SEARCH

SEARCH BY CITATION

Keywords:

  • Lyme disease;
  • Gene expression;
  • Vaccine;
  • Borrelia burgdorferi

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Lyme disease pathogenesis
  5. 3Changes in gene expression during the B. burgdorferi life cycle
  6. 4Strategies to survive mammalian immune responses
  7. 5Signals that trigger gene expression changes and recombination
  8. 6Concluding remarks
  9. Acknowledgements
  10. References

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.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Lyme disease pathogenesis
  5. 3Changes in gene expression during the B. burgdorferi life cycle
  6. 4Strategies to survive mammalian immune responses
  7. 5Signals that trigger gene expression changes and recombination
  8. 6Concluding remarks
  9. Acknowledgements
  10. References

Borrelia burgdorferi, the causative agent of Lyme disease, is an example of a microorganism that has adapted through evolution to survive in different 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 differentially 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 effective therapeutics and second generation vaccines targeting antigens expressed in the mammalian host. Recently, significant 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.

2Lyme disease pathogenesis

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Lyme disease pathogenesis
  5. 3Changes in gene expression during the B. burgdorferi life cycle
  6. 4Strategies to survive mammalian immune responses
  7. 5Signals that trigger gene expression changes and recombination
  8. 6Concluding remarks
  9. Acknowledgements
  10. References

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 Pacific 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 flu-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 affected 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 different 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 inflammation of the joints with a temporal and histological pattern that partially resembles human disease. The inflammation peaks at 2–4 weeks of infection, and resolves over a period of months [24,25]. It is characterized by a neutrophilic infiltrate 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 influence the pathogenesis of Lyme disease. The number of B. burgdorferi in the affected organs [29], spirochetal virulence [30–32], and the humoral and cellular responses arising during infection [33–40], affect 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 deficiencies that lead to impaired antibody production maintain a high spirochetal burden throughout the infection period and do not resolve the inflammatory 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 cell-mediated 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 different environmental conditions [54,55]. Changes that occur in vitro reflect the effect 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 differentially regulated during the life cycle of the bacterium. These studies have benefited 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 efforts of investigators in gene regulation and protein function of the numerous differentially regulated genes during the life cycle of the microorganism.

3Changes in gene expression during the B. burgdorferi life cycle

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Lyme disease pathogenesis
  5. 3Changes in gene expression during the B. burgdorferi life cycle
  6. 4Strategies to survive mammalian immune responses
  7. 5Signals that trigger gene expression changes and recombination
  8. 6Concluding remarks
  9. Acknowledgements
  10. References

It is well known that B. burgdorferi alters its gene expression during its life cycle. Intuitively, these changes are due to the different 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 differentially expressed genes, it seems obvious that proteins that bind tick or mammalian proteins are differentially expressed in the vector and the reservoir host, respectively (see Table 1). These include OspA, DbpA, Bbk32 and the gene family that encodes OspE/F-related proteins (Erps), among others.

Table 1.  Some of the B. burgdorferi genes differentially expressed in the tick and the mammalian host
  1. The function, when known and their mode of regulation are also given.

GeneFunctionRegulation
Tick
ospAanchor to the tick midgutexpressed in unfed ticks; downregulated during blood meal by unknown signals
ospCtarget of the spirochete to the salivary glandsregulated by temperature and pH
Mammalian host
dbpA (bba24)decorin-binding proteinexpressed in the mammalian host; upregulated by a decrease in pH
bbk32fibronectin-binding proteininduced upon engorgement of the tick
bbk50unknown functionsame kinetics as bbk32
ospE/Fcomplement inhibitionexpressed in the mammalian host
Bba64P35, of unknown functionregulated by pH, temperature and cell density
vlsimmune escape; highly variablerecombination induced by unknown factors; induced by inflammatory signals
mlpunknown function; multicopy lipoprotein genesinduced by body or nearly body temperature and some unknown mammalian factor(s)
revunknown functioninduced upon feeding of ticks

3.1Gene 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 definitive 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 efficient 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 specifically 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 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 findings 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.

image

Figure 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.

Download figure to PowerPoint

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].

3.2Gene expression in the mammalian host

After transmission to the mammalian host, the spirochetes remain in the skin for several days before they colonize different tissues, including the joints and the heart in the mouse, where they can induce inflammatory 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 fibronectin-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 infectivity both in the vector and the vertebrate host, since antibodies that target Bbk32 and 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 cross-reaction 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 inflammatory 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 different environments. One of such techniques consists of the use of immune sera from mice infected during different 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 different 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 identification 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 identification 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 different 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 differentially 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 different 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 immunodeficient 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 effect that has been shown to enhance infectivity [83], and at least partially be associated with pathogenicity [78]. Bbk32 is a fibronectin-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 mammalian 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 findings reported by Liang and collaborators regarding lipoprotein expression during infection of immunocompetent mice [69].

4Strategies to survive mammalian immune responses

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Lyme disease pathogenesis
  5. 3Changes in gene expression during the B. burgdorferi life cycle
  6. 4Strategies to survive mammalian immune responses
  7. 5Signals that trigger gene expression changes and recombination
  8. 6Concluding remarks
  9. Acknowledgements
  10. References

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 findings 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 influencing 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 insufficient knowledge on specifics 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.

4.1Recombination 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 different 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 defined 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 regions for immune evasion and persistence in the mammalian host.

image

Figure 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 flank 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.

Download figure to PowerPoint

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].

4.2Erps 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 different degrees of homology. Initially, the members of the family were identified 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 identified 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 affinities of individual Erps to factor H proteins from different animal species differ, thus allowing a single bacterium to confront the complement system of different species and ensuring their survival along their enzootic cycle [110].

5Signals that trigger gene expression changes and recombination

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Lyme disease pathogenesis
  5. 3Changes in gene expression during the B. burgdorferi life cycle
  6. 4Strategies to survive mammalian immune responses
  7. 5Signals that trigger gene expression changes and recombination
  8. 6Concluding remarks
  9. Acknowledgements
  10. References

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 definite 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.

5.1Environmental 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 influenced by the blood meal and that have been shown to affect 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 influence its expression. Indeed, the growth phase has been also described to affect the presence of this lipoprotein [117]. It is possible, though, that the regulation of the protein is more complex and affected by other factors besides environmental cues.

The family of proteins known generically as Erps are 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.

5.2Immune pressure

Antibody responses against B. burgdorferi appear to be an important factor affecting 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 immunodeficient mice revealed some differences in the degree of gene downregulation. The administration of immune sera to immunodeficient 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 different organs, including the joints and the hearts, appear to be highly affected 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 N40-75-specific gene that could be responsible for the difference [78]. A more detailed examination at the mRNA level of spirochetes infecting their mammalian host should provide an answer.

5.3Host factors

A number of genes are not regulated by environmental factors or immune pressure, although they are differentially 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 specific locus. Other changes not associated with environmental modifications 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 proinflammatory responses as its parental isolate, cN40. The lack of pathogenicity of this isolate, its lower ability to induce proinflammatory cytokines, its poor adaptation to immunocompetent mice and the lack of specific antigens that could result in antibody-mediated killing led to the study of proinflammatory factors affecting recombination events in the spirochete [119]. Indeed, the infection of mice that lack the proinflammatory cytokine IFNγ or its specific receptor induced a lower degree of recombination at the vls locus, pointing to host signals related to inflammation as survival factors for the spirochete [119]. In this context, the induction of proinflammatory 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 definitive function for the Vls protein, besides its role in immune escape, this hypothesis would suggest that the proinflammatory cytokine production of B. burgdorferi is aimed at their perpetuation in the mammalian host, until the enzootic cycle can be completed.

5.4Molecular mechanisms of gene regulation

Three different sigma factors have been described in B. burgdorferi B31, including σ70, σ54 (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 the spirochete, it opens new venues to study gene regulation at the molecular level.

6Concluding remarks

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Lyme disease pathogenesis
  5. 3Changes in gene expression during the B. burgdorferi life cycle
  6. 4Strategies to survive mammalian immune responses
  7. 5Signals that trigger gene expression changes and recombination
  8. 6Concluding remarks
  9. Acknowledgements
  10. References

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 differentially 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 efficient prevention of infection. The difficulties associated with the study of this microorganism have forced researchers to develop novel strategies to overcome the limitations inherent to the system, which will undoubtedly be at least partially applicable to other microorganisms that are difficult to study, including other spirochetes that cause disease in humans.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Lyme disease pathogenesis
  5. 3Changes in gene expression during the B. burgdorferi life cycle
  6. 4Strategies to survive mammalian immune responses
  7. 5Signals that trigger gene expression changes and recombination
  8. 6Concluding remarks
  9. Acknowledgements
  10. References

Supported by grants from the National Institutes of Health (E.F.) and the American Heart Association (J.A.). E.F. is the recipient of a Clinical-Scientist Award in Translational Research from the Burroughs Wellcome Fund.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Lyme disease pathogenesis
  5. 3Changes in gene expression during the B. burgdorferi life cycle
  6. 4Strategies to survive mammalian immune responses
  7. 5Signals that trigger gene expression changes and recombination
  8. 6Concluding remarks
  9. Acknowledgements
  10. References
  • [1]
    Barbour, A.G., Fish, D. (1993) The biological and social phenomenon of Lyme disease. Science 260, 16101616.
  • [2]
    Schulze, T.L., Bowen, G.S., Lakat, M.F., Parkin, W.E., Shisler, J.K. (1985) The role of adult Ixodes dammini (Acari: Ixodidae) in the transmission of Lyme disease in New Jersey, USA. J. Med. Entomol. 22, 8893.
  • [3]
    Lane, R.S., Piesman, J., Burgdorfer, W. (1991) Lyme borreliosis: relation of its causative agent to its vectors and hosts in North America and Europe. Annu. Rev. Entomol. 36, 587609.
  • [4]
    Anderson, J.F. (1989) Epizootiology of Borrelia in Ixodes tick vectors and reservoir hosts. Rev. Infect. Dis. 11, S14511459.
  • [5]
    Anderson, J.F., Magnarelli, L.A., Stafford, K.C. (1990) Bird-feeding ticks transstadially transmit Borrelia burgdorferi that infect Syrian hamsters. J. Wildl. Dis. 26, 110.
  • [6]
    Steere, A.C., Bartenhagen, N.H., Craft, J.E., Hutchinson, G.J., Newman, J.H., Rahn, D.W., Sigal, L.H., Spieler, P.H., Stenn, K.S., Malawista, S.E. (1983) The early clinical manifestations of Lyme disease. Ann. Intern. Med. 99, 7682.
  • [7]
    Steere, A.C., Batsford, W.P., Weinberg, M., Alexander, J., Berger, H.J., Wolfson, S., Malawista, S.E. (1980) Lyme carditis: cardiac abnormalities of Lyme disease. Ann. Intern. Med. 93, 816.
  • [8]
    Steere, A.C. Lyme disease. N. Engl. J. Med. 321, 1989. 586
  • [9]
    Paparone, P.W. (1997) Cardiovascular manifestations of Lyme disease. J. Am. Osteopath. Assoc. 97, 156161.
  • [10]
    Haass, A., Treib, J. (1996) Neurologic manifestation and classification of borreliosis. Infection 24, 467469.
  • [11]
    Koch, F., Stanzl, U., Jennewein, P., Janke, K., Heufler, C., Kampgen, E., Romani, N., Schuler, G. (1996) High level IL-12 production by murine dendritic cells: upregulation via MHC class II and CD40 molecules and downregulation by IL-4 and IL-10. J. Exp. Med. 184, 741746.
  • [12]
    Nadelman, R.B., Nowakowski, J., Forseter, G., Goldberg, N.S., Bittker, S., Cooper, D., Aguero-Rosenfeld, M., Wormser, G.P. (1996) The clinical spectrum of early Lyme borreliosis in patients with culture-confirmed erythema migrans. Am. J. Med. 100, 502508.
  • [13]
    Kindstrand, E. (1995) Lyme borreliosis and cranial neuropathy. J. Neurol. 242, 658663.
  • [14]
    Wang, W.Z., Fredrikson, S., Sun, J.B., Link, H. (1995) Lyme neuroborreliosis: evidence for persistent up-regulation of Borrelia burgdorferi-reactive cells secreting interferon-gamma. Scand. J. Immunol. 42, 694700.
  • [15]
    Evans, J. (1995) Lyme disease. Curr. Opin. Rheumatol. 7, 322328.
  • [16]
    Steere, A.C. (1995) Musculoskeletal manifestations of Lyme disease. Am. J. Med. 98, 44S–48S; discussion 48S–51S.
  • [17]
    Pachner, A.R., Delaney, E., O'Neill, T. (1995) Neuroborreliosis in the nonhuman primate: Borrelia burgdorferi persists in the central nervous system. Ann. Neurol. 38, 667669.
  • [18]
    Nadelman, R.B. and Wormser, G.P. (1995) Erythema migrans and early Lyme disease. Am. J. Med. 98, 15S–23S; discussion 23S–24S.
  • [19]
    Fallon, B.A., Nields, J.A. (1994) Lyme disease: a neuropsychiatric illness. Am. J. Psychiatry 151, 15711583.
  • [20]
    Logigian, E.L., Kaplan, R.F., Steere, A.C. (1990) Chronic neurologic manifestations of Lyme disease. N. Engl. J. Med. 323, 14381444.
  • [21]
    Steere, A.C. (2001) Lyme disease. N. Engl. J. Med. 345, 115125.
  • [22]
    Evans, J. (1996) Lyme disease. Curr. Opin. Rheumatol. 8, 327333.
  • [23]
    Pachner, A.R., Duray, P., Steere, A.C. (1989) Central nervous system manifestations of Lyme disease. Arch. Neurol. 46, 790795.
  • [24]
    Barthold, S.W., De Souza, M.S., Janotka, J.L., Smith, A.L., Persing, D.H. (1993) Chronic Lyme borreliosis in the laboratory mouse. Am. J. Pathol. 143, 959971.
  • [25]
    Schaible, U.E., Wallich, R., Kramer, M.D., Museteanu, C., Simon, M.M. (1991) A mouse model for Borrelia burgdorferi infection: pathogenesis, immune response and protection. Behring Inst. Mitt. 88, 5967.
  • [26]
    Steere, A.C., Dwyer, E., Winchester, R. (1990) Association of chronic Lyme arthritis with HLA-DR4 and HLA-DR2 alleles. N. Engl. J. Med. 323, 219223.
  • [27]
    Kalish, R.A., Leong, J.M., Steere, A.C. (1993) Association of treatment-resistant chronic Lyme arthritis with HLA-DR4 and antibody reactivity to OspA and OspB of Borrelia burgdorferi. Infect. Immun. 61, 27742779.
  • [28]
    Gross, D.M., Forsthuber, T., Tary-Lehmann, M., Etling, C., Ito, K., Nagy, Z.A., Field, J.A., Steere, A.C., Huber, B.T. (1998) Identification of LFA-1 as a candidate autoantigen in treatment-resistant Lyme arthritis. Science 281, 703706.
  • [29]
    Yang, L., Weis, J.H., Eichwald, E., Kolbert, C.P., Persing, D.H., Weis, J.J. (1994) Heritable susceptibility to severe Borrelia burgdorferi-induced arthritis is dominant and is associated with persistence of large numbers of spirochetes in tissues. Infect. Immun. 62, 492500.
  • [30]
    Carroll, J.A., Dorward, D.W., Gherardini, F.C. (1996) Identification of a transferrin-binding protein from Borrelia burgdorferi. Infect. Immun. 64, 29112916.
  • [31]
    Hughes, C.A., Engstrom, S.M., Coleman, L.A., Kodner, C.B., Johnson, R.C. (1993) Protective immunity is induced by a Borrelia burgdorferi mutant that lacks OspA and OspB. Infect. Immun. 61, 51155122.
  • [32]
    Norris, S.J., Howell, J.K., Garza, S.A., Ferdows, M.S., Barbour, A.G. (1995) High- and low-infectivity phenotypes of clonal populations of in vitro-cultured Borrelia burgdorferi. Infect. Immun. 63, 22062212.
  • [33]
    Anguita, J., Persing, D.H., Rincón, M., Barthold, S.W., Fikrig, E. (1996) Effect of anti-interleukin 12 treatment on murine Lyme borreliosis. J. Clin. Invest. 97, 10281034.
  • [34]
    Brunet, L.R., Spielman, A., Telford, S.R. (1995) Short report: density of Lyme disease spirochetes within deer ticks collected from zoonotic sites. Am. J. Trop. Med. Hyg. 53, 300302.
  • [35]
    Keane-Myers, A., Nickell, S.P. (1995) Role of IL-4 and IFN-gamma in modulation of immunity to Borrelia burgdorferi in mice. J. Immunol. 155, 20202028.
  • [36]
    Keane-Myers, A., Nickell, S.P. (1995) T cell subset-dependent modulation of immunity to Borrelia burgdorferi in mice. J. Immunol. 154, 17701776.
  • [37]
    Lengl-Janssen, B., Strauss, A.F., Steere, A.C., Kamradt, T. (1994) The T helper cell response in Lyme arthritis: differential recognition of Borrelia burgdorferi outer surface protein A in patients with treatment-resistant or treatment-responsive Lyme arthritis. J. Exp. Med. 180, 20692078.
  • [38]
    Matyniak, J.E., Reiner, S.L. (1995) T helper phenotype and genetic susceptibility in experimental Lyme disease. J. Exp. Med. 181, 12511254.
  • [39]
    Szczepanski, A., Benach, J.L. (1991) Lyme borreliosis: host responses to Borrelia burgdorferi. Microbiol. Rev. 55, 2134.
  • [40]
    Yang, L., Ma, Y., Schoenfeld, R., Griffiths, M., Eichwald, E., Araneo, B., Weis, J.J. (1992) Evidence for B-lymphocyte mitogen activity in Borrelia burgdorferi-infected mice. Infect. Immun. 60, 30333041.
  • [41]
    Barthold, S.W., De Souza, M., Feng, S. (1996) Serum-mediated resolution of Lyme arthritis in mice. Lab. Invest. 74, 5767.
  • [42]
    Fikrig, E., Barthold, S.W., Chen, M., Chang, C.H., Flavell, R.A. (1997) Protective antibodies develop, and murine Lyme arthritis regresses, in the absence of MHC class II and CD4+ T cells. J. Immunol. 159, 56825686.
  • [43]
    Simon, M.M., Gern, L., Hauser, P., Zhong, W., Nielsen, P.J., Kramer, M.D., Brenner, C., Wallich, R. (1996) Protective immunization with plasmid DNA containing the outer surface lipoprotein A gene of Borrelia burgdorferi is independent of an eukaryotic promoter. Eur. J. Immunol. 26, 28312840.
  • [44]
    Padilla, M.L., Callister, S.M., Schell, R.F., Bryant, G.L., Jobe, D.A., Lovrich, S.D., DuChateau, B.K., Jensen, J.R. (1996) Characterization of the protective borreliacidal antibody response in humans and hamsters after vaccination with a Borrelia burgdorferi outer surface protein A vaccine. J. Infect. Dis. 174, 739746.
  • [45]
    Fikrig, E., Barthold, S.W., Chen, M., Grewal, I.S., Craft, J., Flavell, R.A. (1996) Protective antibodies in murine Lyme disease arise independently of CD40 ligand. J. Immunol. 157, 13.
  • [46]
    Garcia-Monco, J.C., Seidman, R.J., Benach, J.L. (1995) Experimental immunization with Borrelia burgdorferi induces development of antibodies to gangliosides. Infect. Immun. 63, 41304137.
  • [47]
    Ma, J., Gingrich-Baker, C., Franchi, P.M., Bulger, P., Coughlin, R.T. (1995) Molecular analysis of neutralizing epitopes on outer surface proteins A and B of Borrelia burgdorferi. Infect. Immun. 63, 22212227.
  • [48]
    Aydintug, M.K., Gu, Y., Philipp, M.T. (1994) Borrelia burgdorferi antigens that are targeted by antibody-dependent, complement-mediated killing in the rhesus monkey. Infect. Immun. 62, 49294937.
  • [49]
    Fikrig, E., Bockenstedt, L.K., Barthold, S.W., Chen, M., Tao, H., Ali-Salaam, P., Telford, S.R., Flavell, R.A. (1994) Sera from patients with chronic Lyme disease protect mice from Lyme borreliosis. J. Infect. Dis. 169, 568574.
  • [50]
    Barthold, S.W., Bockenstedt, L.K. (1993) Passive immunizing activity of sera from mice infected with Borrelia burgdorferi. Infect. Immun. 61, 46964702.
  • [51]
    Callister, S.M., Schell, R.F., Case, K.L., Lovrich, S.D., Day, S.P. (1993) Characterization of the borreliacidal antibody response to Borrelia burgdorferi in humans: a serodiagnostic test. J. Infect. Dis. 167, 158164.
  • [52]
    Barthold, S.W., Feng, S., Bockenstedt, L.K., Fikrig, E., Feen, K. (1997) Protective and arthritis-resolving activity in sera of mice infected with Borrelia burgdorferi. Clin. Infect. Dis. 25 (Suppl. 1), 917.
  • [53]
    Schaible, U.E., Wallich, R., Kramer, M.D., Nerz, G., Stehle, T., Museteanu, C., Simon, M.M. (1994) Protection against Borrelia burgdorferi infection in SCID mice is conferred by presensitized spleen cells and partially by B but not T cells alone. Int. Immunol. 6, 671681.
  • [54]
    Barbour, A.G. (1984) Isolation and cultivation of Lyme disease spirochetes. Yale J. Biol. Med. 57, 521525.
  • [55]
    Pollack, R.J., Telford, S.R., Spielman, A. (1993) Standardization of medium for culturing Lyme disease spirochetes. J. Clin. Microbiol. 31, 12511255.
  • [56]
    Babb, K., El-Hage, N., Miller, J.C., Carroll, J.A., Stevenson, B. (2001) Distinct regulatory pathways control expression of Borrelia burgdorferi infection-associated OspC and Erp surface proteins. Infect. Immun. 69, 41464153.
  • [57]
    Stevenson, B., Schwan, T.G., Rosa, P.A. (1995) Temperature-related differential expression of antigens in the Lyme disease spirochete, Borrelia burgdorferi. Infect. Immun. 63, 45354539.
  • [58]
    Carroll, J.A., Cordova, R.M., Garon, C.F. (2000) Identification of 11 pH-regulated genes in Borrelia burgdorferi localizing to linear plasmids. Infect. Immun. 68, 66776684.
  • [59]
    Margolis, N., Rosa, P.A. (1993) Regulation of expression of major outer surface proteins in Borrelia burgdorferi. Infect. Immun. 61, 22072210.
  • [60]
    Indest, K.J., Ramamoorthy, R., Sole, M., Gilmore, R.D., Johnson, B.J., Philipp, M.T. (1997) Cell-density-dependent expression of Borrelia burgdorferi lipoproteins in vitro. Infect. Immun. 65, 11651171.
  • [61]
    Ramamoorthy, R., Philipp, M.T. (1998) Differential expression of Borrelia burgdorferi proteins during growth in vitro. Infect. Immun. 66, 51195124.
  • [62]
    Fraser, C.M., Casjens, S., Huang, W.M., Sutton, G.G. (1997) Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi. Nature 390, 580586.
  • [63]
    Pal, U., De Silva, A.M., Montgomery, R.R., Fish, D., Anguita, J., Anderson, J.F., Lobet, Y., Fikrig, E. (2000) Attachment of Borrelia burgdorferi within Ixodes scapularis mediated by Outer surface protein, A. J. Clin. Invest. 106, 561569.
  • [64]
    Pal, U., Montgomery, R.R., Lusitani, D., Voet, P., Weynants, V., Malawista, S.E., Lobet, Y., Fikrig, E. (2001) Inhibition of Borrelia burgdorferi–tick interactions in vivo by Outer surface protein A antibody. J. Immunol. 166, 73987403.
  • [65]
    Gilmore, R.D., Piesman, J. (2000) Inhibition of Borrelia burgdorferi migration from the midgut to the salivary glands following feeding by ticks on OspC-immunized mice. Infect. Immun. 68, 411414.
  • [66]
    Ohnishi, J., Piesman, J., De Silva, A.M. (2001) Antigenic and genetic heterogeneity of Borrelia burgdorferi populations transmitted by ticks. Proc. Natl. Acad. Sci. USA 98, 670675.
  • [67]
    Probert, W.S., Johnson, B.J. (1998) Identification of a 47 kDa fibronectin-binding protein expressed by Borrelia burgdorferi isolate B31. Mol. Microbiol. 30, 10031015.
  • [68]
    Fikrig, E., Feng, W., Barthold, S.W., Telford, S.R., Flavell, R.A. (2000) Arthropod- and host-specific Borrelia burgdorferi bbk32 expression and the inhibition of spirochete transmission. J. Immunol. 164, 53445351.
  • [69]
    Liang, F.T., Nelson, F.K., Fikrig, E. (2002) Molecular adaptation of Borrelia burgdorferi in the murine host. J. Exp. Med. 196, 275280.
  • [70]
    Fikrig, E., Barthold, S.W., Sun, W., Feng, W., Telford, S.R., Flavell, R.A. (1997) Borrelia burgdorferi P35 and P37 proteins, expressed in vivo, elicit protective immunity. Immunity 6, 531539.
  • [71]
    Montgomery, R.R., Malawista, S.E., Feen, K.J., Bockenstedt, L.K. (1996) Direct demonstration of antigenic substitution of Borrelia burgdorferi ex vivo: exploration of the paradox of the early immune response to outer surface proteins A and C in Lyme disease. J. Exp. Med. 183, 261269.
  • [72]
    Schwan, T.G. (1996) Ticks and Borrelia: model systems for investigating pathogen–arthropod interactions. Infect. Agents Dis. 5, 167181.
  • [73]
    Schwan, T.G., Simpson, W.J. (1991) Factors influencing the antigenic reactivity of Borrelia burgdorferi, the Lyme disease spirochete. Scand. J. Infect. Dis. Suppl. 77, 94101.
  • [74]
    Hodzic, E., Feng, S., Freet, K.J., Borjesson, D.L., Barthold, S.W. (2002) Borrelia burgdorferi population kinetics and selected gene expression at the host–vector interface. Infect. Immun. 70, 33823388.
  • [75]
    Akin, E., McHugh, G.L., Flavell, R.A., Fikrig, E., Steere, A.C. (1999) The immunoglobulin (IgG) antibody response to OspA and OspB correlates with severe and prolonged Lyme arthritis and the IgG response to P35 correlates with mild and brief arthritis. Infect. Immun. 67, 173181.
  • [76]
    Benoist, C., Mathis, D. (2001) Autoimmunity provoked by infection: how good is the case for T cell epitope mimicry. Nat. Immunol. 2, 797801.
  • [77]
    Suk, K., Das, S., Sun, W., Jwang, B., Barthold, S.W., Flavell, R.A., Fikrig, E. (1995) Borrelia burgdorferi genes selectively expressed in the infected host. Proc. Natl. Acad. Sci. USA 92, 42694273.
  • [78]
    Anguita, J., Samanta, S., Revilla, B., Suk, K., Das, S., Barthold, S.W., Fikrig, E. (2000) Borrelia burgdorferi gene expression in vivo and spirochete pathogenicity. Infect. Immun. 68, 12221230.
  • [79]
    Hurtenbach, U., Museteanu, C., Gasser, J., Schaible, U.E., Simon, M.M. (1995) Studies on early events of Borrelia burgdorferi-induced cytokine production in immunodeficient SCID mice by using a tissue chamber model for acute inflammation. Int. J. Exp. Pathol. 76, 111123.
  • [80]
    Jonsson, M., Elmros, T., Bergstrom, S. (1995) Subcutaneous implanted chambers in different mouse strains as an animal model to study genetic stability during infection with Lyme disease Borrelia. Microb. Pathog. 18, 109114.
  • [81]
    Akins, D.R., Bourell, K.W., Caimano, M.J., Norgard, M.V., Radolf, J.D. (1998) A new animal model for studying Lyme disease spirochetes in a mammalian host-adapted state. J. Clin. Invest. 101, 22402250.
  • [82]
    Fikrig, E., Chen, M., Barthold, S.W., Anguita, J., Feng, W., Telford, S.R., Flavell, R.A. (1999) Borrelia burgdorferi erpT expression in the arthropod vector and murine host. Mol. Microbiol. 31, 281290.
  • [83]
    Brown, E.L., Wooten, R.M., Johnson, B.J., Iozzo, R.V., Smith, A., Dolan, M.C., Guo, B.P., Weis, J.J., Hook, M. (2001) Resistance to Lyme disease in decorin-deficient mice. J. Clin. Invest. 107, 845852.
  • [84]
    Grab, D.J., Givens, C., Kennedy, R. (1998) Fibronectin-binding activity in Borrelia burgdorferi. Biochim. Biophys. Acta 1407, 135145.
  • [85]
    Revel, A.T., Talaat, A.M., Norgard, M.V. (2002) DNA microarray analysis of differential gene expression in Borrelia burgdorferi, the Lyme disease spirochete. Proc. Natl. Acad. Sci. USA 99, 15621567.
  • [86]
    De Silva, A.M., Fikrig, E., Hodzic, E., Kantor, F.S. S.R. Telford III Barthold, S.W. (1998) Immune evasion by tickborne and host-adapted Borrelia burgdorferi. J. Infect. Dis. 177, 395400.
  • [87]
    Zhang, J.R., Hardham, J.M., Barbour, A.G., Norris, S.J. (1997) Antigenic variation in Lyme disease borreliae by promiscuous recombination of VMP-like sequence cassettes. Cell 89, 275285.
  • [88]
    Iyer, R., Hardham, J.M., Wormser, G.P., Schwartz, I., Norris, S.J. (2000) Conservation and heterogeneity of vlsE among human and tick isolates of Borrelia burgdorferi. Infect. Immun. 68, 17141718.
  • [89]
    Wang, G., Van Dam, A.P., Dankert, J. (2001) Analysis of a VMP-like sequence (vls) locus in Borrelia garinii and Vls homologues among four Borrelia burgdorferi sensu lato species. FEMS Microbiol. Lett. 199, 3945.
  • [90]
    Liang, F.T., Nowling, J.M., Philipp, M.T. (2000) Cryptic and exposed invariable regions of VlsE, the variable surface antigen of Borrelia burgdorferi sl. J. Bacteriol. 182, 35973601.
  • [91]
    Liang, F.T., Jacobs, M.B., Philipp, M.T. (2001) C-terminal invariable domain of VlsE may not serve as target for protective immune response against Borrelia burgdorferi. Infect. Immun. 69, 13371343.
  • [92]
    Eicken, C., Sharma, V., Klabunde, T., Lawrenz, M.B., Hardham, J.M., Norris, S.J., Sacchettini, J.C. (2002) Crystal structure of Lyme disease variable surface antigen VlsE of Borrelia burgdorferi. J. Biol. Chem. 277, 2169121696.
  • [93]
    Zhang, J.R., Norris, S.J. (1998) Genetic variation of the Borrelia burgdorferi gene vlsE involves cassette-specific, segmental gene conversion. Infect. Immun. 66, 36983704.
  • [94]
    Sung, S.Y., McDowell, J.V., Marconi, R.T. (2001) Evidence for the contribution of point mutations to vlsE variation and for apparent constraints on the net accumulation of sequence changes in vlsE during infection with Lyme disease spirochetes. J. Bacteriol. 183, 58555861.
  • [95]
    Restrepo, B.I., Barbour, A.G. (1994) Antigen diversity in the bacterium B. hermsii through ‘somatic’ mutations in rearranged vmp genes. Cell 78, 867876.
  • [96]
    Kotarsky, H., Hellwage, J., Johnsson, E., Skerka, C., Svensson, H.G., Lindahl, G., Sjobring, U., Zipfel, P.F. (1998) Identification of a domain in human factor H and factor H-like protein-1 required for the interaction with streptococcal M proteins. J. Immunol. 160, 33493354.
  • [97]
    Neeleman, C., Geelen, S.P., Aerts, P.C., Daha, M.R., Mollnes, T.E., Roord, J.J., Posthuma, G., Van Dijk, H., Fleer, A. (1999) Resistance to both complement activation and phagocytosis in type 3 pneumococci is mediated by the binding of complement regulatory protein factor, H. Infect. Immun. 67, 45174524.
  • [98]
    Ram, S., Sharma, A.K., Simpson, S.D., Gulati, S., McQuillen, D.P., Pangburn, M.K., Rice, P.A. (1998) A novel sialic acid binding site on factor H mediates serum resistance of sialylated Neisseria gonorrhoeae. J. Exp. Med. 187, 743752.
  • [99]
    Ram, S., McQuillen, D.P., Gulati, S., Elkins, C., Pangburn, M.K., Rice, P.A. (1998) Binding of complement factor H to loop 5 of porin protein 1A: a molecular mechanism of serum resistance of nonsialylated Neisseria gonorrhoeae. J. Exp. Med. 188, 671680.
  • [100]
    Ram, S., Mackinnon, F.G., Gulati, S., McQuillen, D.P., Vogel, U., Frosch, M., Elkins, C., Guttormsen, H.K., Wetzler, L.M., Oppermann, M., Pangburn, M.K., Rice, P.A. (1999) The contrasting mechanisms of serum resistance of Neisseria gonorrhoeae and group B Neisseria meningitidis. Mol. Immunol. 36, 915928.
  • [101]
    Diaz, A., Ferreira, A., Sim, R.B. (1997) Complement evasion by Echinococcus granulosus: sequestration of host factor H in the hydatid cyst wall. J. Immunol. 158, 37793786.
  • [102]
    China, B., Sory, M.P., N'Guyen, B.T., De Bruyere, M., Cornelis, G.R. (1993) Role of the YadA protein in prevention of opsonization of Yersinia enterocolitica by C3b molecules. Infect. Immun. 61, 31293136.
  • [103]
    Kraiczy, P., Skerka, C., Kirschfink, M., Zipfel, P.F., Brade, V. (2001) Mechanism of complement resistance of pathogenic Borrelia burgdorferi isolates. Int. Immunopharmacol. 1, 393401.
  • [104]
    Lam, T.T., Nguyen, T.P., Montgomery, R.R., Kantor, F.S., Fikrig, E., Flavell, R.A. (1994) Outer surface proteins E and F of Borrelia burgdorferi, the agent of Lyme disease. Infect. Immun. 62, 290298.
  • [105]
    Stevenson, B., Tilly, K., Rosa, P.A. (1996) A family of genes located on four separate 32-kilobase circular plasmids in Borrelia burgdorferi B31. J. Bacteriol. 178, 35083516.
  • [106]
    Stevenson, B., Casjens, S., Rosa, P. (1998) Evidence of past recombination events among the genes encoding the Erp antigens of Borrelia burgdorferi. Microbiology 144 (Pt 7), 18691879.
  • [107]
    Stevenson, B. (2002) Borrelia burgdorferi erp (ospE-Related) gene sequences remain stable during mammalian infection. Infect. Immun. 70, 53075311.
  • [108]
    Sung, S.Y., McDowell, J.V., Carlyon, J.A., Marconi, R.T. (2000) Mutation and recombination in the upstream homology box-flanked ospE-related genes of the Lyme disease spirochetes result in the development of new antigenic variants during infection. Infect. Immun. 68, 13191327.
  • [109]
    Hellwage, J., Meri, T., Heikkila, T., Alitalo, A., Panelius, J., Lahdenne, P., Seppala, I.J., Meri, S. (2001) The complement regulator factor H binds to the surface protein OspE of Borrelia burgdorferi. J. Biol. Chem. 276, 84278435.
  • [110]
    Stevenson, B., El-Hage, N., Hines, M.A., Miller, J.C., Babb, K. (2002) Differential binding of host complement inhibitor factor H by Borrelia burgdorferi Erp surface proteins: a possible mechanism underlying the expansive host range of Lyme disease spirochetes. Infect. Immun. 70, 491497.
  • [111]
    Hubner, A., Yang, X., Nolen, D.M., Popova, T.G., Cabello, F.C., Norgard, M.V. (2001) Expression of Borrelia burgdorferi OspC and DbpA is controlled by a RpoN–RpoS regulatory pathway. Proc. Natl. Acad. Sci. USA 98, 1272412729.
  • [112]
    Cassatt, D.R., Patel, N.K., Ulbrandt, N.D., Hanson, M.S. (1998) DbpA, but not OspA, is expressed by Borrelia burgdorferi during spirochetemia and is a target for protective antibodies. Infect. Immun. 66, 53795387.
  • [113]
    Hagman, K.E., Lahdenne, P., Popova, T.G., Porcella, S.F., Akins, D.R., Radolf, J.D., Norgard, M.V. (1998) Decorin-binding protein of Borrelia burgdorferi is encoded within a two-gene operon and is protective in the murine model of Lyme borreliosis. Infect. Immun. 66, 26742683.
  • [114]
    Schwan, T.G., Piesman, J., Golde, W.T., Dolan, M.C., Rosa, P.A. (1995) Induction of an outer surface protein on Borrelia burgdorferi during tick feeding. Proc. Natl. Acad. Sci. USA 92, 29092913.
  • [115]
    Stevenson, B., Bono, J.L., Schwan, T.G., Rosa, P. (1998) Borrelia burgdorferi Erp proteins are immunogenic in mammals infected by tick bite, and their synthesis is inducible in cultured bacteria. Infect. Immun. 66, 26482654.
  • [116]
    Yang, X., Popova, T.G., Hagman, K.E., Wikel, S.K., Schoeler, G.B., Caimano, M.J., Radolf, J.D., Norgard, M.V. (1999) Identification, characterization, and expression of three new members of the Borrelia burgdorferi Mlp (2.9) lipoprotein gene family. Infect. Immun. 67, 60086018.
  • [117]
    Obonyo, M., Munderloh, U.G., Fingerle, V., Wilske, B., Kurtti, T.J. (1999) Borrelia burgdorferi in tick cell culture modulates expression of Outer surface proteins A and C in response to temperature. J. Clin. Microbiol. 37, 21372141.
  • [118]
    Zhang, J.R., Norris, S.J. (1998) Kinetics and in vivo induction of genetic variation of vlsE in Borrelia burgdorferi. Infect. Immun. 66, 36893697.
  • [119]
    Anguita, J., Thomas, V., Samanta, S., Persinski, R., Hernanz, C., Barthold, S.W., Fikrig, E. (2001) Borrelia burgdorferi-induced inflammation facilitates spirochete adaptation and variable major protein-like sequence locus recombination. J. Immunol. 167, 33833390.