Quorum sensing by the Lyme disease spirochete

Review

Quorum sensing by the Lyme disease spirochete

Brian Stevenson *, Kate von Lackum, Rachel L. Wattier, Jason D. McAlister,Jennifer C. Miller, Kelly Babb

Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine,MS 415 Chandler Medical Center, Lexington, KY 40536-0298, USA

Abstract

The Lyme disease spirochete, Borrelia burgdorferi, utilizes a LuxS/autoinducer-2-dependent quorum sensing mechanism to control aspecific subset of bacterial proteins. It is hypothesized that this system facilitates transmission of B. burgdorferi from feeding ticks intowarm-blooded hosts.

© 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.

Keywords: Borrelia burgdorferi; LuxS; Autoinducer; Tick; Relapsing fever

1. Introduction

Lyme disease is an important arthropod-borne disease ofhumans in many parts of the globe, with major endemic fociin North America, Europe and Asia. This disease can mani-fest in many organ systems, with symptoms that include skinrashes, arthritis, meningitis, optic neuritis, facial nerve palsyand atrioventricular nodal block. Failure to treat infectionpromptly and adequately can result in long-term debilitatingeffects on the patient’s health.

The causative agent of Lyme disease, Borrelia burgdor-feri, is maintained in nature by cycles of infection betweenwarm-blooded vertebrates and Ixodes spp. ticks (Fig. 1). Thebacteria persistently infect reservoir vertebrates, such as ro-dents or birds, which serve as hosts for vector ticks. Larvalticks acquire B. burgdorferi during feeding on infected hosts,maintain the infection throughout the molt to the nymphalstage, then transmit to reservoir animals when the nymphsfeed. B. burgdorferi regulates expression of many differentproteins throughout this cycle, presumably synthesizing onlythose proteins specifically required for each step along theway. As do other infectious organisms, B. burgdorferi senseschanging environmental conditions as the bacterium passesthrough its cycle, and transmits that information throughregulatory pathways to control gene and protein expressionpatterns. Studies conducted over the past 8 years have begun

to illuminate these pathways. Temperature appears to be animportant environmental cue affecting protein synthesis:several proteins associated with mammalian infection areproduced at low levels by bacteria cultivated at 23 °C, but areproduced at significantly higher levels when cultures areshifted from 23 to 34 °C [1,2]. This temperature change isthought to mimic the conditions experienced by bacteria inan unfed tick (ambient temperature) and during tick feedingon a warm-blooded animal (changing from ambient to bloodtemperature). The pH of the culture medium is another im-portant signal affecting protein synthesis in vitro [3], pre-sumably related to the acidification of the tick midgut during

* Corresponding author. Tel.: +1-859-257-9358; fax: +1-859-257-8994.E-mail address: [email protected] (B. Stevenson).

Fig. 1. The natural cycle of the Lyme disease spirochete. Bacteria cancontinuously infect reservoir animals (A), apparently persisting throughoutthe lifetime of those animals. B. burgdorferi are transmitted from reservoirto vector during feeding of larval ticks (B). Bacteria colonize the tickmidgut, and persist there through the molt from larva to nymph (C). Infectednymphs then transmit B. burgdorferi to warm-blooded animals on whichthey feed (D). In locations where Lyme disease is endemic, both larval andnymphal ticks feed on the same host species, allowing maintenance of B.burgdorferi in a cycle between the ticks and that host. Fed nymphs molt intoadults and retain B. burgdorferi infections through that molt, although adultsgenerally do not feed on the same vertebrate hosts as do larvae and nymphs,and thus do not contribute to the maintenance of B. burgdorferi in mostgeographic areas. Transmission of B. burgdorferi infection is exceeding rarebetween adult ticks and their eggs or from vertebrate to vertebrate, necessi-tating the arthropod–vertebrate cycle for its maintenance.

Microbes and Infection 5 (2003) 991–997

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feeding [4]. B. burgdorferi also alters gene expression inresponse to various unidentified chemicals, which probablycorrespond with components of host blood or other tissues[5–8]. The mechanisms by which B. burgdorferi senses anyof these stimuli or transmits such information to regulateprotein synthesis are still largely unknown.

A steadily growing list of bacterial species have beendemonstrated to use quorum sensing mechanisms to regulategene expression. Such bacteria secrete a specific compound,termed an autoinducer, into their surroundings, while simul-taneously sampling the environment for its presence. Whenautoinducer concentrations reach a specific threshold, bacte-ria are triggered to alter gene expression. A population ofbacteria can thus use quorum sensing to coordinate certaincharacteristics, such as expression of virulence factors. Insome bacterial species, autoinducer molecules appear to besynthesized constitutively, the result being that autoinducerconcentrations increase as bacterial population grows. Insuch cases, the effects of quorum sensing are observed duringlate exponential or stationary phases of culture growth. How-ever, many bacterial species regulate synthesis of their auto-inducer, producing the signal only under certain conditions.For example, a number of different species synthesize highlevels of autoinducer during the mid-exponential growthphase in culture, but produce significantly less autoinducerduring late exponential and stationary phases, presumablyusing bacterial growth rate and nutrient supply as signals toregulate autoinducer synthesis [9–11]. Other bacteria regu-late autoinducer production in response to various externaland internal signals [11–14].

Autoinducer-2 (AI-2) is a recently described signalingmolecule utilized by many different bacterial species [15].AI-2 is produced through the same pathway employed bymany microorganisms to methylate substrates and recycleproducts of those reactions (Fig. 2) [15,16]. This autoinducermolecule is so well conserved across the kingdom Bacteriathat AI-2 produced by one bacterial species can stimulate aresponse from unrelated bacteria [15,17]. For example, the

marine bacterium Vibrio harveyi utilizes AI-2 in a quorumsensing mechanism to regulate bioluminescence, and AI-2induces V. harveyi to produce light regardless of autoinducersource. This characteristic enables the use of V. harveyi forhighly specific bioassays of AI-2 production [15,17,18].

2. B. burgdorferi quorum sensing

Analysis of the genome sequence of B. burgdorferi typestrain B31 identified a potential homolog of luxS, the geneencoding the last enzyme utilized in AI-2 biosynthesis[15,19,20]. Comparison of the predicted amino acid se-quences of the B. burgdorferi protein with LuxS proteinsfrom V. harveyi and Bacillus subtilis indicates that whileoverall identities among the three proteins are somewhatlimited, residues involved in enzymatic function are com-pletely conserved (Fig. 3). Furthermore, the B. burgdorferigene appears to be located in an operon with likely homologsof metK and pfs, genes encoding other enzymes required forAI-2 synthesis (Figs. 2 and 4). Notice of this genetic juxta-position led to the recent determination of both the mecha-nism of bacterial AI-2 synthesis and its chemical structure[15,16].

Our laboratory has investigated the postulated B. burgdor-feri luxS gene in detail, and demonstrated that it does in factencode a functional LuxS enzyme [20]. The commonly usedDH5$ strain of Escherichia coli contains a mutation in luxS,and cannot synthesize AI-2, although autoinducer synthesiscan be restored by complementation with a functional luxShomolog from another bacterial species [18]. The putative B.burgdorferi luxS gene was cloned into E. coli DH5$, andculture supernatants were assayed by induction of V. harveyiluminescence, which indicated that AI-2 was indeed pro-duced [20]. Furthermore, induction of bioluminescence bysupernatants of luxS-complemented cultures was dose-dependent (our unpublished results). In contrast, culture su-pernatants from either untransformed E. coli DH5$ or DH5$

Fig. 2. Biosynthetic pathway of autoinducer-2 (AI-2) [15,16]. Abbreviations: ATP, adenosine triphosphate; SAM, S-adenosylmethionine; SAH,S-adenosylhomocysteine; SRH, S-ribosylhomocysteine; DPD, 4,5-dihydroxy-2,3-pentanedione.

Fig. 3. Alignment of the predicted amino acid sequences of the LuxS proteins of B. burgdorferi, V. harveyi, and B. subtilis. Residues involved in LuxS functionare indicated by asterisks [41–44].

992 B. Stevenson et al. / Microbes and Infection 5 (2003) 991–997

carrying a control plasmid failed to induce luminescence byV. harveyi [20]. More recently, recombinant B. burgdorferiLuxS protein was purified and demonstrated to synthesizeAI-2 in vitro (our unpublished results).

It is important to note that the presence of a functionalLuxS enzyme does not ipso facto prove that an organismutilizes AI-2 as a signaling molecule [21]. Cellular methyla-tion reactions produce S-adenosylhomocysteine (SAH,Fig. 2), which is toxic. Some bacteria detoxify SAH toproduce adenine and S-ribosylhomocysteine (SRH) througha Pfs nucleosidase. SRH can then be cleaved by LuxS toproduce homocysteine and 4,5-dihydroxy-2,3-pentanedione(DPD), the precursor of AI-2. Since homocysteine may thenbe enzymatically converted by either MetE or MetH to me-thionine, it has been suggested that the function of LuxS inmany bacteria is actually to salvage homocysteine [21].However, that is not likely to be the case with the Lymedisease spirochete. B. burgdorferi possesses very limitedmetabolic capabilities, and is auxotrophic for almost allamino acids. Examination of the B. burgdorferi genomesequence indicates that this organism lacks homologs ofeither MetE or MetH, enzymes required for conversion ofhomocysteine to methionine [19]. Thus, degradation of SAHto homocysteine is probably of no value to B. burgdorferi,suggesting that the important function of LuxS is actually theproduction of DPD (and AI-2).

If B. burgdorferi encodes LuxS to produce AI-2 for use incell–cell communication, one would expect that exogenouslyadded AI-2 would affect B. burgdorferi protein expression.To this end, AI-2 from culture supernatants of E. coli DH5$

complemented with the B. burgdorferi luxS gene was addedto mid-exponential-phase cultures of B. burgdorferi. As con-trols, B. burgdorferi were cultured either without any addedE. coli supernatant or with culture supernatants from non-complemented DH5$. All supernatants were sterilized byfiltration through 0.22-µm membranes prior to use. Effects ofthese treatments on B. burgdorferi protein expression pat-terns were then examined by two-dimensional polyacryla-mide gel electrophoresis. It has been consistently observedthat addition of AI-2-containing supernatants results in al-tered expression levels of specific borrelial proteins (ourunpublished results and [20]). It was also noted that a numberof proteins, including OspA and FlaB, were not altered intheir expression levels, indicating that the effects of AI-2were specific to a particular subset of B. burgdorferi proteins.

No discernable differences in protein profiles were observedbetween B. burgdorferi grown without any added E. coliculture supernatant and those grown in the control E. colisupernatant, indicating that E. coli DH5$ does not itselfproduce a substance that alters B. burgdorferi protein expres-sion. It is possible, however, that AI-2 synthesis by E. colicauses those bacteria to produce metabolites or other sub-stances not made in the absence of AI-2, and that it is one ormore such substances that cause B. burgdorferi to alter itsprotein profile, rather than AI-2. Studies are currently underway in our laboratory to conclusively determine whetherAI-2 or another molecule is responsible for quorum sensingin B. burgdorferi.

Some of the B. burgdorferi proteins whose expressionprofiles were altered by AI-2-containing supernatant havebeen identified. Among these are members of the Erp multi-gene family [20]. These proteins, also known as OspE–OspFrelated proteins, are outer surface lipoproteins that bind hostcomplement regulator factor H, and are therefore hypoth-esized to protect B. burgdorferi from killing by vertebratehost innate immune responses [22–24]. Proteomic and mi-croarray approaches are currently being employed to identifyadditional AI-2 modulated genes and proteins. Our prelimi-nary data suggest that expression of a number of other knownand putative outer surface proteins is regulated through AI-2(our unpublished results). It appears that this regulatory sys-tem performs an important role in organizing the surfaceproperties of B. burgdorferi.

Hübner et al. [25] recently produced a strain of B. burg-dorferi deleted for luxS. Those bacteria retained infectivitywhen administered to laboratory animals via needle inocula-tion, suggesting that LuxS is not essential for mammalianinfection. However, a significant caveat to such studies is thatthe results are all or none, and subtleties can be missed. Theauthors did not examine relative infectious doses, dissemina-tion rates, or bacterial loads in infected tissues, so it cannotyet be determined whether or not LuxS plays a role inmammalian infection. It is also important to note that thestudy examined only one segment of the B. burgdorferiinfectious cycle, so conclusions cannot be made about theroles of LuxS and AI-2 during tick infection or in transmis-sion of bacteria between arthropod vector and mammalianhost.

It is not yet known how B. burgdorferi detects AI-2. V.harveyi utilizes a dual two-component regulatory mecha-nism to detect AI-2 and transmit a signal through the cell,LuxPQ and LuxUD [15,26]. LuxP is a periplasmic proteinthat binds AI-2; LuxQ is a transmembrane protein that con-tains both a histidine protein kinase and a response regulatordomain; and LuxU and LuxD are cytoplasmic proteins com-prising a second two-component system with separate pro-tein kinase (LuxU) and response regulator (LuxD) proteins.In contrast, Salmonella typhimurium apparently uses anABC transporter-like system to detect AI-2, involving theLsrABCD proteins [27]. Analysis of the genome of B. burg-dorferi type strain B31 reveals two potential two-component

Fig. 4. Arrangement of luxS and surrounding genes on the B. burgdorferichromosome. luxS appears to be the final gene in a four-gene operon thatincludes probable homologs of pfs and metK, along with an open readingframe lacking homology to any previously described gene. This putativeoperon is flanked by divergently transcribed genes. Open reading frameswithout proven functions are designated by the temporary nomenclatureused by the Institute for Genomic Research in their characterization of the B.burgdorferi strain B31 genome [19].

993B. Stevenson et al. / Microbes and Infection 5 (2003) 991–997

regulatory protein pairs and a large number of ABC trans-porters, most of which do not have proven functions [19]. It isinteresting to note that AI-2 is derived from a ribose-containing molecule (Fig. 2) and that the periplasmic AI-2-binding proteins of both V. harveyi and S. typhimurium arehomologous to the E. coli RbsB ribose-binding protein[26,27]. The B. burgdorferi strain B31 chromosome encodesa probableABC transporter in which the putative periplasmicprotein resembles RbsB [19]—might this be the AI-2 recep-tor? Alternatively, since the Gram-negative bacteria V. har-veyi and S. typhimurium each utilize different mechanisms torespond to AI-2, it is quite possible that the distantly relatedspirochete B. burgdorferi uses a completely unique sensorymechanism. In the past few years, efficient recombinantgenetic tools have been developed for use in B. burgdorferithat will allow researchers to dissect the molecular mecha-nisms by which Lyme disease spirochetes sense and respondto AI-2.

3. B. burgdorferi regulates luxS expression levels

Bioassays conducted in our laboratory have consistentlyfailed to detect production of AI-2 by B. burgdorferi in vitro[20]. In those studies, several different strains of bacteriawere cultured at a variety of temperatures, in media withdifferent pH values, and at different cell densities, yet noAI-2 was detected by V. harveyi bioluminescence assay.These results led us to speculate that B. burgdorferi mightregulate production of the autoinducer, as do many otherbacteria [20]. Two recent studies provided evidence to sup-port that hypothesis.

Comparative microarray analyses by Ojaimi et al. [28]demonstrated that transcription of luxS can be regulated by B.burgdorferi in vitro. Bacteria cultured at 34 °C containedsignificantly greater levels of mRNA for both luxS and theputative metK than did bacteria grown at 23 °C. RT-PCRanalyses by our laboratory have confirmed temperature-dependent regulation of luxS (our unpublished results). Asnoted above, these temperatures model those experienced byB. burgdorferi in fed and unfed ticks, respectively. It appearsthat B. burgdorferi cultured at 34 °C produces AI-2 at levelsbelow the detection threshold of V. harveyi bioluminescenceassays, or biosynthetic enzyme levels are also regulated post-transcriptionally.

A second study demonstrated that B. burgdorferi regu-lates expression of the luxS gene during its natural infectioncycle. Narasimhan et al. [29] used gene arrays and RT-PCRto examine B. burgdorferi gene expression in unfed and fedinfected ticks. Among the regulated genes detected by thoseresearchers was luxS, transcript of which was undetectable inunfed ticks, but present during tick feeding.

B. burgdorferi grow significantly faster when cultivated at34 °C than they do at 23 °C [2]. Bacteria within infected ticksalso divide quite rapidly during tick feeding [30,31]. The datadescribed above indicate that these increases in growth rate

are accompanied by increases in luxS expression levels.Since all evidence indicates that B. burgdorferi cannot useLuxS for homocysteine salvage, we conclude that the spiro-chetes upregulate luxS to increase production of AI-2 duringperiods of rapid growth. Xavier and Bassler [11] observedthat several other bacterial species also increase productionof AI-2 when dividing rapidly, and speculate that autoinducerlevels may be used by bacteria to monitor a population’sgrowth rate and growth potential.

Taken together, these data suggest that B. burgdorferipossesses an AI-2-dependent quorum sensing system that isactivated during feeding of vector ticks. We propose that sucha regulatory system would provide a great benefit to B.burgdorferi at the critical point of transmission from vectorto warm-blooded host. In this model, when a bacterium in atick midgut detects signals that the tick is feeding, such asnutrients or other components of host blood and increasedtemperature, the bacterium initiates rapid cell division, en-hances transcription of luxS, and secretes AI-2. If other B.burgdorferi in the midgut likewise produce autoinducer, allthose bacteria could correctly “conclude” that the vector tickis feeding on a potential host, and coordinate synthesis ofproteins essential for transmission. Thus, a large populationof bacteria will be transmitted simultaneously, in numberssufficient to overwhelm host immune responses and success-fully establish infection. Our laboratory is continuing to testand refine this hypothesis.

4. Does B. burgdorferi possess additional quorumsensing mechanisms?

In addition to AI-2, bacteria utilize a variety of organicmolecules as quorum sensing autoinducers, including ho-moserine lactones and polypeptides. To our knowledge, no-body has thoroughly examined Lyme disease spirochetes todetermine whether they produce and secrete any of thosecompounds. It is of interest to note that some bacteria whichuse polypeptides in cell–cell signaling detect those autoin-ducers through specific ABC transporters. While B. burgdor-feri encodes such a probable ABC transporter on its chromo-some, it encodes three different oligopeptide-bindingsubunits on the chromosome and two additional subunits onsmaller “plasmid” DNAs [19,32]. Perhaps one or more ofthem functions as part of an additional, polypeptide-basedquorum sensing mechanism? As many other bacteria possessmultiple quorum sensing mechanisms, exploring B. burgdor-feri for evidence of additional systems might prove fruitful.

It was earlier suggested that B. burgdorferi possesses acell density-dependent form of quorum sensing [33,34], al-though later studies disproved that hypothesis [35,36]. Thebases for that speculation were observations that culturedbacteria in late exponential phase may produce greater quan-tities of certain proteins than do bacteria in early exponentialphase cultures. However, those phenomena are actually dueto changes in pH of the culture medium, which acidifies


during bacterial growth. Proteins previously reported as be-ing induced only at high cell density are instead produced bybacteria at low culture density if grown in pre-acidified me-dia. Furthermore, protein expression levels remain constantwhen bacteria are grown to high densities if the culturemedium is buffered to prevent pH change [35,36]. Thus,there is no evidence that B. burgdorferi utilizes a density-dependent quorum sensing mechanism that functions in theartificial environment of culture medium.

In addition to B. burgdorferi, Ixodes spp. ticks can becolonized by numerous other species of bacteria [37]. Quo-rum sensing between different bacterial species appears to bea component of some polymicrobial infections [10,38]. Itwill be interesting to see if B. burgdorferi is likewise capableof communication with co-infecting bacteria.

5. Do other spirochetes utilize quorum sensing?

Along with the Lyme disease spirochete B. burgdorferi,the genus Borrelia includes many other species, such as theagents of tick-borne relapsing fever (B. hermsii, B. parkeri,and others), the agent of louse-borne relapsing fever (B.recurrentis), and the agent of avian borreliosis (B. anserina).Genetic analysis of B. hermsii revealed that it contains ahomolog of luxS on its chromosome (our unpublished re-sults). An ancestral bacteria must therefore have acquiredluxS at some distant time preceding the divergence of Lymedisease and relapsing fever borreliae. Since the differentBorrelia species share many genetic and phenotypic similari-ties, it will be intriguing to explore whether other members ofthe genus synthesize AI-2 and use that molecule in quorumsensing.

The spirochete phylum is a diverse bacterial group thatincludes several important pathogens affecting humans anddomestic livestock. The genome of the syphilis spirochete,Treponema pallidum, has been completely sequenced [39].This bacterium carries physically separated homologs ofboth metK and pfs, but lacks a luxS homolog. The chromo-some of a periodontal disease-associated treponeme,T. denticola, has also been sequenced but is not yetannotated (http://hgsc.bcm.tmc.edu/microbial/Tdenticola/).A T-Blast-N analysis of the chromosome on 27 May 2003revealed that T. denticola likewise contains homologs ofmetK and pfs but lacks luxS. Thus, the treponemes appear todetoxify SAH via Pfs without subsequent production of AI-2and homocysteine. The genome of a third spirochete, theleptospirosis agent Leptospira interrogans, has also beencompleted, and contains a metK homolog but lacks both luxSand pfs [40]. L. interrogans apparently detoxifies SAHthrough a one-step mechanism utilizing SAH hydrolase, asdo eukaryotes, archaea, and many other bacteria [40]. Thesedata strongly suggest that neither leptospires nor treponemescan produce AI-2, raising the possibility that AI-2-dependentquorum sensing among spirochetes may be limited to thegenus Borrelia. Perhaps the arthropod-dependent transmis-sion strategies of borreliae require the coordination afforded

by quorum sensing, whereas other infectious spirochetes canefficiently “go it alone”? Continued examination of thepathogenic properties of B. burgdorferi and comparisonswith other spirochetes will get to the bottom of this question.Additionally, as the genomes of more spirochete species areexamined, a picture will develop as to when bacteria of thisphylum acquired the LuxS/AI-2 system, or whether onlycertain spirochetes have lost that capability.

6. Conclusions and perspectives

Significant evidence indicates that Lyme disease spiro-chetes use quorum sensing as an important mechanism tocontrol protein expression. The bacteria encode a functionalLuxS protein and are thus capable of producing AI-2. Asevidenced by dramatic changes in protein profiles upon ex-posure to AI-2, B. burgdorferi also has the ability to recog-nize that molecule as a signal to alter expression of specificproteins. B. burgdorferi regulates expression of luxS, sug-gesting that it can also regulate biosynthesis of AI-2, produc-ing the autoinducer only at specific times during the bacterialinfectious cycle.

The complexity of its infectious cycle mandates thatB. burgdorferi precisely sense the environment and regulateprotein expression. An individual bacterium must interactwith many different tissue types during that cycle, derivenutrition from both warm-blooded host and vector tick, andavoid clearance by host and vector immune systems. Theremay be additional benefits in having the whole population ofbacteria coordinate certain functions. For instance, simulta-neous transmission of large numbers of B. burgdorferi from atick into a warm-blooded animal will increase the odds that atleast some bacteria survive the host’s immune system andestablish infection. As luxS expression is enhanced duringtick feeding, AI-2-dependent quorum sensing may servesuch a function. If so, counteracting this quorum sensingmechanism may prove to be a useful target for prevention ofB. burgdorferi transmission into humans and other animals.

The few studies so far conducted on B. burgdorferi quo-rum sensing only scratch the surface of this topic, and manyquestions remain to be answered about this system. Contin-ued studies of quorum sensing and other mechanisms bywhich B. burgdorferi controls gene expression will be essen-tial to understanding the biology of this bacterium and itspathogenic properties, and for development of improvedvaccines and therapies for treatment of Lyme disease.

Acknowledgements

Research in our laboratory is funded by U.S. NationalInstitutes of Health grants R01–AI44254, R01–AI53101,and 5T32–AI49795. We thank Natalie Mickelsen for techni-cal assistance and John Carmen for comments on this manu-script.


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Quorum sensing by the Lyme disease spirochete