University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2007-12-20 Surface of Yersinia pestis: LCRV, F1 Production, Invasion and Oxygen: A Dissertation Kimberly Lea Pouliot University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Amino Acids, Peptides, and Proteins Commons, Bacteria Commons, Bacterial Infections and Mycoses Commons, Biological Factors Commons, and the Inorganic Chemicals Commons Repository Citation Pouliot KL. (2007). Surface of Yersinia pestis: LCRV, F1 Production, Invasion and Oxygen: A Dissertation. GSBS Dissertations and Theses. https://doi.org/10.13028/5ayn-7c77. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/358 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. THE SURFACE OF YERSINIA PESTIS: LCRV, F1 PRODUCTION, INVASION AND OXYGEN A Dissertation Presented Kimberly Lea Pouliot Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY December 20 2007 Program of Molecular Genetics and Microbiology December 20 , 2007 THE SURFACE OF YERSINIA PESTIS: LCRV, F1 PRODUCTION, INVASION AND OXYGEN A Dissertation Presented KIMBERLY LEA POULIOT The signatures of the Dissertation Defense Committee signifies completion and a proval as to style and content of the Dissertation Jon D. Goguen , Thesis Advisor Joa n Mecsas, Member of Committee Egil Lien , Member of Committee Madelyn Schmidt, Member of Committee Neal Silverman , Member of Committee The signature of the Chair of the Committee signifies that the written dissertation meets the requirements of the Dissertation Committee John M. Leong, Chair of Committee The signature of the Dean of the Graduate School of Biomedical Sciences signifies that the student has met all graduation requirements of the school An1hony Carruthers PhD. Dean of the Graduate School of Biomedical Sciences Program in Molecular Genetics and Microbiology December 20 , 2007 COPYRIGHTS Chapter II is copyrighted by the American Society for Microbiology, Washington DC. Chapter II. Evaluation of the role of LcrVITLR2-mediated immunomodulation in the virulence of Yersinia pestis. Kimberly Pouliot, Ning Pan, Shixia Wang, Shan , Egil Lien , and Jon D, Goguen. 2007. Infection and Immunity 75: 3571-3580. ACKNOWLEDGEMENTS I must first thank my mentor, Jon Goguen , for his unwavering patience support and wisdom. He truly fostered my love of the biological sciences. I thank my thesis committee, Egil Lien , John Leong, Madelyn Schmidt and Neal Silverman , for all their support and advice. Thanks also go out to outside committee member, Joan Mecsas , for agreeing to participate in my defense. I thank my collaborators: Ning Pan , Shan Lu , Shixia Wang, Eicke Latz Leslie Shaw and Brian Akerley from UMMS; Ralph Isberg and Vicki Auerbach from Tufts. I give special thanks to past lab members: Chrono Lee , Nancy Deitemeyer, and Karen Gingris for their technical assistance. I would like to thank my family and friends for their continuing support and encouragement. Special thanks go to my parents and grandmother, my brother Matthew Copeland , Roman Berman , Vanessa Melanson and Thomas Pfeiffer. Abstract Of the eleven species of bacteria that comprise the genus Yersinia of the family Enterobacteriaceae three speCIes are pathogenic for humans. Yersinia pseudotuberculosis and Yersinia enterocolitica usually cause a mild, self-limiting mesenteric lymphadenitis or ileitis. Yersinia pestis causes a highly invasive often fatal disease known as plague. All three elaborate a type three secretion system that is essential for virulence and encoded on closely related plasmids. In Y pestis all the effectors, structural components and chaperones are encoded on the 70kb plasmid, pCD I. Of these , LcrV from enterocolitica has been implicated in playing an immunosuppressive role through its interaction with host Toll-like receptor 2 (TLR2) and induction of IL- IO. Through expression and purification of recombinant LcrV from Escherichia coli we show that only high molecular weight species of rLcrV are able to stimulate TLR2. In a highly sensitive subcutaneous mouse infection model we demonstrate no difference in the time to death between TLR2-sufficient or deficient mice. Analysis of cytokine levels between these two genotypes also shows no significant difference between splenic IL- IO and IL-6 or levels of bacteria. We conclusively show that this interaction, if it does occur, plays no significant role in vivo. In a separate set of experiments, we also determined that the expression of , a peptide shown to be responsible for 37 dependent inhibition of invasion by pestis in vitro was significantly decreased under high oxygen conditions. This led us to re-examine the invasion phenotype both in vitro and in vivo. These results give new insights into virulence gene expression in Y pestis by environmental cues other than temperature Table of Contents Signature Page Copyrights II. Acknowledgements i i i. Abstract IV. Table of Contents List of Tables VII. List of Figures IIX. Chapter One. Introduction Plague and the biology of Yersinia pestis Evasion of innate immunity through thermally-inducible virulence factors Other recognized Virulence Factors of Yersinia pestis 12. Chapter Two. Evaluation of the role of LcrV/TLR2-mediated immunomodulation in the virulence of Yersinia pestis 17. Introduction 18. Materials and Methods 20. Results 26. Discussion 38. Acknowledgements 45. Addendum 46. Chapter Three. A Brief Review of Natural and Experimental 47. Transmission of Y. pestis List of Figures Chapter Two. Figure 1. Activation of TLR2 by rLcrV 28. Figure 2. Analysis of rLcrV by native PAGE 30. Figure 3. Analysis of rLcrV by gel filtration 32. Figure 4. Analysis of an alternative rLcrV construct by 34. gel filtration Figure 5. TLR2 deficiency and survival of Y. pestis- 35. infected mice Figure 6. TLR2 deficiency has little effect during infection 37. with Y. pestis Chapter Four. Figure 7. Transmission Electron Microscopy Images of 63. Yersinia pestis- infected cell monolayers. Figure 8. Enhanced Invasion of WI26 cells by Y. pestis 65. grown at 37 C at air interface Figure 9. Low oxygen conditions inhibit high efficiency invasion 66. Figure 10. Requirement for very high oxygen levels for 66. efficient invasion during 37 C growth in liquid culture Figure 11. Detection of F1 67. Figure 12. An F1 deletion mutant retains invasive ability 68. at 37 C independent of oxygen ability Figure 13. Y. pestis grown under microaerophillic conditions 70. are significantly more virulent than aerobically-grown bacteria V11 Figure 14. Intracellular survival following infection of WI26 cells Chapter Five. Figure 15. Pia-mediated invasion by E. coli 89. Figure 16. The role of protease activity of Pia in invasion of 90. cultured epithelial cells Figure 17. Invasion of 30 grown Y. pestis is not 93. influenced by the presence of the 4 subunit of the laminin receptor. Figure 18. Pia expression is repressed by 95. Figure 19. F1 expression is repressed under conditions 97. of low pH Figure 20. Tunicamycin treatment of WI26 cells specifically 99. inhibits invasion by 30 grown , but not 37 grown Y. pestis. Figure 21. Y. pestis utilizes a separate invasion pathway 100. from Y. pseudotuberculosis Chapter One. Introduction Plague and the biology of Yersinia pestis Of the eleven species of bacteria that comprise the genus Yersinia of the family Enterobacteriaceae, three species are pathogenic for humans. Yersinia pseudotuberculosis and Yersinia enterocolitica usually cause a mild, self-limiting mesenteric lymphadenitis or ileitis. In contrast, Yersinia pestis causes a highly invasive often fatal disease known as plague. Plague has been responsible for three major pandemics, each involving millions of deaths. The second of these, often known as the “Black Death” (1347-1351), killed close to one third of the European population. There are currently 1,000 to 3,000 cases of plague a year, mostly in Africa, Asia and South America, with 10-15 cases occurring in rural areas of the Southwestern United States. It is clear that Y. pestis is derived from a single clone of Y. pseudotuberculosis, and estimates based on a “mutational clock” theory indicate that this divergence occurred only 1500 to 20,000 years ago [1]. While the enteric yersiniae are transmitted to humans via the oral-fecal route, transmission of Y. pestis mainly occurs through the bite of infected fleas, an adaptation facilitated by the acquisition of two plasmids pMT1, pPCP1. pMT1 encodes genes necessary for colonization of the flea gut, while pPCP1 enhances dissemination from flea bite to bloodstream [2, 3]. Yersinia pestis is endemic in rodent populations in the southwestern United States, Africa, South America and Asia. The rat flea, Xenopsylla cheopis, feeds on infected rodents and other small mammals, engulfing bacteria-laden blood meals. Within 2 the flea gut, the bacteria produce two products of particular importance. A phospholipase D (Ymt) that is essential for protection from flea antibacterial defenses, and a surface polysaccharide that promotes aggregation of the bacteria and