[Frontiers in Bioscience 14, 3051-3063, January 1, 2009]
Molecular characterization of the tick-Borrelia interface
Aravinda Manu de Silva1, Katharine Rose Tyson1, Utpal Pal2
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TABLE OF CONTENTS
1. ABSTRACT
Spirochetes in the genus Borrelia are responsible for tick-borne relapsing fever and Lyme disease. Borrelia-tick interactions are highly specific as each species of Borrelia is only transmitted by one or a few closely related species of ticks. Borrelia colonize the gut or salivary glands of ticks. Several Borrelia genes required for tick colonization or transmission have been identified. Borrelia genes required for transmission are induced by a pathway controlled by the alternate sigma factors RpoN (54) and RpoS (S). A protein in the gut of I. scapularis ticks that functions as a receptor for B. burgdorferi has been identified. In addition, Ixodes tick saliva has proteins that alter host hemostasis and immunity, and some of these salivary proteins directly interact with Borrelia to facilitate transmission and host infection, whereas others appear to assist Borrelia indirectly by suppressing host defense mechanisms. The exciting discoveries on Borrelia-tick interactions are also being translated into novel preventive measures such as transmission blocking vaccines.
2. INTRODUCTION
Spirochetes are a diverse group of helical shaped bacteria that can be free-living, symbiotic or parasitic to many different host species. Spirochetes in the genus Borrelia have an especially complex life cycle as they are transmitted by arthropod vectors, primarily ticks, to different vertebrate hosts. The genus Borrelia can be further separated into genetically distinct groups related to the Borrelia that cause either Lyme disease or relapsing fever (Figure 1). Within each group some species of Borrelia cause disease in people and animals, whereas other species appear not to be pathogenic. Borrelia-tick interactions are highly specific as each species of spirochete is only transmitted by one or few closely related tick species. The whole genome sequence of several Borrelia species has been determined and this information has been used to profile the transcriptome and proteome of Borrelia at different stages of the life cycle. Moreover, new tools have been developed to genetically manipulate Borrelia, and it is now feasible to create and complement gene-specific mutants. As a result of these advances, we have gained novel insights into the biology of Borrelia-tick interactions, which are reviewed here. (Figure 1: A phylogram of selected Borrelia species)
3. THE LYME DISEASE BORRELIA
Lyme disease is an emerging infectious disease that has steadily increased in prevalence over the past 30 years (1). The disease is now common in parts of North America, Europe and Asia. Lyme disease is caused by at least 7 different species of closely related Borrelia that are transmitted by prostriate hard ticks (2). The three major species commonly associated with human infections are B. burgdorferi sensu stricto, distributed throughout the United States and Europe, and B. afzelii and B. garinii, which have overlapping ranges in Europe and parts of Asia (2).
3.1. Genome of B. Burgdorferi
The sequence of the B. burgdorferi sensu stricto strain B31M1 genome provided important clues about the physiology and complex life cycle of this spirochete (3). The organism contains a segmented genome of approximately 1.5 megabases, consisting of a linear chromosome of 950 kb and 21 extra-chromosomal DNA elements (12 linear and 9 circular plasmids) ranging in size from 5 to 56 kb that add an additional 620 kb of DNA (3, 4). Interestingly, the organization and functional annotations of chromosomal and plasmid genes differ dramatically. The chromosome has fewer non-coding sequences and pseudogenes than the plasmids. The chromosomal DNA harbors a total of 853 genes that mainly code for house keeping proteins, such as those associated with cell cycle, transport, growth and metabolism. The plasmids encode 535 genes and 90% of these genes have no similarity to genes outside the Borrelia genus, suggesting that they perform specialized functions possibly related to the adaptation of the spirochete to different hosts. Some plasmids are unstable and readily lost during propagation of the bacteria in vitro, indicating they are required for specialized functions in the vector and/or host and not for growth in culture (5). It is difficult to genetically manipulate Borrelia because of the highly segmented, unstable genome and the presence of plasmid encoded restriction modification systems that degrade foreign DNA. Nevertheless, recent experimental efforts to introduce foreign DNA via allelic exchange or on shuttle vectors have been successful (5).
3.2. B. burgdorferi distribution within vector
Borrelia are maintained in nature through complex enzootic cycles that vary depending on the species and the geographical location (6, 7). In the Eastern United States, Ixodes scapularis is the main vector while Peromyscus leucopus, the white-footed mouse, is the primary reservoir host. Larval ticks acquire the pathogen while feeding on an infected mouse. The spirochetes are transstadially maintained in ticks through the molt to the nymphal and adult stages. The bacteria primarily colonize the luminal space of the gut, where they are found in association with the gut epithelium (8, 9). During a second blood meal, the spirochetes multiply in the gut, enter the hemocoel and disseminate to multiple organs including the salivary glands through which the bacteria enter the host (9-11) (Figure 2). The dissemination within the tick is transient and there is no evidence that bacteria can colonize any sites other than the lumen of the gut. Transmission is complex and requires approximately 48 to 60hrs following tick attachment to the host, during which period the spirochetes experience complex changes in gene expression. (Figure 2: Distribution of B. burgdorferi within a feeding nymphal tick.)
3.3.Regulation of B. burgdorferi gene expression at the vector-host interface
In contrast to many pathogenic bacteria, B. burgdorferi devotes large portions of its genome (more than 8% of coding genes, or approximately 150 genes) towards producing lipoproteins, many of which are exposed on the bacterial outer surface (3). Within the feeding tick, the spirochetes alter the expression of many genes in preparation for transmission and infection of a new host (12, 13). B. burgdorferi outer surface proteins (Osp) A and C have served as a paradigm for understanding the regulation of bacterial gene expression within feeding ticks. In ticks, ospA is predominantly expressed before the blood meal, whereas ospC is induced during the blood meal (14-17). The functions of these two proteins are consistent with their pattern of expression, where OspA is required for colonizing the vector and OspC is required for infecting the host (see below).
Physiochemical clues, such as temperature, pH and cell density act as signals for regulating the expression of ospA and C in culture and these signals are likely to play a role in the feeding tick as well (17-20). Proteomic and microarray studies with cultured spirochetes grown in "tick" (low temperature, high pH) or "host" (high temperature, low pH) like conditions have led to the identification of large subsets of Borrelia proteins and genes with "OspA-like" or "OspC-like" patterns of expression (19, 21-23). The bacterial signaling pathway regulating the expression of ospC and ospC-like genes has been characterized in some detail (24-27). The pathway is activated by a two component system consisting of a sensor with a histidine kinase domain (HK2) and a cytoplasmic response regulator protein (Rrp2) (28). Activated Rrp2 together with the alternative sigma factor, RpoN, induces expression of the rpoS that encodes the second alternative sigma factor, RpoS (28), and possibly other B. burgdorferi genes (29). RpoS activates the transcription of ospC and ospC-like genes associated with tick transmission and host infection.
Less is known about the pathways and signals regulating ospA and ospA-like genes mainly expressed in the vector. Some studies indicate a simple reciprocal relationship where the signals and pathways that induce ospC within feeding ticks also decrease the production of ospA (16, 30). Other studies have found that even under ideal conditions for ospC expression, the bacterial population is heterogeneous with many spirochetes producing both proteins and others producing either one or neither of the two proteins, indicating a more complex regulation of ospA and C (31, 32). More recent studies have demonstrated that at the level of single bacterial cell, ospA and ospC are, indeed, reciprocally regulated by the RpoN/RpoS pathway (24, 25). In summary, the emerging view is that external signals and the RpoN/RpoS sigma factors play a key role in regulating gene expression at the vector/host interface, including the transcriptional activation of ospC and repression of ospA.
3.4. B. burgdorferi antigenic variation within ticks
Lyme disease spirochetes display high frequency antigenic variation that is driven by recombination at the vls locus on lp28-1. This locus has an expression site encoding a surface exposed lipoprotein, vlsE, and 15 silent cassettes located upstream of the expression site (33). Recombination between portions of the silent cassettes within a central variable domain of the expression site leads to the expression of novel vlsE alleles encoding antigenic variants of VlsE (34). Novel recombination events are detectable within days after infection of the mammalian host and likely help the spirochete evade host defense mechanisms (35, 36). When ticks feed on a mouse infected with B. burgdorferi, the ticks are colonized with a population of spirochetes that contain many different vlsE alleles (37). Studies have been conducted to determine if the vlsE locus recombines and generates novel antigenic variants within ticks as well. Investigators used artificial feeding methods to introduce clonal populations of spirochetes expressing a single allele of vlsE into ticks (37-39). When these infected ticks were fed on naïve mice, only the original, single vlsE allele was detected in the ticks, whereas multiple new vlsE alleles were detected in the mice within a few days of tick transmission. One explanation for these results is that the recombination frequency is similar in mice and ticks, but strong selective pressure in the host amplifies rare recombinants. Alternatively, it is possible that recombination at the vlsE locus is regulated and that the recombination rate is higher in mice compared to ticks.
3.5. B. burgdorferi genes of functional significance in the vector
The annotated genome of B. burgdorferi has approximately 1780 genes. Investigators have used gene expression patterns as a surrogate to understand the function of Borrelia genes. Specific genes that were selectively expressed in the vector or host were predicted to encode proteins of functional significance in the vector or host, respectively. Recently some of these predictions have been confirmed by creating Borrelia mutants and studying their phenotype in ticks or the host. Below, we summarize the studies that have led to the identification and genetic characterization of some B. burgdorferi genes required at different stages in the vector (Table 1: Representative B. burgdorferi genes required at specific stages of the vector)
3.5.1. B. burgdorferi ospAB operon
Two of the best characterized B. burgdorferi genes, ospA and ospB, constitute a single operon in the linear plasmid lp54, and are amongst the major outer membrane proteins produced by B. burgdorferi grown in vitro. These proteins show differential expression in the life cycle of spirochetes (13). ospA and ospB are expressed when spirochetes exit the infected mammalian host and enter feeding ticks (15, 40). Recombinant OspA and OspB specifically bind to the tick gut, and passive transfer of non-Borreliacidal OspA or OspB antibodies in the murine host prevents tissue adherence of spirochetes in feeding ticks in vivo (41-43). This information provided the first clue that these proteins may possibly be involved in spirochete colonization of the tick gut. More convincing evidence was obtained when infection studies were performed with a mutant strain of B. burgdorferi containing an insertionally inactivated ospAB operon (44). The mutant without OspA and OspB remained fully infectious to mice but was unable to infect ticks. The ospAB mutant was able to migrate into the feeding ticks from an infected mouse, but the mutant failed to persist in the tick gut, indicating a critical role for the ospAB operon in the maintenance of Borrelia in ticks. An independent study using an OspB-deficient B. burgdorferi isolate indicated that like OspA, OspB is also actively involved in colonization and persistence of B. burgdorferi in the arthropod vector (45). Together, these experiments demonstrate the vital importance of OspA and OspB in mediating B. burgdorferi colonization of the tick gut. B. burgdorferi strains containing a copy of ospA fused to the constitutive flaB promoter have also been created and tested in mouse infection studies. Spirochetes constitutively producing OspA failed to infect immuno-competent mice, indicating that ospA expression in the host is deleterious to the bacteria (46).
3.5.2. B. burgdorferi ospC
OspC is a prominent Borrelial outer membrane protein that is swiftly upregulated by spirochetes during transmission from the feeding tick to the host (15, 17). Studies with ospC null mutants have unequivocally demonstrated that OspC is required for infecting mice (47, 48). These studies have also led to conflicting results about a role for the protein in the vector. One group created an ospC null mutant and observed a defect in tick salivary gland invasion as well as host infection (48). In a follow up study (49), the same group reported that OspC recruited a tick protein to the bacterial surface that facilitated salivary gland invasion and host infection (see below). Another group independently created ospC null mutants and used them in tick and mouse infection studies. They concluded that OspC was required for establishing an infection in a host but not for the migration or dissemination within the tick (47, 50-52). A recent study with B. afzelii, which is responsible for Lyme disease in Europe and Asia, supports the hypothesis that OspC is required for dissemination within the tick (53). The investigators complemented a natural ospC mutant and observed that the complemented strain invaded salivary glands more efficiently than the ospC mutant.
3.5.3. B. burgdorferi genes on lp25 required for tick infection
As mentioned previously, B. burgdorferi has at least 21 different linear and circular plasmids. As several plasmids are lost during in vitro culture, it is possible to assemble panels of isolates missing non-essential plasmids in order to determine if specific plasmids are required for infecting the vector and host. Linear plasmid 25 (lp25) has been found to be essential for both host and tick infection (54-57). Bbe22 on lp25 encodes a nicotinamidase that is most likely required for the biosynthesis of NAD. Bbe22 alone was sufficient to restore the ability of strains lacking lp25 to infect mice (54). However, complementation with Bbe22 only partially restored tick infectivity, indicating that additional genes on lp25 were required for tick infection (56). Revel and colleagues recently used in vitro microarray expression data to predict genes on lp25 required in the vector (58). They focused on bbe16, as this gene appeared to be selectively expressed when Borrelia were grown in vitro under conditions mimicking the tick. By creating bbe16 null mutant and complemented strains, they demonstrated BBE16 was required for persistent infection of ticks (58). BBE16 is most likely a surface exposed lipoprotein and the exact function of the protein remains to be elucidated. These studies indicate that both bbe22 and bbe16 on lp25 are required for tick infection.
3.5.4. Chromosomally encoded Borrelia genes required in the vector
As described earlier, the chromosomally encoded RpoN and RpoS sigma factors activate and repress many genes in feeding ticks, implying a role for the RpoN/RpoS pathway in B. burgdorferi tick transmission. Fisher et al. created a rpoN null mutant and demonstrated that mutant spirochetes successfully colonized tick guts but were unable to invade salivary glands and infect the vertebrate host, indicating that RpoN regulated genes were involved in transmission from the vector to the host (29).
Bb0690 is a chromosomally encoded gene that is selectively expressed to high levels in ticks and encodes a protein that bears strong similarity to a family of DNA-binding proteins induced during starvation (Dps proteins) of the bacteria (59). B. burgdorferi Dps expression is low throughout murine infection but increases during the tick inter-molt periods. A B. burgdorferi Dps null mutant was able to infect mice (59). When naïve ticks were allowed to engorge on these mice, the dps mutant was able to infect the ticks but was unable to survive the extended period between tick molts. Dps protects bacterial DNA against starvation or oxidative stress-induced damage. Therefore, BB0690 may protect B. burgdorferi against DNA damage during the long inter-molt period between blood meals.
An infectious mutant of B. burgdorferi lacking another chromosomal gene, bb0365, has also recently been created (Pal et al, Manuscript in press). Spirochetes lacking BB0365, also known as p22 or lipoprotein LA7, were able to infect mice. When naïve ticks engorged on spirochete-infected mice, the bb0365 mutant had a markedly decreased survival rate within ticks when compared to wild type B. burgdorferi. These studies suggest that BB0365 could be important for spirochete survival in feeding ticks. In summary, the ability to create specific mutants of B. burgdorferi has allowed researchers to identify Borrelia proteins that function at different stages in vector phase of the life cycle. This list, which includes both plasmid and chromosomally encoded proteins, is still relatively small and many more proteins required in the tick are likely to be identified in the near future.
3.6. Tick proteins that facilitate Borrelia transmission
Ixodes tick saliva has molecules with anti-hemostatic, anti-inflammatory, vasodilatory, and immunosuppressive activities, which assist the tick to feed on a host for prolonged periods without immune recognition and rejection (Table 2). Beyond serving the tick during feeding, salivary proteins can also directly contribute to the efficient transmission of Borrelia to a new host. In vitro studies have demonstrated that salivary gland extracts from I. recinus ticks reduce the phagocytosis and killing of B. afzelii by murine macrophages (60, 61). Other groups have also demonstrated that needle inoculation of B. burgdorferi into naïve mice in the presence of I. recinus or I. scapularis salivary gland extracts (SGE) allows more efficient survival and dissemination of the spirochetes as demonstrated by increases in the numbers of spirochetes present in various tissues when compared to mice needle inoculated with B. burgdorferi in the absence of SGE (62, 63). More recently, Lima and co-workers demonstrated that B. burgdoferi in tick salivary glands had an ID50 that was 10 times lower than Borrelia in the tick gut (64). Together these studies indicate that I. scapularis salivary components enhance the infectivity of B. burgdorferi.
(Table 2: Ixodes tick proteins likely to play a direct or indirect role in facilitating B. burgdorferi transmission)
3.6.1. Salp15- a tick protein that binds to OspC
Ramamoorthi and co-workers reported that I. scapularis salivary protein 15 (Salp15) bound to OspC and facilitated transmission of spirochetes from the tick to the host, suggesting that OspC may have a role in the vector as well as during early host infection (49) (Table 2). Through in vitro and in vivo binding assays, Salp15 was shown to directly bind to B. burgdorferi through OspC, and this binding protected B. burgdorferi from antibody mediated killing in vitro. When mice previously exposed to B. burgdorferi and displaying an antibody response against the spirochete were infected by needle inoculation with B. burgdorferi in the presence or absence of recombinant Salp15, the levels of spirochetes in the skin, joints, and bladder were significantly higher in mice that received Salp15. Additionally, when salp15 was silenced in I. scapularis nymphs by RNA interference, the amounts of spirochetes transmitted to naïve mice were significantly reduced when compared to mock treated infected ticks (49). The results from this study indicate that Salp15 assists B. burgdorferi in transmission to and infection of the host during tick feeding.
The Salp15 binding to Borrelia might be responsible for a puzzling observation about OspC production by spirochetes moving from the tick to the host. We have reported that many Borrelia in the salivary glands and host skin are OspC negative by immuno- florescence microscopy, which argues against a role for this protein in transmission (32). Spirochetes in the salivary glands and host's skin may appear OspC negative because Salp15 masks OspC and prevents antibody from staining the bacteria.
3.6.2. The tick receptor for OspA (TROSPA)
Recent studies have identified a tick gut receptor that interacts with B. burgdorferi OspA (65). Pal et al. have established that B. burgdorferi OspA binds the tick receptor for OspA (TROSPA), which is expressed in the gut of I. scapularis ticks (65). In vitro, OspA specifically bound recombinant TROSPA, while TROSPA antisera partially blocked binding of OspA to tick gut extracts. Interestingly, TROSPA expression within the tick was upregulated during infection with B. burgdorferi and decreased during engorgement, correlating with ospA downregulation and transmission of the spirochetes to a new host. When uninfected ticks were allowed to feed on infected mice administered with TROSPA antisera, acquisition of B. burgdorferi by the ticks was significantly reduced when compared to infected mice treated with pre-immune sera. Similarly, RNA interference of TROSPA in nymphal ticks significantly diminished the ability of the ticks to acquire B. burgdorferi from infected mice. The results of Pal et al. demonstrate that B. burgdorferi colonizes the tick gut by adhering to TROSPA through OspA. This attachment allows the spirochete to persist within the tick until the next blood meal occurs and transmission begins. Although TROSPA has been shown to be actively engaged in supporting B. burgdorferi during its life cycle within the arthropod, the native, physiological function of TROSPA in ticks is currently unknown. TROSPA is not consistently expressed in all stages of I. scapularis ticks, and its expression is highly influenced by environmental signals, such as tick engorgement or B. burgdorferi infection, suggesting a developmental or transient rather than a housekeeping function. TROSPA bears weak amino acid sequence homology (31%) to anti-freeze glycoproteins (AFGP) in the protein database. AFGP have extensive sequence divergence across phyla and normally function by binding ice particles, thereby preventing ice crystal formation and freeze-induced tissue damage in plants, fish and arthropods. Studies addressing the role of TROSPA in tick physiology might contribute to a better understanding of B. burgdorferi pathogenesis in ticks.
3.6.3. The Isac family of proteins
Recently, researchers have identified a large family of Ixodes scapularis anti-complement (Isac) proteins that block the alternative complement pathway (66-68). At least one member of this family (Salp20) protects serum sensitive strains of Borrelia from lysis in in vitro assays, indicating this protein may protect Borrelia from host complement within the vector and during transmission (68). Furthermore, Soares et al. used RNA interference to silence Isac and related family members in ticks infected with B. burgdorferi (67). When control and Isac silenced nymphs were fed on mice, Isac treated ticks fed poorly and the Borrelia load within these ticks was lower than in the control ticks, indicating that Isac is needed for efficient tick feeding as well as for the growth of Borrelia within ticks. Taken together, these results demonstrate a possible role for the Isac-like family of proteins in Borrelia survival and transmission. As more work is done in this area, additional tick proteins required for Borrelia infection and transmission will be identified.
3.7. Tick immune system and B. Burgdorferi
Arthropods have innate immune systems that recognize and destroy invading pathogens. Surprisingly, little work has been done to determine how Borrelia evade tick immune responses. In one study, B. burgdorferi were injected into the hemocoel of I. scapularis and Dermacentor variabilis, which is not a competent tick vector of Borrelia (69). The spirochetes readily survived in the Ixodes hemolymph, whereas they were rapidly killed by hemocytes in the D. variabilis hemolymph. This study indicated that Ixodes ticks, unlike Dermacentor ticks, were immuno-tolerant towards B. burgdorferi.. Even though they appear immuno-tolerant and permissive to B. burgdorferi infection, I. scapularis and related ticks are not completely tolerant of B. burgdorferi as some tick genes are induced following infection. Ribeiro et al. recently identified several salivary genes that were up-regulated in infected I. scapularis nymphs when compared to uninfected nymphs (66), and these genes may encode proteins that limit Borrelia infection. Rudenko et al. have also demonstrated by subtractive hybridization that an I. recinus defensin-like gene is up-regulated in the gut after infection with B. burgdorferi (70, 71). Additionally, Rudenko et al. determined that putative genes potentially involved in an oxidative stress response are also up-regulated after infection. We currently do not understand the mechanism behind the relative immuno-tolerance of I. scapularis to B. burgdorferi and the role of tick immune responses in determining vector competence.
4. TICK-BORNE RELAPSING FEVER BORRELIA
Tick-borne relapsing fever (TBRF) is a rare but widespread disease present in the Americas, Europe, Africa and Asia. The Borrelia species responsible for relapsing fever are mainly transmitted by Argasid ticks, otherwise known as soft ticks. B. recurrentis, responsible for louse-borne relapsing fever, is transmitted by an insect vector. TBRF is characterized by episodes of fever separated by 1-2 week intervals of mild or no fever. Untreated disease can lead to serious neurological complications and a mortality frequency of 4 to 10%. Few studies have been done to determine the distribution of TBRF Borrelia within ticks. Burgdorfer compared B. duttonii distribution within the nymphal and adult Ornithodorus ticks and found that the spirochetes disseminated within both the nymphal and adult vectors within 24 hrs of infection and colonized multiple tissues including the salivary glands (72). Interestingly, nymphal ticks transmitted B. duttonii through the salivary glands whereas adult ticks appeard to transmit the organisms through coxial fluid. More recently, Schwan and Hinnebusch examined the distribution of B. hermsii in ticks and observed that in most cases, multiple organs including the salivary glands were colonized by the spirochetes (73). Thus, unlike the Lyme disease spirochetes, which mainly colonize the tick gut lumen, the relapsing fever spirochetes disseminate in the vector and colonize multiple tissues including the salivary glands.
4.1. Relapsing Fever Borrelia and tick interactions
In comparison to Lyme disease Borrelia, we know very little about the molecular interactions between relapsing fever Borrelia and their tick vectors. Relapsing fever spirochetes use multiple mechanisms to vary the dominant antigen expressed on the bacterial surface in mammals. Antigenic variation leads to cyclic spirochetemias in mammals that reach a density of 108 bacteria/mL of blood. Each new cycle of spirochetemia is characterized by the expression of a new antigenic variant on the surface. Schwan and colleagues compared the major surface proteins expressed by B. hermsii in mice and in ticks (73). They observed that the antigenic composition of the spirochetes within ticks was relatively stable, and most B. hermsii in the salivary glands expressed a single variable surface protein designated Vsp33. Moreover, the replacement of the variable surface antigens expressed in mice with the invariant Vsp33 appeared to be triggered by the low temperature encountered in the tick vector. Interestingly, B. hermsii Vsp33 is related to B. burgdorferi OspC at the amino acid level and antibodies against OspC cross react with Vsp33. Schwan has argued that Vsp33 and OspC have related functions in their respective Borrelia species and that the differences in the expression patterns of these proteins relate to the different feeding behaviors of the tick vectors (74). Lyme disease spirochetes infect hard ticks that feed continuously for several days, and there is ample time for the spirochetes to turn on the expression of ospC, which is required for transmission and host infection. In contrast, Argasid ticks only feed for a matter of minutes. Consequently, B. hermsii colonizing the salivary glands express vsp33 and are poised for transmission during the short blood meal.
Projects are underway to sequence the genomes of relapsing fever spirochetes. DNA cross hybridization studies indicated that B. hermsii has at least 81% of the chromosomal and 43% of the plasmid encoded genes of B. burgdorferi, implying that these organisms have similar genomes despite the differences in the diseases they cause and the vectors they infect (75). The genetic tools developed for B. burgdorferi are also being used to study relapsing fever spirochetes. More comparative studies are needed to understand how Lyme disease and relapsing fever spirochetes have adapted to enter, colonize and leave their respective vectors, which have markedly different life cycles and feeding patterns.
5. TICK-TRANSMISSION BLOCKING VACCINES
Studies on Borrelia-tick interactions are likely to contribute to the development of novel transmission blocking vaccines. Bacterial pathogens, such as Borrelia, that cause persistent infections in mammals have evolved sophisticated mechanisms of antigenic variation and differential gene expression to evade host immunity. As a result, antigens expressed in the host are, often, not good vaccine candidates. As the vector does not have an adaptive immune system, Borrelia antigens expressed in the vector are conserved and antigenic variation mechanisms appear to be inactive in the vector (39). In fact, the FDA-approved OspA Lyme disease vaccine induces antibodies in the host that enter the guts of infected feeding ticks and prevent the transmission of spirochetes from the vector to the host (14). Although an OspA-based vaccine is still in use to prevent B. burgdorferi infection in some animals, the human vaccine is no longer available. Therefore, further studies are needed to develop the next generation of vaccines for protecting people from Borrelia infections. As transmission blocking vaccines have the potential to block ticks from both acquiring and transmitting Borrelia, studies are also being conducted to determine if these vaccines can be used in reservoir hosts to reduce the proportion of infected ticks. In addition to bacterial antigens, tick proteins also have the potential to be developed into vaccines. Vaccines that target tick salivary proteins expressed early in the blood meal could inhibit tick feeding and block pathogen transmission. Alternatively, vaccines based on tick molecules that interact with Borrelia might be effective in blocking transmission without having any adverse effects on the vector.
6. SUMMARY, OUTSTANDING QUESTIONS AND FUTURE DIRECTIONS
Arthropod-borne diseases are fascinating biological systems where specific components from the vector, pathogen and host interact to enable pathogens to move between hosts. Since the discovery of the Borrelial etiology of Lyme disease in 1982, much progress has been made in developing tools to study these spirochetes. These tools are now being applied to understand both Borrelia-host and Borrelia-vector interactions. As highlighted above, recent work has led to exciting discoveries of both Borrelia and tick molecules required at different stages in the vector. What is also clear is that we have only identified a very small subset of interactions and much remains to be discovered. We need to identify additional microbial as well as vector gene products required for the acquisition, maintenance and transmission of the microbe by the arthropod. How does Borrelia evade tick immunity or survive in the feeding gut when the bloodmeal is being digested by the vector? How does Borrelia persist in ticks through the intermolt periods dominated by temperature extremities and a scarcity of nutrients? Studies should also address details of how B. burgdorferi invades solid physical barriers, such as the chitinous gut peritrophic membrane, and then selectively migrates through the gut, hemocoel and salivary gland epithelia during its transmission to mammals. Comparative studies are needed with different species of Borrelia and ticks to identify common themes, as well as species-specific differences.
Given recent technological advances, the next few years are likely to be particularly productive with respect to defining the function of additional Borrelia genes in the vector and host. There are still formidable challenges to uncovering tick molecules regulating Borrelia infection. The long life span (approximately 2 years) and slow post-embryonic development of Ixodes ticks, coupled with limited genomic information severely blocks application of gene manipulation tools to tick research. Currently it is also not possible to analyze the entire tick transcriptome or proteome. In 2004, the I. scapularis Genome Project (IGP), a collaborative effort between the international community of tick researchers and two genome sequencing centers, was approved and is currently supported by the National Institutes of Health (76, 77). The goal of the IGP is to perform whole genome shotgun sequencing (WGS) to approximately six-fold coverage of the genome (76). Compared to sequenced fly genomes, I. scapularis has a larger genome (2.1 x 103 Mbp), which is uniquely organized into 27% highly repetitive, 39% moderately repetitive and 34% unique DNA (78). Currently, more than 18 million trace reads, representing approximately five-fold coverage of the genome, have been deposited at the National Center for Biotechnology Information (NCBI) trace archive (77). In addition, 20 Ixodes BAC clones, 370,000 BAC-end reads, and more than 80,000 ESTs have also been sequenced. The Ixodes mitochondrial genome has been assembled and annotated. By 2008, sequencing, assembly, and annotation of six-fold coverage of the Ixodes genome are expected. The tick genome will be best utilized if the community of tick researchers also develops tools for creating transgenic ticks.
7. ACKNOWLEDGEMENTS
We thank Joseph Piesman (CDC, Fort Collins, CO) and Frank Yang (Indiana University, IN) for their critical comments on this review. Borrelia and tick studies in our laboratories have been supported by the National Institute of Allergy and Infectious diseases (U01AI058263-04), National Institute of Arthritis and Musculoskeletal Diseases (RO1 AR047948) and the Arthritis Foundation. Utpal Pal is the recipient of Scientist Development Grant from American Heart Association.
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Key Words: Borrelia, Tick, Lyme disease, Relapsing fever, Spirochetes, Ixodes, Review
Send correspondence to: Aravinda de Silva, Department of Microbiology and Immunology, CB#7290 University of North Carolina, Chapel Hill, NC 27599, Tel: 919-843-9963, Fax: 919-962-8103, E-mail:desilva@med.unc.edu