Gene Regulation During the Enzootic Cycle of the Lyme Disease Spirochete

Abstract

Borrelia burgdorferi, the spirochete that causes Lyme disease, exists in an enzootic cycle, alternating between a tick vector and a vertebrate host. To adapt to and survive the environmental changes associated with its enzootic cycle, including nutrient availability, B. burgdorferi uses three different systems to regulate the expression of genes: RpoN-RpoS, histidine kinase (Hk)1/response regulator 1 (Rrp1), and Rel_Bbu. The RpoN-RpoS alternative sigma factor cascade activates genes required for transmission from the tick to the vertebrate, maintenance of the vertebrate infection, and persistence in the tick. Rel_Bbu controls the levels of the alarmones guanosine pentaphosphate and guanosine tetraphosphate, which are necessary for surviving the nutrient-deficient conditions in the midgut of the tick following absorption of the blood meal and the subsequent molt. The Hk1/Rrp1 two-component system produces cyclic dimeric guanosine monophosphate that regulates the genes required for the transitions between the tick and vertebrate as well as protective responses to the blood meal.

I. INTRODUCTION

Lyme disease, a multisystem disorder, is the most common tick-transmitted infection in the northern hemisphere, with ~300,000 cases reported per year in the United States alone.¹ The spirochete Borrelia burgdorferi and closely related genospecies, including Borrelia afzelii and Borrelia garinii in Europe and Asia as well as the recently discovered Borrelia mayonii, are the causative agents of Lyme disease.^2–4 An enzootic cycle involving a tick vector and a vertebrate host maintains the spirochete in nature (Fig. 1).^5–7 Ixodes larvae acquire B. burgdorferi by feeding on a mammalian host, and the spirochetes inhabit the midgut. The larvae molt and, when the resulting nymphs feed, the spirochetes leave the midgut, enter the hemocoel, migrate to the salivary glands, and transmit to the vertebrate source of the blood meal.⁸ Therefore, the spirochete must not only survive in two very different milieus but also be able to efficiently move between them at the appropriate time, which requires systems to sense the biological cues and transduce the signals to express the relevant regulon.

FIG. 1.

The enzootic cycle of B. burgdorferi. Ixodes larvae acquire the spirochete when they feed on an infected vertebrate host. The RpoN-RpoS alternative sigma factor cascade that was active in the vertebrate begins to shut down, and the Hk1/Rrp1 two-component system produces the second messenger c-di-GMP. Larvae then molt to nymphs; Rel_Bbu produces the alarmones (p)ppGpp to ensure persistence of the spirochete. Nymphs feed on a second vertebrate host and transmit the spirochetes. Protection from the blood meal requires c-di-GMP, and vertebrate host-specific genes become turned on via the alternative sigma factor RpoS. Nymphs molt to adults that feed, mate, and lay eggs to continue the cycle (adapted from Caimano et al.).⁷

Numerous genes are differentially expressed as B. burgdorferi cycles from tick to vertebrate and vertebrate to tick.⁹ Outer-surface lipoprotein (Osp)A and OspC, the most plentiful outer-membrane lipoproteins,^10–13 represent the most dramatic change in the phase-specific proteome, with OspA levels dropping and OspC levels rising during transmission from tick to vertebrate.^14–21 OspA is crucial to colonize the tick;²² it binds to a tick midgut protein²³ and plasminogen²⁴ and protects the spirochete from antibodies in the blood meal.²⁵ OspC is a transmission factor required to establish infection;^26–32 it also binds to plasminogen,^33,34 although its precise molecular function remains unknown and its specific role in the enzootic cycle is somewhat controversial. However, OspC synthesis is turned off soon after establishing residence in the vertebrate as part of the spirochetal mechanism to evade adaptive immunity.^29,35–38

The environmental signals informing the spirochete of its location in the enzootic cycle, enabling it to suitably respond, are still not fully defined. The first cue described was temperature: Schwan et al.²⁰ showed that the increased temperature of the blood meal reciprocally regulates OspA and OspC levels during tick feeding. Other environmental factors associated with the phases of the enzootic cycle that affect gene expression in B. burgdorferi include pH,^39,40 oxygen,⁴¹ carbon dioxide,⁴² transition metals,⁴³ nutrients,⁴⁴ and osmolarity.⁴⁵ Other signals that are involved and the mechanisms by which they are integrated and transduced to produce the phase-specific regulatory response are not currently known. However, the regulation of gene expression during the enzootic cycle is controlled by the RpoN-RpoS alternative sigma factor cascade, the histidine kinase (Hk)1/response regulator (Rrp)1 two-component system and its second messenger cyclic dimeric guanosine monophosphate (c-di-GMP), Rel_Bbu, and the alarmones guanosine pentaphosphate and guanosine tetraphosphate ([p]ppGpp). Here, we review gene regulation during the enzootic cycle and recommend other publications for greater in-depth coverage.^6,7,46–53

II. RPON-RPOS ALTERNATIVE SIGMA FACTOR CASCADE

Expression of ospC requires the alternative sigma factor RpoS, and rpoS transcription requires the alternative sigma factor RpoN,⁵⁴ at least for transcription of a short rpoS messenger RNA (mRNA) hypothesized to be expressed during maintenance of the vertebrate-specific regulon.^47,55,56 In addition, RpoS represses ospAB transcription.⁵⁷ The RpoS-dependent transcriptome, which would be expressed by B. burgdorferi in the vertebrate, includes many other genes and encoding proteins involved in cell envelope biogenesis, transport, and metabolism and the chemotaxis and motility that are required during transmission and in the vertebrate host.^9,58–60 Therefore, RpoS has been proposed to serve as a “gatekeeper” of the enzootic cycle.^59,61

The regulation of RpoS is in turn surprisingly complicated. rpoS transcription and RpoS protein levels increase in response to environmental signals associated with tick feeding.^{40,45,54,55,62,63} The rpoS gene has a consensus RpoN-dependent promoter^55,63,64; transcription of a short rpoS mRNA requires the alternative sigma factor RpoN,⁵⁴ along with the enhancer-binding protein and response regulator Rrp2.^63,65–67 The mechanism by which Rrp2 is activated, presumably by phosphorylation, is enigmatic, because neither any of the annotated Hks, including its cognate Hk2,^63,68 nor acetyl phosphate⁶⁹ are culpable. In addition, rpoS transcription is activated by the PerR/Fur homolog Borrelia burgdorferi oxidative stress regulator (BosR)^56,70–74 and repressed by the XylR/NagC homolog BadR.^75,76 BosR not only binds to the rpoS promoter region⁷⁴ but also regulates, including via autoactivation⁷⁷ and ospAB repression, the expression of many RpoS-independent genes.^72,78,79

RpoS production is also posttranscriptionally regulated by the small RNA DsrA_Bb.⁵⁵ DsrA_Bb is complementary to the upstream region of an RpoD-dependent long rpoS mRNA that can form a hairpin stem-loop structure that is hypothesized to obstruct ribosome binding and translation; therefore, DsrA_Bb is predicted to relieve this secondary-structural sequestration in a temperature-dependent fashion.⁵⁵ The interaction between rpoS mRNA and DsrA_Bb is most likely mediated by the RNA chaperone Hfq.⁸⁰ The B. burgdorferi Hfq homolog is an oddball, but it has RNA chaperone activity (as assessed using a heterologous genetic test) and is required for both RpoS synthesis and mammalian infection.⁸⁰

III. HK1/RRP1 TWO-COMPONENT SYSTEM AND SECOND MESSENGER c-di-GMP

c-di-GMP is a second messenger that regulates lifestyle decisions in bacteria.⁸¹ In B. burgdorferi, the response regulator Rrp1 is a diguanlyate cyclase activated by its cognate Hk1.^82–86 The Hk1/Rrp1 two-component system and c-di-GMP regulate genes required in the tick vector and during enzootic cycle transitions,^83,85,87 as opposed to the RpoN-RpoS alternative sigma factor cascade that regulates genes required in the vertebrate host (see above); both Hk1 and Rrp1 are required for survival of B. burgdorferi in feeding ticks but not in vertebrates.^82–84 The stimuli that activate Hk1 have not yet been discovered,^82–86 and the only known c-di-GMP effector protein in the spirochete is PlzA.^88–90 c-di-GMP activates, usually through PlzA, genes whose products are involved in using alternate carbon sources, such as glycerol via the products of the glp operon, and building the cell envelope, including outer-membrane lipoproteins that probably protect B. burgdorferi from the antibacterial components of the blood meal.⁸⁵ Glycerol and the glp operon are required for B. burgdorferi to maximally survive in the tick.^83,91

IV. REL_Bbu AND ALARMONES (p)ppGp

The stringent response to nutrient limitation in bacteria is regulated by the alarmones (p)ppGpp, whose levels are controlled by RelA and SpoT homologs.^92–94 RelA synthesizes (p)ppGpp, and SpoT either synthesizes or hydrolyzes (p)ppGpp in several bacteria; however, in B. burgdorferi, these two enzymatic activities are combined in the single bifunctional enzyme Rel_Bbu that responds to nutrient stress.^44,95,96 (p)ppGpp is not produced in rel_Bbu mutants.^44,97 The rel_Bbu can infect vertebrates, but they are compromised for survival during the intermolt in the tick.⁴⁴ (p)ppGpp influences the expression of many genes associated with persistence of the spirochete in the tick vector, including the genes of the glp operon^44,98; curiously, (p)ppGpp activates glpF and glpK (encoding the glycerol uptake facilitator and glycerol kinase, respectively) but represses glpD (encoding glycerol-3-phosphate dehydrogenase).⁴⁴ This observation suggests that (p)ppGpp directs glycerol from the tick into membrane biogenesis instead of energy production in B. burgdorferi. (p)ppGpp also affects the expression of other genes whose products are associated with persistence in the tick as well as the late operon of a prophage that exists as a family of 32-kb circular plasmids.⁴⁴

V. CONCLUSIONS

B. burgdorferi regulates its gene expression while traversing its enzootic cycle to prepare for the transitions between vector and host and adapt to the unique environments of the tick and vertebrate. The spirochete uses three regulatory systems that include a pair of alternative sigma factors, a pair of two-component systems, and a pair of purine second messengers to precisely modulate gene expression, ensuring efficient maintenance of the enzootic cycle in nature. Quite a few of the gene products regulated by these systems have a function that is clearly related to either mobility among environments or survival in a specific environment; however, probably most have no homology to known gene products, so their role in the enzootic cycle remains mysterious.

ABBREVIATIONS

c-di-GMP

Cyclic dimeric guanosine monophosphate

Hk

histidine kinase

Osp

outer-surface (lipo)protein

(p)ppGpp

guanosine pentaphosphate and guanosine tetraphosphate

Rrp

response regulator