Temperature-Dependent Variations and Intraspecies Diversity of the Structure of the Lipopolysaccharide of Yersinia pestis ²,‡ Yuriy A. Knirel, § Buko Lindner, # Evgeny V. Vinogradov, §,⊥ Nina A. Kocharova, § Sof’ya N. Senchenkova, § Rima Z. Shaikhutdinova, | Svetlana V. Dentovskaya, | Nadezhda K. Fursova, | Irina V. Bakhteeva, | Galina M. Titareva, | Sergey V. Balakhonov, O Otto Holst, # Tat’yana A. Gremyakova, | Gerald B. Pier,* ,& and Andrey P. Anisimov | N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow 119991, Russia, Research Center Borstel, Leibniz Center for Medicine and Biosciences, D-23845 Borstel, Germany, State Research Center for Applied Microbiology, Obolensk 142279, Moscow Region, Russia, Antiplague Research Institute of Siberia and Far East, Irkutsk 664047, Russia, and Channing Laboratory, Brigham and Women’s Hospital, HarVard Medical School, Boston, Massachusetts 02115 ReceiVed July 22, 2004; ReVised Manuscript ReceiVed October 18, 2004 ABSTRACT: Yersinia pestis spread throughout the Americas in the early 20th century, and it occurs predominantly as a single clone within this part of the world. However, within Eurasia and parts of Africa there is significant diversity among Y. pestis strains, which can be classified into different biovars (bv.) and/or subspecies (ssp.), with bv. orientalis/ssp. pestis most closely related to the American clone. To determine one aspect of the relatedness of these different Y. pestis isolates, the structure of the lipopolysaccharide (LPS) of four wild-type and one LPS-mutant Eurasian/African strains of Y. pestis was determined, evaluating effects of growth at mammalian (37 °C) or flea (25 °C) temperatures on the structure and composition of the core oligosaccharide and lipid A. In the wild-type clones of ssp. pestis, a single major core glycoform was synthesized at 37 °C whereas multiple core oligosaccharide glycoforms were produced at 25 °C. Structural differences occurred primarily in the terminal monosaccharides. Only tetraacyl lipid A was made at 37 °C, whereas at 25 °C additional pentaacyl and hexaacyl lipid A structures were produced. 4-Amino-4-deoxyarabinose levels in lipid A increased with lower growth temperatures or when bacteria were cultured in the presence of polymyxin B. In Y. pestis ssp. caucasica, the LPS core lacked D-glycero-D-manno-heptose and the content of 4-amino-4-deoxyarabinose showed no dependence on growth temperature, whereas the degree of acylation of the lipid A and the structure of the oligosaccharide core were temperature dependent. A spontaneous deep-rough LPS mutant strain possessed only a disaccharide core and a slightly variant lipid A. The diversity and differences in the structure of the Y. pestis LPS suggest important contributions of these variations to the pathogenesis of this organism, potentially related to innate and acquired immune recognition of Y. pestis and epidemiologic means to detect, classify, control and respond to Y. pestis infections. Bubonic and pneumonic plague is caused by the Gram- negative bacterium Yersinia pestis circulating in natural foci in Eurasia, Africa, and the Americas, which involve a rodent reservoir and an insect vector (1-7). Enzootic Y. pestis infection is due to continual transmission among susceptible rodents by various flea vectors, which results in acute infection with bacteremia in their enzootic rodent hosts. The high lethality of plague in rodent reservoirs is necessary for the organism’s continued transmission in nature. Fleas ingesting infected blood during preagonal bacteremia must depart from the host and feed on a new rodent, which subsequently becomes infected (1-7). It has been assumed that selective pressures within different host species and fleas contribute to the emergence of variant strains of Y. pestis which have been variously classified as biovars (bv.), based on differences in glycerol fermentation, nitrate reduction, and ammonia oxidation, or as subspecies (ssp.) or ecotypes (4), wherein strains are differentiated due to variations in fermentative activity, nutritional requirements, and ability to cause infectious bacteremia and death in diverse mam- malian species. The pathogenicity of Y. pestis is determined, in part, by a number of bacterial features that counteract mammalian (1- 8) and insect (1, 2, 5-9) antimicrobial factors, ensuring ² Research performed within the framework of the International Science and Technology Center (ISTC) Partner Project #1197, sup- ported by the Cooperative Threat Reduction Program of the US Department of Defense (ISTC Partner). Research also supported by the Russian Ministry for Industry, Science and Technology Contract #43.600.1.4.0031 and the German Research Foundation Grant LI-448- 1. ‡ Different parts of this work were presented at the eighth Interna- tional Symposium on Yersinia, September 4-8, 2002 in Turku, Finland, the 12th European Carbohydrate Symposium, July 6-12, 2003 in Grenoble, France, and the 104th General ASM Meeting, May 23-27, 2004 in New Orleans, LA. * Corresponding author. Mailing address: Channing Laboratory, 181 Longwood Av., Boston, MA 02115. Telephone: 617-525-2269. Fax: 617-525-2510. E-mail: gpier@rics.bwh.harvard.edu.- § Zelinsky Institute of Organic Chemistry. # Leibniz Center for Medicine and Biosciences. ⊥ Present address: Institute for Biological Sciences, National Re- search Council, 100 Sussex Dr., Ottawa ON, Canada K1A 0R6. | State Research Center for Applied Microbiology. O Antiplague Research Institute of Siberia and Far East. & Harvard Medical School. 1731 Biochemistry 2005, 44, 1731-1743 10.1021/bi048430f CCC: $30.25 © 2005 American Chemical Society Published on Web 01/08/2005