MLST of housekeeping genes captures geographic population structure and suggests a European origin of Borrelia burgdorferi Gabriele Margosa,b, Anne G. Gatewoodc, David M. Aanensend, Kla´ ra Hanincova´ e, Darya Terekhovae, Stephanie A. Vollmera, Muriel Cornetf, Joseph Piesmang, Michael Donaghyh, Antra Bormanei, Merrilee A. Hurnj, Edward J. Feila, Durland Fishc, Sherwood Casjensk, Gary P. Wormserl, Ira Schwartze, and Klaus Kurtenbacha Departments of aBiology and Biochemistry and jMathematical Sciences, University of Bath, Bath BA2 7AY, United Kingdom; cDepartment of Epidemiology and Public Health, Yale School of Medicine, Yale University, New Haven, CT 06520; dDepartment of Infectious Disease Epidemiology, Imperial College London, St. Mary’s Hospital, London W2 1PG, United Kingdom; eDepartment of Microbiology and Immunology and lDivision of Infectious Diseases, Department of Medicine, New York Medical College, Valhalla, NY 10595; fCentres Nationaux de Re´fe´ rence des Borrelia et de la Leptospirose, Institut Pasteur, 75724 Paris Cedex 15, France; gBacterial Diseases Branch, Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, Ft. Collins, CO 80521; hDepartment of Clinical Neurology, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, United Kingdom; iPublic Health Agency, LV-1012, Riga, Latvia; and kDivision of Cell Biology and Immunology, Department of Pathology, University of Utah Medical School, Salt Lake City, UT 84132 Edited by Barry J. Beaty, Colorado State University, Fort Collins, CO, and approved April 23, 2008 (received for review January 16, 2008) Lyme borreliosis, caused by the tick-borne bacterium Borrelia where B. burgdorferi is now prevalent: the Northeast, the upper burgdorferi, has become the most common vector-borne disease in Midwest, and northern coastal California. B. burgdorferi is North America over the last three decades. To understand the relatively rare in Europe compared with the northeastern United dynamics of the epizootic spread and to predict the evolutionary States and has not been recorded in Asia (4, 35–37). trajectories of B. burgdorferi, accurate information on the popu- The genome of B. burgdorferi is remarkable among bacteria, lation structure and the evolutionary relationships of the pathogen in that it consists of a linear chromosome, which encodes Ϸ50% is crucial. We, therefore, developed a multilocus sequence typing of the predicted proteome, and a large number of linear and (MLST) scheme for B. burgdorferi based on eight chromosomal circular plasmids (38, 39). Both chromosomal and plasmid- housekeeping genes. We validated the MLST scheme on B. burg- located loci have been used for typing B. burgdorferi, such as the dorferi specimens from North America and Europe, comprising chromosomal rrs-rrlA intergenic spacer (IGS) (40, 41), the gene both cultured isolates and infected ticks. These data were com- encoding the membrane protein p66 (42), or the plasmid- pared with sequences for the commonly used genetic markers located, hypervariable gene encoding the immunodominant rrs-rrlA intergenic spacer (IGS) and the gene encoding the outer outer surface protein C (ospC) (25, 43). surface protein C (ospC). The study demonstrates that the concat- Unambiguous genotyping systems are key to describing epi- enated sequences of the housekeeping genes of B. burgdorferi demiological and ecological patterns and illuminating the evo- provide highly resolved phylogenetic signals and that the house- lutionary processes that shape microbial populations. Several keeping genes evolve differently compared with the IGS locus and recent studies have used sequence information of multiple loci ospC. Using sequence data, the study reveals that North American to characterize Lyme borreliosis spirochetes (15, 17, 41, 44, 45). and European populations of B. burgdorferi correspond to genet- However, these typing approaches deviate from typical multilo- ically distinct populations. Importantly, the MLST data suggest that cus sequence typing (MLST) schemes or multilocus sequence B. burgdorferi originated in Europe rather than in North America as analysis (MLSA) (46) developed for other microbial pathogens proposed previously. in that different categories of loci were combined, such as hypervariable genes encoding outer surface proteins, conserved ͉ ͉ evolution Lyme borreliosis ticks housekeeping genes, or noncoding loci. Most MLST/MLSA schemes are based on housekeeping yme borreliosis is the most prevalent vector-borne disease in the genes, which are subject to purifying selection and slow evolu- Ltemperate zone of the Northern Hemisphere. It is a tick-borne tion, and the variation within these genes is nearly neutral (47). bacterial zoonosis, with hard ticks of the genus Ixodes acting as Although there are normally fewer polymorphic sites in indi- vectors (1) and various vertebrate species serving as reservoir hosts vidual housekeeping genes compared with hypervariable genes, (2–6). At present, Lyme borreliosis spirochetes constitute a group using the combined sequences of multiple housekeeping genes of 13 named species (7–18). Several of these, namely Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii, and Borrelia spielmanii, are associated with disease in humans (8, 19–22). B. burgdorferi Author contributions: G.M., D.F., and K.K. designed research; G.M., A.G.G., K.H., and S.A.V. (sometimes referred to as B. burgdorferi sensu stricto) occurs in both performed research; A.G.G., D.M.A., K.H., D.T., M.C., J.P., M.D., A.B., S.C., and G.P.W. contributed new reagents/analytic tools; G.M., M.A.H., E.J.F., I.S., and K.K. analyzed data; Europe and North America (23). In the United States, Lyme and G.M., E.J.F., I.S., and K.K. wrote the paper. disease is caused by B. burgdorferi only (24). The authors declare no conflict of interest. Despite almost three decades of research and control efforts, the numbers of new human cases of Lyme borreliosis in the This article is a PNAS Direct Submission. United States continue to increase (24). It is likely that this Freely available online through the PNAS open access option. reflects increasing population sizes and geographic ranges of Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. EU375814–EU375823 and EU377743–EU377781 for ospC and pathogenic genotypes of B. burgdorferi (25) because it is known EU375824–EU375833 and EU377782–EU377822 for the IGS). Sequences of the housekeep- that populations of the principal vector tick in eastern North ing genes have been submitted to the Multi Locus Sequence Typing web site, www.mlst.net America, Ixodes scapularis, have been spreading from past and can be accessed via strain ID or ST. refuges into new woodland habitats (26–34). In addition, it is bTo whom correspondence should be addressed. E-mail: [email protected]. likely that the wide host range of B. burgdorferi has been This article contains supporting information online at www.pnas.org/cgi/content/full/ facilitating its epidemic dispersal in the northeastern United 0800323105/DCSupplemental. States (2, 3). There are three main regions in the United States © 2008 by The National Academy of Sciences of the USA 8730–8735 ͉ PNAS ͉ June 24, 2008 ͉ vol. 105 ͉ no. 25 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0800323105 Downloaded by guest on September 30, 2021 has been shown to provide high discriminatory power while Table 1. STs, IGS types, and ospC major groups of B. burgdorferi retaining signatures of longer-term evolutionary relationships or Strain MLST ST IGS type* ospC group† clonal stability (47–50) (see also www.mlst.net). Furthermore, analyses of multiple loci can buffer against potentially skewed B31, BL206, B515, 16812UT 1 1 A evolutionary pictures obtained by single-locus analyses (49, 51). Ca4, Ca5, Ca6 2 1 A Despite their power, typical MLST/MLSA schemes have not yet 297, B504, 498801UT 3 2 K been applied as tools in population or landscape genetics studies B509 4 2 H of vector-borne pathogens. Ca࿝WTB27 5 2 A To contribute to the understanding of the emergence of Lyme Ca92-0953 6 2 H borreliosis, we aimed to test whether B. burgdorferi is structured MR623, B373 7 3 B geographically and to infer the evolutionary origin of this B156 8 4 F bacterial species. We, therefore, have developed a MLST scheme MR661, MR654, 15506UT 9 4 N for B. burgdorferi based on housekeeping genes to characterize Ca࿝WTB32 10 4 F B. burgdorferi populations at the different phylogenetic levels JD1, BL538, BL515, 114311UT 11 5 C required for evolutionary, epidemiological, and population ge- BL522, B356 12 6 M netics analyses. We show that North American and European Ca92-1337 13 6 M populations of B. burgdorferi constitute distinct lineages and that MR616, 15912UT 14 6 G the housekeeping genes evolve differently compared with the B500 15 7 I IGS locus and ospC. Importantly, phylogenetic analyses of the B331, B361 16 7 I concatenated housekeeping genes suggest that B. burgdorferi Ca92-1096 17 7 I originated in Europe. B485, MR607, MR662 18 8 U N40, B418, MR640, B348, 15903UT 19 9 E Results IPT2, IPT69, IPT191 20 1 B B. burgdorferi Samples and Housekeeping Genes Analyzed. Speci- IPT23, IPT190 20 1 M1 mens of B. burgdorferi from the Northeast and Midwest United 20604LT 20 1 ND States, California, and Europe that were analyzed by MLST are 22521LT 20 ND ND listed in supporting information (SI) Table S1. These included IPT19 21 1 Q field-collected infected ticks and isolates from patients and ticks. 21509LT 21 ND ND The characteristics of the eight housekeeping genes selected, i.e., IPT135 22 1 Q clpA, clpX, nifS, pepX, pyrG, recG, rplB, and uvrA, such as GC IPT137 23 1 B content (10) or dN/dS ratio, are shown in Table S2. IPT39 24 5 S IPT58 24 5 L MLST of B. burgdorferi and Comparison with the Major IGS Genotypes IPT193, IPT198 25 10 N1 and ospC Major Groups. In total, 33 sequence types (STs) were NE49 26 10 Q defined among the 64 samples of B.