THE UNIVERSITY OF CHICAGO THE GLUTATHIONYLATION OF YERSINIA PESTIS LCRV AND ITS EFFECTS ON PLAGUE PATHOGENESIS A DISSERTATION SUBMITTED TO THE FACULTY OF THE DIVISION OF THE BIOLOGICAL SCIENCES AND THE PRITZKER SCHOOL OF MEDICINE IN CANDIDACY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY COMMITTEE ON MICROBIOLOGY BY ANTHONY KEITH MITCHELL CHICAGO, ILLINOIS AUGUST 2018 COPYRIGHT ©2018 BY ANTHONY KEITH MITCHELL TABLE OF CONTENTS PAGE LIST OF FIGURES ....................................................................................................................... iv LIST OF TABLES ......................................................................................................................... vi ACKNOWLEDGEMENTS .......................................................................................................... vii ABSTRACT ................................................................................................................................... ix CHAPTERS I. INTRODUCTION ...................................................................................................1 II. GLUTATHIONYLATION OF YERSINIA PESTIS LCRV AND ITS EFFECTS ON PLAGUE PATHOGENESIS ........................................................18 III. CONCLUSION ......................................................................................................57 APPENDICES A. LCRV MUTANTS THAT ABOLISH YERSINIA TYPE III INJECTISOME FUNCTION.................................................................................63 B. YFBA, A YERSINIA PESTIS REGULATOR REQUIRED FOR COLONIZATION AND BIOFILM FORMATION IN THE GUT OF CAT FLEAS ....................................................................................................84 C. FIGURES .............................................................................................................103 D. TABLES ..............................................................................................................148 REFERENCES ............................................................................................................................159 iii LIST OF FIGURES PAGE 1. LcrV secreted by Y. pestis is glutathionylated at Cys273 ..................................................104 2. Mass determination and Edman sequencing of LcrVS228 ................................................105 3. The lcrVC273A mutation abolishes LcrV glutathionylation and accelerates Y. pestis-mediated macrophage death ..............................................................................107 4. The codon substitution Cys273Ala, which precludes LcrV glutathionylation, does not affect Y. pestis type III secretion of Yop effectors ............................................109 5. Glutathionylation of LcrV enhances bubonic plague pathogenesis .................................111 6. Kinetics of disease progression and host adaptive immune responses in bubonic plague-infected rodents ......................................................................................112 7. The codon substitution Cys273Ser abolishes Y. pestis posttranslational modification of LcrV and attenuates virulence in a mouse model of bubonic plague .................................................................................................................114 8. LcrV binds to macrophage RPS3 and modulates host inflammatory responses .............116 9. yopJ is dispensable for lcrVC273A-mediated killing of Y. pestis-infected macrophages, and type III secretion is not impacted by loss of glutathione synthetase (gshB) .............................................................................................................118 10. Extracellular glutathione modifies secreted LcrV and promotes Y. pestis survival in blood ..............................................................................................................120 A1. Yersinia pestis lcrV mutants with the dominant negative low-calcium response (LCR−) phenotype ............................................................................................................122 A2. Short extensions at the C terminus of LcrV cause a dominant negative LCR− phenotype in Y. pestis ......................................................................................................124 A3. Strep tag insertions in LcrV .............................................................................................125 A4. Affinity chromatography of Strep-tagged LcrV ..............................................................127 A5. Strep-tagged LcrV and Yersinia enterocolitica effector translocation ............................128 iv PAGE A6. LcrVS324 caps YscF needles that lack YopD ....................................................................130 A7. Strep-tagged LcrVs and their LCR phenotypes in Yersinia pestis ..................................131 A8. Affinity chromatography of Strep-tagged LcrV expressed in Y. pestis ...........................132 A9. Strep-tagged LcrV and Yersinia pestis effector translocation .........................................134 − A10. LcrVS324 causes a dominant negative LCR phenotype in Y. pestis harboring calcium-blind alleles yscFD28A and/or yscFD46A ...............................................................136 B1. Y. pestis colonization of cat fleas requires the hmsF locus ..............................................137 B2. Y. pestis induced aggregates and gut blockade in cat fleas ..............................................139 B3. Y. pestis forms biofilms in the proventriculus and in the gut of infected cat fleas ............................................................................................................................141 B4. Three LysR-type transcriptional regulators contribute to Y. pestis in vitro biofilm formation .............................................................................................................143 B5. The yfbA gene is required for Y. pestis CO92(ΔpCD1) cat flea colonization and biofilm formation ......................................................................................................145 B6. The yfbA gene is required for Y. pestis CO92(ΔpCD1) biofilm formation in cat fleas ............................................................................................................................147 v LIST OF TABLES PAGE 1. Summary of mass spectrometry analysis of tryptic peptides from Y. pestis LcrVS228 and E. coli rLcrVS228 .........................................................................................149 2. Tandem mass spectrometry of the 1,985.79-Da LcrVS228 peptide ...................................151 3. Peptide mass fingerprinting by LC-MS/MS identifies macrophage RPS3 as a ligand of translocated LcrV ......................................................................................152 4. MALDI-TOF MS analysis of LcrVS228 and LcrVS228 C273S purified from Y. pestis supernatants or E. coli extracts ..........................................................................153 5. Bacterial strains and plasmids used in this study .............................................................154 6. Primers used in this study ................................................................................................155 A1. Missense mutations in lcrV codon 327 block Yersinia pestis type III secretion .............156 A2. Insertions of the eight-codon Strep tag at various positions into lcrV and its effect on Yersinia pestis type III secretion of YopE or LcrV .....................................157 A3. LcrVS324 blocks type III secretion of YopH in Y. pestis calcium-blind mutants with yscFD28A and yscFD46A mutations .............................................................................158 vi ACKNOWLEDGEMENTS Many mentors, colleagues, friends, and family members have provided me with invaluable assistance throughout my tenure as a graduate student. I am especially grateful for the enthusiastic supervision, commitment, and generous resources of Dr. Olaf Schneewind. It is often said that the most important phase of any scientific endeavor occurs prior to the start of a single experiment, namely, by asking the right questions. In this respect, as in numerous others, nowhere could I have found a better mentor to guide me to a more comprehensive understanding of the process and methodology that underpins fruitful scientific inquiry. I would also like to express my sincere gratitude to the other members of my Dissertation Committee—Dr. Sean Crosson, Dr. Dominique Missiakas, Dr. Howard Shuman, and Dr. Alexander Chervonsky—for their support and guidance over the past several years; for their beneficial insights and commentary on my research projects; and for sharing their scientific expertise on topics ranging from genetics and biochemistry to immunology and bacterial pathogenesis. Members of the Schneewind–Missiakas laboratory, both past and present, are likewise deserving of many thanks for their willingness to offer thoughtful scientific advice, to engage in stimulating discussions on any number of subjects, and to provide assistance with, and training in, new experimental techniques. In particular, I would like to thank Dr. Bill Blaylock who not only trained me in the technical and intellectual aspects of experimental microbiology upon my arrival at the laboratory but also exhibited an ongoing passion and dedication to research and teaching that has been a lasting inspiration. I would also like to acknowledge the Pritzker School