adhesion molecule [28]. The resistance of these patients to antibiotic treatment [27] suggests that the in ammatory condition, although triggered by the spirochetal protein, is maintained in the absence of infection. While this hypothesis is provocative, it has not yet been generally accepted [76]. Until the development of gene array techniques that permit the study of the expression of several hundred genes at a time, few techniques had explored with success changes that occur when B. burgdorferi migrate to di erent environments. One of such techniques consists of the use of immune sera from mice infected during di erent periods of time or hyperimmune sera from mice immunized with culture-grown spirochetes. The use of this differential screening technique allowed the recognition of several genes that are preferentially expressed in the mammalian host, as compared to the in vitro environment, or at di erent time points of infection. Thus, the use of sera at 2^4 weeks of infection would allow obtaining a representation of proteins present during the initial phases of infection [77]. Its comparison with later time point sera (several months of infection) would also permit the identi cation of genes that are upregulated late during infection of the mammal. The use of this technique, combined with the generation of non-pathogenic derivatives of spirochetes allowed the identi cation of several antigens that seem to be associated with infectivity and/or pathogenicity [70,77,78]. A useful model of mammalian-adapted B. burgdorferi was developed by Hurtenbach and collaborators [79] and used thereafter by other groups [80,81]. It is based on the growth of spirochetes in chambers located surgically in the peritoneum of rodents. The chamber allows the passage of solutes from the peritoneal cavity but retains the spirochetes inside, allowing their recovery for further study. This method minimizes problems associated with the lower number of spirochetes present on infected animal tissues. However, it does not represent gene expression of di erent tropisms, as shown, for example, for ErpT that is preferentially expressed in extra-cutaneous tissues [82]. The use of these chambers has allowed the description of genes that are di erentially expressed in the mammalian host, compared to in vitro growth conditions [81]. The analysis of B. burgdorferi gene expression through the use of the microarray technique has also provided cues on the genes expressed during di erent phases of infection of the mammal and the role of immune pressure on the changes that take place. Thus, a comparison between gene expression of spirochetes infecting immunode cient and immunocompetent mice, or SCID mice treated with immune sera and controls, has revealed that the majority of putative lipoproteins present in the spirochete are downregulated at a time when immune responses are active, strongly indicating that immune selection plays an important role in these changes [69]. Several lipoproteins remain expressed throughout the infection period in the mammalian host, however. These include DbpA and B, Bbk32 and several Erps. The continuous expression of these lipoproteins in the mammal suggests that they perform important functions for the maintenance of the infection. DbpA and DbpB bind decorin, a protein component of the extracellular matrix, an e ect that has been shown to enhance infectivity [83], and at least partially be associated with pathogenicity [78]. Bbk32is a bronectin-binding protein [67], which would also help the spirochete attach to extracellular matrices [84]. The continuous expression of these genes suggests that they may be required for the dissemination and localization of the spirochete within the mammalian host. Their function may also explain the tropism of the bacterium for certain tissues, in which the expression of these extracellular matrix proteins is enhanced. The study of gene expression during the life cycle of the spirochete using microarrays that represent the whole genome of B. burgdorferi B31 has revealed that the changes in gene expression that occur upon engorgement of the tick are of transient nature and tempered during mamma-

6 498 J. Anguita et al. / FEMS Microbiology Reviews 27 (2003) 493^504 Cassette DNA replicated DNA promiscuously recombination 17 bp DR 17 bp DR N-terminus IR1 VR1 IR2 VR2 IR3 VR3 IR4 VR4 IR5VR5 IR6 VR6 C- terminus Original VR Telomere DNA degraded Fig. 2. Structure and recombination at the vls locus of B. burgdorferi. The locus contains 15 silent cassettes of DNA located upstream of the vlse gene. The VlsE protein is composed of two IRs, the N- and C-termini, and six IRs alternated with six VRs. Two 17-bp direct repeats ank the variable domain. Recombination occurs through the replication of short segments of DNA in the silent cassettes and their recombination with the VRs promiscuously. The segment of DNA that is replaced in the VR is subsequently degraded. lian infection [85]. At least for spirochetes that are grown in chambers implanted in the peritoneum of rats, gene expression patterns do not vary greatly with time [85], which coincides with the ndings reported by Liang and collaborators regarding lipoprotein expression during infection of immunocompetent mice [69]. 4. Strategies to survive mammalian immune responses An important function that B. burgdorferi needs to accomplish during infection of the mammalian host is the evasion of immune responses. The ability of the spirochete to accomplish this task is underscored by experiments in which sera from infected mice are administered to mice on the same day or 4 days after challenge with the microorganism [78,86]. Although protective when co-administered with the bacteria, the antisera fail to prevent infection when given to the animals several days after infection. Furthermore, only mice infected with clonal in vitro grown spirochetes, but not by skin transplant or tick challenge, are protected with the antisera when given at the time of infection [86]. A possible interpretation of these ndings may include the emergence of spirochetal phenotypes that do not express antigens that are recognized by the antibodies present in the immune sera. However, microarray analysis of B. burgdorferi gene expression in SCID mice treated with immune sera provided an incomplete number of downregulated genes, compared to spirochetes present in immunocompetent mice. Thus, the adaptation that takes place during the time period that lags between protection and lack of protection by the immune sera is probably based on multiple factors and may include the participation of host molecules in uencing gene expression or recombination events. The mechanism by which B. burgdorferi is able to evade immune responses has been the object of intensive investigation and speculation, partly because insu cient knowledge on speci cs of gene expression and function of particular genes. Besides downregulation of antibody-targeted surface proteins, two other potential mechanisms allow the spirochetes to survive in their host in the presence of immune responses. These include a recombination system that allows the spirochete to change antigenic determinants of a surface immunogenic protein, VlsE, and the inhibition of complement-based phagocytosis by the expression of surface proteins that bind factor H Recombination as a means of immune escape One mechanism that is potentially essential for spirochetal immune escape is the recombination that takes place at the variable major protein-like sequence (vls) locus [87]. The vls system has been characterized in B. burgdorferi B31. It has been described in other strains of B. burgdorferi sensu stricto, B. garinii and B. afzelii [88,89] with di erent degrees of homology. The locus consists on a vls expression site (vlse) located near the right telomere of the linear plasmid lp28-1 and 15 silent cassettes upstream. vlse encodes a surface-exposed protein of 34 kda with three de ned domains: two invariable regions (IRs) at the amino- and carboxyl-termini and an internal variable domain, which is composed of six IRs and six variable regions (VRs) (Fig. 2) [90,91]. The crystal structure of the protein has been resolved [92]. It shows that only the VRs of the variable domain are surface exposed, which is in complete concordance with previous studies performed by several groups. These studies indicated that immunodominant IRs are not accessible to antibodies generated during infection [90]. Similarly, the C-terminal invariable domain has been shown to be immunodominant, although it does not show protective capabilities in vivo or bind to in vitro grown spirochetes, indicating too that these regions of the molecule are not surface exposed in the spirochete [91]. In contrast, the use of polyclonal antibodies raised against the whole polypeptide in proteinase K-treated and untreated spirochetes indicated surface exposure of the protein [87]. Together, these data suggest that only the VRs of the protein are surface exposed, and emphasize the importance of genetic variation at these

7 J. Anguita et al. / FEMS Microbiology Reviews 27 (2003) 493^ regions for immune evasion and persistence in the mammalian host. Evidence indicates that the mechanisms used by the spirochete to induce genetic variation include gene conversion [93] and point mutations [94]. Gene conversion mechanisms are unidirectional and consist of the copy of the silent cassette sequences and their exchange with the VRs of the variable domain of the gene (Fig. 2) [93]. Thus, the sequence of the silent cassettes remains unaltered during the process, in contrast with other Borrelia spp. recombination mechanisms, such as those performed by vmps of Borrelia hermsii [95] Erps and complement inhibition The complement cascade is an important part of the innate immune system as a barrier method to prevent the invasion of microorganisms. Complement activation can be elicited by the classical (antigen antibody-mediated), lectin and alternative (pathogen surface) pathways. The three pathways converge at the level of C3 convertase, a proteinase that cleaves complement component C3. As a result, the larger fragment, C3b, binds to the surface of the bacteria and induces internalization by phagocytes. C3b bound to C3 convertase also binds C5 with the formation of C5b forming an attack complex that can damage the bacterial cell surface. Several microorganisms have devised mechanisms to evade the action of the complement cascade, including Streptococcus pyogenes [96], Streptococcus pneumoniae [97], Neisseria gonorrhoeae [98,99], Neisseria meningitides [100], Echinococcus granulosus [101] and Yersinia enterocolitica [102]. All these microorganisms use the same approach to evade complement activity: binding of the plasma protein factor H and factor H-like protein 1 (FHLP-1), with subsequent promotion of factor I-mediated degradation of C3b [103]. Erps constitute a family of surface-exposed lipoproteins with di erent degrees of homology. Initially, the members of the family were identi ed as outer surface proteins E and F and their homologs [104,105]. The name Erp is more commonly used to refer to the family members present in strain B31, while the alternate names are still used for other strains, including N40. There are evidences that suggest that sequence variability among the members of the family has arisen from recombination events [106]. However, no recombination has been identi ed in the vertebrate during infection with B. burgdorferi [107] despite a recent report in the contrary [108]. Similarly to other bacterial species that evade complement action, B. burgdorferi is able to bind to its surface factor H and FHLP-1 through OspE/F and Erps [109,110]. The study from Stevenson and collaborators has also provided clues to understand the presence of several Erp homologs co-expressed by the spirochetes [110]. The a nities of individual Erps to factor H proteins from di erent animal species di er, thus allowing a single bacterium to confront the complement system of di erent species and ensuring their survival along their enzootic cycle [110]. 5. Signals that trigger gene expression changes and recombination The changes in gene expression that occur during the life cycle of B. burgdorferi are not completely understood at the regulation level. It seems intuitively obvious to assign changes in environmental conditions as one of the main factors that trigger these changes, although they have just started to be explained at the molecular level with detail [111]. When the tick starts feeding, several changes associated with the entrance of the blood meal occur. These include shifting in temperature, changes in ph and contact with mammalian host factors. Once in the mammalian host, the immune response, mainly in the form of antibody production, has a de nite role in the changes that occur in the spirochete, especially in lipoprotein expression, since these are the antigens that are exposed to the action of the immunoglobulins Environmental factors and cell density Changes that occur in the vector and the mammalian host are responsible for the regulation of a number of genes in B. burgdorferi. These include ospc, the erp family, the mlp gene family, rev, bba64, and others [112^116]. Among the environmental factors that are in uenced by the blood meal and that have been shown to a ect B. burgdorferi gene expression, temperature and ph are the best studied. The spirochete that resides in a tick midgut prior to feeding encounters an increase of the temperature and a decrease in ph that result from the ingestion of blood from the mammalian host. The most likely genes to be upregulated by these changes are those associated with the migration of the spirochete to the salivary gland and their transmission to the mammal, as well as the genes that encode antigens responsible for the initial encounter with the innate immune system (i.e. complement). OspC is a protein transiently expressed in the transition period across the hemocele [66]. Its upregulation by temperature increases has been extensively studied in vitro [56,57,117] and other studies have observed the same upregulation in its expression in the tick once the blood meal starts [66,114]. Although ospc is upregulated by increases in temperature in vitro, it is downregulated once the spirochetes are in the salivary gland, strongly suggesting that other signals in uence its expression. Indeed, the growth phase has been also described to a ect the presence of this lipoprotein [117]. It is possible, though, that the regulation of the protein is more complex and a ected by other factors besides environmental cues. The family of proteins known generically as Erps are

8 500 J. Anguita et al. / FEMS Microbiology Reviews 27 (2003) 493^504 also upregulated by temperature elevations and ph [56,115]. Thus, their level of expression is increased during the tick blood meal, which probably represents a preparation for the spirochete to encounter the complement system in the mammalian host. An interesting group of genes are those belonging to the paralogous family represented by p35 (bba64). Several members of this family of genes have been shown to be upregulated by decrease in ph, which could be associated with the mammalian host [58]. Bba64, Bba65 and Bba66 have been shown not to be expressed in the mammalian host by a non-pathogenic high passaged derivative of the clonal strain N40 (cn40 passage 75, named N40-75) [78], which associates signals arriving form the mammalian host to the ability of the spirochete to upregulate genes that are necessary for their adaptation/pathogenicity to the host Immune pressure Antibody responses against B. burgdorferi appear to be an important factor a ecting the expression of some of the surface lipoproteins. The comparison of lipoprotein gene expression in SCID mice treated with normal and immune mouse sera indicated that antibodies present in the sera induced the downregulation of several surface proteins [69]. However, the evaluation of these results in the context of those obtained comparing immunocompetent and immunode cient mice revealed some di erences in the degree of gene downregulation. The administration of immune sera to immunode cient mice failed to induce the same degree of gene downregulation than that obtained in immunocompetent mice. It is possible that the antibody titer against certain lipoproteins was below the threshold level to be active, or alternatively, other signals are required to attain this downregulation [69]. The ability of B. burgdorferi to evade antibody responses is also related to the pathogenic potential of the spirochete. Thus, high passaged spirochetes that are not pathogenic in immunocompetent mice are able to induce disease in SCID mice with no distinction in the degree of disease compared to the parental strain [78]. Furthermore, the number of spirochetes in di erent organs, including the joints and the hearts, appear to be highly a ected by the antibody response [78]. These data suggest that immune evasion, achieved through recombination or downregulation of surface proteins plays a major role in the survival of the bacterium in the mammalian host. The exact contribution of each phenomenon (recombination and gene downregulation) remains to be completely elucidated. An immunoscreening procedure using a cn40 genomic library and sera from mice infected with pathogenic and non-pathogenic isolates failed to yield any N speci c gene that could be responsible for the di erence [78]. A more detailed examination at the mrna level of spirochetes infecting their mammalian host should provide an answer Host factors A number of genes are not regulated by environmental factors or immune pressure, although they are di erentially expressed during the life cycle of the spirochete. Moreover, little is known about the signals that trigger recombination events at the vls locus. Alterations in temperature are not related to these changes [118], although they occur in vivo as soon as 4 days after experimental infection of mice, but not in vitro [118], indicating that the mammalian host provides the signal to recombine at this speci c locus. Other changes not associated with environmental modi cations or only partially associated include the regulation of OspC expression and the Erp P21 in B. burgdorferi N40. Experiments carried out with N40-75 demonstrated that this high passaged isolate is unable to induce as strong proin ammatory responses as its parental isolate, cn40. The lack of pathogenicity of this isolate, its lower ability to induce proin ammatory cytokines, its poor adaptation to immunocompetent mice and the lack of speci c antigens that could result in antibody-mediated killing led to the study of proin ammatory factors a ecting recombination events in the spirochete [119]. Indeed, the infection of mice that lack the proin ammatory cytokine IFNQ or its speci c receptor induced a lower degree of recombination at the vls locus, pointing to host signals related to in ammation as survival factors for the spirochete [119]. In this context, the induction of proin ammatory cytokines and their downstream targets would provide a favorable environment for B. burgdorferi to survive, even in the presence of strong antibody production. We still do not know the spatial organization of the surface of the bacterium. Nevertheless, for lack of a de nitive function for the Vls protein, besides its role in immune escape, this hypothesis would suggest that the proin ammatory cytokine production of B. burgdorferi is aimed at their perpetuation in the mammalian host, until the enzootic cycle can be completed Molecular mechanisms of gene regulation Three di erent sigma factors have been described in B. burgdorferi B31, including c70, c54 (RpoN) and RpoS [62]. The regulation of gene expression conditioned by environmental changes has been recently studied by Hubner and collaborators [111]. These authors described a regulatory system in which RpoN controls the expression of the sigma factor RpoS, which in turn is responsible for the induction of downstream genes including ospc and dbpa. This regulatory mechanism would then act as a switch in response to environmental condition changes, possibly one of the main regulatory factors for the spirochete in the transition from the arthropod to the mammalian host. Although this mechanism of gene regulation is not applicable to all the changes observed during the life cycle of

9 J. Anguita et al. / FEMS Microbiology Reviews 27 (2003) 493^ the spirochete, it opens new venues to study gene regulation at the molecular level. 6. Concluding remarks B. burgdorferi is a well-adapted pathogen and a model system to study gene regulation. Over the past several years intensive investigations have revealed details about their adaptation process in the tick and the mammal, their mechanisms of survival in both environments and the factors that regulate both events. Future work will determine the function of the genes that are di erentially regulated and will draw a clearer picture of the biology of the spirochete. With this knowledge, the control of the morbidity associated with the disease will be closer and the generation of second generation vaccines will allow the e cient prevention of infection. 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