Investigating the role of CR3 in trogocytosis of Trichomonas vaginalis cells by Neutrophil-like cells. Senior Thesis California State Polytechnic University, Department of Biology Aljona Leka Team Members: Jose Moran Mercer Lab Spring 2020 Table of Contents 1. Abstract 2. Introduction 3. Results a) PLB-985 cells differentiate into Neutrophil-like cells b) Strategy for functional deletion of CD11b c) Generation of CD11b knockout cell lines with CRISPR-Cas9 gene editing system showed low cell viability post transfection d) NLCs kill T. vaginalis in the presence of human serum e) NLCs kill T. vaginalis in the presence of heat inactivated human serum 4. Methods a. Promyelocytic cell lines and culture b. Immunolabeling for CD11b and CD18 c. Generation of genetically modified cells d. Culturing transfectants e. Single cell dilution f. Culturing T. vaginalis cells g. Cytotoxicity assay h. Plasmid Construction i. Isolating the px459construct for transfection 5. Discussion 6. References 7. Acknowledgements 8. Supporting information Abstract Trichomonas vaginalis (T. vaginalis) causes the non-viral sexually transmitted infection (STI), trichomoniasis. Trichomoniasis affected almost 276.4 million people globally in 2008 alone, with most incidents occurring in underserved communities. The main curative treatment for T. vaginalis is an antibiotic, Metronidazole, though antibiotic resistance is on the rise. Neutrophils are the first responders against T. vaginalis, killing the parasite through a recently discovered process called trogocytosis, in which the neutrophils “nibble” on the parasite’s membrane leading to killing of the parasite. However, current literature lacks the knowledge of which molecular components are involved in trogocytosis of T. vaginalis. Trogocytosis is a contact- dependent process mediated through opsonins, specifically iC3b, which serves as a tag to make the pathogens “tasty” for the neutrophils. Complement receptor (CR) 3, which is composed of dimer CD11b/CD 18, is known to bind iC3b leading to phagocytosis, but has yet to be correlated with trogocytosis. We hypothesize that CR3 plays a role in mediating trogocytosis of T. vaginalis. To test our hypothesis, we will knockout CR3 on neutrophil like cells using, CRISPR- Cas9 and test whether the CR3 knockout cells are able to kill the parasite with the same efficiency as the unmutated cells, when cultured with T. vaginalis through a cytotoxicity assay. Introduction Trichomoniasis is a common sexually transmitted infection (STI) caused by the highly motile anaerobic flagellated protozoan Trichomonas vaginalis (T. vaginalis), affecting nearly 276.4 million people globally in 2008 alone (Kissinger et al., 2015). A population study reported that nearly 90% of incidents of T. vaginalis occur in underserved and low-income communities, with 10.5% of African American women affected compared to only 1.1% of white women (Kissinger et al., 2015). Trichomoniasis can be treated and cured, but nearly half of diagnosed patients do not show any symptoms and consequently do not seek treatment (Schwebke et al., 2004), which suggests that infection rates might be much higher than reported. T. vaginalis infections can have much worse consequences in women than men, especially for those of an older age. Women who test positive for T. vaginalis show increased rates of cervical cancers, spontaneous abortions, and low-weight births, probably due to genital inflammation caused by T. vaginalis and concomitant human papillomavirus (HPV) infection (Yang et al., 2018). Even though infection rates are high, trichomoniasis is classified as a “neglected infection,” due to a limited public health response, and little research being conducted on the parasite (Taylor et al., 2014, Muzny et al., 2018). The current treatment against trichomoniasis is a non-specific antiparasitic and antibiotic, Metronidazole (Kissinger et al., 2015). Metronidazole does not prevent reinfection, can have many side effects like nausea and headaches, and up to 10% of T. vaginalis strains have become resistant (Kissinger et al., 2015). Studies are gearing towards understanding how our immune system fights T. vaginalis and hopefully help pave the way for the development of preventative treatments in the future. Neutrophils are part of the innate immune system and play an essential role as the first responders to sites of injury and infection (Bardoel et al., 2014). Studies have shown that neutrophils are the major inflammatory cells in the vaginal discharges of patients infected with T. vaginalis (Ryu et al., 2004). It has already been demonstrated that neutrophils kill pathogens through 3 different mechanisms: Neutrophil Extracellular Traps (NETosis), in which they eject their DNA and toxic granules, ensnaring the pathogen in a “net” of DNA making it difficult for them to move and much easier to catch (Bardoel et al., 2014). Phagocytosis, in which they engulf the pathogen whole and digest it, and degranulation, in which they release toxic granules that fuse with the pathogen’s cytoplasmic membrane but they can also affect surrounding host tissue (Bardoel et al., 2014). Once differentiated in the bone marrow, neutrophils come out terminally differentiated and have a short circulatory lifespan, and after they are isolated apoptosis will occur within 6 to 12 hours (Kubes at al., 2013). Their short lifespan makes them challenging to work with, which is why researchers often use promyelocytic cells, such as PLB-985 cells (Rosales et al., 2018) as model to study neutrophil functions. PLB-985 cells were obtained from a patient suffering from myeloblastic leukemia (Tucker et al., 1987), and can be differentiated into neutrophil-like cells (NLCs) through dimethylsulfoxide (DMSO) (Rincon et al., 2018). Though the conventional mechanisms for neutrophil-mediated pathogen removal are NETosis, phagocytosis and degranulation, there is a newly discovered, and less studied method through which neutrophils kill pathogens: trogocytosis. Trogocytosis is a contact-dependent killing mechanism in which neutrophils take “nibbles” of the parasite’s cell membrane (Mercer et al., 2018). Current literature has yet to define the molecular components of trogocytosis, but it has already shown that antibodies and iC3b complement factor, both found in human serum opsonize the parasite (Mercer et al., 2018). It has also been established that trogocytosis is partially mediated by antibody–Fc receptor engagement as part of the adaptive immune system (Mercer et al., 2018), though it has yet to be established whether the complement system does take part in the mediation of trogocytosis and through which molecular player. Studies have shown that iC3b binds to opsonin receptors on neutrophils, such as CR3, which is a complement receptor, part of the innate immune response, composed of dimer CD18/CD11b (Diamond et al., 1993). CD11b is also a differentiation marker for NLCs derived from PLB-985 cells (Pedruzzi et al., 2001, Ashkenazi et al., 2009), and it contains a highly conserved protein domain, called the I domain that binds to iC3b (Diamond et al., 1993). The interaction between CD11b and iC3b has already been shown to trigger phagocytosis (Baldwin et al., 1998), though it has not yet been correlated with trogocytosis. CD11b/CD18 has been shown to be important for the conjugate formation between effector and target cell in trogoptosis, as well as the “nibbling” process leading to trogoptosis (Matlung et al., 2018). Trogoptosis is a trogocytosis-related necrotic process, which happens in antibody opsonized tumor cell death, where an accumulation of trogocytic events, “nibbles”, leads to cell death due to plasma membrane damage (Matlung et al., 2018). It has been shown that the efficiency of trogoptosis, and therefore cell necrosis of cancer cells, can be inhibited when CD11b is blocked (Matlung et al., 2018), though CD11b has yet to be correlated with trogocytosis of T. vaginalis. We hypothesize that CD11b as part of the CR3 receptor is involved in the triggering of trogocytosis of T. vaginalis. To test the enabling effect of CD11b on trogocytosis of T. vaginalis, we will mutate the CD11b gene, more specifically the I domain, through CRISPR-Cas9 with the help of a uniquely designed guide RNA (gRNA) (Xu et al., 2019). We plan to induce a complete functional deletion of CD11b I domain to test whether CD11b triggers trogocytosis , using CD11b knock out NLCs in a cytotoxicity assay, where NLCs and T .vaginalis are coincubated in the presence of human serum (Mercer et al., 2018). To test whether the complement system does play a role in killing of T. vaginalis we performed the cytotoxicity assay in the presence of heat inactivated human serum. Heat inactivates serum complement proteins allowing us to examine the role of the complement system in innate immune interactions (Mankovich et al.,2013), such as those between neutrophils and T. vaginalis. We have already confirmed the presence of CD11b and CD18 surface markers on the membrane of NLCs through antibody staining and optimized the cytotoxicity assay, though we are still working on optimizing our transfection assay to generate the CD11b knockout NLCs. It has been shown that trichomoniasis is one of the most prevalent STIs partially due to the lack of symptoms presenting in the host and lack of treatment, it can also help facilitate transmission of many secondary infections including HIV(Kissinger et al., 2015). The development of preventative treatment is critical and understanding the molecular mechanism