Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
DSpace
UBCV
Li, Haoran
Master of Science - MSc
University of British Columbia
Phase separation is involved in the organization and regulation of membraneless cellular components and their functions. We showed previously that the cytoplasmic regulatory module of the ABC transporter Rv1747 of Mycobacterium tuberculosis, which is involved in cell wall biosynthesis, is able to phase separate in vitro. Here we demonstrate that this regulatory module forms static foci and full length Rv1747 mobile ones dependent on the presence of the regulatory module in Mycobacterium smegmatis. Disrupting the key interactions by the FHA domains in this regulatory module, in particular FHA-1, and phosphorylated threonines in the linker between the FHA domains demolish the resulting foci in both constructs. Specifically, we show that the S47A single mutant and the S47A, S248A double mutant exhibit no foci formation while the S248A single mutant exhibits a mixed phenotype dominated by diminished clustering. Complementarily, the regulatory module of Rv1747 carrying phosphor-ablative mutations of all Thr/Ser to Ala in the linker also shows no sign of foci formation. In addition, charges of the intrinsically disordered linker also contribute to foci formation by the Rv1747 regulatory module, as the mutant carrying Arg/Lys to Ala mutations displays no foci in M. smegmatis. Collectively, these data strongly suggest that Rv1747 phase separates in M. smegmatis and highlight the role of phosphorylation and non-specific charge-charge interactions by the FHA domains and the ID linker in regulating phase separation of Rv1747 in M. smegmatis.
INVESTIGATING MOLECULAR CLUSTERING OF RV1747 IN M. SMEGMATIS by HAORAN LI B.Sc., The University of British Columbia, 2019 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Biochemistry and Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) July 2021 © Haoran Li, 2021 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Investigating molecular clustering of Rv1747 in M. smegmatis submitted by Haoran Li in partial fulfillment of the requirements for the degree of Master of Science in Biochemistry and Molecular Biology Examining Committee: Dr. Jörg Gsponer, Professor, Biochemistry and Molecular Biology, UBC Supervisor Dr. Lawrence McIntosh, Professor, Biochemistry and Molecular Biology, UBC Supervisory Committee Member Dr. Yossef Av-Gay, Professor, Microbiology and Immunology, UBC Supervisory Committee Member Dr. Thibault Mayor, Professor, Biochemistry and Molecular Biology, UBC Additional Examiner iii Abstract Phase separation is involved in the organization and regulation of membraneless cellular components and their functions. We showed previously that the cytoplasmic regulatory module of the ABC transporter Rv1747 of Mycobacterium tuberculosis, which is involved in cell wall biosynthesis, is able to phase separate in vitro. Here we demonstrate that this regulatory module forms static foci and full length Rv1747 mobile ones dependent on the presence of the regulatory module in Mycobacterium smegmatis. Disrupting the key interactions by the FHA domains in this regulatory module, in particular FHA-1, and phosphorylated threonines in the linker between the FHA domains demolish the resulting foci in both constructs. Specifically, we show that the S47A single mutant and the S47A, S248A double mutant exhibit no foci formation while the S248A single mutant exhibits a mixed phenotype dominated by diminished clustering. Complementarily, the regulatory module of Rv1747 carrying phosphor-ablative mutations of all Thr/Ser to Ala in the linker also shows no sign of foci formation. In addition, charges of the intrinsically disordered linker also contribute to foci formation by the Rv1747 regulatory module, as the mutant carrying Arg/Lys to Ala mutations displays no foci in M. smegmatis. Collectively, these data strongly suggest that Rv1747 phase separates in M. smegmatis and highlight the role of phosphorylation and non-specific charge-charge interactions by the FHA domains and the ID linker in regulating phase separation of Rv1747 in M. smegmatis. iv Lay Summary Tuberculosis remains a threat to the public due to its drug resistance. Hence, understanding the physiology of its causative reagent, Mycobacterium tuberculosis, may reveal new therapeutic targets and promote treatment development. In this thesis, I present my research of Rv1747, a transmembrane transporter of cell wall components, which is required for the pathogen to grow and infect human. Rv1747 can form liquid droplets enriched of this protein through a process named phase separation which is similar to the separation of oil and water. I have shown that clusters enriched of Rv1747 are formed in cells dependent on the regulatory module of the protein. I have further uncovered the specific components in the regulatory module of Rv1747 responsible for clusters formation. My research thus reveals phase separation as the organizing principle for Rv1747 function in cells. This may inspire development of therapies against phase separation of Rv1747 to treat tuberculosis infection. v Preface The majority of the research and results presented in this thesis are for Master program in Biochemistry and Molecular Biology. The conceptualization of this project is accredited to my supervisor Jörg Gsponer, I established all the methods and performed all the experiments under the guidance of my supervisor. Jörg Gsponer contributed a significant portion of writing to this thesis. vi Table of Contents Abstract ............................................................................................................................ iii Lay Summary....................................................................................................................iv Preface ................................................................................................................................v Table of Contents...............................................................................................................vi List of Tables...................................................................................................................viii List of Figures....................................................................................................................ix List of Abbreviations ..........................................................................................................x Acknowledgements............................................................................................................xi Dedication.........................................................................................................................xii Chapter 1: Introduction.......................................................................................................1 1.1 Mycobacteria tuberculosis virulence and infection...............................................1 1.1.1 Tuberculosis epidemiology and history.........................................................1 1.1.2 Host infection by Mtb and immune escape mechanism.................................3 1.1.3 Infection progress and granuloma..................................................................6 1.2 Signaling system ...................................................................................................8 1.2.1 Prokaryotic signaling system..........................................................................8 1.2.2 Eukaryotic STPKs........................................................................................10 1.2.3 STPKs in Mtb...............................................................................................11 1.3 Rv1747 structure and relevance in Mtb infection................................................14 1.4 Phase separation..................................................................................................16 1.4.1 Increasing understanding of phase separation in biology.............................17 1.4.2 Determining factors in phase separation......................................................18 1.4.3 Phase separation in eukaryotic processes.....................................................20 1.4.4 Phase separation in bacteria.........................................................................21 1.4.4.1 Examples of bacterial phase separation.................................................22 vii 1.4.4.2 Rv1747, a Mtb protein that can phase separate.....................................23 1.5 Summary and working hypothesis......................................................................25 Chapter 2: Materials and Methods....................................................................................28 2.1 Plasmids preparation...............................................................................................28 2.1.1 Construction of pJC52 backbone.....................................................................29 2.1.2 Construction of mEos2-tagged plasmids..........................................................31 2.2 Expression of mEos2-tagged Rv1747 constructs in M. smegmatis........................32 2.3 Microscopy of transformed M. smegmatis.............................................................33 2.4 Data analysis and figure preparation......................................................................34 Chapter 3: Results.............................................................................................................36 3.1 Rv1747 clustering in M. smegmatis is dependent on the regulatory module….....36 3.2 Key interaction sites in the FHA domains and linker are necessary for foci formation by Rv1747 clustering in M. smegmatis.................................................39 3.3 Linker charge affects Rv17471-310 clustering in M. smegmatis..............................43 Chapter 4. Discussion........................................................................................................45 4.1 Regulatory domain dependent Rv1747 phase separation in M. smegmatis ...…...45 4.2 Key interactions by FHA domains and linker are necessary for Rv1747 phase separation in M. smegmatis..................................................................................47 4.3 Linker charge affects Rv1747 phase separation in M. smegmatis.........................50 4.4 Conclusion.............................................................................................................51 4.5 Significance, limitations and future direction........................................................52 References.........................................................................................................................56 viii List of Tables Table 1 List of plasmid constructs.................................................................................28 Table 2 List of primers....................................................................................................29 ix List of Figures Figure 1. Forkhead-associated (FHA) domains: FHA-2 replace beta-Hairpin of the Canonical Fold between strand 1 and 2 in FHA-1 with a less stable beta strand 1.........................................................................................................................................15 Figure 2. Rv17471-310 phase separation under phosphorylated and unphosphorylated conditions.........................................................................................25 Figure 3. Comparison of clustering formation across mEos2 vector, mEos2-Rv1747, mEos2-Rv17471-310, and mEos2-Rv1747∆1-310 in M. smegmatis....................................38 Figure 4. Comparison of clustering formation across mEos2 vector, mEos2-Rv17471-310, mEos2-Rv17471-310S47A, mEos2-Rv17471-310S248A, mEos2-Rv17471-310S47,248A, and mEos2-Rv17471-310STA in M. smegmatis ................................................41 Figure 5. Comparison of clustering formation across mEos2 vector, mEos2-Rv1747, mEos2-Rv1747S47A, mEos2-Rv1747S248A, and mEos2-Rv1747S47,248A in M. smegmatis..........................................................................................................................42 Figure 6. Comparison of clustering formation across mEos2 vector, mEos2-Rv17471-310, and mEos2-Rv17471-310RKA in M. smegmatis........................................... 44 x List of Abbreviations ABC ATP binding cassette BR-bodies Bacterial ribonucleoprotein bodies CTD C-terminal domain ECF Extracytoplasmic function FHA Forkhead associated FRAP Fluorescence recovery after photobleaching HIV Human immunodeficiency virus IDR Intrinsically disordered region LAT Linker for activation of T cell LTBI Latent tuberculosis infection MDR-TB Multi drug resistant tuberculosis Mtb Mycobacteria tuberculosis NA Numerical aperture NBD Nucleotide binding domain Ni-NTA Nickel-nitrilotriacetic agarose N-WASP Neuronal Wiskott-Aldrich Syndrome Protein PALM Photoactivated localization microscopy PBS Phosphate buffer saline PIM Phosphatidyl-myo-inositol mannosides PI3P Phosphatidylinositol 3-phosphate Pkn Protein Kinase PRM Proline-rich motifs pThr Phosphorylated threonine RBP RNA binding protein RD Regulatory domain RNAP RNA polymerase RR-TB Rifampicin resistant tuberculosis SDM Site directed mutagenesis SMLM Single molecule localization microscopy STPK Serine threonine protein kinase TB Tuberculosis TCR T cell receptor TIRFM Total internal reflection fluorescence microscope TMD Transmembrane domain TNF-α Tumour necrosis factor alpha V-ATPase Vacuolar ATPase WHO World health organization xi Acknowledgements I want to thank Dr. Jörg Gsponer for the opportunities, support, and trust during my Master program. He genuinely cares about my personal development, my success in the project, and my future career. When I first started in his lab, he patiently listened to my goal of completing my Master in a year and half and helped me started a cancer prediction project that fit the timeline. After seeing my struggle in the project, he fully supported my decision to change to my current project in Rv1747, which renewed and elevated my passion for research in biochemistry. During the special period of Covid-19, I was not able to continue my project due to lab restrictions. However, with Dr. Jörg Gsponer’s help, I was able to return to my project at the earliest time possible. I thank Dr. McIntosh and Dr. Av-Gay for advice on my research project and allowing me to use their lab space and materials for my project. I thank Dr. Libin Abraham for his mentorship and dedication in microscopic data collection and data analysis. I thank Mr. Joseph Chao for his mentorship and support in training and improving my techniques in molecular cloning and cell culture. I thank Dr. Karlton Scheu and Mr. Loktin Hui for their advice and help. I thank all the current and past lab members for help and inspirations. xii Dedication To my parents 1 Chapter 1: Introduction 1.1 Mycobacteria tuberculosis virulence and infection 1.1.1 Tuberculosis epidemiology and history Mycobacterium tuberculosis (Mtb) is an obligate human pathogen and the causative reagent of tuberculosis (TB) that was thought to be hereditary until the isolation and identification of Mtb in 1882 by Robert Koch. He successfully identified this pathogen in experimentally infected animal tissue structures through alcohol-methylene blue staining and paved the way to isolation of Mtb in pure culture. He received the 1905 Nobel Prize of medicine for his “investigation and discoveries in relation to tuberculosis”[1]. The earliest historical records describing TB infection could be dated back to 1550 BC in Egypt with its appearance in ancient Egyptian art. From a male Egyptian mummy, strong macroscopic evidence suggested pulmonary and spinal tuberculosis, which was further supported with the detection of Mycobacterium tuberculosis DNA in the infected tissue and bones[2]. However, the earliest existence of Mtb dates back more than 17,000 years as fragments of Mtb DNA[3] and various mycobacterial virulence biomarkers[4] were detected in the bone lesion from a bison skeleton in Wyoming. Although present during most of human history thereafter, TB reached an unprecedented epidemic peak during 17th to 19th century when it swept Europe and was known as ‘consumption’. 2 Around the turn of the 19th century, it was estimated that TB openly infected over 50 million people worldwide and caused over 7 million deaths per year. Pulmonary TB and extrapulmonary TB are the two types of clinical manifestation of TB disease. Extrapulmonary TB takes up 20% of TB cases and refers to TB development in organs other than the lungs, including the pleura, the central nervous system, the lymphatic system, the genitourinary system, and the bones and joints[5]. Furthermore, TB presents a dynamic spectrum of infections and is clinically classified as latent TB infection (LTBI) or active TB disease. LTBI is normally asymptomatic and non-transmissible whereas active TB disease is typically associated with fever, fatigue, lack of appetite, and weight loss. Patients with active pulmonary TB disease can transmit the infection and may develop symptom of coughing up blood in advanced stages or death if left untreated[6]. Increasing evidence has also suggested that Mtb is not only associated with TB but is also related to chronic obstructive pulmonary disease, lung cancer, autoimmune diseases, and metabolic syndromes, which will not be discussed here[7]. Despite reduced morbidity and mortality of TB infections due to improvement in hygiene and socioeconomic conditions, the usage of the BCG vaccine, and the discovery of anti-TB drugs[8], it was estimated that over 10 million people fell ill and around 1.4 million people died due to TB worldwide in 2019. According to the World Health Organization (WHO), TB remains among the top 10 causes of death and is the leading cause of death from a single infectious agent (above HIV) [9]. 3 Although the first line anti-TB drugs isonizid, rifampicin, pyrazinamide and ethambutol provide an effective treatment strategy, the emergence of drug resistance has brought up major concerns. The most predominant resistant strains are rifampicin resistant TB (RR-TB) and multidrug-resistant TB (MDR-TB) , which is resistant to at least isoniazid and rifampicin. According to the WHO, half a million new RR-TB cases (of which 78% were MDR-TB) were identified in 2019. India, China, and the Russian Federation were the top 3 countries with 27%, 14%, and 9% of reported RR-TB cases respectively[9]. Collectively, the emergence of these drug-resistant strains poses critical needs for new and effective treatments around the globe. 1.1.2 Host infection by Mtb and immune escape mechanism The early stages of Mtb infection have been extensively investigated using animal models such as mice, guinea pigs and rabbits [10]. One of the most widely used attenuated strains in the animal studies is Mtb H37Ra, which can infect animal hosts at various doses given[11]. Mtb enters the respiratory tract during inhalation and reaches the alveolar space. Alveolar macrophages can uptake Mtb through non-specific pinocytosis or well-defined receptors[12]. Other phagocytic cells including, but not limited to, neutrophils, monocytes, and dendritic cells may also ingest Mtb once recruited to the locus of infection. The microenviroment in the alveolar space may influence this host- 4 pathogen interaction, for example, surfactant protein D is abundant in the fluid lining the epithelium and can prevent macrophages from internalizing Mtb[13]. After engulfed by phagocytic cells, Mtb utilizes immune escape strategies that have evolved to exploit host immune defense, intruding and surviving inside of phagocytic cells. Moreover, in the case of pulmonary tuberculosis, Mtb relocates itself to lung interstitium (or the parenchyma) to achieve long-term dormancy without causing severe tissue damage or transmission and wait for reactivation when the host immune system becomes compromised[14][15]. Together, this strategy of Mtb infection poses an extreme challenge to eliminate this pathogen. Here, by using the extensively researched macrophage-Mtb interaction, I give a brief summary of how Mtb ensures its survival through inhibition of phagolysosome maturation, inhibition of antigen presentation, and modification of macrophage death modality. The phagosomal maturation is a tightly controlled two-step process which enables progressive acidification of macrophage phagosomes and their terminal fusion with lysosomes. Acidification of phagosomes is primarily achieved via recruitment of the vacuolar-H+-ATPase (V-ATPase) , which actively pumps proton into the lumen, to the phagosmal membrane. Next, phagosomes are trafficked into, and merged with membrane-bound lysosomal vesicles that contains lytic enzymes including proteases and lipases. The acidic environment of the terminal phagolysosome facilitates pathogen 5 degradation, enhances the activation of pH-dependent proteases and antigen-presenting molecules[16]. Mtb blocks phagosomal acidification by secreting the mycobacterial protein tyrosine phosphatase PtpA, which excludes the host V-ATPase from the phagosomal surface through binding to its H-subunit[16]. Other mycobacterial proteins including early secretory antigen-6, culture filtrate protein, and secreted secA1/2 proteins can also inhibit phagosomal acidification by preventing accumulation of V-ATPase on the phagosomal membrane[14]. Mtb also disrupts the fusion between phagosomes with lysosomes to ensure its survival. Phosphatidylinositol 3-phosphate (PI3P) is one of the important components for the fusion event, which is targeted by Mtb. Mtb infection can downregulate and upregulate PI3P biosynthesis and hydrolysis respectively and ultimately suppresses maturation of phagolysosome[14]. Other virulence factors including PtpA, SecA2, PknG, SapM are also found to directly or indirectly prevent fusion between phagosomes and lysosomes through various mechanisms[14][15][16]. Antigen presentation by MHC class II is essential in mounting the adaptive immune response against intracellular parasites, so there is no surprise this process is targeted by various Mtb proteins during its intracellular survival[15]. Mycobacterial protein PE_PGRS47A can be secreted into the cytosol of macrophages to disrupt the formation 6 of autophagosomes which processes bacterial antigens and displays them on MHC II [15]. Apoptosis of the infected macrophages acts as the last line of defense to protect the host during failure to eradicate intracellular Mtb infection, as it results in killing of the parasites and priming of adaptive immunity. Therefore, it is not surprising that Mtb can regulate macrophage apoptosis to achieve optimal survival and dormancy. It was found that the wildtype Mtb strains H37Rv and M. bovis induce less intense apoptotic events in differentiated THP-1 cells, which is an immortalized cell line of monocytes commonly used as model system, compared to the weakly toxic MTBH37R strain and the BCG strain of M. bovis[17]. Furthermore, studies have shown that virulent Mtb secretes proteins including superoxide dismutase, KatG (hydrogen peroxidase), protein kinase (Pkn) E (serine threonine kinase), NuoG (dehydrogenase), Rv3654c, and Rv3655c, all of which can inhibit TNF-α induced macrophage apoptosis via different regulatory pathways [14]. 1.1.3 Infection progress and granuloma After successfully infecting alveolar macrophages, Mtb ultimately makes its way to lung interstitium (or parenchyma). The most prevalent ideas on how Mtb gains access to the parenchyma suggest that it either directly infects epithelial cells or utilizes infected macrophages to translocate across the epithelium[6]. At the same time, infected alveolar 7 monocytes and macrophages would transport Mtb to pulmonary lymph nodes for T cell priming, which leads to the recruitment of more immune cells including B and T cells to the locus of infection (lung interstitium) forming a granuloma. From the host’s perspective, granulomas keep Mtb under control and prevent it from spreading. However, studies have shown that Mtb is able to initiate the type I IFN response, which ultimately results in recruitment of a specific population of myeloid cells that are prone to be infected by Mtb. Therefore, from the pathogen’s perspective, granuloma also establishes a niche for Mtb to survive and replicate[6]. LTBI is defined when the granuloma successfully contains the infection and prevents further tissue damage. The duration of latent infection can vary between weeks and decades, and among LTBI patients, small proportion of patients (5% to 15%) would encounter reactivation and progress to active TB disease[18]. Therefore, the majority of LTBI patients would remain asymptomatic throughout the course of life. The mechanism for reactivation is yet unknown, but many risk factors have been identified. The most potent risk factor for reactivation is human immunodeficiency virus (HIV) infection. Patients co-infected with Mtb and HIV develop active TB disease at a 100-fold higher risk. Patients under hemodialysis due to chronic renal failure, patients on immune suppression due to transplantation, and patients with silicosis are also of higher risk for Mtb reactivation. Patients suffering from autoimmune disease and treated with tumour necrosis factor alpha (TNF-α) inhibitors, patients with diabetes, and children under four 8 years old are placed in a moderate-risk category, while malnutrition, alcohol use, and smoking confer low risk for reactivation[19]. 1.2 Signaling system of mycobacterium tuberculosis 1.2.1 Prokaryotic signaling system Signal transduction ensures essential activities of all living cells and can be broadly defined as sensing an input or signal, which can be either intracellular or extracellular, and converting it into an output or response that may alter cellular activity, morphology, or physiology. The sensor can be either a molecule or a domain of a molecule (normally a protein) that senses the signal, and the transducer can be either a molecule or a domain that converts the signal into a response. Most commonly, signal transduction systems are responsible to sense extracellular or environmental signals and transduce them across the cytoplasmic membrane to mount adaptive changes in cell activities to ensure survival in different conditions. This is extremely important in the case of a intracellular pathogen like Mtb, as sensing the dangerous environment inside the macrophage and expressing adaptive virulence factor proteins are essential to ensure its survival. Different signaling systems have been identified in prokaryotes. Here I provide a short introduction of the most important ones. 9 Two-component systems, which consist of paired sensors and response regulators, constitute the best-studied transmembrane signaling systems in bacteria[20]. Classically, the transmembrane sensor histidine kinase receives a signal and autophosphorylates the His residue, followed by the transfer of that phosphate from pHis to the Asp residue on the cytoplasmic response regulator, typically a transcription factor, which induces downstream changes in gene expression[21]. In the cytoplasm of prokaryotes, evolutionarily older and simpler one-component systems exist and involve proteins such as the lactose operon repressor that contains both a sensor domain and a regulator domain. One component systems receive signals through modifications such as binding of small molecules or transfer of phosphate groups on the sensor domain and induce transcriptional changes of the cell through the binding of the regulator domain to DNA[22]. The third known signaling systems in prokaryotic cells are called the extracytoplasmic function (ECF) σ factors, which are components of the RNA polymerase complex that convey specific binding affinity to different promoter sequences[23]. Their inactivating binding partners, anti-σ factor proteins, are typically transmembrane or cytoplasmic sensors which, upon reception of signaling stimuli, release ECF σ factors, which in turn regulates transcription[24]. Last but not the least, Serine Threonine Protein Kinase (STPK)-based signaling plays an important role in some prokaryotes. Its first discovery in Mtb was made by Av-Gay et 10 al. who combined a variety of molecular techniques including Southern analysis, PCR and genome database screening in 1997[25]. The initial 8 eukaryotic-like kinases found by Av-Gay et al. expanded to 11 in 2000[26], indicating an important role of STPKs in Mtb. In contrast to two-component systems that are the major signal transduction systems in nearly all prokaryotic cells, STPKs are only found in certain groups of bacteria including Acidobacteria, Actinobacteria (which includes mycobacteria), Cyanobacteria and Deltaproteobacteria (where the first prokaryotic STPKs were found)[26][27][28]. As STPKs play a very important role in Mtb signaling and are key to the activity of the protein studied here, I will now provide further insights into STPK-based signaling. 1.2.2 Eukaryotic STPKs In Eukaryotes, signaling is primarily accomplished via a network of protein phosphorylation cascades that involve coordinated action of many STPKs, tyrosine kinases, and their associated phosphatases. In contrast to tyrosine kinases that primarily interact with tyrosine residuals, STPKs function by transferring a phosphate group, which can be derived from an ATP or GTP, onto serine or threonine groups of their substrates. The outcome of these phosphorylation events normally involve activation or deactivation of specific protein activities, changes in protein localization, or further transfer of the phosphate group to downstream effector molecules. Phosphatases perform the opposite function of kinases and reverse the effects of phosphorylation to restore the substrates 11 and effectors to their initial state, terminating the signaling cascade or preparing them for new signaling event. Therefore, kinases and phosphatases resemble the function of switches that regulate signal transduction pathways[29]. 1.2.3 STPKs in Mtb The ability of Mtb to evade the host immune system through various mechanisms originates from its complex signal transduction pathways. The signaling system of Mtb is composed of 5 major families of kinases and phosphatases including histidine kinases (comprises the classic two component system), serine/threonine protein kinases (STPKs), serine/threonine phosphatase, protein tyrosine kinase, and protein tyrosine phosphatases[30]. Here, I mainly focus on STPKs. The Mtb genome encodes 11 STPKs, including PknA, PknB, PknD, PknE, PknF, PknG, PknH, PknI, PknJ, PknK, and PknL, which is almost the same as the number of two-component systems, emphasizing the potential usefulness and significance of STPKs in Mtb signaling. All STPKs, except PknG and PknK, contain a C-terminal extracellular domain that is believed to sense the environmental signal. Furthermore, all have a transmembrane domain, and a N-terminal kinase domain, which is activated upon signal recognition and phosphorylates serine or threonine residues on substrates, indicating their role in transducing external signals. The remaining two cytoplasmic STPKs are able to sense signals and mount responses within the cell[31]. Mtb STPKs are highly similar to 12 their eukaryotic counterparts in terms of structure, despite having only 30% sequence similarity. They are described as ‘eukaryotic-like’[26]. Despite the structural and functional similarities, significant differences between eukaryotic and Mtb STPKs exist. In contrast to eukaryotic STPKs, which are part of complex signaling networks and cascades, most of the Mtb STPKs have paired relationships with their substrates, resembling the signaling of two-component systems [32][33][34][35]. Some in vitro studies tested the interaction between Mtb STPKs and came up with promising results indicating cross-talk between them, which may imply their role in signaling networks, but more studies and evidence are needed to reach clear conclusions[36]. Moreover, while eukaryotic STPKs form the most important and extensive signaling systems in humans, interacting with over 60% of all proteins[37][38], their role in Mtb is somewhat limited, as only 7.5% of Mtb proteins are found to be phosphorylated by them[39]. Furthermore, according to phosphoproteome data, Mtb’s STPKs phosphorylate threonine and serine with a ratio of 3:2, while eukaryotic STPKs are more biased on serine over threonine with a 9:1 ratio[39]. The most significant difference between STPKs and other major bacterial signaling systems is that the phosphorylated downstream effectors are rarely regulating transcription directly[31]. In the following, a brief introduction is provided for PknA and PknB, the only two STPKs essential for Mtb growth, as well as PknF, which is the most relevant to Rv1747, the protein of interest in this thesis. 13 PknA and PknB are encoded by the same operon. This operon also encodes PBPA, which is a cell wall synthesis enzyme, and RodA, which determines cell shape[40]. Although interactions between PknA/PknB and PBPA/RodA have not been established, the genetic linkage of these two STPKs with PBPA/RodA implies that PknA and PknB play a role in cell wall synthesis and cell shape regulation. Consistent with this idea, it has been shown that overexpression of these two kinases had significant effects on cell branching, elongation, and septation[41]. Other key findings regarding the function of these two STPKs include their interaction with Wag31 and Rv3910. Wag31 is the homologue of the cell division protein DivIVA in Mtb and was found to be phosphorylated by PknA[41]. Rv3910 is the Mtb homologue of the E. coli protein MviN, which is responsible to flip the lipid-linked peptidoglycan precursor lipid II into the periplasmic space from the cytoplasm and facilitate the incorporation of this muropeptide into peptidoglycan[42]. PknF is part of the same operon as Rv1747, an ABC transporter that is discussed in 1.3 and 1.4. Previous studies have shown that autophosphorylated PknF can recruit and phosphorylate Rv1747 by interacting with its FHA-1 domain[43][44]. Furthermore, according to a study where PknF overexpressing and antisense expressing strains were used, PknF was found to have a significant effect on Mtb growth, morphology, and septum placement, suggesting a role in Mtb growth and shape regulation[45]. 14 1.3 Rv1747 structure and relevance in Mtb growth Rv1747 is a Mtb ATP-binding cassette (ABC) transporter encoded by the open reading frame Rv1747 and characterized as a transporter involved in cell wall biosynthesis. Rv1747, starting from the N-terminus, consists of a regulatory module (RD), a cytoplasmic nucleotide binding domain (NBD) where ATP hydrolysis happens, potentially providing energy for morphological change and substrates’ transportation, and a helical transmembrane domain (TMD) through which substrates are transported[43]. The N-terminal RD contains the FHA-1 (Forkhead associated) and FHA-2 domains connected by an intrinsically disordered linker of about 100 amino acid residues. The linker contains multiple threonine groups that can be phosphorylated by PknF [43][46]. FHA domains are small phosphopeptide recognition domains that are involved primarily in signaling processes in all life forms. They have a conserved fold and exhibit high binding specificity for phosphothreonine (pThr) [47]. Consistent with the fact that FHA domains recognize pThr, it has been shown that both FHA-1 and FHA-2 can bind pThr residues in the linker of the RD. A recent study determined the structures of FHA-1 and FHA-2 with the help of X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, respectively, and elucidated that although both domains share highly similar structures, FHA-2 replaces the beta-hairpin of the canonical fold between strand 1 and 2 in FHA-1 with a less stable beta strand 1 (Figure 1) [48]. FHA-2 has a higher affinity for phosphorylated 15 threonines over FHA-1. The underlying reason may be that a higher entropic barrier during binding is compensated by a larger and favorable enthalpy change[48]. Extensive NMR studies have shown that in the context of the RD, the phosphorylated linker, enables extensive intra- as well as intermolecular interactions [48]. Figure 1. Forkhead-associated (FHA) domains: FHA-2 replace beta-Hairpin of the Canonical Fold between strand 1 and 2 in FHA-1 with a less stable beta strand 1. FHA domains are from the regulatory module of the Rv1747 of M. tuberculosis, FHA-1 (A) is a crystallographic structure with backbone in green, strand 1 in orange, strand 2 in cyan, beta-Hairpin of the Canonical Fold in yellow. FHA-2 (B) is from NMR ensemble of Heinkel et al., (2018) with backbone in purple, strand 1 in red, the terminus corresponding to the beta-Hairpin in FHA-1 is colored pink to show difference. The corresponding strands in different FHA domains are labeled with number 1-11, FHA-2 does not have a strand 2. Although the exact substrates of this protein still need to be identified, a decreased level of phosphatidyl-myo-inositol mannosides (PIMs) in lipid extracts and an increased expression of the efflux pump-related iniBAC operon in the Rv1747 knock-out mutant strongly suggest that Rv1747 is associated with the transport of cell wall 16 materials[49][50]. Furthermore, there have been studies establishing the association between Rv1747 and Mtb pathogenesis. Rv1747 knockout mutants have an attenuated growth in both mice and macrophages, indicating the requirement of Rv1747 for Mtb growth[49][51]. Strains with phosphor-ablative T152A or T210A linker mutations and strains with the S47A mutation in the FHA-1 phosphothreonine binding site also show a reduced growth in mice and macrophages, which collectively suggest that interactions between FHA domains and phosphorylated linker are important to the function of Rv1747 [44]. 1.4 Phase separation Phase separation is well-known in polymer chemistry which describes the phenomenon where a homogenous mixture of polymers undergoes spontaneous phase segregation to form two distinctive phases that are enriched or depleted of certain polymeric chains, respectively [52]. The resulting phases usually exhibit liquid-like characteristics such as more-or-less spherical shapes, ability to fuse upon contact to form larger spheres, internal rearrangements, and selective molecule exchanges across the boundaries, and therefore this phenomenon is also referred to as liquid phase condensation[53]. 17 1.4.1 Increased understanding of phase separation in biology Despite being quite commonly observed in polymer chemistry, the significance of phase separation in biology has not been well recognized until recently. In 2005, a group of researchers studied the density and molecule movement in and out of Cajal bodies in the eukaryotic nucleus and pointed out that Cajal bodies are membraneless semifluid-like spheres suspended in the nucleoplasm[54]. In 2009, another group of researchers successfully showed that P granules containing RNA and proteins in Caenorhabditis elegans embryos are formed by phase separation and exhibit liquid-like properties [55]. Ever since then, phase separation has been associated with the formation of an increasing number of membraneless organelles and nuclear compartments including but not limited to the nucleolus, nuclear speckles, Cajal bodies, stress granules, and P bodies[52]. With increasing evidence showing that phase separation is the driving force behind the formation of many membraneless organelles, the question arises how phase separation contributes to the regular function of these cellular compartments. In 2012, Rosen and colleagues proposed the idea that phase separation is not only the organizing principle for organelle formation but also for their biological functions. They reconstituted multi valent protein-containing bodies with purified components and successfully reproduced their phase separation. Furthermore, they showed that the sharp phase transition of mixtures containing N-WASP (neuronal Wiskott-Aldrich Syndrome 18 Protein) and its biological partners Nck and phosphorylated nephrin, enabled an increase in activity of Arp2/3 complex-dependent actin nucleation[56]. In the past decade, a steadily increasing number of studies have associated the formation of appropriate condensates with different cellular compartments and their functions. Furthermore, studies have also suggested that aberrant condensate assembly or disassembly can cause dysfunction of cellular processes and even disease[57]. 1.4.2 Determining factors in phase separation Successful formation of liquid-like condensates is dependent upon continuous rearrangement of the interactions between the components. Therefore, understanding the driving force behind condensate formation requires an in-depth look into the types of protein interactions present in condensates [57]. Proteins that are able to phase separate as well as those that are found enriched in membranless organelles have a key characteristic: they are multi-valent. Multivalency has two main origins: the multidomain character of modular proteins and a high content in intrinsically disordered regions (IDRs)[57]. Rosen and colleagues performed extensive research on the interaction between folded SH3 domains and proline-rich motifs (PRMs) with various constructs carrying different numbers of SH3 domains and PRMs. They showed that phase separation is dependent upon the number of SH3 domains and PRMs present in protein constructs; where a minimum of 3 repeats of each was required for the formation of condensates. 19 Furthermore, increasing number of repeats of both modules lowers the protein concentrations required for phase separation[56]. IDRs usually adopt a collection of different conformations in solution unlike independently folding protein parts. IDRs found in proteins that phase separate are usually enriched in short sequence motifs or have sequence patterns that enable transient charge-charge, dipole-dipole, and/or pi-pi interactions. In most cases, amino acids that enable these type of interactions are scattered through the IDRs, thus have spacers between each other. Weak interactions between the scattered residues dynamically “crosslink” nearby biomacromolecules, while the non-interacting residues between them, the spacers, provide flexibility and promote dynamic liquid-like properties to the condensates[57]. Although protein-protein interactions play an important role in many phase separating systems, interactions with and between RNA molecules are believed to be just as important in some condensates. For instance, BR-bodies (bacterial ribonucleoprotein bodies) are bacterial condensates where mRNA degradation happens (for more details see below). Unfolded mRNA has been shown to enhance BR-body formation and modulate the size of the condensates formed by the protein RNAse E.[58]. 20 1.4.3 Phase separation in eukaryotic processes The best-studied cytoplasmic condensates are stress granules, which consist primarily of RNA and RNA-binding proteins (RBPs). Upon cellular stress, the key translation initiation factor eIF2α is phosphorylated, which results in downstream release of mRNAs from polysomes. The contact between RBPs such as G3BP1 and the unfolded mRNAs promotes dephosphorylation of RBPs and facilitates the initiation of condensate formation, eventually leading to the formation of mature stress granules. As soon as the stress is resolved, eIF2α is dephosphorylated, which then promotes the disassembly of the stress granules and the release of mRNA. Stress granules are not only involved with the storage of mRNA and regulation of translation upon cellular stresses, but also with many other cellular processes including signal transduction or the innate immune response, all of which require appropriate condensate formation[57]. T cell receptor (TCR) signaling is another example of a cellular process in which condensate formation via phase separation has been shown to play an important role. TCR signaling in cells starts with TCR being phosphorylation by the membrane-bound kinase Lck. The cytoplasmic module of TCR, when phosphorylated, recruits tyrosine kinase ZAP70 from the cytosol which in turn phosphorylates multiple tyrosine sites on the transmembrane protein LAT (linker for activation of T cells). These phosphorylated tyrosine residues are recognized and bound by SH2 domains from Grb2. Grb2 utilizes its SH3 domains to further interacts with proline-rich motifs of Sos1 to form the signaling 21 complex. The activated signaling complex can further propagate the signal and regulate cellular processes including calcium mobilization and actin polymerization. Rosen and colleagues reconstituted part of the TCR signaling pathway by anchoring phosphorylated LAT (pLAT), Grb2, and Sos1 on a supported lipid bilayer in vitro. Intriguingly, they observed that the formed clusters exhibited dynamic, liquid-like properties including round edges, the ability of fusion, and diffusive exchanges. Furthermore, to examine the signaling function of this reconstituted pathway, they integrated a downstream actin polymerization module which is composed of N-WASP, Nck, Arp2/3, and actin monomers and successfully showed actin polymerization from the LAT clusters[59]. These experiments clearly demonstrated the functional importance of condensate formation in TCR signaling. 1.4.4 Phase separation in bacteria The growing recognition for the importance of phase separation in the assembly of membraneless organelles in eukaryotes has also inspired investigations of subcellular membraneless structures in bacteria in recent years. However, exploration of liquid-like condensates in prokaryotic system has faced its own challenges[60]. This is majorly due to the small size of bacteria, which is below the diffraction limit of most of the techniques commonly used for observing phase separation in eukaryotes. This inherent difficulty gives rise to further challenges as most of the criteria used to characterize condensates 22 including spheric shapes, fusion, and molecule exchanges might be hard to determine when it comes to the much smaller bacteria. Instead, alternative criteria are adapted in the characterization of phase condensates in bacteria, which includes the observation of cluster of fluorescently tagged proteins in cells, hexanediol sensitivity of clusters, and dynamic properties of the clusters verified with single-molecule tracking[61]. 1.4.4.1 Examples of bacterial phase condensates To date, the best characterized bacterial condensates using these approaches are BR-bodies. BR-bodies concentrate RNA degradosome proteins and their substrates locally to promote and regulate mRNA degradation[62][63]. The CTD (C-terminal domain) of RNAse E is the major component of BR-bodies and forms liquid-like condensates in vitro[62]. Upon formation, these condensates are able to selectively recruit unstructured RNAs that can in turn enhance droplet formation and regulate droplet size in vitro[63]. RNAse E has been found to form foci that disappear under mRNA depletion in alpha-proteobacteria[62]. RNAse E’s CTD also forms foci that fuse into larger clusters in Escherichia coli[63]. Furthermore, single-molecule tracking has revealed molecular mobility of RNAse E in C. crescentus where RNAse E molecules within the condensates have exhibited confined motion with non-negligible diffusion coefficients[58]. Taken together, these experimental findings are consistent with the idea that BR-bodies are bacterial condensates formed via protein phase separation. 23 A variety of other bacterial proteins have been found to phase separate in vitro, respectively form dynamic foci[60]. Among these proteins are the RNA polymerase (RNAP), which is responsible for RNA transcription[64], the polar organizing protein Z (PopZ), which promotes polar localization and recruits protein components for the pole formation[65], the DNA-binding protein from starved cells (Dps), which compacts and protects the nucleoid from damage in the stationary phase[66], the single-stranded DNA binding protein (SSB), which stabilizes single-stranded DNA and promotes DNA replication and repair[67], and FtsZ, which promotes the assembly of the divisome during bacterial cell division[68]. All these and other bacterial proteins that have been shown to phase separate are involved in the critical cellular processes including mRNA degradation, transcription, cell division, DNA compaction, DNA repair, and starvation response, which emphasizes the increasingly important role of phase separation in bacterial cellular functions. 1.4.4.2 Rv1747, a Mtb protein that can phase separate As discussed in Section 1.3, the ABC transporter Rv1747 is a transmembrane protein in Mtb that has previously been shown to affect Mtb’s growth in both mice and macrophages and thus may play an important role in Mtb infection[49][51]. Work from our lab has previously shown that multiple intramolecular and intermolecular interactions are formed between two FHA domains and phospho-acceptor threonines in the 24 connecting linker[69] upon phosphorylation by PknF. Moreover, it was found that Rv17471-310, the construct that contains the two FHA domains and the connecting linker, thus constituting the RD, can undergo phosphorylation-enhanced phase separation in vitro. In vitro condensates have been found to exhibit liquid-like properties such as spherical shapes, the ability to fuse upon contact, and diffusive exchanges of components in and out of the condensates. Specifically, unphosphorylated Rv17471-310 was found to phase separate at high protein concentration of 250 µM, while the phosphorylated Rv17471-310 was found to form liquid-like condensates at a much lower protein concentration of 50 µM (Figure 2). Regarding the diffusive exchange property of Rv17471-310, condensates were assessed by FRAP (fluorescence recovery after photobleaching). Fluorescent recovery of both unphosphorylated and phosphorylated Rv17471-310 was found to be around 60%, though condensates formed by phosphorylated Rv17471-310 exhibited slower recovery than the ones formed by unphosphorylated Rv17471-310 , implying an impaired mobility of molecules in the phosphorylated sample possibly resulting from the enhancement of the interaction between the linker and FHA domains upon phosphorylation[69]. Mtb’s STPKs and its sole phosphatase modulate the ability of Rv17471-310 to phase separate, respectively, reverse it and differentially colocalize with condensates. Overall, these previously collected data clearly demonstrate that the cytoplasmic regulatory part of Rv1747 has the ability to phase separate in vitro. 25 Figure 2. Rv17471-310 phase separation under phosphorylated and unphosphorylated conditions. Unphosphorylated and phosphorylated Rv17471-310 phase separated at different threshold concentrations. Figure shown includes fluorescent images of unphosphorylated (Top) and phosphorylated (Bottom, incubated with 0.5 μM PknF and 100 μM ATP) Oregon Green-tagged Rv17471-310 (Scale bars: 40 μm). Figure adapted from: Heinkel et al., 2019. 1.5 Summary and working hypothesis The ABC transporter Rv1747 affects growth of Mtb and may play a role in Mtb infection. It has been shown that the Rv1747 knockout mutant has an attenuated growth in both mice and macrophages[49][51]. Furthermore, its impact on Mtb growth appears to be dependent on interactions between the FHA domains and the phosphorylated linker in its cytoplasmic regulatory module, since strains with ablative, linker T152A or T210A mutations and strains with the S47A mutation in the FHA-1 phosphothreonine binding site have reduced growth in mice and macrophages [44]. How the function of Rv1747 as a ABC exporter affects Mtb growth is still unknown, nor are the substrates that are transported. However, a recent study revealed that its activity impacts cell wall levels of PIMs, immunoregulatory glycolipids known to impact on Mtb intracellular fate[49][50]. 26 Our lab recently revealed that Rv1747’s regulatory module with its two FHA domains and the linker reversibly phase separates into liquid condensates upon phosphorylation by multiple Mtb STPKs, while the addition of Mtb’s sole phosphatase PstP resolves the condensates. Together with the observation that regulatory module mutations that prevent FHA-pThr interactions lead to impaired Mtb growth in mice and microphages, and the fact that STPKs are key players on Mtb signal integration, these findings suggest that phosphorylation-induced phase separation regulates Rv1747’s activity as an exporter of cell wall components. Thus, a model was proposed according to which phosphorylation of Rv1747 promotes reversible phase separation that then induces clustering of this ABC transporter on the membrane. Such clustering may increase transporter efficiency or generate a scaffold structure enabling interactions with components of cell wall biosynthesis pathways[69]. Consistent with this model, single-molecule localization microscopy (SMLM) experiments from our lab have shown that endogenous Rv1747 forms nanoclusters in the Mtb membrane[69]. Although the data collected thus far suggest that Rv1747 is likely to phase separate also in cells, further evidence is required. Thus, the foundational in vitro observations of Rv1747 phase separation need to be further supported in cells, which gives inspiration to my study of Rv1747 clustering in M. smegmatis. I hypothesized that Rv1747 forms higher level subcellular structures in Mtb as a result of phase separation of its regulatory module. My goal is to utilize M. smegmatis, a commonly used model organism that is 27 safer, more transformable, and faster growing than Mtb, to express various fluorescence-tagged Rv1747 constructs and characterize foci (refer to 2.4 for detailed definition) formation by these constructs. 28 Chapter 2: Materials and Methods 2.1 Plasmids preparation Table 1 List of plasmid constructs. For fast reference, this list includes the name of construct, backbone used in molecular cloning, gene inserted, and mutations. Name Backbone Gene Mutation(s) pJC48 customized none none pJC49 customized none none pJC52 customized none none pC1 pUC57 Rv17471-310 Linker Thr/Ser to Ala pC2 pUC57 Rv17471-310 Linker Arg/Lys to Ala mEos2 pJC52 none none mEos2-Rv1747∆1-310 pJC52 Rv1747∆1-310 Rv1747∆1-310 mEos2-Rv17471-310 pJC52 Rv1747 none mEos2-Rv17471-310S47A pJC52 Rv17471-310 FHA-1 S47A mEos2-Rv17471-310S248A pJC52 Rv17471-310 FHA-2 S248A mEos2-Rv17471-310S47,248A pJC52 Rv17471-310 FHA-1, 2 S47A, S248A mEos2-Rv17471-310STA pJC52 Rv17471-310 Linker Ser/Thr to Ala1 mEos2-Rv17471-310RKA pJC52 Rv17471-310 Linker Arg/Lys to Ala2 mEos2-Rv1747 pJC52 Rv1747 none mEos2-Rv1747S47A pJC52 Rv1747 FHA-1 S47A mEos2-Rv1747S248A pJC52 Rv1747 FHA-2 S248A mEos2-Rv1747S47,248A pJC52 Rv1747 FHA-1, 2 S47A, S248A 1. Ser: 109/118/125/142/161/178/211; Thr: 116/117/131/137/139/152/153/169/191/203/210/222 2. Arg: 105/107/112/120/154/166/182/188/200/217/220; Lys: 214 Plasmids used in this study are listed in Table 1. All plasmids were constructed with customized pJC52 vector backbone obtained from Mr. Joseph Chao, a senior research associate in the laboratory of Dr Av-Gay lab[69]. All non-vector plasmids were manually 29 constructed, except for mEos2-Rv17471-310 and mEos2-Rv1747, which were obtained from Mr. Joseph Chao from Dr Av-Gay lab[69]. 2.1.1 Construction of pJC52 vector backbone Table 2 List of primers. For fast reference, this list includes the name of primers, brief description of the identify, restriction site and corresponding sequence. Name Description Restriction site Sequence J58 Forward primer for acetamide promoter none CGTCGATCCCGTGTAGTCTG J59 Reverse primer for acetamide promoter BamHI GAGTGGATCCGGCATGTGGACTCCCTTTCT J60 Forward primer for mEos2 BamHI GTGCGGATCCCATGAGTGCGATTAAGCCAGAC J61 Reverse primer for mEos2 none TTCAGGTTCAGGGGGAGGTG J62 Linker sense sequence BspEI, AflII, EcoRI, ClaI, KpnI from 5’ to 3’ CCGGAGGCGGTGGATCGGGCGGAGGTGGCTCGGGCGGTGGAGGCCTTAAGGAATTCTTAATTAAATCGATGGTAC J63 Linker antisense sequence BspEI, AflII, EcoRI, ClaI, KpnI from 5’ to 3’ CATCGATTTAATTAAGAATTCCTTAAGGCCTCCACCGCCCGAGCCACCTCCGCCCGATCCACCGCCT J64a Forward primer for Rv1747 AflII CCGACTTAAGGTGCCGATGAGCCAACC J65a Reverse primer for Rv17471-310 EcoRI CCACGAATTCCTAGTTCTCTTCACGGCGCG J65b Reverse primer for Rv1747, Rv1747∆1-310 EcoRI GAGCGAATTCCTGGGTTGGGCAGTGAAC C1 Forward primer for Rv1747∆1-310 AflII CCGACTTAAGCTGCTGGAGACCCGCGT C2 Forward primer for FHA-1 S47A none CACCCCCTGATCGCCCGGGCACACC 30 C3 Reverse primer for FHA-1 S47A none GGTGTGCCCGGGCGATCAGGGGGTG C4 Forward primer for FHA-2 S248A none CGAGGTGTTGGCCGCACGTCACCACGC C5 Forward primer for FHA-2 S248A none GCGTGGTGACGTGCGGCCAACACCTCG All primers used in this study are listed in Table 2. Primers J58 and J59 were used to amplify the acetamidase promoter with an internal HindIII site from the pALACE vector (Addgene #31853) and introduce a BamHI restriction site downstream of the translational start site. The amplified product and a pALACE vector were cut with HindIII and BamHI separately and ligated back together to generate the pALACE-like vector with the His6x-tag originally downstream of the acetamidase promoter removed, and the resulted vector was named pJC48. Primers J60 and J61 were used to amplify the mEos2 gene with an internal BspEI site from mEos2-C1 (Addgene #54510) and introduce a BamHI restriction site upstream of the mEos2 gene. Primers J62 and J63 were denatured and annealed to generate double stranded linker DNA including an internal ClaI site and sticky ends compatible with BspEI and KpnI restricition sites. The amplified mEos2 gene product cut with BamHI and BspEI was ligated with double stranded linker DNA generated from J62 and J63, and the resulted product was further ligated with pMV261 cut with BamHI and KpnI to generate pMV261-like vector with inserted mEos2 and linker and was named pJC49. 31 pJC48 digested with XbaI and BamHI (acetamidase promoter) was ligated with pJC49 digested with BamHI and ClaI (mEos2 and linker), and the resulting product was further ligated with pMV261 digested with XbaI and ClaI (original HSP60 promoter removed) to generate pJC52, which is essentially pMV261 with the HSP promoter replaced with an acetamidase promoter followed by mEos2 and a linker. 2.1.2 Construction of mEos2-tagged Rv1747 plasmids pJC52 is used as mEos2 vector in this study. Primer pairs J64a&J65a, J64a&J65b, C1&J65b were used to amplify the regulatory domain of Rv1747 encoding the first 310 amino acids of Rv1747, full length Rv1747, and Rv1747∆1-310, which is a construct that lacks the first 310 residues. i.e., the regulatory module, respectively, to generate amplicons with upstream AflII and downstream EcoRI restriction sites. The resulted amplicons digested with AflII and EcoRI were ligated with pJC52 digested with AflII and EcoRI to generate mEos2-Rv17471-310, mEos2-Rv1747, mEos2-Rv1747∆1-310. Primer pairs C2&C3 and C4&C5 were used along with Agilent SDM (site directed mutagenesis) Kit and XL-1 Blue super competent cells to introduce the S47A and S248A mutations in mEos2-Rv17471-310 to generate mEos2-Rv17471-310S47A and mEos2-Rv17471-310S248A, respectively, via site directed mutagenesis. The double mutant mEos2- 32 Rv17471-310S47,248A was generated by performing another site directed mutagenesis on mEos2-Rv17471-310S47A with the primer pair C4&C5. Primer pairs C2&C3 and C4&C5 were used along with Agilent SDM Kit and XL-1 Blue super competent cells to introduce S47A and S248A mutations in mEos2-Rv1747 to generate mEos2-Rv1747S47A and mEos2-Rv1747S248A, respectively, via site directed mutagenesis. The double mutant mEos2-Rv1747S47,248A was generated by performing another site directed mutagenesis on mEos2-Rv1747S47A with the primer pair C4&C5. Plasmids pC1 and pC2 were ordered from BioMatik and were designed to carry Rv17471-310 with all linker Thr/Ser residues mutated to Ala and all linker Arg/Lys residues mutated to Ala, respectively, in the pUC57-Kan vector with upstream AflII and downstream EcoRI sites. The customized pJC52 digested with AflII and EcoRI was ligated with pC1 and pC2 digested with AflII and EcoRI to generate mEos2-Rv17471-310STA and mEos2-Rv17471-310RKA, respectively. 2.2 Expression of mEos2-tagged Rv1747 constructs in M. smegmatis All mEos2-tagged Rv1747 plasmids were sequence-verified and transformed into M. smegmatis (strain mc2155) via electroporation according to F. Heinkel et al. paper[69]. 33 2.3 Microscopy of transformed M. smegmatis cells M. smegmatis harboring the mEos2 vector and mEos2-tagged plasmids including mEos2-Rv17471-310, mEos2-Rv17471-310S47A, mEos2-Rv17471-310S248A, mEos2-Rv17471-310S47,248A, mEos2-Rv17471-310STA, mEos2-Rv17471-310RKA, mEos2-Rv1747, mEos2-Rv1747S47A, mEos2-Rv1747S248A, mEos2-Rv1747S47,248A, and mEos2-Rv1747∆1-310 were grown in Middlebrook 7H9 broth with 6.7% OADC, 0.05% Tween-80, 20 µg/mL kanamycin at 37 °C for 3-5 days to an OD600 ~ 0.4 – 0.6. Cells were diluted to OD600 ~ 0.02 in Middlebrook 7H9 broth with 0.2 % succincate, 0.05 % Tween-80, 20 µg/mL kanamycin and induced with acetamide (0.2 % for vector, 1 % for regulatory domain wildtype and mutants, and 2 % for full length wildtype and mutants) and incubated at 37 °C for 16 hours to an OD600 ~ 0.4 - 0.6. Coverslips (Marienfeld #1.5H, 18x18 mm; #0107032, Lauda-Königshofen, Germany) were incubated with 70% ethanol overnight, washed with phosphate buffer saline (PBS) twice, coated with 0.01% poly-L-lysine (Sigma-Aldrich; #P4707) for 0.5 – 1 hour followed by aspiration, incubated with induced live M. smegmatis for 0.5 – 1 hour, washed with PBS, and assembled into a top-stage incubation system with Chamlide magnetic chamber (Quorum Technologies; #CM-B18-1). All imaging was done at 37 °C using Olympus TIRFM (Total internal reflection fluorescence microscope) system based on an inverted microscope (Olympus IX81) equipped with a 150x NA (numerical aperture) 1.45 TIRFM objective (Olympus), 34 motorized filter wheel (Olympus), high performance electron multiplier (EM)-charge-coupled device (CCD) camera (Photometrics Evolve), and realtime data acquisition software (Metamorph). mEos2 were excitted with 491 nm solid-state diode lasers. The TIRF plane was adjusted to yield a penetration depth of 150 nm and TIRF imaging was conducted for 10 s at 33 frames/s. Icy Bioimaging analysis software were used for detection of individual particles/foci. Binary (0 or 1) images of particle detection was done with the UnDecimated Wavelet Transform Detector plugin, which was set to detect bright spots over a dark background, exhibit 50 % sensitivity to spots < 3 pixels, and apply size filtration in order to detect objects ranging from 2 pixels to 3000 pixels. 2.4 Data analysis and figure preparation All representative images were taken from either frame 1 or 2 of a 10-second movie recording. All bright cells had either “foci” or “no foci” based on the following signal intensity quantification. The pixel intensity of the fluorescent signals was quantified by drawing a line using the line tool in ImageJ across the entire length of the bacterium and used to generate fluorescent intensity graph. Cells expressing vectors control (mEos2) had uniform signal intensities with minimal fluctuations. Foci are characterized by high fluorescent intensity values (peaks) that are distinct and prominent when compared to the rest of the cell body. Any spurious high-intensity peaks that last only one frame in the movie were not included as foci, i.e. intensity peaks have to last for several frames to be 35 identified as foci. In conditions where a mixed phenotypes (with cells having either foci or no foci) were seen, the percentage of cells with either phenotype is reported in the representative images. The percentage was quantified on > 30 individual cells. Kymographs were generated using the time evolution of fluorescence signals across the length of bacterium. A total of 330 frames were generated during 10 seconds of TIRF imaging for each cell; in each frame, a straight line across the entire length of cell was drawn to record the distribution of fluorescent signal at that time and used as the horizontal line in the kymograph. Stacks of such horizontal lines in the kymograph clearly indicated the movement of fluorescence signal across the length of cell over 10 seconds. Further descriptions of kymographs can be found in Munro et al.[70] and Heinkel et al[69]. 36 Chapter 3: Results 3.1 Rv1747 clustering in M. smegmatis is dependent on the regulatory module As previously demonstrated, Rv17471-310 is able to phase separate in a phosphorylation-dependent manner in vitro[69]. If phase separation plays a role in Rv1747’s activity regulation, Rv1747 should also be able to phase separate within bacteria, and this phenomenon should depend on the presence of the regulatory module. Thus, overexpression of Rv1747 in Mtb should lead to the formation of nanoclusters that can be identified as foci of the fluorescently labeled protein. To test this, we expressed mEos2 vector (fluorescent label), mEos2-Rv1747, mEos2-Rv17471-310, and mEos2-Rv1747∆1-310 in M. smegmatis and characterized the distinctive phenotypes among cells carrying those constructs. Cells expressing mEos2 control vector displayed uniform distributions of fluorescence intensity across the cells and no signs of foci formation with minimal fluorescent fluctuation (Figure 3A, top panel and middle panel). Moreover, no obvious changes in fluorescence intensity were observed in kymographs (Figure 3A, bottom panel), which illustrate the 10-second time evolution of fluorescence signals across the length of the bacterium. However, cells expressing mEos2-Rv17471-310, the cytoplasmic RD of Rv1747, exhibited a phenotype that differs greatly from the one observed in cells 37 expressing the mEos2 control vector. One or two dominant foci were clearly visible in each bacterium, as illustrated in the representative images and the fluorescence intensity graphs (Figure 3B, top and middle panel). Interestingly, kymographs revealed a static feature for these foci as they remained fixed in place for the majority of the frames collected (Figure 3B, bottom panel). FRAP experiments carried out on these foci by Dr. Libin Abraham showed that mEos2-Rv17471-310 underwent diffusive exchanges (data not included), consistent with the idea that these foci are dynamic condensates[69]. Next, we examined the phenotype of full length Rv1747 tagged with mEos2 (mEos2-Rv1747) (Figure 3C). We discovered another unique phenotype that was different from the one observed for cells expressing either the mEos2 control vector or mEos2-Rv17471-310. Cells expressing full length Rv1747 had multiple foci that were smaller compared to those formed in cells expressing mEos2-Rv17471-310 (Figure 3B). Kymograph analyses revealed that these foci are more mobile/dynamic rather than static as they were assembling and disassembling during the 10s of imaging. Nevertheless, foci formation of full length Rv1747 is consistent with our hypothesis that phase separation of the regulatory module induces clustering of the entire transmembrane protein. To test this hypothesis further, we investigated the phenotype of cells expressing mEos2-Rv1747∆1-310, a construct that lacks the RD (Figure 3D). We observed a phenotype that resembles more the mEos2 control than the one found for mEos2-Rv17471-310 or mEos2-Rv1747, respectively. Neither signs of foci formation, nor changes 38 in the fluorescence signal were observed in collected images, fluorescence intensity graphs and kymographs (Figure 3D). This finding strongly suggests that foci formation by mEos2-Rv1747 is dependent on the presence of the regulatory domain, i.e. residues 1-310. Figure 3. Comparison of foci formation across different Rv1747 constructs. Top panels: Internal reflection fluorescence (TIRF) microscopy images of representative live M. smegmatis cells expressing the indicated proteins are shown (scale bar: 5 μm). Two representative cells are shown for each plasmid construct. Asterisks indicate the cells used for analysis in middle and bottom panels. Middle panels: fluorescence intensity graph generated from the representative image in order to identify foci (intensity peaks indicated by magenta arrowheads) or no foci (minimal fluctuation). Bottom panels: kymographs showing the change in fluorescence signal across the length of cells over 10 seconds. Foci that remained throughout the entire sequence of imaging (10 seconds) are indicated by red arrowheads. Foci that appear during the course of imaging and later disappeared are indicated by yellow arrowheads. mEos2-Rv17471-310▲▲▲ ▲mEos2-Rv1747▲1-310mEos2A10 sec kymographFluorescence intensity (a.u)mEos2-Rv1747▲▲▲▲▲▲▲▲ ▲▲▲▲▲▲▲▲▲▲▲▲B C D* 39 3.2 Key interaction sites in the FHA domains and linker are necessary for foci formation by Rv1747 in M. smegmatis Our lab has confirmed that phosphorylation enables interactions between the FHA domains and the linker in vitro [48]. Moreover, we observed reduced threshold concentration for Rv17471-310 condensate formation under phosphorylated conditions using STPKs and reversibility of this condensation process using PstP[69]. Inspired by this knowledge, we decided to use different approaches in order to disrupt the ability of the FHA domains to interact with pThrs and the disordered linker to be phosphorylated, followed by an assessment of foci formation in M. smegmatis. As interactions between phosphorylated threonines and FHA domains depend on conserved serines in the binding pocket of FHA domains, we mutated the active site serines in one or both FHA domains in our first approach. Specifically, we introduced the S47A and S248A mutation individually or combined in the mEos2-Rv17471-310 constructs (mEos2-Rv17471-310S47A, mEos2-Rv17471-310S248A, mEos2-Rv17471-310S47,248A) and expressed the mutants in M. smegmatis to measure effects on foci formation. In the second approach, we expressed a mutant in M. smegmatis that had all of the phoshpho-acceptor threonines in the disordered linker replaced by alanine (mEos2-Rv17471-310STA) and visualized foci formation. As control (Figure 4 A and B) we used cells expressing the mEos2-tagged vector only (negative control) and cells expressing mEos2-Rv17471-310 (positive control). The same analysis criteria as before were used to generate 40 representative images, fluorescent intensity graphs (not shown in the figures), and kymographs for all the constructs. Cells expressing mEos2-Rv17471-310S47A, mEos2-Rv17471-310S47,248A, and mEos2-Rv17471-310STA exhibited drastic phenotypic changes away from the positive control toward a more vector-like phenotype, where no clear signs of foci formation were present (Figure 4 C, E and F, top panel). Moreover, kymographs showed no appearance of sites of high fluorescence intensity during the 10 second of recording (Figure 4 C, E and F, bottom panel). Interestingly, cells expressing mEos2-Rv17471-310S248A displayed two different phenotypes with about 90% of the cells showing no foci and universal fluorescence distributions, while about 10% of the cells exhibited foci formation (Figure 4D, top panel). Furthermore, the foci formed in the minority of cells were more mobile when compared with those seen in cells expressing mEos2-Rv17471-310 (Figure 4D, bottom panel). Together, these observations demonstrate that canonical interactions by the FHA domains, specifically FHA-1, as well as the phosphorylations of the linker are necessary for foci formation by the regulatory module. 41 Figure 4. Comparison of foci formation across different mutant constructs of mEos2-Rv17471-310 that prevent FHA interactions and linker phosphorylation. Top panels: Internal reflection fluorescence (TIRF) microscopy images of representative live M. smegmatis cells expressing the indicated proteins are shown (scale bar: 5 μm); Two representative cells are shown for each plasmid construct. Bottom panels: kymographs showing the change in fluorescence signal across the length of cells over 10 seconds. Red arrows indicate static foci. Next, we designed and prepared full length Rv1747 mutants of mEos2-Rv1747S47A, mEos2-Rv1747S248A, and mEos2-Rv1747S47,248A to investigate whether the same shift from foci forming to vector-like phenotypes would be observed for the FHA mutants in the context of full length Rv1747. As control (Figure 5 A and B) we used cells expressing the mEos2-tagged vector only (negative control) and cells expressing full length mEos2-Rv1747 (positive control). Cells expressing mEos2-Rv1747S47A and mEos2-Rv1747S47,248A showed clear phenotypic shifts away from the positive control and became vector-like with minimal signs of foci formation (Figure 5 C and E, top panel). For both these mutants, fluorescence intensity distributions in the kymographs are much more uniform than in the 42 positive control for the large majority of cells (Figure 5 C and E, bottom panel). Cells expressing mEos2-Rv1747S248A displayed a more mixed phenotype with about 90% of the cells showing no clear signs of clustering (top panel) and more vector-like kymographs (bottom panel). However, the remaining cells ( ~ 10%) showed clear signs of foci formation. Interestingly, these foci displayed a more static character than the positive control, as can be seen from the kymographs (Figure 5D, bottom panel). Together, these data further validate that the key interaction sites in the FHA domains, specifically in FHA-1, and linker phosphorylation are necessary for foci formation by Rv1747. Figure 5. Comparison of foci formation across different mutant constructs of full length mEos2-Rv1747 that prevent FHA interactions and linker phosphorylation. Top panels: Internal reflection fluorescence (TIRF) microscopy images of representative live M. smegmatis cells expressing the indicated proteins are shown (scale bar: 5 μm); Two representative cells are shown for each plasmid construct. Bottom panels: kymographs showing the change in fluorescence signal across the length of cells over 10 seconds. 43 3.3 Linker charge affects Rv17471-310 clustering in M. smegmatis As previously mentioned, proteins that contain IDRs with scattered interaction motifs can phase separate through formation of different charge-charge, dipole-dipole, or pi-pi interactions[57]. Furthermore, the effect of charge-charge interactions on Rv17471-310 phase separation was previously established by changing the ionic strength in vitro. It was found that the saturation concentration of non-phosphorylated Rv17471-310 increase with ionic strength[69]. We believe that the ID linker (pI 11.9) could contribute to phosphorylation-independent phase separation due to possible weak multivalent charge-charge interactions with the FHA domains (pIs: 6.4 and 5.0). In a first attempt, we approached this idea by mutating all lysine and arginine residues in the linker of mEos2-Rv17471-310 to alanine (mEos2-Rv17471-310RKA). As controls (Figure 6 A and B), we used cells expressing the mEos2-tagged vector only (negative control) and cells expressing mEos2-Rv17471-310 (positive control). Cells expressing mEos2-Rv17471-310RKA exhibited vector like phenotypes with little signs of foci formation (Figure 6C, top panel) and an overall quite uniform fluorescence distribution along the bacteria during the 10 s of recording for the kymographs (Figure 6C, bottom panel). This observation of diminished foci formation by mEos2-Rv17471-310RKA suggests that electrostatic interactions between the FHA domains and the linker could indeed contribute to Rv17471-310 clustering in M. smegmatis. 44 Figure 6. Foci formation by a mEos2-Rv17471-310 mutant that has all positively charged linker residues mutated to alanine. Top panels: Internal reflection fluorescence (TIRF) microscopy images of representative live M. smegmatis cells expressing the indicated proteins are shown (scale bar: 5 μm); Two representative cells are shown for each plasmid construct. Bottom panels: kymographs showing the change in fluorescence signal across the length of cells over 10 seconds. Red arrows indicate static foci. 45 Chapter 4: Discussion 4.1 Regulatory domain dependent Rv1747 phase separation in M. smegmatis Investigating phase separating systems in bacteria is challenging due to the small size of bacteria. Therefore, criteria and methods complementary to those used for eukaryotes have been proposed to study phase separating systems in bacteria such as the identification of dynamic cluster/foci and the monitoring of the diffusive properties of molecules within these foci using single-molecule tracking approaches [71][61]. We decided to test first whether our system, Rv1747, forms foci in M. smegmatis. Consistent with our expectation based on the RD’s ability to phase separate in vitro, we observed foci formation by mEos2-Rv17471-310 in M. smegmatis (Figure 3B). More importantly, also full length Rv1747 formed mobile foci in M. smegmatis (Figure 3C), and this dependent on the presence of the regulatory module. These findings are consistent with our hypothesis that phase separation of the RD drives clustering of full length Rv1747 in the mycobacterial membrane. Despite both Rv1747 and Rv17471-310 forming foci when expressed in M. smegmatis, their foci exhibit distinctive characteristics that are worth discussing. mEos2-Rv17471-310 formed only a few, large and rather static foci, i.e. foci that remained at the same position during the 10 seconds of recording. In contrast, full length Rv1747 formed many more and highly dynamic foci that appeared and disappeared during the imaging process. A 46 key difference between these two construct is the cytosolic localisation of mEos2-Rv17471-310. The cytosolic localization is likely allowing the construct to more freely diffuse and initially formed condensates to coalesce and ripe to a one or two dominant foci. In addition, cytosolic localization may enable unexpected interactions that normally don’t occur when the RD is confined to the membrane. In fact, these unexpected interactions may in turn affect the localization of Rv17471-310 if the binding partners localize to specific places in the cytosol. For instance, many foci formed by mEos2-Rv17471-310 were located in the middle of the cells. When correlated with the fact that Rv1747 is most likely involved in cell wall biosynthesis, and the divisome of mycobacteria being regulated in part via STPKs-mediated phoshorylation, one can speculate that maybe a binding partner that is involved in synthesizing the new cell wall in dividing cells and/or members of the divisome interact and recruit Rv17471-310 to septa (one of the places where the new cell wall is synthesized)[31]. Full length Rv1747, by contrast, is in a very different environment of the cell membrane. This said, we did not confirm the transmembrane localization for this construct. The membrane itself has intrinsic fluidity[72], which is likely affecting the ability of Rv1747 to diffuse and coalesce. It should be noted that although our findings are consistent with the idea that Rv1747 also phase separates in M. smegmatis, further studies are required to fully establish RV1747’s ability to phase separate in bacteria. For instance, the dynamic property of the 47 proteins in the foci could be validated using single-molecule tracking methods (will be discussed in more detail in 4.5.) 4.2 Key interactions by the FHA domains and linker are necessary for Rv1747 foci formation in M. smegmatis Phase separation of the RD in vitro is enhanced by phosphorylation of the linker, Therefore, we aimed to investigate the dependence of foci formation on the ability of the FHA domains to interact with pThrs and the linker to be phosphorylated. We prepared FHA active sites mutants as well as a construct in which all linker Thr/Sers were mutated to Ala. We observed a clear phenotypic shift from foci forming to vector-like cells when expressing mEos2-Rv17471-310S47A and mEos2-Rv17471-310S47,248A in M. smegmatis. Moreover, 90% of the cells expressing mEos2-Rv17471-310S248A also lost the ability to form foci (Figure 4). Interestingly around 10% of mEos2-Rv17471-310S248A single mutant still formed foci. Very similar observations were made when all these mutants were introduced in the context of the full length Rv1747 protein (Figure 5). These findings are overall in good agreement with recent in vitro studies carried out by Loktin Hui, a member of Dr. McIntosh’s laboratory. He introduced the S47A and S248A single mutants as well as the S47, 248A double mutant in Rv17471-310 in order to assess effects of these mutants on phase separation in vitro. 50 μM of each protein was incubated with 5 48 μM PknF and 1mM ATP for an hour before imaging under light microscopy. He found that the Rv17471-310S47A single mutant as well as the Rv17471-310S47,248A double mutant were unable to phase separate under these conditions, in contrast to the wildtype RD. However, the Rv17471-310S248A single mutant was still able to phase separate and form condensates in vitro[73], which contrast our finding of the majority of M. smegmatis cells not forming mEos2-Rv17471-310S248A foci. This said, about 10% of the cell did form foci. One of the reasons that may contribute to this observational difference is the concentration of the Rv1747 constructs in M. smegmatis. Rv17471-310S248A present in M. smegmatis may be close or below the threshold concentration for phase separation in some cells [69]. A more detailed discussion regarding this issue is provided in 4.5. Overall, these observations reveal that pThr-FHA interactions, specifically those that involve FHA-1, are required for phase separation of the RD in vitro, at least at protein concentrations of 50 μM, and for the formation of foci by the RD and the entire Rv1747 protein under the induction conditions used. Previous experiments from the lab strongly suggest that these pThr-FHA interactions involve phosphorylated threonines from the linker that connects FHA-1 and FHA-2. Therefore, we tested next how phosphor-ablative mutations in the linker affect foci formation of the RD. The mEos2-Rv17471-310STA mutant that has all Thr/Ser residues in the linker replaced by Ala exhibited no signs of foci formation (Figure 4F), consistent 49 with the idea that removal of all phosphor-acceptor threonine in the linker would prevent binding to the FHA domains and thus phase separation. However, it needs to be stressed that mutating all the Thr and Ser in the ID linker may have disrupted many other interactions with different binding partners that potentially contribute to the formation of foci by wildtype RD and full length Rv1747. It is well established that interactions with other biomolecules (e.g proteins or RNA) in the right concentration range can promote phase separation of certain systems. A similar concern can be raised for the mutations of the FHA domains, because the FHA domains of Rv1747 are known to recruit partner proteins. One of the known interaction partners of Rv1747 is the STPK PknF, which gets recruited via binding to FHA-1 and then phosphorylates the RD linker [44]. Intriguingly , in vitro experiments conducted by Hui showed that Rv17471-310 incubated with PknF is able to phase separate in the absence of ATP[73], i.e. interactions with PknF appear to induce phase separation even when phosphorylation is absent or limited. Thus, PknF could be a partner that promotes phase separation and foci formation. Disrupting this interaction via mutations could have contributed to the phenotypic changes that we observed. If so, the fact that PknF is recruited to Rv1747 by FHA-1 and not FHA-2 may also contribute to some of the phenotypic differences that we observed for the S47A and S248A mutants, i.e, that impeding interactions by FHA-1 abolished foci formation but not so mutations of the interaction site of FHA-2. 50 4.3 Linker charge affects Rv1747 foci formation As previously mentioned, in vitro experiments have shown that increasing the ionic strength leads to increases in the saturation concentration for phosphorylation-independent phase separation of the RD, indicating the contribution of electrostatic interactions to the process. We hypothesised that the oppositely charged ID linker (pI 11.9) and FHA domains (pIs: 6.4 and 5.0) may be involved in non-specific electrostatic interactions (not involving the pThr-FHA pairing) that promote phosphorylation-independent phase separation at high RD concentrations. Manipulating ionic strength is challenging in live cells, and therefore, we directly disrupted potential charge-charge interactions by replacing all positively charged Arg/Lys in the disordered linker with Ala (mEos2-Rv17471-310RKA). We observed a vector-like phenotype for this mutant (Figure 6). This drastic change in phenotype compared to the foci-forming mEos2-Rv17471-310 is consistent with the idea that non-specific electrostatic interactions could contribute to phase separation and foci formation. However, more studies, specifically in vitro investigation of the impact on phase separation, are required to establish a better understanding of the contribution of charge-charge interactions to phase separation of Rv1747. It should be noted that, although this phenotypic shift can be explained by disrupted charge-charge interaction between the ID linker and FHA domains, it may also arise from conformational changes of the ID linker as charge pattern have been shown to affect space orientation and 51 compactness of the interaction motifs and is essential for protein-solvent and protein-protein interactions[53]. 4.4 Conclusion Mtb is the etiological factor of TB which infects over 10 million people and causes over 1.4 million death worldwide in 2019 and remains the leading cause of death from a single infectious agent. The ABC transporter and virulence factor Rv1747 is associated with cell wall biosynthesis in Mtb and can undergo spontaneous phosphorylation and concentration dependent phase separation in vitro. Phase separation is involved in organization and regulation of the functions of membraneless cellular compartments and may be the underlying mechanism regulating the function of Rv1747. In this thesis, the goal was to show that Rv1747 forms foci in M. smegmatis and investigate factors contributing to this process. We used M. smegmatis as model organism and revealed that both the RD and full length of Rv1747 forms static and mobile clusters, respectively, dependent on the presence of regulatory module in M. smegmatis. Different localization of cytoplasmic RD and transmembrane full length construct and therefore interactions with different binding partners may contribute to the observed phenotype difference. We demonstrated that the key interactions by the FHA domains, in particular FHA-1, and phosphorylated threonines in the linker are essential for cluster formation in both RD 52 and full length of Rv1747 in M. smegmatis, as we showed S47A single mutant and S47A, S248A double mutant exhibiting no foci formation while S248A single mutant exhibiting majorly vector-like phenotype with a minority cells forming clusters. We further supported this by showing the phosphor-ablative mutant of the regulatory module of Rv1747 with all Thr/Ser to Ala in the linker exhibiting no sign of foci formation. In addition, the charge-charge interaction by the intrinsically disordered linker may contribute to foci formation by Rv1747 regulatory module, as the mutant carrying all Arg/Lys to Ala mutations demolished clustering in M. smegmatis. However, the FHA domains and the linker may contribute to phase separation through interactions with other binding partners, and future work is required to provide a thorough understanding. In summary, we have provided strong evidence of Rv1747 phase separation in M. smegmatis. We have investigated possible contributing factors including phosphorylation dependent and non-specific charge-charge interactions, respectively, by the FHA domains and the ID linker and suggested their roles in regulating phase separation dependent Rv1747 function in M. smegmatis. 4.5 Significance, limitations and future direction Although we have achieved promising progress in supporting the hypothesis of phase separation playing a role in Rv1747’s function, limitations of this study still exist and future work is required for a thorough understanding of its role in Mtb virulence. Here, I 53 briefly talk about issues regarding expression levels, phosphorylation, construct design, and single-molecule tracking. First of all, we expressed Rv1747 wildtype and mutant proteins in M. smegmatis without a clear assessment of the expression levels of the different constructs. This raises the concern that foci formation may be biologically irrelevant and result from overexpression of the protein, as non-phosphorylated Rv17471-310 has been shown to phase separate at high concentration[69]. Therefore, to ensure the biological significance of this study, various protein quantification methods should be used in the future. For instance, His-tagged Rv1747 purified with nickel-nitrilotriacetic agarose (Ni-NTA) beads followed by quantification by Western blotting[74]. Although susceptible to errors related to sample preparation, SDS-PAGE loading, protein transfer and antibody detection, this method in combination with cell number quantification by OD600 should be able to generate estimated of protein concentration per cell[74]. Secondly, we did not assess whether any of the constructs that we expressed in M. smegmatis were phosphorylated in the bacterial cells . The results for the different mutants, however, suggest that the linker in the RD gets phosphorylated and that interfering with this phosphorylation, respectively, interactions that are mediated by pThr-FHA pairing impedes foci formation. For convenient recognition of pThr, polyclonal rabbit anti-phosphothreonine antibodies can be used in the process of Western blotting following protein purification mentioned in the paragraph above[75]. For further 54 characterization, mass spectrometry can be used to investigate the phosphorylation status of Thr in the ID linker [39]. Thirdly, the construct design in this study has its own limitations. The wildtype and mutant forms of Rv17471-310, although conveniently complementing the in vitro findings from Heinkel and Hui, were cytoplasmic and, therefore, may lead to interactions and localizations that are not biologically relevant. Another limitation arises from the design of the linker mutant mEos2-Rv17471-310STA. As mentioned before, mutating all Thr/Ser in the linker may affect interactions between linker Thr/Ser with binding partners. Therefore, although conclusion can be drawn that linker phosphorylation is necessary for Rv1747 foci formation, future experiments are needed to investigate how these phosphorylations exactly promote phase separation and foci promoting interactions. Similarly, future work is required to examine how electrostatic interactions mediated by the linker contribute to foci formation. Furthermore, although assumed to be incorporated in the membrane, the localization of the wildtype and mutants of full length Rv1747 as well as Rv1747∆1-310 was not confirmed. Fortunately, this issue can be resolved by generating super-resolved images of the foci in M. smegmatis. For instance, photoactivated localization microscopy (PALM) is a super-resolution method that can be used in the future for this purpose. To achieve super resolution and distinguish the location of a single molecule, the intensity of each fluorophore must not overlap with each other. PALM stochastically activates 55 photoactivable fluorophores, mEos2 for example, and images only a small subset of fluorophores at a time to temporally separate molecules that would be otherwise spatially indistinguishable. Thousands of subset of fluorophores undergo cycles of activation, imaging, and photobleaching to generate super-resolved image[76]. All of the plasmid constructs used in this study are tagged with photoactivable fluorophore mEos2, which makes PALM the most convenient method to use in the future. Last but not least, a key step in determining that foci are indeed liquid-like condensates is to reveal the diffusive behavior of foci constituents by single-molecule tracking[71][61]. Despite observation of mobile foci formation in full length Rv1747 (Figure 3C) and dynamic property of the foci shown from FRAP[69], single-molecule tracking is necessary to show dynamic properties of foci at molecular level. Single-particle tracking can be conveniently combined with PALM where the location of the same molecule from different frames can be connected to form trajectories in order to track its movement. 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Thesis/Dissertation
eng
Biochemistry and Molecular Biology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Gsponer, Jörg
Investigating molecular clustering of Rv1747 in M. smegmatis
Text