MIAMI UNIVERSITY the Graduate School

MIAMI UNIVERSITY The Graduate School

Certificate for Approving the Dissertation

We hereby approve the Dissertation

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

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

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

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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.

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 genes 59 mype1520-1570 and M. iowae genes p271_111-106 and protein sequence alignment of MYPE1570 and P271_106.

Figure 8 Map of transposon vector pOO77. 62

Figure 9 Immunoblot analysis of GFP in E. coli and 65 M. iowae transformants.

Figure 10 Proposed model for M. penetrans AO 93 development throughout cell growth.

ACKNOWLEDGEMENTS

My journey through graduate school has been one of the most challenging and rewarding endeavors of my life. I am so grateful to have had the opportunity to meet and surround myself with some truly amazing, humble, funny, respectable, and incredibly smart individuals. The friendships and connections that I have formed have helped shape me as a student and person and without them I would not be the person I am today, and for that I am extremely thankful.

I would like to fully and wholeheartedly thank my wonderful advisor and friend Dr. Mitchell Balish. You have been such a great mentor whose unwavering efforts turned a nervous and apprehensive student into a developing scientific scholar with a thirst for knowledge and love of bacterial cell biology. I know I have frustrated you many times with my pestering questions and inquiries and I thank you for being extremely patient with me and never giving up. You have not only equipped me with a solid foundation of scientific thought and problem-solving skills but have also imparted and cultivated a sense of self-confidence. For all of this, along with the many laughs we have shared and much more I sincerely thank you.

Next, I would like to thank my committee members, Dr. Natosha Finley, Dr. Joe Carlin, Dr. Kelley Abshire and Dr. Jack Vaughn. They have been very supportive and encouraging throughout my graduate studies. Together and individually they have each given me helpful advice regarding my research as well as providing continual motivation and always challenging me to do better and grow as a scientist. Special thanks, also, to Dr. Carlin and Dr. Abshire for being my readers and providing helpful and constructive edits and reviews of my dissertation prior to formal submission.

I would especially like to thank the late Dr. Gary R. Janssen. Although I did not end up working in your lab, you never held that against me. You provided so much support and care for me as if you were still my advisor. You were a wonderful

vi person, mentor, scientist, and friend. I cannot ever fully express everything that you have given me. I think of you everyday and miss you dearly. From our scientific talks and inquiries, to our talks about music, cheers and shots during the fun times, to the wonderful times we listened to records together there are so many memories and wonderful times that I was so blessed to share with you. You had so much passion and integrity for science and so much love for students. I hope that I can aspire to be even half the man that you were and please know that your memory and love will always be with and apart of me.

A special thank you to Dr. Brandi Baros who was a wonderful mentor, professor, and friend. Dr. B, you introduced me to microbiology and it was your passion and love for it that got me interested in microbiology in college. If not for you I don’t know if I would have chosen this path, so thank you so much introducing me and providing me with the initial foundation for microbiology research.

I also want to thank all of my former and current lab mates; Dominika Jurkovic, Ryan Relich, Rachel Pritchard, Natalie Clines, Nicole Marotta, Monica Feng, Hailey Riggs, Nathan Schwab, and Jananie Rockwood. You have all been helpful at some point throughout my time in lab and I am happy to have shared my growth and friendship with you all. I especially appreciate all of the many many laughs and wonderful, often times dirty, conversations we have had, some of which I will never forget. I would also very much like to thank all of the undergrads that I have had the privilege to interact with, all of whom have also added to the fantastic and diverse lab environment. I would especially like to thank Neena Patel who dedicated so much of her time to help me with my projects. I don’t know why she stayed or managed to put up with me as her graduate student for so long but I am very grateful. Neena, I know I was not always the easiest to work with but I am glad that we had the chance to work together. It was wonderful to watch you grow as a person and researcher and I know that you are bound for greatness.

Throughout my time in the microbiology department I have met so many wonderful and smart people. I owe a huge thank you to Barb, Darlene, Amy, and Bev,

vii the micro department would not be able to function without you. I loved getting to know all of you and you were all so helpful for all of the many many times at which I asked you questions, needed something, or came down for a fun chat. A special thank you to Barb and Joe, as well, for providing a large amount of firewood for free; I had many a nice fire during cold nights all thanks to you! I owe a huge thank you to all of the faculty, staff, and students that make up the Miami University microbiology department for their individual help towards me and for making the department such a great place. I would like to specifically acknowledge a few special friends who made my time here incredible and whom have become life-long friends: Bill P (and Marry), Dan Z, Liz F, Amber B, Sarah S (aka “mer”), Amber T, Wei L, Morgan L, Jananie R, Jenna D, Tzvia S, Heather B, and my Canadian homeboy Chris S! From late nights at the bars to broomball games (and other intramural sports) to cabrewing and all of the other fun things we did together, it was all amazing and I am so happy to have met all of you and call you my friends, I love you all (in a non romantic way)!

To my parents, I owe you so much! Thank you for always being there for me and supporting all of my life decisions. I know I had to have been a pain to deal with at times but thank you for your unending love. You have been with me at every point in my life and I could not ask for anything more, I love both so much! To Christine (aka my 2nd mom), this also applies to you as well, you have treated me as if I were your own son, thank you so much, I love you too.

And lastly but most significantly, I want to thank from the bottom of my heart and the depths of my soul my dearest, closest, and best friend Ryann. I would not have survived graduate school if it weren’t for you. I could write so so so much about what you have done for me and how much I love you but it would never be enough and could never truly or completely convey what you mean to me. You are, and always will be, my rock, colleague, companion, and the love and light of my life. I am forever grateful and happy that we crossed paths and can’t wait to start a new chapter together with you in Houston and where ever else life takes us! I love you so much and I cannot wait to spend the rest of my life with you!!!!!!

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INTRODUCTION

Proteins are fundamental and integral components that govern and drive all cellular activity and life. Although proteins exhibit vast diversity, with involvement in processes including metabolism and energy production, signaling and transport, synthesis of cellular precursors, and more, they serve two main objectives: to catalyze biochemical reactions and to provide structural support for cell morphology and subcellular structures. Both roles depend on the correct assembly and interaction of proteins and their proper localization. Many proteins and proteinaceous complexes require specific spatial positioning within cells in order to function. The resulting deliberate uneven subcellular distribution of biomolecules can be defined as polarity (Cove, Hope & Quatrano, 1999). Cytoskeletal proteins play vital roles in the control of cell polarity and are key elements in establishing and maintaining structural integrity. Like eukaryotes, bacteria possess a variety of cytoskeletal proteins, even species of the genus Mycoplasma, which is known for drastic reduction in genome size and complexity compared to other organisms, illustrating the fundamental nature of the cytoskeleton and cell polarity.

Some species of Mycoplasma exhibit distinct morphological polarity, with the presence of a polar prosthecal structure called an attachment organelle (AO). This unique differentiated tip structure, which is specific to mycoplasmas, plays an important role in host colonization as it directly mediates host cell attachment (Balish, 2014a). Several studies have focused on the organization and composition of AOs from different Mycoplasma species and concluded that they each contain a set of novel cytoskeletal proteins important for the formation, localization, and structural integrity of this polar complex (Meng & Pfister, 1980; Göbel, Speth & Bredt, 1981; Balish, 2006a, 2006b; Nakane & Miyata, 2007; Jurkovic, Newman & Balish, 2012). Analyses from these studies along with genomic data indicate that the components, organization, and mechanics of AOs differ across phylogeny, likely as a product of convergent evolution (Himmelreich et al., 1996; Sasaki et al., 2002; Jaffe et al., 2004; Jurkovic, 2012). Given these data and the clear functional and morphological polarity that the AO imparts, mycoplasmas that have these

structures can serve as good model systems for studying and understanding how bacteria use different cytoskeletal proteins to acquire cellular organization and to establish and maintain cell polarity, especially given the reduced genomic nature of these organisms (Balish, 2014b). The studies described in this dissertation address these topics to enable better understanding of mycoplasma AOs in the greater context of elucidating the mechanisms of bacterial cell polarity.

A. Significance of bacterial cell polarity Despite typically being small in size, bacterial cells exhibit intricate organization of proteins both internally and externally (Shapiro, McAdams & Losick, 2002). Like their eukaryotic counterparts, bacteria exhibit polarity. Examples of bacterial polarity among different species include the protein DivIVA in Streptomyces coelicolor, which serves as the nucleation site and recruitment factor for polar hyphal growth, and the protein PopZ of Caulobacter crescentus, which anchors the chromosome to the cell pole and temporally recruits proteins necessary for cell cycle progression (Ebersbach & Jacobs-Wagner, 2007; Bowman et al., 2008, 2010; Ebersbach et al., 2008; Schofield, Lim & Jacobs-Wagner, 2010; Flärdh et al., 2012; Laloux & Jacobs-Wagner, 2014). Because subcellular localization is dependent on correct cell shape, which is dictated by polarity, the importance of spatial dynamics in bacteria is quite evident. In fact, polarization of bacterial cells plays essential roles in most cellular processes and activities such as growth, cell division, attachment, motility, chromosome segregation, differentiation, and more (Treuner-Lange & Søgaard- Andersen, 2014). The prevalence of polarity is supported by evidence that more than 10% of the encoded proteins of C. crescentus show non-uniform subcellular localization (Werner et al., 2009; Bowman, Lyuksyutova & Shapiro, 2011).

The mechanisms used by cells for localization of proteins and other compounds are multifaceted and intricate due to the highly organized, dynamic, and dense nature of cells (Gitai, 2005). Polarity must be regulated not only spatially and temporally but also at the level of the stoichiometry of certain proteins needed for assembly of macromolecular structures. Given this complexity it is no surprise that bacteria have evolved multiple

mechanisms to establish and maintain polarity (Bowman et al., 2011; Treuner-Lange & Søgaard-Andersen, 2014).

Like eukaryotes, bacteria use cytoskeletal elements as major players in establishing, regulating, and maintaining the polarization of cells. Unlike eukaryotic cells, however, bacteria have a greater diversity of cytoskeletal proteins. Although bacteria have homologs of the proteins that comprise the canonical eukaryotic microfilaments, microtubules, and intermediate filaments, they also produce multiple bacteria-specific cytoskeletal proteins that are necessary for localization of biomolecules and polarization of cells (Lin & Thanbichler, 2013). For example, ParA, which facilitates the segregation of chromosomes in dividing bacterial cells (Gerdes, Howard & Szardenings, 2010). This expanded repertoire of cytoskeletal elements suggests that establishment and maintenance of cell polarity among bacteria is a high priority given the evolutionary drive to maintain multiple proteins and mechanisms with such similar functions. Although the form in which cellular polarization is manifested differs across the bacterial domain, the theme of using cytoskeletal elements to accomplish polarization is recurrent (Bowman et al., 2011).

B. Polarity among mycoplasmas The importance of subcellular organization and localization of components to generate polarity is evident from the presence of cytoskeletal proteins in genomically streamlined organisms like mycoplasmas. The genus Mycoplasma is a group of bacteria that have undergone reductive evolution from a low-GC Gram-positive bacterial ancestor and contain the smallest genomes of organisms capable of being grown axenically. Two major characteristics of mycoplasmas are that they lack a cell wall and have limited biosynthetic capabilities. As a result, these fastidious organisms are host-dependent. Yet despite their minimalistic genomes and lack of a cell wall, these organisms have considerable morphological complexity and diversity (Jurkovic, 2012). A clear example is the AO, a polar, prosthecal structure that mediates attachment to host cells via recruitment and localization of adherence-related proteins. In this regard the AO is similar to the stalk of C. crescentus cells, with the appendage being an extension of the cell body rather than an external

proteinaceous structure, such as pili or flagella (Ong, Wong & Smit, 1990; Jurkovic, 2012). This structure is also often involved in gliding motility, which is the smooth movement of individual cells across solid surfaces. In mycoplasma species that exhibit gliding motility, the AO is always at the leading end of the cell, indicating that this structure also houses the motor proteins. The AO serves as a consolidated and organized structure necessary for cytadherence and gliding motility, which is consistent with roles in establishing and maintaining cell polarity.

C. Importance of Mycoplasma attachment organelles Although there are many ways to establish host cell attachment, as indicated by the large variety of adherence mechanisms used by other bacteria, the production and use of AOs by certain mycoplasma species is one solution to a mechanism for cytadherence. Although the primary role of AOs may be attachment, further examination and analysis have indicated other functions for this structure. Gliding motility tends to be a common feature among mycoplasma species that have AOs. Although some species with AOs have not been demonstrated to be motile, conditions conducive to their gliding mechanisms in the absence of their specific host cells may not have been identified. Furthermore, gliding motility of Mycoplasma pulmonis was lost after nine passages (Nelson & Lyons, 1965; Balish, 2006a), suggesting that absence of motility may occur from mutation of genes for components of the motility machinery in culture. Therefore, it is possible that under the proper conditions most mycoplasmas that utilize an AO exhibit gliding motility.

As gliding motility appears to be a common feature specific to mycoplasmas that have an AO, it stands to reason that gliding plays an important role for the cell. One possibility that has been suggested is the use of gliding motility for efficient cell division (Balish, 2014b). In support of this role for motility, cells of a Mycoplasma pneumoniae adherence mutant have a branched morphology, suggesting that these cells have trouble with cytokinesis (Romero-Arroyo et al., 1999; Balish & Krause, 2006; Balish, 2014a). Gliding motility might provide a force on the cell membrane during cell division to promote separation of daughter cells (Balish, 2014a, 2014b). In other bacteria, membrane

constriction, effectuated by FtsZ, is coordinated with peptidoglycan synthesis at the site of constriction to prevent the constriction from reversing, favoring efficient cell division (Li et al., 2007). However, the absence of a cell wall in mycoplasmas renders this mechanism inapplicable. Thus, gliding motility might act in concert with FtsZ as a substitute for peptidoglycan to allow for efficient cell division in mycoplasma species that exhibit this characteristic (Balish, 2014a, 2014b). In support of this model, ftsZ, which is an essential gene in most other bacteria, can be knocked out in Mycoplasma genitalium (Lluch-Senar, Querol & Piñol, 2010). While they are viable, these M. genitalium mutant cells exhibit phenotypes indicative of problems associated with cell division (Lluch-Senar et al., 2010). Unlike wild-type cells, in which cytadherence mutants occur frequently, M. genitalium ΔftsZ mutants do not exhibit spontaneous loss of cytadherence (Lluch-Senar et al., 2010; Balish, 2014a). These observations suggest that FtsZ and gliding motility work synergistically and further support the importance of the AO in cell division of motile mycoplasmas (Balish, 2014a).

It has also been observed in M. pneumoniae cells that duplication and migration of the new AO to the opposite pole occurs concomitantly with the replication of the chromosome (Seto et al., 2001). The presence of DNA associated with the base of the AO core was observed in seven species of the M. pneumoniae cluster (Hatchel & Balish, 2008), suggesting that the AO might also play a role in chromosome segregation.

Taken together, these results indicate that mycoplasma AOs play a significant role beyond host cell attachment. The localization of adhesins, motor, and cytoskeletal proteins, not only facilitates polar attachment, but also serves to orient the cells, provide structural support, and enhance the efficiency of cellular developmental and division mechanisms, along with other potential functions that have yet to be discovered.

D. Attachment organelles of other Mycoplasma species Although most Mycoplasma species do not have any apparent differentiated tip structure, the species that do are dispersed among different mycoplasma phylogenetic clusters (Balish, 2006a, 2014b). Out of 14 phylogenetic clusters there are a total of 5 within

the Mycoplasma genus that contain at least one representative species that has an AO: the Mycoplasma sualvi, M. pulmonis, Mycoplasma muris, M. pneumoniae, and Mycoplasma fastidiosum clusters (Jurkovic, 2012). M. pneumoniae, a representative of the M. pneumoniae cluster, has become a model species in terms of studying AO composition and structure due to its impact as a human pathogen. The M. pneumoniae AO is a highly organized and intricate structure of assembled proteins that control adherence, motility, and architecture (Balish, 2006b). Proteins P1 and P30 are the main adhesins of M. pneumoniae. Both are transmembrane proteins that localize to the AO. Although P1 is distributed across the cell surface it is found at higher densities in clusters at the AO (Baseman et al., 1982; Feldner, Göbel & Bredt, 1982; Collier, Hu & Clyde, 1983; Balish, 2006b). Localization of P1 to the AO is necessary for cytadherence, as evidenced by mutants that express but fail to concentrate P1 at the tip being unable to attach (Baseman et al., 1982; Hahn, Willby & Krause, 1998; Balish et al., 2003a). P30 is also required for cytadherence but is found solely at the tip of the AO (Morrison-Plummer, Leith & Baseman, 1986; Baseman et al., 1987; Seto & Miyata, 2003; Balish, 2006b). The function of P30 as an adhesin is supported by the lack of adherence in M. pneumoniae mutants lacking P30 production and in cells incubated with P30 monoclonal antibodies (Morrison-Plummer et al., 1986; Romero-Arroyo et al., 1999). However, this protein also appears to play a role in gliding motility that is independent of its contribution towards attachment. This role is evidenced by a M. pneumoniae mutant that expresses a variant of P30 in which the resulting cells are capable of cytadherence but deficient in gliding motility (Hasselbring, Jordan & Krause, 2005; Balish, 2014b). The proteins that confer motility are contained within the AO, as indicated by an M. pneumoniae mutant in which the AO may detach from the cell body and remain motile for a period of time while the remaining cell body is immobile (Hasselbring & Krause, 2007; Balish, 2014a). These data suggest that upon proper assembly the AO is a self-contained functional unit.

The components involved in formation, localization, stability, and structure of the M. pneumoniae AO are located on its interior (Balish & Krause, 2002). A major feature of the M. pneumoniae AO is the presence of a proteinaceous electron-dense core. This core, which occupies the majority of the internal space, consists of a double rod capped with a

button structure at the distal end and a bowl-like structure at the proximal end (Biberfeld & Biberfeld, 1970; Henderson & Jensen, 2006). Directly surrounding the core is an electron-sparse clearing that is devoid of ribosomes and other large particles (Biberfeld & Biberfeld, 1970; Balish, 2014a). Intact structural components of the AO core can be harvested through extraction with the non-ionic detergent Triton X-100. The insolubility of these core proteins in Triton X-100 along with ultrastructural examination and phenotypic analyses demonstrate that these components have biochemical and physical characteristics of cytoskeletal proteins and are necessary for normal assembly and function of the AO (Meng & Pfister, 1980; Göbel et al., 1981; Regula et al., 2001; Balish, 2014a). There are at least eight cytoskeletal proteins that form the backbone of the M. pneumoniae AO (Balish, 2014a; Nakane et al., 2015; Balish & Distelhorst, 2016). These core proteins serve as both structural and functional components and their spatial and temporal organization and assembly is essential for proper development and activity of the AO (Krause & Balish, 2004; Balish, 2006b, 2014a; Nakane et al., 2015; Balish & Distelhorst, 2016). Furthermore, these cytoskeletal proteins play pivotal roles in establishing cellular polarity by orchestrating the proper localization of the AO that allows for aspects of other cell functions, underscoring the importance of these core proteins. The significance of these proteins to AO composition and structure is also suggested by the fact that the majority of these proteins are conserved in the closely related species M. genitalium and Mycoplasma gallisepticum. (Balish, 2014a). There are eight species within the M. pneumoniae cluster and all have an AO that contains a cytoskeletal core (Gourlay, Wyld & Leach, 1977; Hatchel & Balish, 2008). Furthermore genomic data from the seven other species shows them all to have orthologs of the M. pneumoniae core proteins (unpublished data; (Fraser et al., 1995; Himmelreich et al., 1996; Papazisi et al., 2003; Clark et al., 2016)

Mycoplasma mobile is another species that also uses a polar AO, but, it is a distant relative of M. pneumoniae belonging to the M. sualvi cluster (Johansson & Pettersson, 2002). The AO of M. mobile shares the same functions as those of M. pneumoniae in that it is necessary for cytadherence and gliding motility (Miyata et al., 2000; Miyata & Uenoyama, 2002). However, the M. mobile AO morphology and cytoskeletal core are distinct from that of M. pneumoniae in both structure and composition (Himmelreich et al., 1996; Jaffe et al.,

2004; Balish, 2014a; Miyata & Nakane, 2014). Rather than forming a rod-like polar protrusion, like M. pneumoniae, the M. mobile AO has a rounded cone-like extension (Kirchhoff & Rosengarten, 1984; Balish, 2014a). The main adhesin of M. mobile, transmembrane protein Gli349, is distributed along the lateral side of the AO rather than at the tip and can be seen as spikes protruding from the cell membrane in this region (Miyata & Petersen, 2004; Uenoyama, Kusumoto & Miyata, 2004; Adan-Kubo et al., 2006; Miyata, 2010). Gli349 also appears to be directly involved in gliding motility of M. mobile as antibodies against this protein reduce gliding motility in a concentration-dependent manner (Uenoyama et al., 2004, 2009). Gli349 exists in a quaternary complex with three other proteins that exert a mechanical force that propel cells forward in a proposed catch, pull, and release mechanism that directly depends on ATP hydrolysis (Miyata, 2008, 2010; Miyata & Nakane, 2014). There are approximately 450 of these units along the surface of the AO (Nakane & Miyata, 2007; Miyata & Nakane, 2014). As revealed by treatment of cells with Triton X-100, the interior of the M. mobile AO contains a novel protein structure. This complex, referred to as the jellyfish structure due to its resemblance to that organism, has two major substructures, a bell (like the head of a jellyfish) located at the distal portion of the AO and tentacle-like structures that extend out from the base of the bell and into the cell body (Nakane & Miyata, 2007; Miyata, 2010; Balish, 2014a; Miyata & Hamaguchi, 2016). Based on localization studies and analysis of gliding deletion mutants, the cytoskeletal core, especially the tentacle-like structures, appear to interact with the transmembrane gliding machinery (Nakane & Miyata, 2007; Balish, 2014a). A total of 10 Triton X-100-insoluble proteins from M. mobile have been identified by mass spectrometry, some or all of which are likely components of the AO core (Nakane & Miyata, 2007). Most of the identified proteins are novel, but, two are homologs of components of the membrane ATP synthase, one is a phosphoglycerate kinase, and one is a predicted xylose-binding protein (Nakane & Miyata, 2007; Balish, 2014a). With the exception of the glycolytic pathway enzyme phosphoglycerate kinase, none of these proteins or any of the other proteins of the gliding machinery have homologs in M. pneumoniae (Nakane & Miyata, 2007; Balish, 2014a).

With the exception of sharing between M. mobile and M. pulmonis, no AO protein homologs are shared across the phylogenetic clusters of AO-bearing mycoplasmas (Jurkovic, 2012). It is therefore likely that, with this one exception in which horizontal gene transfer might have occurred, mycoplasma AOs have arisen independently through convergent evolution, suggesting that this feature provides important advantages for survival of some mycoplasma species (Jurkovic, 2012; Balish, 2014a). Despite the diversity in composition and organization of AOs of distantly related organisms, they all provide polarization of particular cellular activities. This independent development of AOs across phylogeny provides opportunities to further understand the various mechanisms used by prokaryotes to establish and maintain cell polarity. Knowledge of the M. pneumoniae and M. mobile AOs can serve as a basis for examining other mycoplasma AOs in species from the other clusters. In order to achieve this level of understanding, however, it is necessary that the species be capable of being maintained in culture and have a sequenced genome to allow for identification and characterization of AO proteins. Given these characteristics, Mycoplasma penetrans serves as a good candidate as a model for a third Mycoplasma cluster, as it uses an AO, can be maintained in culture, and has a sequenced genome.

E. M. penetrans M. penetrans is a representative species of the M. muris cluster and has an AO used for close interaction with host cells (Lo et al., 1991, 1992). However, the proteins involved in its cytadherence are not known. The AO is also important for gliding motility of M. penetrans cells and also appears to be important for efficient cell division (Jurkovic et al., 2012). Gliding cells move unidirectionally with the AO always at the leading end (Jurkovic et al., 2012). The AO is an organized structure necessary for cytadherence, gliding motility, and cell division, similar to the functions observed for the AOs of M. pneumoniae, M. mobile, and other mycoplasmas.

Although many of the identified species of mycoplasmas are commensals, some are pathogens that cause significant impacts to humans and animals (Rosengarten et al., 2000; Citti & Blanchard, 2013). Such examples include M. pneumoniae, a major contributor of

community-acquired pneumonia among humans (Saraya et al., 2014) and Mycoplasma mycoides subsp. mycoides, the causative agent of contagious bovine pleuropneumonia, a severe infectious disease of cattle (Pilo, Frey & Vilei, 2007). Studies on these and other pathogenic mycoplasmas have shed light on some of the molecular mechanisms they use for pathogenesis. However, despite their reduced genome size and number of metabolic pathways, there is still much that is not known about these organisms. Furthermore, beyond the frankly pathogenic mycoplasma species, such as M. pneumoniae and M. genitalium, there are also an increasing number of opportunistic mycoplasma infections that have recently become apparent. One such species, M. penetrans, has emerged as an opportunistic pathogen affecting immunocompromised individuals, most often AIDS patients (Lo et al., 1991, 1992; Wang et al., 1992; Grau et al., 1995, 1998). Epidemiological studies have provided evidence of an association between HIV infection and infection with M. penetrans. In one study the seroprevalence of M. penetrans was found to be 40% among AIDS patients and 20% among asymptomatic HIV-infected individuals but only 0.3% among HIV-negative individuals (Wang et al. 1992; Grau et al. 1995; Brenner, Neyrolles and Blanchard 1996; Grau et al. 1998). Additionally, M. penetrans cells are capable of propagating HIV by stimulating the replication of the HIV genome and acting as mitogenic agents toward B and T lymphocytes, the target cells of HIV (Nir-Paz et al., 1995; Sasaki et al., 1995; Iyama et al., 1996; Shimizu, Kida & Kuwano, 2004). During experimental infection of chicken embryos, M. penetrans had high mortality rates between 70-100% (Hayes et al., 1996). M. penetrans has also been found to cause extensive vacuolation in multiple cell lines, has high binding affinity for IgA, shows hemolytic and hemoxidative activities, and expresses multiple other virulence factors such as nucleases, phospholipases, and an ADP- ribosylating and vacuolating toxin that has similarity to the M. pneumoniae CARDS toxin (Salman & Rottem, 1995; Shibata, Sasaki & Watanabe, 1995; Borovsky et al., 1998; Bendjennat et al., 1999; Kannan & Baseman, 2000; Tarshis et al., 2004; Johnson, Kannan & Baseman, 2009; Moussa, Nir-Paz & Rottem, 2009). This organism also has the capability to invade and survive within host cells, a feature whose commonness among mycoplasmas is unclear (Borovsky et al., 1998; Dallo & Baseman, 2000; Tarshis et al., 2004). Given these data it has been suggested that M. penetrans may be a cofactor in the progression of AIDS (Grau et al., 1995). However, there has also been a documented case of systemic M.

penetrans infection in a HIV-negative immunocompromised patient with primary antiphospholipid syndrome (Yáñez et al., 1999). Therefore, the incidence of M. penetrans infection may be greater and more impactful to immunocompromised individuals aside from HIV/AIDS patients. Potentially, the number of people susceptible to M. penetrans infection could be very high and the possibility that these infections could result in further complications or disease progression is of concern. For this reason it is necessary to further understand the cellular processes and pathogenic mechanisms of M. penetrans.

F. Importance of studying the M. penetrans attachment organelle The inability to treat bacterial infections is becoming a significant concern given the rise of antibiotic resistance. The incredible speed with which bacteria derive resistance to new antibiotics makes facing this challenge even more difficult. Furthermore, beta-lactam antibiotics, which target cell wall synthesis, cannot be used for treatment of mycoplasma infections, resulting in even fewer treatment options. Resistance to macrolides, fluoroquinolones, and tetracycline, the main classes of antibiotics used to treat mycoplasma infections, have already been reported in some mycoplasma species (Gerchman et al., 2008; Bébéar, Pereyre & Peuchant, 2011; Meyer Sauteur, van Rossum & Vink, 2014; Redelinghuys et al., 2014; Balish & Distelhorst, 2016). This suggests the likelihood that other mycoplasma species will soon develop resistance to these antibiotics as well. Given the role that M. penetrans may play in the progression of AIDS and/or other immunocompromised individuals, the potential inability to treat an M. penetrans infection due to antibiotic resistance could become extremely problematic.

In order to develop novel therapeutic agents to overcome this major problem, a thorough understanding of bacterial cell biology is needed. Knowledge of the fundamental components and organization of bacterial cells will be vital for identifying the key elements in the function and dynamics of cellular processes that should be targeted for optimal and efficient inhibition of cellular activity. The large variety of mechanisms used by different species that yield the same or similar functional outputs, however, makes this endeavor more challenging. For example, both M. pneumoniae and M. penetrans use an AO, yet the

components used for assembly and function are different. In this regard it is important and necessary to study the M. penetrans AO itself, not only to understand the assembly process and mechanism of function but also to provide more information about mycoplasma AOs to allow for comparative analysis. In this way we can begin to address and identify the similarities and differences between the components, assembly mechanisms, and functionality of mycoplasma AOs. Studying the M. penetrans AO and comparing it with other mycoplasma AOs will provide greater insight into how mycoplasmas establish polarity, what the characteristics of their cytoskeletal proteins are, how they achieve attachment and gliding motility, the mechanisms they use for subcellular localization and organization of AO components, and the extent to which there is shared functional or structural conservation across distantly related mycoplasma species. All of this information will be helpful for development of therapeutic agents specific to M. penetrans and other mycoplasmas in addition to expanding the knowledge of bacterial cell biology and evolution.

G. Hypotheses The goal of this work was to characterize the M. penetrans AO regarding its composition, cellular localization, and role in pathogenesis. The overall hypothesis of this dissertation was that the M. penetrans AO, which is involved in the earliest phases of host interaction, is built upon a novel cytoskeletal structure. To address this we focused on three major aims throughout three chapters. In chapter one we analyzed the composition of the AO core of M. penetrans using detergent extractions and MALDI-TOF analysis with the hypothesis that the AO core is composed of novel proteins that share a specific set of characteristics with properties of static cytoskeletal elements like intermediate filaments. In chapter two we addressed the hypothesis that components of the AO core are localized to both poles of M. penetrans by making a green fluorescent protein (GFP) fusion to a previously identified AO core protein and examining its localization in M. iowae, a closely related genetically tractable relative of M. penetrans. In the third chapter we examined the differential gene regulation of M. penetrans cells when grown in culture versus in the presence of host cells to address the hypothesis that expression of M. penetrans AO

proteins are increased during early stages of host cell interactions. The data from these experiments are expected to provide further insight into the mechanisms of cell polarity and pathogenicity of M. penetrans. Furthermore, these data will expand our knowledge of the cellular and pathogenic mechanisms that mycoplasma may utilize for infection of host cells which will be useful in the design of therapeutic agents against M. penetrans and other virulent mycoplasmas.

CHAPTER 1

The variable internal structure of the Mycoplasma penetrans attachment organelle revealed by biochemical and microscopic analyses

Steven L. Distelhorst, Dominika A. Jurkovic, Jian Shi, Grant J. Jensen, and Mitchell F. Balish

Submitted to the Journal of Bacteriology

Abstract

Although mycoplasmas have small genomes, many of them, including the HIV- associated opportunist Mycoplasma penetrans, construct a polar attachment organelle (AO) used for both adherence to host cells and gliding motility. However, the irregular phylogenetic distribution of similar structures within the mycoplasmas as well as compositional and ultrastructural differences among these AOs suggest that AOs have arisen several times by convergent evolution. We investigated the ultrastructure and protein composition of the cytoskeleton-like material of the M. penetrans AO by several forms of microscopy and biochemical analysis to discern whether the M. penetrans AO was constructed on principles similar to those of other mycoplasmas like Mycoplasma pneumoniae and Mycoplasma mobile. We found that the M. penetrans AO interior was generally dissimilar from other mycoplasmas in that it exhibited considerable heterogeneity in size and shape, suggestive of a gel-like nature. Despite these differences several M. penetrans detergent-insoluble proteins, identified by mass spectrometry, shared certain distinctive biochemical characteristics with M. pneumoniae AO proteins, though not with M. mobile AO proteins. We conclude that convergence between M. penetrans and M. pneumoniae AOs extends to the molecular level.

Introduction

Mycoplasmas are cell wall-lacking bacteria that live parasitically or commensally in nature but can be cultured axenically. These genomically reduced organisms occupy a unique space within the field of bacterial cell polarization that makes them potentially informative concerning mechanisms by which polarization is achieved as well as the evolutionary origins of these mechanisms. Some mycoplasmas have a differentiated, prosthecal tip structure known as an attachment organelle (AO) at one pole (Balish, 2006a). The AO houses adhesin proteins for interaction with host cells and surfaces, as well as machinery that carries out unidirectional gliding motility, and in some cases appears to be physically associated with the chromosome (Balish, 2014b). The interior of the AO contains a complex of structural proteins that serves as a cytoskeletal scaffold for AO assembly (Balish, 2014b). In the sense that the AO is constructed at a cell pole and involved in functions associated with polarity, this structure parallels other polar structures of bacteria like the Caulobacter crescentus stalk (Balish, 2014b). The majority of information regarding the components, assembly, and substructures of mycoplasma AOs comes from studies of two different species, Mycoplasma pneumoniae and Mycoplasma mobile. These two species are members of distantly related phylogenetic clusters within the genus (Johansson & Pettersson, 2002). Characterization of the structures and proteins that make up the interior of the AO of each of these species has shown them to be different both ultrastructurally and compositionally, with no shared AO protein homologs (Jaffe et al., 2004; Balish, 2006a), suggesting that like the C. crescentus stalk, these structures have evolved independently and recently as compared with other members of their respective phyla. Mycoplasma penetrans also has a polar AO, representing a third mycoplasma phylogenetic cluster (Lo et al., 1991, 1992; Jurkovic et al., 2012). M. penetrans is known for its association with AIDS patients, in whom it is commonly found in the urogenital tract, but it has also been isolated from an HIV-negative patient with antiphospholipid syndrome (Lo et al., 1991, 1992; Yáñez et al., 1999). The major focus of studies of M. penetrans has been its putative role as a cofactor that may accelerate the progression of AIDS (Nir-Paz et al., 1995; Sasaki et al., 1995; Iyama et al., 1996; Shimizu et al., 2004). We previously

showed that the M. penetrans AO contains material distinct in structure to that of M. pneumoniae and M. mobile (Jurkovic et al., 2012). Furthermore, M. penetrans lacks homologs of AO structural proteins of M. pneumoniae or M. mobile (Himmelreich et al., 1996; Sasaki et al., 2002; Jaffe et al., 2004). Difficulty in working with this organism stems from its genetic intractability, leaving biochemical and cell biological routes as the best options for its study. The ability to establish cellular polarization and to localize specific biomolecules to non-uniformly distributed subcellular positions is critical for many bacteria. Many fundamental processes such as DNA segregation, cell division, and placement of specialized structures rely on morphological polarity as a cue for appropriate spatial organization. The cytoskeleton, composed of a network of polymeric proteins, plays an integral role in maintaining the morphological and functional integrity of bacterial cells by establishing polarity (Cho, 2015). In addition to the widely phylogenetically distributed MreB and FtsZ, which form dynamic filaments that contribute to the nearly universal bacterial processes of peptidoglycan synthesis and cell division (Margolin, 2009), there is increasing evidence of a diverse array of other cytoskeletal elements with narrow phylogenetic distribution that facilitate more recently evolved functions (Pilhofer & Jensen, 2013). In contrast to the highly dynamic MreB and FtsZ, many of these proteins form static structures. The alpha- helical coiled coil-rich, intermediate filament-like protein crescentin from C. crescentus is associated with the establishment of cell shape (Ausmees, Kuhn & Jacobs-Wagner, 2003; Lin & Thanbichler, 2013). Many bacterial scaffold proteins contain an abundance of coiled coil domains, important for oligomerization and close protein-protein interactions, that are referred to as coiled coil-rich proteins (CCRPs) (Burkhard, Stetefeld & Strelkov, 2001). CCRPs serve in an array of functions such as maintaining cell morphology and enabling cell motility, often by participating in polar localization of proteins (Lin & Thanbichler, 2013). The widely distributed bactofilins are characterized by the conserved DUF583 domain, and form stable nucleotide-independent filaments that serve as spatial landmarks (Kühn et al., 2010; Koch, McHugh & Hoiczyk, 2011). Other bacterial cytoskeletal proteins include DivIVA and PopZ, which assemble into 2- or 3-dimensional arrays at extant or nascent cell poles and interact with proteins involved in pole-associated processes (Bowman et al., 2008; Ebersbach et al., 2008; Lenarcic et al., 2009; Ramamurthi & Losick, 2009; Kühn et al., 2010;

Koch et al., 2011; Lin & Thanbichler, 2013; Ptacin et al., 2014). It is essential to understand processes that rely on bacterial cell polarization, which can be achieved by establishing an inventory of cytoskeletal proteins across bacterial phylogeny and cataloging their characteristics and determining how these properties enable them to carry out their functions. We sought to identify the proteins of the interior of the M. penetrans AO and examine their properties in relation to other proteins of mycoplasma AOs. Because mycoplasma AOs seem to have evolved in a convergent manner we hypothesized that the proteins involved in structure and assembly of the M. penetrans AO are distinct from those of other mycoplasmas, and we predicted that their composition would reveal important information about the properties of the AO. We investigated the protein content of the detergent-insoluble material of M. penetrans, which comprises the AO interior, and characterized the structure of this material.

Materials and Methods

Bacterial culture Hyperadherent Mycoplasma penetrans strain HP88 cells (Jurkovic, Hughes & Balish, 2013) were grown to mid-log phase at 37°C in plastic tissue culture flasks containing SP-4 broth (Tully et al., 1979).

Detergent extraction and protein analysis Cultures of M. penetrans were decanted and washed twice with warm potassium- free phosphate-buffered saline (PBS; 145 mM NaCl, 2.83 mM NaH2PO4-H2O, 7.20 mM

NaHPO4-7H2O, pH 7.2). For detergent extraction, Triton X-100 or Tween-20 in 20 mM Tris- HCl –150 mM NaCl (pH 7.2) was added to the cultures to a final concentration of 1% in 25 ml PBS. Detergent extracts were then incubated for 30 minutes at 37° C, scraped, and centrifuged for 20 minutes at 17,050 x g. Whole cell lysates were treated the same without the addition of detergent. Following centrifugation the pellets were washed three times and resuspended in PBS. Protein concentration was determined using a bicinchoninic acid protein assay kit (Pierce). Equal amounts of protein of each fraction were subjected to 9% SDS-PAGE (Laemmli, 1970) and stained with Coomassie Brilliant Blue or Sypro Ruby (Molecular Probes). Detergent-extracted proteins were separated by SDS-PAGE and select bands were excised, reduced and alkylated, digested with trypsin, and analyzed by ABSciex 4800 MALDI-TOF/TOF at the University of Cincinnati Proteomics Laboratory as previously described (Eismann et al., 2009). MultiCoil (Wolf, Kim & Berger, 1997) was used to test predicted amino acid sequences for the presence of coiled coil domains; the presence of regions with >50% probability of forming alpha-helical coiled coils was recorded as positive.

Scanning electron microscopy (SEM) Cells were prepared for scanning electron microscopy as previously described (Hatchel et al., 2006). Briefly, cells were grown on glass coverslips for 6 to 24 h in SP-4 media supplemented with 3% gelatin at 37°C. For examination of Triton X-100-insoluble

(TXI) structures or Tween-20-insoluble (TWI) structures, detergent in 20 mM Tris-HCl, pH 7.5/150 mM NaCl was added to cells at a final concentration of 1% and incubated at 37°C for 30 min. The samples were fixed for 30 minutes at room temperature in 1.5% glutaraldehyde/1% paraformaldehyde/0.1 M sodium cacodylate, pH 7.2. Following fixation the coverslips were washed four times in 0.1 M sodium cacodylate, pH 7.2 and dehydrated through a series of ethanol washes from 25% to 100%. The coverslips were then critical point-dried, gold sputter-coated, and viewed on a Zeiss Supra 35 FEG-VP scanning electron microscope at the Miami University Center for Advanced Microscopy and Imaging.

Electron cryotomography (ECT) M. penetrans was grown on Quantifoil Au-finder grids (Quantifoil Micro Tools GmbH) in SP-4 broth at 37˚C until mid-log growth, indicated by a color change in the media from red to orange. The EM grids with attached M. penetrans cells were removed from the media, loaded onto tweezers, and washed with fresh media. Colloidal gold (10 nM) was added to the grids and they were blotted (Whatman, grade 40) prior to being plunge-frozen in liquid ethane on a gravity plunger freezer. Grids were loaded into a 300 kV FEI Polara G2 electro cryo-transmission electron microscope equipped with a field emission gun, a lens- coupled 4k x 4k Gatan UltraCam, and a Gatan energy filter (GIF). Samples were maintained in liquid nitrogen temperature as tilt series were captured of whole cells. The tilt-series was recorded from -60° to +60° with 1° increments at a 10-µm defocus using Leginon (Suloway et al., 2009). All tilt series were collected through GIF around zero-loss energy with a slit-width of 20 keV for a cumulative dose of 180 e-/Å2 used for each tilt series.

Measurements and analysis To measure the lengths of nucleoid-free zones, M. penetrans cells were grown overnight on cover slips, fixed as previously described (Jurkovic et al., 2012), and mounted on slides with Vectashield containing DAPI (Vector Laboratories). Fields were imaged using a 100X objective as previously described (Jurkovic et al., 2012). Phase-contrast and DAPI fluorescence images were overlaid and the lengths of 84 polar nucleoid-free zones were measured using SPOT software. To measure the lengths of TXI and TWI objects, SEM

images of the objects from multiple fields, captured as described above, were measured along their long axes. Objects that appeared to be clusters of individual objects were excluded. For TXI objects, 70 were measured, and for TWI objects, 79 were measured. Statistical comparison of lengths was done by one-way ANOVA after using a Shannon-Wilk test to establish that the distributions of lengths were not normal.

Reverse transcriptase (RT)-coupled polymerase chain reaction (PCR) The RT reaction was carried out as previously described for Mycoplasma iowae (Pritchard & Balish, 2015). Briefly, cDNA synthesis was performed with 100 ng of total RNA from M. penetrans and random hexamers using the Verso cDNA synthesis kit (Thermo Scientific) according to the manufacturer’s instructions. One microliter of cDNA was then used as template in a 50-μl PCR with Taq polymerase (New England Biolabs) using EasyStart PCR tubes (Molecular Bioproducts) according to the manufacturer’s instructions. The primers used in each reaction were designed to span the adjacent gene junctions (Table 1). Reactions were amplified by incubating at 94° C for 2 minutes, followed by 30 cycles of 94°C for 30 seconds, 53°C for 1 minute, and 68°C for 1 minute, with a final extension of 5 minutes at 68°C. The PCR products were then subject to gel electrophoresis. Reactions with genomic DNA as well as without addition of RT or template were performed as positive and negative controls, respectively.

Table 1. Primers used for RT-PCR.

Primer Product Target Sequence (5’-3’) designation size (bp) 1510end(up) AATGAGCAGTAAGTGCTAGC MYPE1510-20 603 1520begin(down) GTCTTAGGAATTTGAACAGGTG 1520end(up) GGAAAAAGAAGCTGTCACAG MYPE1520-30 566 1530begin(down) GGTTGATTGTCAGCAGAACC 1530end(up) TTCCAGCTCCTGCATATGAC MYPE1530-40 527 1540begin(down) GTCAGTTCCCTGAAATTGTTC 1540end(up) CTAAATTGTTCCAATATTGAATAATCGC MYPE1540-50 454 1550begin(down) ATAGATGAGATTCTTTATCAAAGTTCTC 1550end(up) ATCAAACCCAAGCGATTCTG MYPE1550-60 541 1560begin(down) TCAACATCGTCTTGTCTTGG 1560end(up) TGCTCCTAGTAGTGAGTTGC MYPE1560-70 696 1570begin(down) CTTCTCTAGCTTGAACTTGG 1570end(up) TTTGGAAGACCAGCAGGAAG MYPE1570-80 380 1580begin(down) GTGTTGCCAGGTCTAAGATC NA 1580RT(down) CATAGCCGTTTGAACAGTGT NA

Results

Internal structure of the M. penetrans AO As previously described, M. penetrans cells are elongated (Figure 1B), with a polar tip structure that functions as an AO. The AO confers attachment and gliding motility, which contributes to cell division; dividing cells often take the form of two cell bodies linked by a membranous connecting filament (Figure 1A; Jurkovic et al., 2012). M. penetrans cells attach to host cells by means of a pole, whose interior is distinctly differentiated from the cytoplasm of the rest of the cell despite being adjacent to it, as previously established by transmission electron microscopy (TEM) (Lo et al., 1991, 1992). Treatment of M. penetrans cells attached to plastic with the nonionic detergent Triton X- 100 results in solubilization of a large amount of cellular material, leaving behind discrete detergent-insoluble objects with a range of dimensions as visualized by SEM (Figure 1C; Jurkovic et al., 2012). Unlike the AO structures obtained similarly from species of the M. pneumoniae cluster (Hatchel & Balish, 2008), treatment with DNase has no effect on the appearance of these objects (not shown), indicating that they do not contain significant amounts of DNA. Close examination of these Triton X-100-insoluble (TXI) structures revealed that despite considerable heterogeneity in size and shape, they generally consisted of a wider, irregular, ball-like object, from which emanated one or more rod-like filaments which were often periodically punctuated with other material along the filaments. They had an average length of 320 ± 100 nm (Figure 2A; n=70). This range of lengths was statistically significantly ~15% smaller than that of the polar nucleoid-free zones of M. penetrans cells (Figure 2B; n=84; p<0.001), as revealed by overlaying phase-contrast images and images of the DNA stained by DAPI (Figure 2D; Jurkovic et al., 2012). The mean length of the nucleoid-free zones was 380 ± 140 nm. To determine whether other nonionic detergents might yield similar structures, we used SEM to examine the structures that remained following extraction of cells grown under identical conditions with the chemically distinct Tween-20 (Figure 1D). These objects were generally similar to those obtained using Triton X-100, although they were larger and had a generally smoother appearance. Indeed, their mean length of 380 ± 130 nm was identical to that of the nucleoid-free zones (Figure 2C;

n=79; p>0.8). The variability in sizes and shapes of the detergent-insoluble objects was recapitulated by ECT images, which showed ribosome-containing zones in the central area of the cell that were distinct from ribosome-free zones at both cell poles. These ribosome- free zones had irregular shapes and boundaries (Figure 3A, B), often extending into the filament connecting cells undergoing division (Jurkovic et al., 2012; Figure 3C). Given the similarities in size distribution, previous TEM images of similar material at M. penetrans poles (Lo et al., 1991, 1992; Neyrolles et al., 1998), and the limited amount of space in an M. penetrans cell for large objects, we conclude that the polar nucleoid-free zones visualized by DAPI staining, the polar ribosome-free zones visualized by ECT, and the DNA-lacking, detergent-insoluble objects visualized by SEM are the same objects, with Triton X-100 solubilizing some component that Tween-20 does not. The overall organization of these structures was quite distinct from those of M. pneumoniae (Henderson & Jensen, 2006; Seybert, Herrmann & Frangakis, 2006), Mycoplasma mobile (Nakane & Miyata, 2007), and Mycoplasma insons (Relich, Friedberg & Balish, 2009), which represent three other phylogenetic lineages or clusters within the genus. Interestingly, whereas the poles of whole M. penetrans cells as visualized by SEM (Figure 1B) or by light microscopy (Figure 2D) were distinctly narrower than the cell bodies, in cells imaged by ECT there was no abrupt narrowing at the poles, with cells exhibiting a more ovoid shape (Figure 3).

Identification and characterization of detergent-insoluble proteins M. penetrans lacks homologs of the AO cytoskeletal proteins of other mycoplasmas (Sasaki et al., 2002). To determine what proteins make up the TXI and Tween-20-insoluble (TWI) material, we examined the protein profiles of whole-cell lysates, TXI proteins, and TWI proteins using SDS-PAGE. The detergent-insoluble material was generated by extracting attached M. penetrans cells in flasks and scraping the material that remained attached to the flasks, ensuring that it was

Figure 1. SEM images of M. penetrans whole cells and detergent-insoluble structures. A) Field of M. penetrans cells; black arrows, connecting filaments; scale bar, 1 μm. B) Individual M. penetrans cell; white arrow, AO; scale bar, 200 nm. C) and D), detergent- insoluble structures from M. penetrans cells extracted with Triton X-100 (C) or Tween-20 (D). *, wide end of structure; scale bar, 200 nm.

Figure 2. AO-associated objects and their lengths. A) Distribution of length of TXI objects observed by SEM; B) distribution of lengths of nucleoid-free zones observed by light and fluorescence microscopy; C) distribution of lengths of TWI objects observed by SEM. D) Example of nucleoid free zones. Black, cell outline viewed by phase-contrast microscopy; white, nucleoid stained by DAPI. Arrows, nucleoid-free zones, measured in (B) along the long axis of the cell; scale bar, 500 nm.

Figure 3. Internal organization of M. penetrans observed by ECT. A-C, sections of different whole cells; D- F, corresponding schematics. A-C, ~20-nm granules are ribosomes (Jensen & Briegel, 2007). The insertion site of a connecting filament was observed at one cell pole (C, F). Black lines, cell membranes and boundaries of ribosome-free zones; circles, ribosomes (positions are schematic and do not represent actual ribosomes). Scale bar, 100 nm.

identical to the material that was visualized by SEM, which consisted almost entirely of structures similar in shape but heterogeneous in size, as described above (Figure 1C, 1D). Several proteins were prominent in in the TXI and TWI fractions, and some were enriched compared to the whole-cell lysate (Figure 4). Consistent with the similar appearances of the structures, the profile of TWI proteins was very similar to that of the TXI ones, with the principal exception of one prominent TWI band migrating at ~40 kDa that was absent in the TXI fraction (Figure 4).

We selected eight bands from SDS-polyacrylamide gels that appeared enriched in both detergent-insoluble fractions compared to whole cell lysate, as well as the one band that was present in the TWI fraction but not the TXI fraction, for identification using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) (Figure 4). The twelve proteins that were identified were associated with a variety of functional characteristics, including four with well-established biochemical roles, three putative lipoproteins, and five of unknown function (Table 2). The four with characterized biochemical roles were lactate dehydrogenase, ribosomal protein S3, pyruvate dehydrogenase subunit E2, and OppF, an oligopeptide ABC transporter ATP-binding protein. Two out of the three putative lipoproteins were identified as P35 and P42, both members of the P35 lipoprotein family; P42 was the protein that was present only in the TWI fraction. The third lipoprotein is a homolog of Mycoplasma genitalium G37 MG309. Of the five identified proteins that were uncharacterized, four are encoded by genes found very close to one another in the genome, MYPE1530, 1550, 1560, 1570. Together with MYPE4000, they share no specific sequence homology with other proteins except in the close relative of M. penetrans, M. iowae (Pritchard et al., 2014). In the original annotation of the M. penetrans genome MYPE1530, 1550, 1560, and 1570 were labeled as cytoskeletal proteins, based on predicted extensive alpha-helical coiled-coil structure, reminiscent of M. pneumoniae AO protein HMW2 (Sasaki et al., 2002), which is predicted to constitute a major structural element within the M. pneumoniae AO cytoskeleton (Balish et al., 2003a; Bose, Balish & Krause, 2009; Nakane et al., 2015)(Balish et al. 2003a; Bose, Balish and Krause 2009; Nakane et al. 2015)(Balish et al., 2003a; Bose et al., 2009; Nakane et al., 2015)(Balish et al., 2003a; Bose et al., 2009; Nakane et al., 2015).

The absence of sequence homology of these five proteins to the proteins of the AO cytoskeleton of M. pneumoniae (Nakane et al., 2015) or the analogous structure in M. mobile (Nakane & Miyata, 2007), together with the disparity in the detergent-insoluble

Figure 4. SDS-PAGE of M. penetrans whole-cell lysate, TWI, and TXI proteins. A total of 22 μg each of protein from whole cell lysate (lane 2), TWI (lane 3), and TXI (lane 4) were separated by SDS-PAGE for comparison and for excision of candidate bands chosen for identification. Lane 1 contains protein standards, with sizes in kDa indicated to the left. A 66-kDa band in the lane 4 is only present in some preparations. Symbols indicate specific bands chosen for MALDI-TOF. +, one protein identified from band; *, two proteins identified from band.

Table 2. TXI and TWI proteins identified by MALDI-TOF.

Gene Functional annotation Size (kDa) MYPE1530 NA 122 MYPE1550 NA 386 MYPE1560 NA 99 MYPE1570 NA 93 MYPE4000 NA 99 MYPE5100 Pyruvate dehydrogenase 51 subunit E2 PdhC MYPE6630 P42 lipoprotein (P35 42 family) MYPE6810 P35 lipoprotein (P35 38 family) MYPE6960 Lipoprotein 138 MYPE8750 Oligopeptide ABC 92 transporter ATP-binding protein OppF MYPE9640 Lactate dehydrogenase 34 Ldh MYPE10090 Ribosomal subunit S3 48 RpsC

structures between M. penetrans and mycoplasmas from other phylogenetic clusters, is consistent with a model in which attachment organelles arose independently during the course of mycoplasma evolution multiple times (Relich et al., 2009; Jurkovic et al., 2012). We examined whether there were nonetheless general features shared in common among the cytoskeletal proteins of M. penetrans, M. pneumoniae, and M. mobile. The five putative M. penetrans cytoskeletal proteins have molecular weights ≥93 kDa, isoelectric points of ~4.5, and predicted alpha-helical coiled-coil regions, all but MYPE4000 with extensive ones. Many of the proteins of the M. pneumoniae attachment organelle, but not of the tip structure of M. mobile, share these features (Table 3).

Cotranscription of putative cytoskeletal genes Examination of the genomic organization of the genes encoding proteins MYPE1530, 1550, 1560, and 1570, as well as predicted structural and compositional similarities among their predicted protein products, suggested that along with the genes encoding the similar proteins MYPE1520 and 1540 they might form a transcriptional unit. All of these genes are each separated by no more than 30 bp. To test whether these genes are cotranscribed we performed RT-PCR. We used a primer complementary to a region near the 3’ end of the 3’-most of these genes, MYPE1570, to create cDNA. From this cDNA we amplified regions spanning the junctions between each of the six candidate genes (MYPE1520-1570) as well as MYPE1510, which is ~180 bp away from MYPE1520 and on the opposite strand. We also made a cDNA using a primer complementary to MYPE1580, which is also ~180 bp away and attempted to amplify cDNA linking MYPE1570 and MYPE1580. There were RT-PCR products for all of the junctions between MYPE1520-1570, but no products for the MYPE1510-1520 junction (Figure 5A). Amplification of the junction between MYPE1570 and MYPE1580 sometimes yielded a faint, poorly reproducible product, suggestive of partial read-through (not shown). These results suggest that MYPE1520-1570 constitute an operon (Figure 5B).

Table 3. Comparison of AO protein features.

Protein Coiled coil MW>70 kDa Extreme pI (<5 Total number or >9) of features M. pneumoniae HMW1 √ √ √ 3 (MPN447) HMW2 √ √ √ 3 (MPN310) HMW3 √ √ 2 (MPN453) P24 (MPN312) 0 P41 (MPN311) √ √ 2 P65 (MPN309) √ √ 2 P200 √ √ 2 (MPN567) TopJ √ √ 2 (MPN119) M. mobile MMOB0150 0 MMOB1620 0 MMOB1630 √ √ 2 MMOB1640 0 MMOB1650 √ 1 MMOB1660 0 MMOB1670 0 MMOB4530 √ 1 MMOB4860 √ 1 MMOB5430 √ 1 M. penetrans MYPE1530 √ √ √ 3 MYPE1550 √ √ √ 3 MYPE1560 √ √ √ 3 MYPE1570 √ √ √ 3 MYPE4000 √ √ √ 3 MYPE5100 0 MYPE6630 √ 1 MYPE6810 √ 1 MYPE6960 √ √ 2 MYPE8750 √ √ √ 3 MYPE9640 0 MYPE10090 √ 1

Figure 5. RT-PCR analysis of putative cytoskeletal operon of M. penetrans. A) Agarose gel containing PCR products from RT-generated transcripts that span across the gene junctions numbered in (B). Top, amplified products from reverse transcribed RNA using a primer complementary to a region at the 3’ end of mype1570; bottom, products amplified from genomic DNA used as a positive control. B) Schematic of genes encoding MYPE1520- 1570 showing genomic orientation and relative size, including the 5’ and 3’ flanking genes. Numbers 1-7 refer to position of primer pairs used to span gene junctions for RT-PCR.

Discussion

As previously described (Jurkovic et al., 2012), there can be little doubt that the detergent-insoluble, DNA-free objects observed by SEM, whose abundance on cover slips is routinely parallel to that of unextracted M. penetrans cells grown from the same inoculum (not shown), are what occupy the space in the nucleoid-free zones of M. penetrans cells and are the material observed in thin sections at cell poles, also observed here by ECT. Indeed, the material underlying the cell poles in ECT images, which closely resemble the material at the cell poles observed in TEM images of thin sections (Lo et al., 1991, 1992; Neyrolles et al., 1998), are strikingly reminiscent of ribosome-free PopZ-containing structures at the poles of C. crescentus cells when PopZ is overproduced (Ebersbach et al., 2008). Our deeper study of these objects reveals that the organization of the TXI and TWI structures of the M. penetrans AO is completely unlike that of M. pneumoniae. Whereas the dimensions of the M. pneumoniae structure appear to be tightly constrained (Hatchel & Balish, 2008), in M. penetrans, as visualized in a number of ways, the dimensions are highly variable. Nonetheless, SEM images of these variable objects revealed some commonalities, including a wider and a narrower portion, the latter often containing one or more flexible rod-like elements. This heterogeneity, which is distinct from the highly regular M. pneumoniae AO structures, suggests the possibility of growth by accretion of material onto, or absorption of material into, a pre-existing structure. As ECT images confirm, both poles contain the same differentiated material, and this material even extends into the filaments connecting dividing M. penetrans cells. These facts are consistent with a model in which nucleation and biogenesis of the AO underlying structure are linked to the cell cycle, perhaps with new material being deposited into spaces in the cytoplasm vacated during chromosome condensation and segregation. The broader size distribution of the nucleoid-free zones and the detergent-insoluble structures suggests that growth of the M. penetrans AO is not capped at a uniform size. The length of the nucleoid-free zone does not correlate with the length of the cell or its division status (not shown), ruling out the possibility that AO growth is directly coordinated with the cell cycle, even though nucleation of the structure seems to be. Possibly growth occurs as long as cell components in the cytoplasm, such as the nucleoid and ribosomes, occupy a small enough volume that there is space for the AO

structures to grow, which includes a stochastic element that accounts for the variation in size. In addition to the heterogeneity in size, the pleomorphy of the detergent-insoluble objects is striking. We noted that whereas our SEM images of whole M. penetrans cells and previously described TEM images of thin sections (Lo et al., 1991, 1992; Neyrolles et al., 1998) show that the region of the cell at the pole is constricted along the long axis, this was not the case in ECT images. One possible interpretation of this discrepancy is that the material within the M. penetrans AO is highly hydrated and therefore subject to shrinkage during the dehydration steps associated with standard SEM and TEM processing techniques, whereas ECT lacks a dehydration step. Conceivably this hydration results from attraction of water molecules to the highly negatively charged proteins present in the structure. If the structural elements are principally organized parallel to the long axis, dehydration would result predominantly in a decrease in width rather than length. Another possibility is that because the cell bodies visualized by ECT are not attached firmly to surfaces, but instead are present in the holes of the carbon-coated grids, they are in a relaxed conformation. In either case, the material within the AO is predicted to have flexible, possibly gel-like, characteristics. Of the proteins we identified in the TXI and similar TWI fractions of M. penetrans by MALDI-TOF, four have characterized biochemical roles. Whether any or all of them are part of the internal structure of the AO is unclear. Pyruvate dehydrogenase subunit E2 normally functions as part of a large structure that could be insoluble in nonionic detergents; on the other hand, none of the other subunits of pyruvate dehydrogenase were identified, suggesting a possible alternative or additional role for this protein, which also moonlights as a surface adhesin in other mycoplasmas (Gründel et al., 2015). OppF is normally a peripheral membrane component of the oligopeptide ABC transporter (Higgins, 2001), including in mycoplasmas (Hopfe & Henrich, 2004). The substrate-binding protein component of this transporter, OppA, doubles as an adhesin in Mycoplasma hominis (Henrich, Feldmann & Hadding, 1993; Henrich et al., 1999), raising the possibility that M. penetrans OppF anchors the material underlying the AO to OppA acting as an adhesin. However, none of the proteins of this complex other than OppF were identified in the M. penetrans detergent-insoluble fractions.

Surprisingly, there were three lipoproteins identified in the detergent-insoluble fraction. Two of these lipoproteins are members of the P35 lipoprotein family, which comprises 44 total M. penetrans genes (Sasaki et al., 2002). The P35 lipoprotein and its paralogs, which are distributed across the surface of M. penetrans cells, are immunodominant (Wang et al., 1992; Neyrolles et al., 1999a, 1999b; Röske et al., 2001). Expression of genes encoding these proteins undergoes extensive antigenic variation, which is believed to be a major factor for immune evasion (Horino et al., 2003). The presence of two of these P35 homologs in the detergent-insoluble fractions may suggest that they indirectly interact with the AO interior, although it is also possible given their extensive distribution across the cell surface that they aggregate or form multiple interactions with each other that prevents their solubilization in nonionic detergents. Indeed, the difference in appearance between the TWI and TXI fractions might be entirely attributable to the presence of P42 in the former. The third lipoprotein identified, MYPE6960, has no known function and its distribution and presence at the cell surface of M. penetrans cells is unknown. However, this protein is a homolog of MG309 a protein in M. genitalium that binds and activates Toll-like receptors (Sasaki et al., 2002; McGowin et al., 2009). Conceivably, this lipoprotein plays a role in adherence of M. penetrans to host cells and is associated with AO proteins. The remaining five proteins that were identified in this study had no predicted function or conserved domains, making them ideal candidates for principal structural elements of the AO. The genes encoding four of these proteins are located very close together in the genome of M. penetrans and were found to be transcribed as a polycistronic message with two other genes. Three of these, MYPE1550, 1560, and 1570, are among the most abundant proteins in the detergent-insoluble fractions. All six of these proteins, as well as MYPE4000, share an unusual set of characteristics: predicted alpha-helical coiled- coil regions, molecular weights greater than 90 kDa, and pIs of approximately 4.5 (Table 3). These characteristics are shared with many of the M. pneumoniae AO cytoskeletal proteins (Table 3) despite lacking sequence homology. It is highly unlikely, given that the M. penetrans proteins lack characteristics of M. pneumoniae AO proteins like proline-rich domains and sequence elements like the EAGR box, that the M. pneumoniae and M. penetrans AO proteins share common ancestry. However, the shared properties are

intriguing when considering the functional analogy of the respective structures that they build.

Acknowledgments

This work was supported by the National Institutes of Health (Public Health Service grant R15 AI073994) to MFB. We thank A. Kiss (Miami University Center for Bioinformatics and Functional Genomics) and K. Pflaum (University of Connecticut) for help with RNA-Seq. We thank R.J. Hickey (Miami University), W. Ambrosius (Wake Forest University), and members of the Balish laboratory for insightful discussions. This work was done in partial fulfillment of SLD’s doctoral dissertation requirements.

CHAPTER 2

Creation of tools to examine attachment organelle protein localization in Mycoplasma iowae, a new genetic model for Mycoplasma penetrans

Steven L. Distelhorst, Neena K. Patel, and Mitchell F. Balish

Abstract

Mycoplasma penetrans is a putative opportunistic pathogen that like some other mycoplasma species has an attachment organelle (AO) used for adherence and gliding motility. Previous characterization of the M. penetrans AO revealed that it contains a novel cytoskeletal structure which lacks homologs of AO core proteins of mycoplasmas from other phylogenetic clusters. We recently identified four Triton X-100-insoluble proteins that are encoded in a six-gene operon that we believe to be involved in formation of the AO core structure. However, characterization of the cellular localization of these proteins has been hampered by the intractability of M. penetrans to genetic manipulation and our lack of success in generating specific antibodies that recognize these proteins. We have recently found that Mycoplasma iowae, a close relative of M. penetrans, is genetically tractable and has an AO with the same functions and similar morphology and ultrastructure of the M. penetrans AO. In this study we identified a set of M. iowae genes that are homologous to the six-gene putative cytoskeletal operon of M. penetrans. Applying the principle that M. iowae could serve as a cellular model for M. penetrans cells, we generated a chimera of one of these proteins via translational fusion to GFP and attempted to examine its cellular localization.

Introduction

The polar localization of proteins is an important feature in the physiology, development, and motility of bacteria. For many bacteria, attachment is important for survival. Proper localization of adhesins is often necessary to allow for correct orientation of bacteria that are dependent on a host, such as mycoplasma species, for which host cell attachment is necessary for survival (Razin & Jacobs, 1992). Furthermore, because attachment is the first step required for pathogenic bacteria like Mycoplasma pneumoniae to colonize the host, the mechanisms necessary to establish attachment are essential for pathogenesis (Atkinson, Balish & Waites, 2008; Waites, Balish & Atkinson, 2008). Given the critical role that polar localization of proteins plays in the attachment of mycoplasmas and other bacteria to host cells it is important to elucidate and understand these mechanisms.

Some mycoplasma species exhibit distinct polar localization of adhesins by generating a terminal prosthecal protrusion known as the attachment organelle (AO) (Balish, 2006a). Given the distinctive morphological and functional polarization of mycoplasma cells with AOs, species that use these structures make good models for understanding some of the mechanisms used to establish polarity. Because of its impact as a pathogen, most studies regarding mycoplasma AOs have been conducted in M. pneumoniae. M. pneumoniae cytadherence is mediated by at least two adhesin proteins, P1 and P30, both of which are concentrated at the AO (Baseman et al., 1982, 1987; Morrison-Plummer et al., 1986; Seto & Miyata, 2003). Multiple proteins are involved in organization of the AO and recruitment and proper localization of these adhesins. The interior of the M. pneumoniae AO is composed of a proteinaceous core that is necessary for structural support, given that mycoplasmas lack a cell wall, and for proper assembly of the AO and recruitment of the adhesins (Balish, 2006a, 2006b). Thus, the AO core serves as a focal point for establishing and maintaining polarity (Balish, 2006b; Balish & Krause, 2006).

Mycoplasma mobile, a distant relative of M. pneumoniae, also has a polar AO, but the composition and morphology of the M. mobile AO and ultrastructure of its core are

completely different (Himmelreich et al., 1996; Shimizu & Miyata, 2002; Jaffe et al., 2004; Balish, 2014a; Miyata & Nakane, 2014). The conserved functions of these AOs, despite their disparate composition, suggest that these species use different mechanisms to establish and maintain polarity. Since the proteins of the AO core play such important roles, identification and comparison of their characteristics can provide insight into the features that may be important for architecture, AO biogenesis, and generation of polarity. Analysis of the AOs from M. pneumoniae and M. mobile has been informative; however, they only represent two phylogenetic clusters, the M. pneumoniae cluster and the Mycoplasma sualvi cluster, respectively. Examination of AOs from a third mycoplasma cluster would provide further insight and comparison of the characteristics of AO core proteins. To this end, we have begun to examine the composition, structure, and characteristics of the Mycoplasma penetrans AO, representing a third cluster, the Mycoplasma muris cluster.

M. penetrans is a putative opportunistic pathogen that has been most studied in the context of its association with AIDS patients. The seroprevalence of M. penetrans among AIDS patients was reported to be at 40%, but only 0.3% among HIV-negative patients (Wang et al., 1992). Additionally, patients with more advanced AIDS were also found to exhibit greater seropositivity for M. penetrans (Grau et al., 1995). Furthermore, the lipoproteins of M. penetrans stimulate the replication of the HIV genome and are mitogenic towards B and T lymphocytes (Nir-Paz et al., 1995; Sasaki et al., 1995; Iyama et al., 1996; Shimizu et al., 2004). The cumulative implications of these results suggest that M. penetrans may act as a cofactor in the progression of AIDS.

Compared to M. pneumoniae and M. mobile, much less is known about the AO of M. penetrans. The M. penetrans AO functions similarly to other AOs in that it is a polar protrusion at which host cell attachment occurs (Lo et al., 1991, 1992). Like M. pneumoniae and M. mobile, the M. penetrans AO has a novel cytoskeletal core that lacks homologs of AO proteins from species outside of the M. muris cluster (Jurkovic et al., 2012). Further analysis of the components that make up this novel AO core in M. penetrans has been greatly hampered by the inability to perform genetic manipulation, despite the availability of a fully sequenced genome. To circumvent these limitations, we have recently identified 12 proteins of M. penetrans found in the Triton X-100 detergent-insoluble (TX-INS)

fraction, which contains objects consistent with the size of the AO core (see Chapter 1). Like most cytoskeletal proteins, the mycoplasma AO core proteins are not solubilized by Triton X-100, so it can be used to extract and concentrate some of these proteins. Analysis of the 12 identified M. penetrans TX-INS proteins revealed 4 proteins whose genes are found in a six-gene transcriptional unit, mype1520-mype1570. The fact that these genes are part of a transcriptional unit suggests that all six gene products could be components of the M. penetrans AO cytoskeleton. Confirmation that these proteins are components of the AO core, however, requires examination of their subcellular location. Previous attempts to localize MYPE1570 by immunofluorescence microscopy were unsuccessful, possibly due to its location being submerged within the core and inaccessible to the antibodies (Jurkovic, 2012). Given the inability to perform immunofluorescence microscopy or any genetic modification to localize these proteins, further characterization of the M. penetrans AO requires alternative approaches.

Mycoplasma iowae, a close relative of M. penetrans within the M. muris cluster, is an agriculturally and economically significant poultry pathogen that predominately infects turkeys but also affects chickens (Al-Ankari & Bradbury, 1996). Infection with M. iowae can cause late embryonic death in turkey and chicken eggs as well as leg abnormalities in turkey pouts (Bradbury & McCarthy, 1983; Trampel & Goll, 1994; Al-Ankari & Bradbury, 1996; Pritchard et al., 2014). The morphology and ultrastructure of the M. iowae AO and its cytoskeletal core are very similar to those of M. penetrans (Jurkovic et al., 2012). We have recently found that M. iowae is transformable with a transposable element, allowing for genetic manipulation (Newman, Clines, and Balish, unpublished). Since M. iowae has an AO that is functionally and structurally similar to M. penetrans and is capable of being genetically modified, we reasoned that this organism can serve as a proxy for examining the localization of AO proteins of M. penetrans. Additionally, beyond its role as a model for understanding the M. penetrans AO, studies of the M. iowae AO have the potential to provide further insight regarding AO composition and organization among species of the same cluster as well as the components and mechanisms that mycoplasma use for polarity. In this study, we identified a putative M. iowae operon with genes homologous to the AO genes identified in M. penetrans and attempted to examine the subcellular localization of

the M. penetrans MYPE1570 ortholog, P271_106, in transformed M. iowae cells containing a C-terminal GFP fusion of this protein using fluorescence microscopy.

Methods

Bacterial strains and growth conditions

M. iowae serovar K strain DK-CPA was used throughout this study. M. iowae was grown at 37° C in 175-cm2 tissue culture flasks containing 50 mL of SP-4 broth to mid-log phase (Tully et al., 1979). For growth of colonies after transformation cells were grown on SP-4 media containing 1% noble agar (Becton Dickinson) and 10 μg/mL tetracycline. Escherichia coli DH5α cells containing plasmid pGLO (Bio-Rad) were grown overnight in Luria-Bertani broth containing 100 μg/ml ampicillin and 2% arabinose.

Construction of plasmids and transformation of M. iowae

In order to obtain the p271_106-gfp gene fusion under the control of the native p271_106 promoter each individual gene and the promoter were amplified and subcloned into plasmid pCR2.1 (Invitrogen) as previously described (Pritchard et al., 2014). The gfp gene was amplified from purified pGLO plasmid (Bio-Rad), using primers designed for amplification of this gene with added restriction sequences (Table 4). The p271_106 gene and promoter were amplified from M. iowae DNA using primers designed for the respective sequences with added restriction sequences (Table 4). All cloned sequences in pCR2.1 were sequenced at the Miami University Center for Bioinformatics and Functional Genomics (CBFG) to ensure 100% identity. The resulting plasmids containing gfp, p271_106, and the p271_106 promoter sequence were named pOO73, pOO70, and pOO72 respectively.

Each of the cloned sequences in pOO73, pOO70, and pOO72 were then sequentially digested and ligated into plasmid pTF20 (Dybvig, French & Voelker, 2000), carrying a modified form of transposon Tn4001, described as follows. Plasmid pOO73 was digested with SalI, purified from a 1% agarose gel, and then ligated into pTF20 that was linearized using SalI, resulting in the plasmid pOO74. The p271_106 gene was then cut out of pOO70 with AscI, gel-purified, and ligated into pOO74, which was linearized using AscI, generating

plasmid pOO75. Plasmid pOO72 was then digested with AsiSI and the promoter sequence was gel purified

Table 4. Primers used for cloning (restriction sites in italics).

Primer Name Sequence (5’-3’) Restriction sites pGLO-GFP(R) GTCGACGGCTGAAAATCTTCTCTCATCC SalI pGLO-GFP(F) GTCGACGGCGCGCCATGGCTAGCAAAGGAGAAGAAC SalI/AscI myio1570FOR(#2) GGCGCGCCGCGATCGCCTTTAAAAAGTGGGAGTGGG AscI/AsiSI myio1570REV(#2) GGCGCGCCACCTGGTCTTAGATCTTTTGC AscI myio1570prm.F(#2) GCGATCGCAACTAAAAGCTATTGGAAGTGTGAG AsiSI MYIO1570prm.R#2-1 GCGATCGCCGCTAGAATTACTGATTTTTAGTCC AsiSI

and ligated into pOO75, which was linearized with AsiSI, resulting in plasmid pOO77 which contains the p271_106-gfp gene fusion under the control of the p271_106 promoter (Figure 6). Plasmid pOO77 was sequenced at the Miami University CBFG to ensure correct orientation of inserts and correct nucleotide sequences.

To produce M. iowae transformants containing pOO77, electrocompotent M. iowae cells were prepared by washing cells three times and resuspending in HEPES-sucrose buffer (8.0 mM HEPES, 272 mM sucrose, pH 7.4) as described previously (Hedreyda, Lee & Krause, 1993). For electroporation 20 μg of pOO77 was added to 100 μl of electrocompetent M. iowae cells and incubated on ice for 15 minutes. Cells were then electroporated in a chilled cuvette under the following conditions; 2.5 kV, 100 Ω, and 25 μF. Immediately following the electroporation 1 ml of cold SP-4 broth was added and the cells were incubated at room temperature for 10 minutes. Cells were then grown for 3 hours at 37° C to allow for recovery prior to plating. After recovery the transformants were plated and grown at 37° C for 10-15 days. Multiple transformants were picked and subjected to three rounds of filter cloning. Cells were incubated for 3 hours in SP-4 broth containing 3% gelatin to allow for attachment (Hatchel et al., 2006) and were then examined by fluorescence and phase-contrast microscopy using a Leica DMIRB microscope.

Immunoblot analysis

The immunoblot was performed as previously described (Relich & Balish, 2011). Whole-cell lysate of E. coli cells carrying the pGLO plasmid (Bio-Rad) were used as a positive control for GFP. Mid-log phase E. coli cells, induced for GFP with arabinose, were pelleted for 15 minutes at 6,000 x g. The cell pellet was resuspended and sonicated for a total of two minutes and the resulting lysate was used for the immunoblot. A total of 50 ug of whole-cell lysate was used for each sample. The protein was separated through a 10% SDS-polyacrylamide gel (Laemmli, 1970) and then transferred to a nitrocellulose membrane for immunoblotting (Towbin, Staehelin & Gordon, 1979). The blot was blocked in 5% (w/v) milk and blotted with a 1:1000 dilution of rabbit monoclonal anti-GFP

Figure 6: Design and construction of plasmid pOO77. The sequences for gfp, p271_106 (represented here as 106), and the p271_106 promoter (represented here as pmtr) were each individually cloned into the transfer vector pCR2.1. These sequences were then cut out of the transfer vector using specific restriction enzymes and sequentially ligated into linearized pTF20. Red numbers under arrows show the order of workflow. Colored scissors indicate the specific restriction enzymes used to cut out sequences or linearize plasmids. Colored lines indicate plasmid backbone (brown or blue) or specific sequences ligated into the plasmids; gfp (green), p271_106 (purple), or p271_106 promoter (orange). Bold letters above black vertical lines represent restriction enzyme cut sites; A, SalI, B, AscI, and C, AsiSI.

(ABcam). The blot was subsequently incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG and the protein bands detected using BCIP and NBT.

Results

Following the recent sequencing, annotation, and analysis of the M. iowae serovar K strain DK-CPA genome (Pritchard et al., 2014) we examined the genome sequence for homologs to M. penetrans genes by performing an amino acid sequence BLAST analysis. We identified a set of genes in the M. iowae genome that looked like orthologs of the mype1520- 1570 genes that compose an operon involved in AO structure in M. penetrans (see Chapter 1). The genomic organization of these six genes in M. iowae, p271_111-106, is very similar to the operon structure in M. penetrans (Figure 7A). Furthermore, a protein alignment of MYPE1570 from M. penetrans and the M. iowae ortholog P271_106 showed these two proteins to be well conserved with 36% identity and 58% similarity (Figure 7B). Because P271_106 is an ortholog of the M. penetrans protein MYPE1570, and unlike M. penetrans, M. iowae is transformable, we sought to examine the subcellular location of P271_106 in M. iowae cells.

We generated a plasmid, pOO77, containing a transposon carrying a p271_106-gfp gene fusion and the region upstream of the first gene of this putative operon (Figure 8), which presumably contains the promoter that governs its expression. Following transformation of competent M. iowae cells and selection for tetracycline resistance, we attempted to visualize the location of the P271_106-GFP fusion product in five filter-cloned transformants by fluorescence microscopy. Unfortunately, we did not observe fluorescence in any cells derived from these five different transformants. To ensure that the desired sequences were present in the genomes of the transformants and that the gene fusion was in frame, we sequenced all five of the transformants examined. This worked showed that the p271_111 promoter and p271_106-gfp gene were in all of the transformants in the correct orientation and that the fusion was in frame. To examine if the P271_106-GFP fusion was being produced, we performed a Western blot, using a commercial antibody against GFP, on whole-cell lysate of three transformants and compared that to lysate from an E. coli strain known to produce GFP. A band consistent with the size of GFP was

Figure 7: Genomic organization of M. penetrans genes mype1520-1570 and M. iowae genes p271_111-106 and protein sequence alignment of MYPE1570 and P271_106. (A) Arrows indicate genes from M. penetrans (top) and M. iowae (bottom) showing gene orientation. Size of arrows is relative to respective gene sizes. The numbers in between the arrows indicates the number of nucleotides between the genes. (B) Protein sequences of MYPE1570 and P271_106 were aligned and compared using BLAST (NCBI). Letters in black boxes show identical amino acids and letters in grey boxes show similar amino acids in the two proteins.

M. penetrans 179 14 2 12 25 30 182 1510 1520 1530 1540 1550 1560 1570 1580

M. iowae 168 22 21 16 24 25 108 112 111 110 109 108 107 106 105

MYPE1570 1 MINEQK------FMTRLEELELNEEFQKLPSDKKIILIAGEKILEMNEYLKNFFLNLE P271_106 1 MNNNKNLDMIFDFDKSDRIEELKSNPEFQKLPYNQQVILLTGEKLLEIDAKIRNQTSQIN

MYPE1570 53 NIAFEKFNAQS------FKQELMDDILSNIQEKLNQSGSISKQEALDLINNRY P271_106 61 N-FFKQFNNSLTDTNPNGIISDELIRSLVREEATKLLEENMNSATVIDEDKIAKIVQEKY

MYPE1570 100 EEEIALLEDKFHYTTEEVVNSANETFNEALERVESLSLFQVQAREEIRKNQDKIYILEEE P271_106 120 NEELYNLEDKFENVTQETIRNFDESISDALEKVENLSQFEIKAREEIQKSQDMIYLLEEE

MYPE1570 160 LTRQKEQNEILRNQVDERIRDLEDLMRFKESELLTGNFNFDGKFESFEDLERHISDKARE P271_106 180 LARQKEENELLKSQIDERFSQIEEANLNGRSDILNYEYH-DVRFETFEDLESFIKQEAQK

MYPE1570 220 IAEEKIEEYLLNQNNSGLGFDDSVTPDMINNFIYNSESAHKEFLKVYEAQIQSGNKLTEL P271_106 239 IAKKEVEDYIHDY----YLFSDDRKDKVIEKLFEENQLKDSEVSKVYQIQIDNNKKLDDL

MYPE1570 280 EKLIDKQTDQINQLSNDRKELIYLIADMIKEKKFNSAEFIKYVESQNIDD------L P271_106 295 ELLLEQQNRQIRSLDEDRQNLILTLEQVFKEKDVDIKKVLDTKPFDSIDNSNIYKEIDVL

MYPE1570 331 TKGEKE------ILKSLNEDDVDFLINNQARELVKEHIENNNTKSISEFEKELLENGF P271_106 355 DDGSDSGSKTYHVNTGNILNKDEIEILVRKEAIELIKNKIS------LLESEKDR-----

MYPE1570 383 GNVEFIVDNDFESLQESLDELKRISETNNKTSDELTELDKKVIELQNQLQKQLDENQILR P271_106 404 ----V--LT-DESVEDKLREIENISHSENEIIKQLEETNIRIKELEDQLKKQIEENINLK

MYPE1570 443 NELFDEITKNA------LYNSNNDNINKDNQLLINEYL-YDGDDMKHNYYNGKTPIPGTK P271_106 457 NDLFDEISRNNGSENINLNLENNDNINNDIESL-NIYSEESEEDMKHNYYNGAFPIPGTK

MYPE1570 496 ITRINNYRINNDLDNISYEESQEIIKSNFAPQN--NFI-EEQKVEPTNWELENQKIIDLE P271_106 516 ITRINNYKVSNDLDDSSFEDSHKVIREDAERISTVNEVETIKPVETTAWQENSQKILDLE

MYPE1570 553 NTVQRQEEEINRLRNQDK------TLSKEEIEFLVKKEATDVVGSNYKQ------K P271_106 576 NVILKQEQEIKRLRDEKTQEQSVDGMSREALEVLVKKEALKIVNEEINQNKKSVGNQVLT

MYPE1570 597 EFMNSLEQTLSQLKELSKIQAQTVADLEKQSKKLEEVEQKVKNANL-----QNQQISPDV P271_106 636 EIDDAIKTTLRKLQELSSMQQKTIDDIEMTNKKIHDIESQLKYTDSGKSSEIEEELARVE

MYPE1570 652 LDEIIEQKYKSK--KDEYDYVDGLKKIEEERRKIDETLELERLRLLAEINLGTKKLTDLQ P271_106 696 LEKLIQEKYKINNQYKEANYADEIRKIEEERRKIEETLELERIRLLTEIENNRRQMQELS

MYPE1570 710 EKSQMNAAPVAPTP-IIIQQPVVQQPAAPAETPAEQPAKKGFFGRPAGSSASSTPSDGPV P271_106 756 EQQKQEENVFQPQPQVIIPQPVNVVEPAPVVVEEPK------P---VVETKKEPEPT

MYPE1570 769 TVNGAPKKKRKQQIFYEVKVHTTPKLTKADLEK P271_106 804 IILEAPKKKRKQQVFYEIKIHSTPKLTRADLEK

Figure 8: Map of transposon vector pOO77. Map shows the order and arrangement of genes and sequences within the plasmid. tetM, tetracycline gene, tnp, transposase gene, oriV, origin of replication of plasmid.

present in the lane containing the E. coli lysate but a band of the predicted size of P271_106-GFP (125 kDa) was absent in lanes containing the M. iowae transformant lysates (Figure 9). These results are consistent with the lack of fluorescence during microscopy and suggest that the P271_106-GFP fusion protein is not being produced by the transformants.

Figure 9: Immunoblot analysis of GFP in E. coli and M. iowae transformants. Whole- cell lysates of induced E. coli DH5α carrying pGLO plasmid and three different isolates of M. iowae transformed with pOO77. Arrow head indicates the location of P271_106-GFP. Arrow indicates the location of GFP. Molecular markers are indicated on the left.

Discussion

The goal of this work was to use M. iowae as a model for M. penetrans to examine the subcellular localization of P271_106, the homolog of MYPE1570. To this end we generated a transposon vector containing a p271_106-gfp fusion gene under control of its native promoter that was successfully integrated into the M. iowae genome, although we ultimately did not observe any fluorescence in any of the M. iowae transformants. The lack of GFP expression in three transformants that were tested indicates that this protein fusion is either not produced or is being degraded very quickly. It is possible that the addition of GFP to the C-terminus of P271_106 prevented the protein from folding correctly, resulting in immediate degradation. This problem may be remedied by fusing GFP to the N-terminus or even sandwiching it in the middle of P271_106 as has been done previously in localization studies of M. pneumoniae HMW2 (Balish et al., 2003b; Kenri et al., 2004).

Another explanation for the lack of P271_106-GFP is that the inserted gene fusion was not transcribed at significant levels. This possibility can be tested by performing RT- PCR using RNA from the transformants and primers that target transcripts of the p271_106-gfp gene fusion. Absence of transcripts would suggest that the lack of fusion protein is due to pretranslational events. The p271_106-gfp gene may have been inserted into regions of the genome in which transcription is limited, resulting in little to no expression. However, given the random nature of insertion of Tn4001 (Mahairas & Minion, 1989), the likelihood that the p271_106-gfp gene was inserted only into poorly expressed regions of the genome in the five transformants examined is low. A more likely explanation for the potential lack of gene expression of p271_106-gfp is that the region upstream of p271_111 contains a weak promoter. p271_106 is likely the last gene of a six-gene transcriptional unit, based on similarity in genomic organization with the homologs of these genes in M. penetrans (Chapter 1). Because the promoter sequences have not been characterized in either M. penetrans or M. iowae, we chose to clone the intergenic region between the 3’ end of the upstream gene that is not part of the operon, p271_112, and the 5’ end of the first gene in the operon, p271_111. This intergenic region is 170 bases in length and almost certainly contains the promoter for this transcriptional unit. We used this

native promoter in an effort to produce an expression pattern similar to wild-type M. iowae cells, and to prevent mislocalization of P271_106-GFP due to overexpression. It is possible that either this promoter is weak or that transcription of the operon does not proceed efficiently to its 3’ end, where p271_106 is located. Under this scenario it is possible that an internal promoter within the operon is principally responsible for transcription of p271_106. Recent evidence from RNA sequencing of M. penetrans provides support for this idea. A much larger number of transcripts of the two 3’-most genes of the operon, mype1560 and 1570, were found compared to the other four genes (see Chapter 3), suggesting the presence of a strong internal promoter within this operon. Indeed, the proteins MYPE1560 and 1570 were consistently seen in higher amounts than MYPE1530 and MYPE1550, whose genes are upstream, and MYPE1520 and MYPE1540 were not detected at all (see Chapter 1). Given the similar genomic organization of these genes in M. iowae, it is likely that the gene expression profile is comparable. As such, the upstream promoter from the 5’ end of the operon may be insufficient for expression of p271_106. If the p271_106-gfp gene is not expressed in these transformants, then an alternative, stronger promoter could be used, such as the tuf promoter, which has been used for localization studies of M. pneumoniae AO proteins (Kenri et al., 2004).

It is also formally possible that the proteins encoded in this operon are present in wild type cells at low concentrations. This could indicate that there is limited transcription of the genes or that transcription of the genes occurs at a specific time in the cell cycle or under certain conditions that were not present under our experimental conditions. However, both of these possibilities are unlikely given that under normal laboratory growth conditions we are able to harvest the AO core structures of which these gene products are likely a part (Jurkovic et al., 2012) and in M. penetrans, we always see the products of four of the six genes of this operon in both the TX-INS material and whole-cell lysate (see Chapter 1). These proteins are apparently present throughout cell growth, therefore, their transcripts are very likely to be expressed and present within the cells at any given time. Furthermore, the presence of mype1560 and mype1570 transcripts in M. penetrans cells in higher abundance than the other 5’ genes in this operon suggests that these genes are likely expressed in higher numbers than the other genes. Given the

similarity in the genes and genomic organization between these two species this trend is likely the same for the p271_107 and p271_106 transcripts in M. iowae cells. Thus, it is unlikely that p271_106 is poorly expressed or only expressed at specific times or under certain conditions; rather the most plausible explanation for the lack of P271_106-GFP is that there is an internal promoter within the p271_111-106 operon that regulates expression of p271_106 and this promoter was not part of our construct.

Although we were unable to examine the subcellular localization of P271_106 in this study, the framework and preliminary genetic tools were established. These tools can be used to establish the localization of P271_106 in M. iowae after alterations to pOO77 are made, such as changing the location of GFP in the protein fusion or using other promoters. Furthermore, success in this area should allow for the study of other putative AO proteins in M. iowae and their orthologs in M. penetrans that will begin to address where they localize, allowing us to further characterize the AOs of these species and better understand the organization of AO components across mycoplasma phylogeny.

Acknowledgments

This work was supported by the National Institutes of Health (Public Health Service grant R15 AI073994) to MFB and the DUOS grant supported by Miami University. We thank the Miami University Center for Bioinformatics and Functional Genomics for help with plasmid construction and DNA sequencing. We thank members of the Balish laboratory for insightful discussions.

CHAPTER 3

Analysis of Mycoplasma penetrans global gene expression in the presence and absence of HeLa cells

Steven L. Distelhorst and Mitchell F. Balish

Abstract

Mycoplasmas are a group of bacteria that have undergone reductive evolution, leaving them with small genomes and a dependence on host cells for survival. Although many mycoplasmas live commensally in nature, some are parasitic. Mycoplasma penetrans is a putative opportunistic pathogen mainly associated with HIV-infected individuals. The majority of research on M. penetrans has focused on its incidence in patients with HIV/AIDS. Despite the identification of putative virulence factors in the sequenced genome of M. penetrans, little is known about the pathogenesis of M. penetrans or its interactions with host cells. Similar to a few other human pathogenic mycoplasmas, such as Mycoplasma pneumoniae or Mycoplasma genitalium, M. penetrans cells have an attachment organelle (AO) used for adherence to host cells and gliding motility. Unlike many other mycoplasmas, however, M. penetrans cells can be internalized by host cells. The proteins of M. penetrans responsible for host cell attachment and uptake are not known. In this study we sought to identify potential candidate genes involved in attachment to and colonization of M. penetrans in host cells. We performed RNA sequencing (RNA-Seq) on M. penetrans cells grown in the presence and absence of HeLa cells to examine the global transcriptome in both conditions and also to compare the gene expression profiles from these conditions to identify differentially expressed genes. Our results show similar expression profiles in these two conditions, suggesting the possibility that neither the presence nor the absence of host cells constitutes a signal for M. penetrans to modulate gene expression.

Introduction

Mycoplasmas are a group of cell wall-less bacteria whose genomes are small due to reductive evolution. In fact, species of this genus contain the smallest genomes of any known organism capable of autonomous replication in pure culture. Due to their limited genomic content, mycoplasmas have become prime subjects of systems biology studies and templates for the design and fabrication of synthetic microorganisms (Fraser et al., 1995; Gibson et al., 2008, 2010; Karr et al., 2012). Despite the fact that mycoplasmas lack the majority of proteins associated with regulation of gene expression in other bacteria, these organisms are capable of differential gene expression and complex genetic regulation (Herrmann & Reiner, 1998; Weiner et al., 2003; Musatovova, Dhandayuthapani & Baseman, 2006; Güell et al., 2009; Zhang & Baseman, 2011; Pflaum et al., 2015; Torres-Puig et al., 2015). For example, both Mycoplasma genitalium and Mycoplasma pneumoniae undergo differential expression of genes when grown at different temperatures (Weiner et al., 2003; Musatovova et al., 2006). M. pneumoniae also exhibits global changes in expression patterns due to the specific activity of certain promoters in the presence of specific carbon sources (Halbedel et al., 2007). Furthermore, in Mycoplasma hyopneumoniae the transcriptome shows differential gene expression during growth at different temperatures and in the presence or absence of certain compounds, such as hydrogen peroxide, iron, and norepinephrine (Madsen et al., 2006a, 2006b; Schafer et al., 2007; Oneal et al., 2008). These studies, along with others, indicate that although mycoplasmas have a small gene pool they still exhibit intricate and stringent control of their expression profiles.

For pathogenic bacteria, including mycoplasmas, regulation of gene expression is a significant factor in pathogenesis. Despite being dependent on host cells for survival outside of laboratory growth conditions, pathogenic mycoplasmas, like other pathogenic organisms, must be able to respond to and withstand the onslaught of immune defenses of their host. Response to host defenses is achieved in different ways by different bacteria, but usually depends on the up-regulation of

certain genes, for example virulence factor genes, that are specific for this purpose. Virulence factors play an integral role in the establishment and proliferation of bacteria within a host and often directly mediate bacteria-host interactions. Identification of virulence factors is a key aspect to understanding the pathogenicity of bacteria and such knowledge can be applied to antibiotic discovery and vaccine development (Wu, Wang & Jennings, 2008).

Mycoplasma penetrans is a presumptive opportunistic pathogen that almost exclusively infects HIV-positive patients. This organism is typically found in the urogenital tract of HIV-positive individuals, although in one case it was isolated from a HIV-negative patient suffering from antiphospholipid syndrome (Yáñez et al., 1999), which, like AIDS, is an immune disorder. One study shows the seroprevalence of M. penetrans among AIDS patients to be as high as 40% (Wang et al., 1992). Furthermore, the lipoproteins of M. penetrans stimulate transcription of the HIV genome and are mitogenic towards B and T lymphocytes (Nir-Paz et al., 1995; Sasaki et al., 1995; Iyama et al., 1996; Shimizu et al., 2004), suggesting that M. penetrans has the capacity to accelerate the progression of AIDS.

The genome of M. penetrans, which is 1.3 Mbp in length and encodes 1065 predicted coding sequences, contains multiple putative virulence genes, including endonucleases, hemolysins, proteases, phospholipases, and an ADP-ribosylating toxin. This toxin has similarity to the M. pneumoniae CARDS toxin, which exhibits strong auto-ADP-ribosylating activity and causes extensive cytoplasmic vacuolation in HeLa cells (Sasaki et al., 2002; Johnson et al., 2009). Beyond the identification of these virulence genes and the characterization of the CARDS-like toxin, little is known about the interactions that occur between M. penetrans and host cells. Attachment of M. penetrans cells occurs via a polar prosthecal structure, known as the attachment organelle (AO), that is functionally similar, yet compositionally distinct, to AOs of other mycoplasmas (Jurkovic et al., 2012). M. penetrans cells are internalized following adherence to host cells and are found in membrane-bound vesicles or free within the cytoplasm (Lo et al., 1993; Girón, Lange & Baseman, 1996; Tarshis et al., 2004). It is clear that the M. penetrans AO mediates the early stages of

host cell attachment in a coordinated polarized manner that precedes and ultimately leads to internalization, yet the specific proteins involved in adherence and colonization of M. penetrans have not been identified. Identification of the proteins involved in adherence and early stages of infection would shed light on M. penetrans pathogenesis and could lead to development of therapeutic agents for treatment of AIDS patients or other immunocompromised individuals infected with M. penetrans. Unfortunately, work in this area has been severely hampered by the inability to carry out genetic manipulation in this organism.

Technology associated with transcriptomics, such as RNA sequencing (RNA- Seq), has provided useful tools for identifying putative virulence genes (Wu et al., 2008; Mandlik et al., 2011; Taveirne et al., 2013; Shah, 2014; Avican et al., 2015; Parreira et al., 2016). The high-throughput nature and large amount of data generated using this method allows for a rapid survey of the expression profile of an organism at a given time or condition. Comparison of expression profiles of a specific organism under different growth conditions can identify differentially expressed genes. In this way, candidate genes involved in certain cellular functions can be identified and subsequently tested in a more targeted and specific manner. RNA-Seq has significantly contributed to the identification of putative virulence factors of many bacteria (Wu et al., 2008; Mandlik et al., 2011; Taveirne et al., 2013; Shah, 2014) and has also provided a means to identify and examine the expression patterns of virulence genes in bacteria in which genetic modification is limited or not possible. Despite its utility, there are currently only a handful of studies in which RNA-Seq has been used to examine the expression profiles in mycoplasmas (Güell et al., 2009; Mazin et al., 2014; Siqueira et al., 2014; Lluch-Senar et al., 2015; Pflaum et al., 2015). Of these, only one described examination of differential gene regulation during infection, focusing specifically on the transcript profile of the vlhA genes, which constitute a lipoprotein gene family, in Mycoplasma gallisepticum (Pflaum et al., 2015).

The interactions between M. penetrans and HeLa cells have been studied to a considerable extent (Andreev et al., 1995; Borovsky et al., 1998; Tarshis et al., 2004;

Zeiman, Tarshis & Rottem, 2008; Johnson et al., 2009). Therefore, we used RNA-Seq to identify candidate genes involved in adherence and early stages of colonization of M. penetrans cells by comparing the expression profiles of M. penetrans grown in culture and with HeLa cells. We anticipated that the data generated from this study could be helpful for further understanding virulence mechanisms of mycoplasmas and, ultimately, in the identification of targets for therapeutic agents against M. penetrans.

Methods

Bacteria culture conditions

Mycoplasma penetrans strain HF-2 was used throughout this study (Yáñez et al., 1999). M. penetrans cells were grown at 37°C in 175-cm2 tissue culture flasks containing 50 ml of SP-4 broth (Tully et al., 1979) to mid-log phase.

RNA extraction RNA was harvested as previously described (Pflaum et al., 2015). M. penetrans cells were harvested by centrifugation at 17,400 x g for 20 min, followed by three washes in PBS. The cell pellet was resuspended in 1 mL of TRI reagent solution (Ambion). Total RNA was then purified using the Direct-zol RNA MiniPrep kit (Zymo Research) according to the manufacturer’s instructions. Remaining DNA was removed from the sample by treating with DNase I twice using the DNA-free kit (Applied Biosystems) according to the manufacturer’s instructions. Quantitative PCR was performed to ensure the RNA was free of DNA. The quality of the RNA was then examined using the Agilent 2100 Bioanalyzer (Agilent Technologies).

RNA-Seq

M. penetrans cells were grown for 6 h in SP-4 broth at 37°C either alone or together with HeLa cells grown to 70-80% confluency at an MOI of 100. Three biological replicates were used for each of these conditions. For each biological replicate, RNA was extracted as described above and divided into three technical replicates. Purified bacterial RNA was enriched by performing poly(A) depletion using the NEBNext Poly(A) mRNA magnetic isolation module (New England Biolabs) on all samples as previously described (Pflaum et al., 2015). The RNA was then depleted of both prokaryotic and eukaryotic rRNA using the Ribo-Zero Gold rRNA

removal kit (Illumina) as directed by the manufacturer’s instructions and then cleaned and concentrated using the Zymo RNA Clean and Concentrator-25 kit (Zymo Research) as described previously (Pflaum et al., 2015). The RNA was eluted in 25 μl RNase-free water, examined for quality using an Agilent 2100 Bioanalyzer (Agilent Technologies), and used for cDNA synthesis. cDNA libraries were created using the Illumina TruSeq stranded mRNA library preparation kit (Illumina) following the manufacturer’s protocol, as previously described (Pflaum et al., 2015). The cDNA libraries were examined for quality using the Agilent 2100 Bioanalyzer and then quantified using a library quantification kit (Kapa Biosystems). Libraries were then normalized to 10 nM, pooled, diluted, denatured, and loaded on to the MiSeq reagent kit v3 cartridge as directed by the manufacturer (Illumina). The samples were sequenced on a MiSeq system using a 75-bp paired-end approach. Data from the RNA-Seq experiment were analyzed as described previously for Mycoplasma gallisepticum data (Pflaum et al., 2015). Briefly, the fastq files were assembled, mapped, and analyzed for differential gene expression using Rockhopper (http://cs.wellesley.edu/~btjaden/Rockhopper/; McClure et al., 2013) with the M. penetrans HF-2 genome as the reference genome. Parameters were as described previously (Pflaum et al., 2015). For comparison of gene expression values the data were first normalized by determining the ratio of reads per kilobase per million (RPKM) and then the fold change was calculated by taking the log2 transformation of the RPKM data between the two biological samples.

Results

To identify genes that undergo differential expression upon interaction with host cells, we performed RNA-Seq on M. penetrans cells when grown alone and when grown in the presence of HeLa cells 6 hours after infection, and compared global gene expression profiles between the two conditions. Surprisingly, there were very little statistically significant differences in gene expression between the two conditions. We found three genes that were up-regulated and one gene that was down-regulated by two-fold (Supplemental Table 1). The three up-regulated genes were mype1035, 8260, and 3985, all of which encode small hypothetical proteins composed of 56, 50, and 65 amino acid residues, respectively. The single down- regulated gene was mype20130, which codes for a tRNA for arginine whose codon is predicted to be commonly used.

We examined the global transcript profile to see which protein-coding genes were most frequently expressed as well as which genes were not expressed. The distribution of gene expression, based on transcript counts for each gene, had a large range from zero to over 7,000 under both conditions. The expression values, reported here as transcript counts, are the normalized read counts of expressed genes in M. penetrans grown in the presence of HeLa cells for 6 hours (Bullard et al., 2010; McClure et al., 2013). Although 260 genes had over 100 transcript counts, the majority of the genes had an expression level less than or equal to 100. A total of 727 protein coding genes, out of 1065, had transcript levels between 1 and 100 (Table 5). There were also 78 genes for which no transcripts were identified, indicating that these genes were expressed below the level of detection. Of the 260 genes with transcript levels above 100, 21 had transcript counts above 1000. The top 20 genes with the highest expression levels included an array of genes involved in metabolism and protein translation as well as genes encoding lipoproteins and hypothetical proteins. These genes included lactate dehydrogenase, multiple subunits of the pyruvate dehydrogenase complex, elongation factor Tu, 3 ribosomal

Table 5: Distribution of transcript counts per gene from M. penetrans cells in the presence of HeLa cells.

Transcript counts Number of genes 0 78 1-10 267 11-50 329 51-100 131 101-300 154 301-500 39 501-1000 46 1001-3000 19 >3001 2

Table 6: Top 20 most expressed genes of M. penetrans incubated with HeLa cells. Transcript count Gene Product 7509 MYPE6570 P35 lipoprotein 6049 MYPE6810 P35 lipoprotein 2523 MYPE6630 P35 lipoprotein homolog reported as lipoprotein IMP13 2110 MYPE5100 branched-chain alpha-keto acid dehydrogenase subunit E2 1913 MYPE5110 dihydrolipoamide dehydrogenase of pyruvate dehydrogenase E3 component 1764 MYPE5080 pyruvate dehydrogenase E1 component subunit alpha 1710 MYPE1690 NADH oxidase 1636 MYPE5090 pyruvate dehydrogenase E1 component subunit beta 1591 MYPE320 elongation factor Tu 1563 MYPE3960 two-component regulator 1313 MYPE9720 hypothetical protein 1234 MYPE10070 50S ribosomal protein L14 1192 MYPE8170 glyceraladehyde-3-phosphate dehydrogenase 1166 MYPE9640 L-lactate dehydrogenase 1123 MYPE6780 P35 lipoprotein homolog reported as gene for P38 lipoprotein 1118 MYPE1340 PTS system glucose-specific enzyme IIABC component 1117 MYPE10040 30S ribosomal protein S14 1114 MYPE820 30S ribosomal protein S9 1112 MYPE3950 hypothetical protein 1101 MYPE9960 methionine aminopeptidase

protein coding genes, and 4 genes for lipoproteins, all of which are members of the P35 lipoprotein family (Table 6).

We also examined the expression profile of the genes that make up the AO cytoskeleton operon, mype1520-1570 (chapter 1). The first four genes of this operon (mype1520-1550) had transcript counts under 100. The last two genes, mype1560 and mype1570 had transcript counts that were 3 or more times higher than the first four genes in the operon.

Discussion

This study is the first to examine the transcriptome of M. penetrans and provides important information regarding the expression profile of M. penetrans cells grown both in broth culture and in the presence of HeLa cells. The objective of this investigation was to identify putative genes involved in infection of host cells, based on the hypothesis that proteins necessary for attachment and other genes involved in colonization and early host cell interactions would be up-regulated in the presence of HeLa cells.

Analyses of the transcriptomes of M. hyopneumoniae and M. gallisepticum during infection showed a total of 79 and 58 genes to be differentially expressed, with 46 and 25 of those genes found to be up-regulated, respectively (Cecchini, Gorton & Geary, 2007; Madsen et al., 2008). Similar trends were identified in studies examining differential gene expression of mycoplasmas in the presence of different environmental conditions, such as temperature or various chemical compounds (Weiner et al., 2003; Madsen et al., 2006b, 2006a; Schafer et al., 2007; Oneal et al., 2008). Among these studies, the smallest number of differentially expressed genes occurred in M. hyopneumoniae experiencing iron depletion, with a total of 27 genes showing differential expression compared to standard growth conditions, 9 of which were up-regulated and 18 down-regulated (Madsen et al., 2006b). Based on these studies and similar trends seen in transcriptomic studies of other bacteria, it was surprising that only 4 genes were differentially expressed in M. penetrans cells under the conditions tested. Furthermore, of these 4 genes only mype20130, which was down-regulated and is not a protein-coding gene, has an assigned function. Examination of the 3 genes that were up-regulated in the presence of HeLa cells showed them all to be small hypothetical proteins with fewer than 70 amino acid residues. Although transcripts of these genes were identified and found in greater numbers in M. penetrans cells exposed to HeLa cells, we suspect that they might not encode authentic translated proteins for a variety of reasons. The products of these transcripts are not only small but also show no homology to any other known gene

products in other sequenced organisms. Further studies are necessary to test whether these proteins are actually produced. Given their lack of homology and small size, we do not regard them as likely candidates for adhesins.

The finding that only 3 M. penetrans protein-coding genes and one tRNA gene, are differentially expressed in the presence of HeLa cells indicates similar cellular activities in both conditions. Although these results were not anticipated there are several possible explanations. The first possibility is that M. penetrans cells exhibit little to no change in gene expression at any stage of infection. As a result of genome reduction that rendered M. penetrans dependent on host cells for survival, M. penetrans could have evolved to constitutively express all genes. The RNA-Seq data, however, indicate otherwise, as there were some genes for which no transcripts were identified (Supplemental Table 1 and Table 5). It is highly unlikely that M. penetrans contains genes that are never transcribed. A more likely explanation is that M. penetrans cells constitutively express the genes necessary for early stages of host interaction and colonization and do not exhibit differential gene regulation until later stages of invasion, upon exposure to some substrate or condition that occurs inside host cells.

Although M. penetrans cells invade HeLa cells as early as two hours post- infection (Borovsky et al., 1998), a second possibility is that within the first six hours of infection too few bacterial cells have been internalized to show a significant change in gene expression in the entire population. The fact that M. penetrans cells are dependent on host cells for survival under natural conditions makes it very likely that they have never evolved an adaptation to living in the absence of host cells and therefore are likely to always be in a mode in which they are deployed for interaction with the host.

A third possible explanation is that regulation in M. penetrans occurs largely through post-translational modification (PTM) of proteins. It has become increasingly clear that PTMs play an important role in the regulation of cellular processes and adaptability (Cain, Solis & Cordwell, 2014; Pisithkul, Patel & Amador-

Noguez, 2015). An intricate array of processes involving the addition or removal of different functional groups at certain amino acids of proteins can alter their structure, functions, and interactions with other proteins or complexes (Cain et al., 2014). Proteolysis also plays a critical role in degrading specific proteins to inhibit their cellular functions or by cleaving certain proteins to produce two or more functional peptides with similar or possibly altered functions (Seymour et al., 2010; Cain et al., 2014; Tacchi et al., 2016). PTM mechanisms are significant in defining and altering the activities of other mycoplasmas and likely allow the cells to respond to changes in growth or environmental conditions more quickly than a global change in gene expression. Studies examining the phosphorylation and lysine acetylation state of the M. pneumoniae proteome showed that addition of these PTMs is very common, in particular affecting the translational machinery (van Noort et al., 2012). Alteration of the translational machinery via PTM may dictate the protein profile of cells under specific conditions and be responsible for changes in protein abundances under different conditions (van Noort et al., 2012). Thus, it is likely that PTMs occur in M. penetrans as well, and may explain the apparent lack in alteration of the M. penetrans transcriptome that we found.

Another possibility is that post-transcriptional regulation is occurring via regulatory RNAs. A large and growing number of small RNAs (sRNA) have been found to be important for regulation in many bacteria, including mycoplasmas (Brantl & Brückner, 2014; Lluch-Senar et al., 2015). These sRNAs typically serve to regulate translation or stability of other RNAs and thus it is possible that the four differentially expressed RNAs identified in this study play a role in regulating certain genes (Brantl & Brückner, 2014). Furthermore, there is increasing evidence that sRNAs play significant roles in regulation of virulence mechanisms during host- pathogen interactions, which may suggest that these RNAs are involved in regulating M. penetrans virulence genes (Harris et al., 2013; Ortega et al., 2014; Pitman & Cho, 2015).

Although the majority of M. penetrans genes exhibited expression levels between 1 and 100, there was a wide range of expression values from no expression to a few genes in which more than 2000 transcript counts were identified. The high expression of p35 paralogs is interesting although not surprising given that the P35 lipoproteins are the most abundant surface antigen of M. penetrans cells (Neyrolles et al., 1999b). The M. penetrans genome contains at least 38 paralogs of p35, most of which are under the control of an invertible promoter that results in phase variation (Sasaki et al., 2002; Horino et al., 2003). Thus, the identification of four highly expressed p35 genes is not unexpected, but the fact that two of these lipoprotein genes, mype6570 and mype6810, exhibited 3-3.5 times the number of transcripts relative to the next most highly expressed gene that was not a p35 homolog is interesting. Although the P35 lipoproteins have typically been considered to function in immune evasion based on their antigenic and phase variation, the large number of transcripts for these two p35 genes might suggest that these proteins have other important roles as well.

Four gene products of the twenty most highly expressed genes were identified as components of either the Triton X-100-insoluble (TXI) fraction or the Tween-20-insoluble (TWI) fraction or both fractions (see Chapter 1). These proteins were P35 (MYPE6810), P42 (MYPE6630), lactate dehydrogenase (MYPE9640), and subunit E2 of pyruvate dehydrogenase (MYPE5100). The high expression values for these genes compared to other M. penetrans genes may suggest that these proteins have multiple functions within the cell and therefore require higher levels of expression. It could also suggest that because they are so highly abundant they interact with each other or with other proteins, preventing their solubilization in Triton X-100 detergent. It is interesting to note that the corresponding protein bands seen in the SDS-PAGE of the TX-INS fraction were prominent (see Chapter 1), consistent with the gene expression data.

The presence of elongation factor Tu (EF-Tu) (mype320), glyceraldehyde-3- phosphate dehydrogenase (GAPDH) (mype8170), and the four subunits of pyruvate dehydrogenase (PDH) (mype5080, 5090, 5100, and 5110) among the genes with the

top expression levels is intriguing considering their potential to be involved in adherence. Although EF-Tu is an important protein for translation and GAPDH and PDH are key metabolic enzymes, they have all also been implicated as moonlighting proteins involved in adherence to host cell proteins in other mycoplasmas. In M. pneumoniae, for example, EF-Tu and the PDH E1β subunit localize to the surface, in addition to their cytoplasmic location, and bind fibronectin, an abundant protein on host cell surfaces (Dallo et al., 2002). The other three PDH subunits of M. pneumoniae are also membrane-bound surface proteins capable of binding to HeLa cells and interacting with and activating human plasminogen (Gründel et al., 2015). Furthermore, GAPDH has also been observed at the surface of M. pneumoniae cells, and recombinant M. pneumoniae GAPDH is capable of binding different human cell lines and fibrinogen (Dumke, Hausner & Jacobs, 2011). These three proteins show similar surface localization and adherence to host proteins in other mycoplasmas and bacteria, such as M. genitalium and some group B streptococci (Alvarez, Blaylock & Baseman, 2003; Seifert et al., 2003). Therefore, we may regard EF-Tu, PDH, and GAPDH, as candidate adhesins of M. penetrans. The high abundance of transcripts of each of these genes may be an indication that these proteins are multifunctional and therefore require greater expression levels.

Examination of the expression profile of the genes that make up the AO cytoskeleton operon, mype1520-1570 showed that mype1560 and 1570, the 3’-most genes of the operon, had the highest transcript counts. The gene products of these 2 genes also showed the highest relative abundance among these 6 cytoskeletal proteins in the detergent-insoluble fractions. Furthermore, the two proteins encoded in this operon which we did not identify, MYPE1520 and MYPE1540, had the lowest transcript counts of the 6 genes. Taken together, these data suggest that there is an internal promoter within this transcriptional unit that allows for increased transcription of mype1560 and 1570.

There were 78 protein-coding genes, representing more than 7% of the total number of protein-coding genes within the M. penetrans genome, that were not expressed in M. penetrans cells when grown in the presence of HeLa cells. Given the

small genome size and limited number of genes of M. penetrans, it would seem highly unfavorable to maintain such a large number of genes if they are never expressed. Therefore, it is likely that these genes are expressed during some yet uncharacterized condition(s) or growth stage(s) and that M. penetrans cells are capable of, and likely exhibit, differential gene expression under specific conditions or times that were not represented in this work.

Acknowledgments

SUMMARY AND CONCLUDING REMARKS

In this study we have characterized a number of the principal components of the Mycoplasma penetrans attachment organelle (AO) and drawn a number of conclusions about the pathway by which this organism constructs this specialized structure. In the work described in chapter 1, we identified 12 proteins from two different nonionic detergent-insoluble fractions that include the AO cytoskeleton. Five of these proteins have no conserved domains or known functions, and because of their composition we consider them to be top contenders as structural proteins of the AO. In the work described in chapter 2, we attempted to localize the Mycoplasma iowae protein P271_106, the ortholog of the M. penetrans protein MYPE1570, by generating a protein fusion with GFP. In the work described in chapter 3, we compared the global transcript profiles of M. penetrans cells grown in the presence and absence of HeLa cells in an effort to understand how the genes for these proteins, as well as other genes, might be regulated during the early stages of infection. The cumulative results from these studies provide further insight regarding AO development in M. penetrans. The data from these studies allow for a comparison of the composition, structure, and development of AOs across mycoplasma phylogeny, including important pathogenic species like Mycoplasma pneumoniae, whose AO is better understood (Balish, 2014b).

Despite superficial similarities in morphology and function of the AOs of M. pneumoniae and Mycoplasma mobile, dissimilarities in both organization and composition of their internal structures are well-documented (Miyata, 2010). In contrast, structure and composition of AO cytoskeletal structures are shared among the close relatives of M. pneumoniae in the M. pneumoniae cluster (Hatchel & Balish, 2008). These differences have been taken to suggest that the AOs of M. pneumoniae and M. mobile arose through convergent evolution (Balish, 2014b), which is not altogether surprising considering that the two species are separated by the deepest phylogenetic division within the genus, with M. pneumoniae in the pneumoniae

group and M. mobile in the hominis group (Johansson & Pettersson, 2002). However, the subsequent finding that Mycoplasma insons, a species in the pneumoniae group that is not a close relative of M. pneumoniae, is unidirectionally motile but lacks a distinct tip structure and instead contains cytoskeletal elements throughout its entire rod-shaped body (Relich et al., 2009) raised the possibility that even within the pneumoniae group there has been independent evolution of polar attachment and motility. If so, then these structures have an even more recent evolutionary origin. Alternatively, the M. insons organization could represent a variation of the M. pneumoniae organization whose homology is not obvious, pending further studies of this organism. M. penetrans is also in the pneumoniae group, but within a different cluster than either M. pneumoniae or M. insons (Brown et al., 2010). The AO of this species, whose motility has been established to be driven by a fundamentally different mechanism than that of M. mobile (Jurkovic et al., 2013), provided an opportunity to test whether there were detectable, conserved elements of AO organization within the pneumoniae group.

The overall organization of the M. penetrans AO core structure identified in this study is quite distinct from those of M. pneumoniae (Henderson & Jensen, 2006; Seybert et al., 2006), M. mobile (Nakane & Miyata, 2007), and M. insons (Relich et al., 2009). However, despite the absence of homology between M. penetrans and M. pneumoniae AO proteins we found that some of their respective proteins share similar characteristics such as high molecular weight, abundance of predicted alpha- helical coiled-coil regions, and low isoelectric points. These shared properties suggest that one solution to forming a polar cytoskeletal structure, arrived at independently in M. pneumoniae and M. penetrans, involves assembly of large, highly charged proteins that interact with each other over an extensive surface area via alpha-helical coiled coils. On the other hand, the lack of any of these characteristics among the M. mobile cytoskeletal proteins indicates that this species has evolved a different approach to generating a protrusive and therefore superficially similar structure. M. insons achieves polarization of adherence and motility without a protrusive structure (Relich et al., 2009), representing a completely different model

for polarization. Thus, our data support the hypothesis that mycoplasma AOs arose independently and further show that, beyond functional and morphological convergence, the M. penetrans and M. pneumoniae AO cores exhibit a deeper level of convergence at the molecular level.

Examination of the organization of the M. penetrans AO using ECT and observations and measurements of the nucleoid-free zones and the TXI and TWI structures, together with previous knowledge regarding its likely role in cytadherence, motility, and cell division, have provided insight into the mechanisms of function and biogenesis of this structure. Analysis of AO development in M. pneumoniae cells has yielded a proposed model in which predivisional cells produce a second AO directly adjacent to the primary motile AO (Balish, 2014b). This new AO, which is capable of attachment but not motility, is driven to the opposite pole as the cell continues to move, allowing AOs to be at opposite poles when cells divide, thereby assisting the cytokinesis process and ensuring the distribution of AOs to daughter cells (Balish, 2014b). In M. penetrans, the presence of AOs always at both poles, as suggested by ECT images, along with the heterogeneity of both the nucleoid-free zones and the detergent-insoluble material suggests that nucleation and development of the M. penetrans AO is different from the model for M. pneumoniae AO growth and duplication. We therefore propose a model of M. penetrans development, elaborating on a previous one (Jurkovic, 2012), that accounts for these observations and also addresses the disparity between the observations of a single narrowed pole in SEM images as opposed to no polar constriction observed in ECT (Figure 10).

In this model we propose that both poles of M. penetrans cells contain AOs capable of growth and attachment. However, due to geometric constraints resulting from the small cell size during early stages of cell growth, only one pole is able to make contact with host cells and establish sustained attachment. In this adherent state the core structure at this pole, designated the old pole, is in a constricted state, which we suggest is a feature that occurs as a result of attachment. Furthermore,

Figure 10: Proposed model for M. penetrans AO development throughout cell growth. During early stages of cell growth of M. penetrans only one AO can be attached and in a constricted state (green shaded area at right pole) due to geometric constraints based on cell size. Growth of core material is stochastic but when attached and constricted the core is in a disfavored state for addition of more material resulting in the majority of new material accumulating at the unattached pole (red shaded area at left pole). As the cell continues to grow the core material of the nonadherent AO also continues to grow until the cell becomes large enough or the AO itself becomes large enough to make contact with and bind to host cells, either of which may occur prior to initiation of division or after the division process is already underway. Like the old AO (right), when the new AO (left) attaches the core material becomes constricted. When both AO are in a constricted state new core material does not get added to either AO and therefore begins to accumulate at midcell (red lines in middle). In the final stages of cytokinesis the core material at the new poles gets split between the daughter cells. Black outline of cells represents the cell membrane and the blue line inside represents DNA.

based on the loose flexibility of the detergent-insoluble structures, indicated by their heterogeneity in size and pleomorphy, we suggest that the core structure is composed of a soft, gel-like material which contains proteins oriented in parallel fashion, constriction of which result in the narrowing of the AO when attached. Although this material is likely present at both poles, we advocate that the narrow pole observed in whole cells in SEM images is a result of the lateral constriction of the parallel filament proteins as a consequence of engagement of the adhesins with the substrate. The absence of constricted AOs in ECT images could result from the M. penetrans cells not being firmly attached to a surface. Furthermore, the presence of soft, gel-like material in the M. penetrans AO could explain the variation in shapes of the TXI and TWI material observed in SEM. We suggest that the growth and development of the core structures at both poles occurs stochastically, explaining the large range of sizes observed for both the nucleoid free zones and the TXI and TWI structures and supporting the idea that these core structures have no fixed size. We further propose that although core structures at either pole can grow, the continued addition of material to the old pole is disfavored due to its constricted state while adherent. In this manner, the opposite non-attached and non-constricted new pole is likely to acquire additional core material more quickly. At some point, however, as the new pole continues to acquire more core material, resulting in increased surface area, and the cell continues to grow, reducing the geometric constraint, this pole is able to come into contact with the host cell allowing it to attach. Like the old pole, when the new pole attaches the core material transitions to a constricted state in which further growth of the core structure is less favorable. This growth model explains the heterogeneity of core sizes as well as the less frequent but occasional appearance of very large structures. Furthermore, the idea that the mass of the core structure is independent of attachment or cell growth helps explain the lack of correlation between the lengths of the nucleoid-free zone and cell size or division status. With both the old and new poles in an adherent state and therefore in a disfavorable state for further growth, new AO core material likely does not get incorporated at either pole. We suggest that new core material accumulates at midcell and, after cytokinesis, will be partitioned into each daughter

cell, becoming its new pole. This model accounts for both the presence of core material in the connecting filament of dividing cells observed in ECT images and the nucleation of M. penetrans AOs.

More microscopy and genetic studies would further elucidate the pathway of AO development of M. penetrans cells. Different microscopy techniques, such as atomic force microscopy or cryo-SEM using a substrate that allows the M. penetrans cells to attach, could be useful for examining morphology of the cells in a native adherent state to address the hypothesis that AOs are constricted when attached. Other methods such as fluorescence microscopy and fluorescence recovery after photobleaching would also be very helpful for further examination of AO protein localization and organization and dynamics of core growth. Unfortunately, however, these techniques require fluorescently tagged proteins, which cannot currently be used in M. penetrans cells because of the inability for genetic manipulation. Due to this roadblock, we have started building a genetic system in the closely related and genetically tractable species, M. iowae. Because M. iowae contains homologs of the M. penetrans cytoskeletal genes, genetic analyses of these genes should provide insight into the organization and development of both the M. iowae and M. penetrans AO. In chapter 2 we described successful transformation of M. iowae cells with a transposable element containing a gene fusion of p271_106 and gfp. The failure to find the protein fusion in the transformed cells was likely due to the use of a weak promoter element or due to the location of gfp at the 3’ end of the p271_106 gene. Now that we have developed a suitable plasmid vector, these issues should be easily addressed and potential cytoskeletal proteins can now be tagged with GFP, or another fluorophore, to allow for localization and further characterization.

In chapter 3 we described examination of the gene expression profiles of M. penetrans cells during early stages of host cell interaction compared to cells grown in media alone. The profiles showed very few differences between the two conditions, suggesting that M. penetrans cells exhibit similar phenotypes under both conditions. An explanation for this is that M. penetrans cells have not evolved response mechanisms for growth in the absence of a host because under non-

laboratory conditions they require host cells. Future studies should further investigate the development of M. penetrans cells throughout the course of interaction with host cells. These expression profiles each provide only a single snapshot of the developmental profile of M. penetrans cells. To advance this work, it will be necessary to examine the expression profiles of M. penetrans cells at other time points throughout the infection process. Longer incubation times of M. penetrans with HeLa cells, such as 12 or 24 hours, is likely to provide necessary time for attachment and internalization of a greater number of M. penetrans cells and may result in changes in the expression profile.

Prior to these studies, the majority of information regarding mycoplasma AOs was from M. pneumoniae and M. mobile. Although there is still much to be learned, the M. penetrans AO is now a third reasonably well-characterized AO. The existence of both similarities and differences between the AOs of these species, as a product of convergent evolution, shows that we cannot simply rely on a single model species to fully understand the structural and functional characteristics of AO proteins and the mechanisms by which these proteins incur polarity. Therefore, the identification and characterization of AOs in other mycoplasmas serves as an important endeavor for the mycoplasma community as well as the field of cell biology. The presence and independent evolution of AOs throughout the Mycoplasma genus is further evidence that a diverse array of cytoskeletal proteins with narrow phylogenetic distribution are present throughout the bacterial kingdom. Global analysis of cytoskeletal structures within and across phylogeny will generate insight into the mechanisms and evolution of cellular polarity that will be invaluable to cell biology studies.

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