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MIAMI UNIVERSITY the Graduate School MIAMI UNIVERSITY The Graduate School Certificate for Approving the Dissertation We hereby approve the Dissertation of Steven Lindau Distelhorst Candidate for the Degree Doctor of Philosophy ______________________________________ Dr. Mitchell F. Balish, Director ______________________________________ Kelly Z. Abshire, Reader ______________________________________ Natosha L. Finley, Reader ______________________________________ Joseph M. Carlin, Reader ______________________________________ Jack C. Vaughn, Graduate School Representative ABSTRACT UNDERSTANDING VIRULENCE FACTORS OF MYCOPLASMA PENETRANS: ATTACHMENT ORGANELLE ORGANIZATION AND GENE EXPRESSION by Steven Lindau Distelhorst The ability to establish and maintain cell polarity plays an important role in cellular organization for both functional and morphological integrity in eukaryotic and prokaryotic organisms. Like eukaryotes, bacteria, including the genomically reduced species of the Mycoplasma genus, use an array of cytoskeletal proteins to generate and maintain cellular polarity. Some mycoplasmas, such as Mycoplasma penetrans, exhibit a distinct polarized structure, known as the attachment organelle (AO), which is used for attachment to host cells and motility. The M. penetrans AO, like AOs of other mycoplasmas, contains a cytoskeletal structure at the core, but lacks any homologs of identified AO core proteins of other investigated mycoplasmas. To characterize the composition of the M. penetrans AO cytoskeleton we purified the detergent-insoluble core material and examined its structure using scanning electron microscopy and cryo-electron tomography. The ultrastructure of the M. penetrans AO core was distinct from those of other mycoplasmas. We identified several proteins from the detergent-insoluble fractions using mass spectrometry. Among twelve proteins identified four likely structural proteins had coding genes that were identified as members of a six-gene operon. Sequence analysis of these six proteins, along with another protein identified as a likely AO component, revealed predicted properties similar to AO cytoskeletal proteins from Mycoplasma pneumoniae, a member of a different phylogenetic cluster, despite a lack of sequence homology. These data support the hypothesis that AOs have independent evolutionary origins, but also suggest convergent evolution of AO organization at the molecular level. The genes encoding these M. penetrans AO proteins were found conserved in the closely related species Mycoplasma iowae. Because M. penetrans is currently genetically intractable whereas M. iowae can be genetically manipulated, we attempted to examine the localization of a homolog of one of the proteins from this cytoskeletal operon. Although our attempts to localize one of these structural proteins were unsuccessful, we constructed a plasmid that can be used to generate a chimera of these proteins via translational fusion with GFP for future studies. We also examined gene expression in M. penetrans cells grown in both the presence and absence of HeLa cells. There was very little significant difference in gene expression between the two conditions, suggesting that M. penetrans cells express the genes needed for early infection even in the absence of HeLa cells. Based on the data from these studies we propose a model for the growth and development of the M. penetrans AO core. UNDERSTANDING VIRULENCE FACTORS OF MYCOPLASMA PENETRANS: ATTACHMENT ORGANELLE ORGANIZATION AND GENE EXPRESSION A DISSERTATION Presented to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Microbiology by Steven L. Distelhorst The Graduate School Miami University Oxford, Ohio 2017 Dissertation Director: Dr. Mitchell F. Balish © Steven Lindau Distelhorst 2017 TABLE OF CONTENTS INTRODUCTION ........................................................................................................................................ 1 A. Significance of bacterial cell polarity ...................................................................................... 2 B. Polarity among mycoplasmas .................................................................................................... 3 C. Importance of Mycoplasma attachment organelles .......................................................... 4 D. Attachment organelles of other Mycoplasma species ...................................................... 5 E. M. penetrans ....................................................................................................................................... 9 F. Importance of studying the M. penetrans attachment organelle ............................... 11 G. Hypotheses ....................................................................................................................................... 12 CHAPTER 1: The variable internal structure of the Mycoplasma penetrans attachment organelle revealed by biochemical and microscopic analyses ........ 14 Abstract ................................................................................................................................................... 15 Introduction .......................................................................................................................................... 16 Materials and Methods ..................................................................................................................... 19 Results ..................................................................................................................................................... 23 Discussion .............................................................................................................................................. 40 Acknowledgments .............................................................................................................................. 44 CHAPTER 2: Creation of tools to examine attachment organelle protein localization in Mycoplasma iowae, a new genetic model for Mycoplasma penetrans ................................................................................................................................................... 45 Abstract ................................................................................................................................................... 46 Introduction .......................................................................................................................................... 47 Methods .................................................................................................................................................. 51 Results ..................................................................................................................................................... 58 Discussion .............................................................................................................................................. 67 Acknowledgments .............................................................................................................................. 70 CHAPTER 3: Analysis of Mycoplasma penetrans global gene expression in the presence and absence of HeLa cells ........................................................................................... 71 Abstract ................................................................................................................................................... 72 Introduction .......................................................................................................................................... 73 Methods .................................................................................................................................................. 77 Results ..................................................................................................................................................... 79 Discussion .............................................................................................................................................. 83 Acknowledgments .............................................................................................................................. 89 SUMMARY AND CONCLUDING REMARKS ................................................................................. 90 REFERENCES ............................................................................................................................................ 98 iii LIST OF TABLES Table 1 Primers used for RT-PCR. 22 Table 2 TXI and TWI proteins identified by MALDI-TOF. 35 Table 3 Comparison of AO protein features. 37 Table 4 Primers used for cloning. 53 Table 5 Distribution of transcript counts per gene from 80 M. penetrans cells in the presence of HeLa cells. Table 6 Top 20 most expressed genes of M. penetrans 81 incubated with HeLa cells. Table S1 RNA sequencing results from Supplemental M. penetrans grown in the presence and absence of HeLa cells. iv LIST OF FIGURES Figure 1 SEM images of M. penetrans whole cells and 25 detergent-insoluble structures. Figure 2 AO-associated objects and their lengths. 27 Figure 3 Internal organization of M. penetrans observed 29 by ECT. Figure 4 SDS-PAGE of M. penetrans whole-cell lysate, 33 TWI, and TXI proteins. Figure 5 RT-PCR analysis of putative cytoskeletal operon 38 of M. penetrans. Figure 6 Design and construction of plasmid pOO77. 55 Figure 7 Genomic organization of M. penetrans
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