Pal－Borrelia adaptation in tick-2003

Review Adaptation of Borrelia burgdorferi in the vector and vertebrate host Utpal Pal, Erol Fikrig * Room 525A, Section of Rheumatology, Department of Internal Medicine, Yale University School of Medicine, 300 Cedar Street, New Haven, CT 06520-8031, USA Abstract Borrelia burgdorferi sensu lato is the causative agent of Lyme disease, which afflicts both humans and some domestic animals. B. burgdorferi, a highly evolved extracellular pathogen, uses several strategies to survive in a complex enzootic cycle involving a diverse range of hosts. This review focuses on the unique adaptive features of B. burgdorferi, which are central to establishing a successful spirochetal infection within arthropod and vertebrate hosts. We also discuss the regulatory mechanisms linked with the development of molecular adaptation of spirochetes within different host environments. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Borrelia burgdorferi; Ticks; Adaptation 1. Introduction Borrelia burgdorferi sensu lato is the group of spirochetes causing Lyme disease and includes at least 10 genospecies [1]. The three genospecies commonly associated with human infections include B. burgdorferi sensu stricto, which is widespread in both the USA and Europe, and B. afzelii and B. garinii, which are primarily distributed in Europe [2]. The clinical manifestations of Lyme borreliosis differ in North America and Eurasia, possibly due to the genetic diversity among different B. burgdorferi genospecies. B. garinii is associated with neurologic diseases, while B. burgdorferi sensu stricto and B. afzelii are more likely to cause arthritis and cutaneous symptoms, respectively [3–5]. Different geno- species seem to vary in their ability to survive in a given host: for example, B. burgdorferi sensu stricto and B. garinii are reported to persist in birds, whereas B. afzelii fails to survive in avian hosts [6]. B. burgdorferi sensu lato is transmitted by Ixodes ticks [7–9]. The nature of the enzootic cycle of B. burgdorferi in specific geographic areas influences the incidence of human infections. In the northeastern and north central United States, B. burgdorferi sensu stricto is principally maintained in a cycle involving larval and nymphal Ixodes scapularis ticks and white-footed mice [2]. Occasionally, I. scapularis nymphs or adults feed and transmit infection to a wide range of vertebrates, including humans. Ixodes species in other parts of the United States also harbor Lyme spirochetes, but few ticks are infected in comparison to areas with a high incidence of human Lyme disease [8,10]. Differences in tick-host preferences may possibly explain the low preva- lence of such infections. For example, I. pacificus in the west and I. scapularis in the southeast feed on reptiles, which are not susceptible to spirochete infection. I. neotomae, another potential vector, feeds on rodents but rarely bites humans [8,10]. In Europe and Asia, the Lyme disease pathogen is generally maintained between small rodents and I. ricinus or I. persulcatus [11]. 2. Structure, genome, gene expression and regulation The structure of B. burgdorferi sensu lato is typical of a spirochete: a spiral or coil-shaped cell that is generally 20–30 µm in length and 0.2–0.5 µm in width. Individual spirochetes, however, can vary in length, diameter, tightness and regular- ity of the coils. The protoplasmic cylinder containing the cytoplasm with its organelles and flagellar apparatus is cov- ered by a periplasm and a lipoprotein-based outer surface membrane [12]. Complete genome sequencing of B. burg- dorferi sensu stricto (strain B31M1) disclosed several unique adaptive features of the spirochete [13]. The genome size is relatively small, approximately 1.5 megabases, consisting of a linear chromosome of 950 kilobases and at least 21 extra- * Corresponding author. Tel.: +1-203-785-2453; fax: +1-203-785-7053. E-mail address: erol.fikrig@yale.edu (E. Fikrig). Microbes and Infection 5 (2003) 659–666 www.elsevier.com/locate/micinf © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. DOI: 1 0 . 1 0 1 6 / S 1 2 8 6 - 4 5 7 9 ( 0 3 ) 0 0 0 9 7 - 2 chromosomal DNA elements or plasmids (620 kilobases within nine linear and 12 circular plasmids) [16]. B. burgdor- feri has the largest numbers of plasmids known for any bacterium to date. Plasmid genes are of special interest, as they contain many of the genes associated with spirochete pathogenicity. The chromosome contains 853 genes coding for a basic set of known eubacterial proteins that drive cell cycle, growth and metabolism, with an unusual absence of genes responsible for cellular biosynthetic reactions [13]. Plasmids encode another 535 genes, and 90% of the genes have no convincing similarity to genes outside Borrelia ge- nus, suggesting that they perform specialized functions pos- sibly related to spirochete adaptation. Further experiments demonstrated that some of the plasmids can be lost during propagation of the bacteria in vitro, and loss of infectivity in mice often parallels the loss of specific plasmids [14–18]. Most of the spirochete genome consist of linear DNA with covalently closed hairpin ends or telomeres. B. burgdorferi has evolved highly efficient enzymes to replicate such telo- meric DNA structures. Recently ResT, a new class of telom- ere resolvase, has been identified to be encoded by B. burg- dorferi locus bbb03, which performs a highly efficient but complex two-step DNA transesterification during replication to generate covalently closed hairpin telomeres [19]. B. burgdorferi has evolved remarkable abilities to survive in a wide range of organisms such as arthropods, and verte- brates like birds and mammals. In contrast to other patho- genic bacteria, B. burgdorferi has devoted a large portion of its genome (more than 8% of coding genes or 150 genes) towards producing lipoproteins [13,16]. Studies have dem- onstrated the ability of the bacterium to alter its surface structure by differential lipoprotein gene expression at vari- ous stages of its life cycle in mammals and ticks, which is likely to aid in host adaptation and immune evasion [20–27]. A few host molecules were also identified that are involved in interactions with spirochete ligands and thus aid in bacterial survivability in diverse host environments (Table 1). In addi- tion to the transcriptional activation of selective genes, events such as variable recombination also contribute to the alter- ations of spirochete structure. As an example, the B. burgdirferi vlsE locus consists of an active telomeric expres- sion site flanked by a number of upstream silent vls cassettes [28]. Recombination events between the vlsE gene and vls cassettes occur in mammals [29] and most likely in feeding ticks [30], producing a genetically diverse population of spirochetes. Apart from controlling transcription or recombi- nation events, spirochetes were also reported to generate antigenetically diverse populations by modulating intracellu- lar (surface vs. periplasmic) translocation of lipoproteins [31]. A series of in vitro studies have identified a number of environmental signals that are thought to regulate gene ex- pression in spirochetes and includes temperature [27,32], pH [33], cell density [34,35] and host factors [36,37]. Interest- ingly, many of these regulated genes are differentially ex- pressed in vivo and include hsp, mlp, dbpA, bbk 32, erp (ospE/F -related), ospC and ospA [21,22,36,38–44]. Induc- tion of lipoproteins belonging to large gene families such as Mlp [45], Erp [44] as well as OspC [27,43] at higher tem- perature may mimic temperature-induced changes in gene expression that occur during tick feeding, when the spiro- chetes are exposed to warm host blood. Likewise, lowered pH changes the expression of a few spirochete genes in vitro [33,46], and these genes may represent regulated genes dur- ing the tick blood meal, because the pH of the tick gut drops during the feeding process. However, the regulation seems complex and is likely to be multifactorial. For example, temperature alone does not appear to be a sufficient signal for ospC induction, because unfed ticks exposed to higher tem- peratures do not induce ospC expression [27]. A recent study suggested that the same environmental stimuli seem to co- regulate spirochete proteins like OspA and OspC in vitro, suggesting that regulatory pathways of differentially ex- pressed genes are interlinked [46]. However, little is known about the mechanism or key regulatory networks that govern spirochete gene regulation in vivo. The B. burgdorferi ge- nome surprisingly contained relatively few homologs of known eubacterial regulatory proteins [13]. For example, although B. burgdorferi displays a classical heat-shock re- sponse, the genome does not contain a known heat-shock sigma factor [13]. A recent microarray analysis also failed to identify significant evidence of adaptive changes in clusters of spirochete regulatory genes under different host or growth conditions, whereas the same conditions caused significant changes in lipoprotein gene expression [37]. It is proposed that B. burgdorferi adapts to exploit relatively minor changes in the expression of regulatory genes in order to affect down- stream target gene expression, so that relatively small alter- ation in regulatory gene expression can control production of large numbers of target lipoproteins [37]. B. burgdorferi Table 1 Examples of differentially expressed B. burgdorferi lipoprotein gene products with suggested functions Gene product Expression Receptor Function Reference DbpA and DbpB Mammal Decorin Colonization [40] Bgp Mammal GAGs Colonization [102] P66 Mammal Integrins Colonization [72] Erp (OspE/F) Mammal Factor H Host defense [62] ErpT (Arp) Mammal ? Colonization? [103] P47 (Bbk32) Mammal Fibronectin Colonization [68] OspC Mammal/tick ? Transmission [51] OspA Tick A gut protein Colonization [91] 660 U. Pal, E. Fikrig / Microbes and Infection 5 (2003) 659–666 constitutively expresses RpoN, a sigma subunit, which is regulated via a post-transcriptional mechanism [47]. RpoN controls the expression of RpoS, an alternative sigma factor, which in turn, is thought to regulate the transcription of several lipoproteins like OspC, OspF, Mlp-8 and DbpA [46,48]. An external or environmental signaling event is proposed to induce an RpoN activator protein which is likely to bind to an enhancer region upstream of where RpoN complexed with the RNA polymerase holoenzyme (–24/–12 region), leading to the synthesis of rpoS mRNA. RpoS then mediates the synthesis of target lipoproteins of B. burgdor- feri [48]. 3. Adaptation in vertebrates B. burgdorferi is transmitted to vertebrates, mostly to mammals, during tick feeding. An Ixodes tick takes approxi- mately 3–4 d to complete the engorgement process, during which a pronounced multiplication of spirochetes takes place in the gut of the tick [49]. The spirochete numbers are reported to increase several hundredfold [50], and the differ- ential gene expression as well as the variable recombination may contribute to the production of new molecules on the spirochete surface [30]. The newly synthesized proteins are believed to aid in the transmission from tick gut via salivary gland to the dermis of the host [51]. Although spirochetes evolve their own mechanism to fight host immune defense, the transmission via tick saliva provides them with certain adaptive advances. A feeding tick secretes molecules that influence the host immune system. For example, tick saliva inactivates the host complement system [52] and inhibits phagocyte function [53], which in turn, could help the adap- tation of the spirochete to its new environment. Recently, an I. scapularis salivary protein, Salp15, has been shown to modulate CD4+ T cells [54]. After transmission, B. burgdor- feri remains localized into the host skin for several days. The spirochetes are reported to invade distant skin sites or a number of organs and are found in high concentrations in the spleen, urinary bladder, joints and heart [8]. Spirochetes can cross the blood-brain barrier in several experimental models, most notably primates, and colonize the nervous system [55–57]. It is interesting to note that the bacteria are able to establish a chronic infection even in the face of the sophisti- cated immune system that exists in mammals. How does the pathogen adapt itself to this challenging environment? Like many other invasive pathogens, B. burgdorferi uses a variety of mechanisms for protection against components of the host innate immune system. The alternate pathway of the complement system is a major primary host defense [58]. The pathway activates with an initial deposition of the C3 protein to the pathogen surface, followed by several amplifi- cation loops, ultimately resulting in the formation of mem- brane attack complex, which kills the pathogen. Some of the Erp (OspE/F-related) proteins have been reported to be syn- thesized by B. burgdorferi during early mammalian infection [59–61]. Studies have revealed that Erps contribute to the ability of B. burgdorferi to infect mammals by blocking host complement-mediated killing [62,63]. The spirochetes are capable of producing different Erps on the surface, and each Erp is reported to exhibit different relative affinities for the complement inhibitors of various potential vertebrate hosts [64]. Therefore, the presence of multiple Erps on the surface can allow for a single B. burgdorferi to resist complement- mediated killing in a wide range of hosts. All known B. burg- dorferi strains can bind C3, although the deposition of the downstream components of complement system like C5b to C9 varies in different strains, resulting in a vast number of strains being resistant to complement attack [65]. The mechanism of resistance to complement is mediated by the binding of two host-derived complement control proteins: factor H and factor H-like protein-1 / reconectin [62,66]. B. burgdorferi surface protein OspE has been identified to bind the complement regulatory factor H [62]. OspE inter- acts with carboxy-terminal of factor H; therefore, the amino- terminal domain of the complement inhibitor remains free to exert its regulatory activities. The spirochete has also evolved other interesting survival strategies against innate immune attack. B. burgdorferi was shown to bypass the need for physiological iron for its growth and survival [67]; many of the iron-containing enzymes are well-known targets for oxi- dative host defenses against pathogens. A large number of genes have been identified in B. burg- dorferi which are selectively expressed, when spirochetes are inside a mammalian host milieu. The differentially expressed genes are likely to contribute to spirochete dissemination and colonization of target tissues. For example, B. burgdorferi synthesizes DbpA, DbpB and BBK 32 early in mammalian infection, which bind to host extracellular matrix proteins like decorin or fibronectin [40,68,69]. Spirochete adhesins such as DbpA or BBK 32 are thought to play critical roles in the early stage of Lyme disease by mediating the tissue adherence of B. burgdorferi. Local adherence ability in the skin extracellular matrix proteins could facilitate the survival of extremely small numbers of spirochetes (estimated to be between 1 and 10 [70]), which first enter the host at the site of tick feeding. B. burgdorferi is thought to replicate in the skin before endovascular dissemination towards distant organs. Theoretically, binding of soluble extracellular matrix pro- teins by spirochetes may also provide this organism with a mechanism for establishing persistent infection in wide host tissues. As shown with B. crocidurae [71], the ability of B. burgdorferi to bind bulky, host-derived proteins, such as fibronectin, may also mask recognition of the spirochete by the host immune system. B. burgdorferi surface molecule P66 has been shown to bind b3-chain integrins [72], which are expressed in a variety of host locations. Thus, multiple adhesin mechanisms through such widespread host receptors as decorin, fibronectin or integrins contribute to the virulence of the organism and aid in the ability of the pathogen to establish chronic infection in multiple tissues. However, little is known about specific host receptors expressed in a local- 661U. Pal, E. Fikrig / Microbes and Infection 5 (2003) 659–666 ized or tissue-specific manner and that bind to B. burgdor- feri. The spirochetes are reported to use host-derived plasmin to facilitate invasion, as demonstrated in cell cultures and in mice genetically deficient in plasminogen [73,74]. Identifi- cation of the guaA gene encoding GMP synthetase in B. burgdorferi, an enzyme responsible for de novo purine biosynthesis, has also been implicated in the survival of bacteria in mammalian blood [75]. A number of recent studies involving microarray analysis of spirochete gene expression within chamber-implants [76] or in hosts at initial or chronic phases of murine infection address the question of how B. burgdorferi evades adaptive immunity in mammals [36,37]. Studies indicated that B. burgdorferi is able to generate multiple phenotypes during the transmission and earlier phases of murine infection [30,77]. Transcription and recombination events have been shown to occur in the feeding tick gut to generate a diverse population of spirochetes with antigenic and genetic variabil- ity [30]. Antibodies generated in the infected host selectively eliminate the targeted immunodominant phenotypes of spi- rochetes, while the adapted spirochetes continue to survive by downregulation of a selected set of proteins targeted by host antibody. A recent study demonstrated that B. burgdor- feri expresses a set of approximately 116 lipoprotein genes during early mammalian infection and, as a part of adaptive immune response, will downregulate more than 80 of these genes [36]. This adaptation is proposed to be fundamental for B. burgdorferi to survive in mammals and establish persis- tent infection. In addition, B. burgdorferi has been proposed to develop alternate strategies to remain hidden from the immune system, such as masking immunodominant surface antigens [78] or persisting in close association with cells at immune privilege sites [79]. B. burgdorferi has been specu- lated to generate specific mechanisms to inhibit phagocytosis or ingestion by host cells [80]. For long-term persistence in host tissues, B. burgdorferi has also been proposed to modu- late a wide range of host cytokines such as TNF-alpha [81], IFN-gamma [82,83], IL-6 [84,85], and IL-12 [86]. IFN- gamma-mediated events have actually been shown to pro- mote vls locus recombination to generate a diverse popula- tion of spirochetes [87], which could potentially help to evade host immune response. 4. Adaptation in ticks B. burgdorferi faces an entirely different environment when transmitted from mammals to Ixodes ticks. Studies have shown that although ticks usually feed for 96 h, both larval and nymphal ticks rapidly acquire spirochetes during the first 24 h of attachment [43], even before ticks are en- gorged with significant amounts of blood. The number of organisms within fed ticks continues to increase during and after the blood meal, possibly by continual entry of spiro- chetes during the blood meal and by replication [43,88]. The bacterium must avoid being digested with the tick blood meal and has to survive in significant temperature extremities of the poikilothermic organism, and also periods of limited nutritional resources and metabolic activity. During a subse- quent blood meal, spirochetes must also have to sense the appropriate stimuli, and cross the gut barrier to travel to the salivary gland at the right time for transmission to a new vertebrate host.