During DNA replication, the strain of the progressing replication fork induces DNA strands to wrap around each other, termed “positive supercoiling”, a significant obstacle to further replication. Type II Topoisomerases (Top2s) are essential and ubiquitous DNA replication enzymes that remove supercoiling to enable replication. However, we do not understand whether Top2s alone are sufficient to relax DNA for replication. We recently discovered that Growth-Associated Protein in Regulation (GapR), an essential DNA binding protein in alphaproteobacteria, binds positive supercoils and stimulates the activity of bacterial Top2s DNA Gyrase and Topoisomerase IV. Although GapR stimulates Top2s in vitro, we do not know the mechanism by which GapR stimulates Top2s. We hypothesize that GapR recruits Top2s to positive supercoils by direct interaction, and we investigated this mechanism by using a phage homolog that we discovered also stimulates Top2s and leveraged the conserved sequences to predict sites of Top2 interaction with GapR. We used the Bacterial Two-Hybrid assay to screen for GapR homolog interaction with Top2 subunits, and formed GapR truncations to interrogate for interaction with Top2s. We identified an interaction between GapR and the homologous Top2 subunits GyrA and ParC. Next, we aim to identify the surface that mediates direct interaction between GapR and Top2s, revealing a previously unknown mechanism of Top2 recruitment. We hypothesize that disrupting the GapR-Top2 interaction will lead to cell death, and as GapR is broadly conserved by alphaproteobacteria, our research could reveal a novel mechanism to inhibit with antibiotics. If a conserved mechanism, our work could identify new anticancer therapeutics, as human Top2 inhibitors are important chemotherapy drugs.

With rising greenhouse gas emissions, both emissions reductions and greenhouse gas capture and conversion are necessary to mitigate the impacts of climate change. Though carbon dioxide comprises the largest proportion of global greenhouse gas emissions, methane causes over 80 times more global warming per unit than carbon dioxide. While methane can be converted catalytically at high temperatures and pressures, bacteria called methanotrophs transform methane into biomass at ambient temperatures and pressures. Engineering these organisms to consume methane globally can help slow climate change. At the Lidstrom Lab, I study the relationship between the methanotroph genome and metabolic regulation. Through this work, I have focused on finding genes that facilitate responses to environmental conditions and am now looking at genes that impact overall growth rate. I design and construct mutant strains, eliminating genes that appear to use the cell’s energy unnecessarily based on transcriptomics data. We expect that eliminating such genes will allow the cells to devote more energy to methane consumption and growth, improving growth rates under low methane conditions. This work is being extended to experiments in a bioreactor to observe how selected mutations and growth conditions impact growth rate at low methane. For this work, I am setting up the bioreactor to maintain the intended reaction conditions and utilizing a gas chromatogram to monitor methane consumption over time. These experiments reveal how the methanotrophs grow under specific conditions. I am also analyzing the results of the experiments using Python, MATLAB, and Excel. Long-term, this research will help prepare methanotrophs for deployment in the field to consume methane in areas of atmospheric methane release including landfills and agricultural sites.

Pseudomonas aeruginosa is a major cause of opportunistic infections in immunocompromised people. P. aeruginosa uses a cell-cell signaling mechanism called quorum sensing (QS) to regulate virulence functions and cooperative behaviors. QS in P. aeruginosa is regulated by two transcription factors, LasR and RhlR. These proteins control the production of extracellular proteases, “public goods” that benefit the entire population. These public goods create an incentive for individuals to cheat by availing themselves of the proteases without incurring their production cost. In fact, when P. aeruginosa is grown in media that requires QS for growth, cheaters emerge in the population and invariably carry mutations in lasR. We were intrigued by the clinical isolate E94, in which we observed the rapid emergence of cheating. E94 contains an inactive LasR via a transposon insertion. Transposons are DNA segments that move between genomic locations. E94 contains dozens of transposable elements. We hypothesized that transposon movement facilitates the adaptability of the E94 genome. I evolved cheaters from wild-type E94 in casein media, which requires QS for growth. I identified cheaters using a phenotypic assay. I then grew cheaters in non-selective media and identified revertant, protease-producing colonies. I used PCR analysis of the cheaters and their protease-producing progeny to determine if the transposon remains inside the lasR gene. I found no transposon movement from lasR upon cheater reversion to a protease-producing phenotype. We also determined that cheaters display no rhlR activity. We then hypothesized that cheating in E94 occurs by disruption of RhlR QS. We did not find mutations in rhlR itself and are performing whole-genome sequencing to identify other genetic targets. Understanding the mechanism of cheating and reversion in E94 gives us insight into the evolution of cooperation and conflict in populations and, potentially, a non-antibiotic approach to controlling bacterial populations a variety of settings.

The dramatic increase in antibiotic-resistant Pseudomonas aeruginosa infections makes it necessary to find new approaches for treatment. P. aeruginosa employs a communication system called quorum sensing (QS) that uses density-dependent accumulation of small diffusible molecules to mediate the production of factors that benefit the entire bacterial community; creating a potential drug target. P. aeruginosa has two QS systems: las and rhl. A previous analysis of the laboratory strain PAO1 revealed that mutations in the las system result in individuals that act as social cheaters that reap the benefits of communally produced goods without expending energy on their production. lasR mutants have a fitness advantage when cultured in a medium requiring the activity of the community goods for survival. I set out to better understand the rhl QS system. I was interested in determining if ΔrhlR mutants act as social cheaters in co-culture with rhlR-competent strains. To make that determination, I screened 12 clinical isolates from the Early Pseudomonas Infection Control (EPIC) study for the ability of their isogenic ΔrhlR mutants to grow in QS and non-QS selective media and their ability to persist in coculture with the parent strain. I also competed these strains against isogenic ΔrhlR mutants by growing them in coculture and using flow cytometry to determine the relative final frequencies of the parent strain and ΔrhlR mutants. Finally, I aimed to determine if rhlR mutants arise spontaneously from the parent strains in a long-term growth experiment by sequencing and functional analysis of the mutants. My preliminary work demonstrates that a small subset of the ΔrhlR mutants dramatically increase relative to concentrations of EPIC strains in coculture and mutations in rhlR do arise in vitro; however, their exact functional effects are still to be determined.  Understanding ΔrhlR cheater dynamics may provide therapeutic targets for antibiotic-resistant P. aeruginosa infections.

Kaposi’s Sarcoma (KS) is among the most common tumors in central Africa and AIDS patients and Kaposi’s Sarcoma-associated herpesvirus (KSHV) is the etiologic agent of KS. While all herpesviruses are capable of both lytic and latent replication programs, KSHV is predominantly in the latent state in the main KS tumor cell,the spindle cell, a cell expressing markers of the endothelium. There is limited viral gene expression during latency so it is difficult to target the virus directly. Therefore, our approach is to target host cellular requirements for KSHV latent infection. Previously, the Lagunoff Lab performed a genome wide CRISPR-Cas9 screen targeting over 18,000 human genes to identify cellular genes essential only to cells latently infected with KSHV. CYP27A1, a gene that encodes a member of the cytochrome p450 family, was one of the top hits identified in the screen. It specifically encodes sterol 27-hydroxylase, which is an enzyme involved in the breakdown of cholesterol. I hypothesize that CYP27A1 is an essential gene for survival of KSHV latently infected cells, as there is evidence that cholesterol is antiviral and KSHV is known to regulate cholesterol. I have successfully cloned CRISPR guide RNAs targeting CYP27A1 into lentiviral vectors, transfected 293T cells with the vectors to make lentivirus, and transduced human tert-immortal endothelial (TIME) cells with the lentivirus to create CYP27A1 knockout cells. ICE analysis was used to confirm strong knockout of the CYP27A1 gene. Currently, we are infecting these cells with KSHV to determine if CYP27A1 is required during KSHV latent infection using cell survival and cell proliferation as readouts. I expect that knockout of CYP27A1 will result in cell death in endothelial cells latently infected with KSHV. By determining genes necessary for KSHV latency, we hope to identify potential therapeutic targets for KS tumors.

The HIV global pandemic has claimed more than 40 million lives so far, and it is an ongoing worldwide health crisis as more than 1.5 million people acquired HIV in 2021 alone. A major barrier towards a cure for HIV is the presence of replication-competent latent HIV-1 proviruses in reservoir cells, which contribute to viral persistence despite years of antiretroviral therapy. One approach to control the latent HIV reservoir is silencing HIV transcription to prevent reactivation. A comprehensive list of host factors that support latency reactivation has not been yet identified. Here, we conduct a CRISPR-Cas9-mediated gene knockout using guide RNAs packaged into budding HIV virions, serving as a readout to identify cellular factors that promote HIV latency reactivation. We used the dependency factor library (HIV-DEP) that contains hundreds of genes involved in proviral transcription. This library was transduced in bulk into two J-lat cell lines, T lymphocytes cell lines which serve as HIV latency models, and we screened for genes that, upon knock-out, prevent latency release after treating the cells with latency reversal agents (LRAs). We identified 47 genes that, when knock-out, prevented HIV latency reactivation in both J-lat models. Our findings suggest that host genes indispensable for HIV transcription also play an important role in HIV latency control. Importantly, these candidate genes could serve as targets for HIV therapeutic intervention.

The emergence of SARS-CoV-2 (CoV-2) and the rapid growth of the global pandemic has necessitated more than ever the need for fully comprehensive studies of viral pathogenesis. Previous studies of CoV-2 highlight the importance of its viral proteins in transmission and adaptability into human hosts, but few have delved into the role that viral RNA structure plays in emergence and pathogenesis. Viral RNA structure is a known contributor to host-jumping replication as seen in related coronaviruses (CoVs), Flaviviruses, and Alphaviruses. Given this phenomenon and that all human CoVs originated from bat CoVs, we hypothesize that genomic and structural differences in the human CoV-2 RNA compared to the bat RatG13 CoV RNA, the believed progenitor of CoV-2, may have contributed to the species-jumping event from bats to humans that caused the emergence of human CoV-2. To test this hypothesis, I used a CoV-2 replicon system to compare replication of the wildtype (WT) replicon versus a mutant replicon (RaTG13-N/3’UTR(syn)) that contains a firefly luciferase reporter gene and synonymous mutations from the 3’ end of RaTG13. In preliminary replication experiments, RaTG13-N/3’UTR(syn) had notably higher replication than the WT replicon in primate fibroblasts. Based on this result, I used electroporation techniques and Renilla luciferase assays to determine the replication kinetics involved in differential replication of these viral replicons in human versus bat epithelial lung cells. I anticipate increased replication of the RaTG13-N/3’UTR(syn) replicon in bat cells compared to human cells when compared against the WT which would entail a mechanistic study of how RNA structures contribute to viral fitness in the two species using genomic and proteomic approaches. If this data supports our hypothesis, this could enhance our understanding of CoVs to allow us to target and survey zoonotic viruses that threaten species-jumping into the human population.

The innate immune system must have a rigorous response to many pathogens in order to successfully defend host cells against infections. During infection, inflammasome forming sensors detect pathogen-specific features which release proinflammatory cytokines such as IL-1B for immune activation. The inflammasome forming sensor NLRP1 directly detects multiple signals indicative of infection, including viral protease activity. Recently, it was shown NLRP1 is indirectly activated by bacterial toxins and UV irradiation that disrupt host protein synthesis, demonstrating it can detect environmental stimuli to cause inflammation. However, much of this activation pathway is poorly understood with only one nonpathogenic cause having been investigated. Our goal is to identify the host proteins required for NLRP1 activation and determine whether NLRP1 can broadly detect disrupted protein synthesis. We hypothesize NLRP1 detection of disruption in protein synthesis is a broad strategy to combat infection and may have an important role in causing inflammation in other diseases associated with disrupted protein synthesis, including cancer and neurodegeneration. Knockout cell lines of proteins suspected of activating NLRP1 are produced through lentiviral transduction of Cas-9, a gene editing tool which cleaves off specific nucleotides corresponding to the target gene of each protease sensor in the inflammasome activation pathway. Each cell line is confirmed to be absent of the target sensor via genotyping, and is followed by a functional assay of each knockout line which induces cellular stress targeting the activation of each cleaved protease. We predict that inflammasome activation as defined by IL-1B concentration will be significantly decreased in knockout cell lines targeting key proteases in the inflammasome signal cascade, showing that the overactivation of these proteases is sufficient for inflammasome activation. These results could provide key targets for drug discovery in the treatment of multiple diseases which cause disruption of protein synthesis, including cancer and neurodegeneration.

Sindbis Virus (SINV) is an alphavirus that is transmitted between birds via mosquitos and causes disease in humans after spillover events. While many alphaviruses have potential to cause severe disease such as Venezuelan Equine Encephalitis Virus (VEEV), SINV is known to cause less life-threatening but still severe and debilitating chronic illness predominantly associated with fever, arthralgia, and myalgia. Given its lower morbidity, SINV often serves as a model system for infectivity and pathogenesis studies of alphaviruses. Macrophages are of particular interest for studying pathogenesis as they are important targets of alphavirus infection. Our aim is to identify viral mutations that contribute to differential replication between avirulent and virulent strains of SINV in macrophages. To test this we exchanged sequences between SINV strains Girdwood (avirulent) and AR86 (virulent) to construct reciprocal chimeras which will be tested alongside parent clones. We observed a significant difference in replication between parent AR86 and Girdwood in macrophages and predict that specific RNA structural differences contribute to this variable replication in macrophages, and we are currently mapping determinants of this phenotype. This work could improve our understanding of host-viral interactions of alphaviruses by discerning the mutations required to shift from avirulent to virulent strains which will play an important role in predicting mutations that contribute to greater pathogenicity in human infections.

Cystic Fibrosis (CF) is a recessive genetic disease that affects roughly 70,000-100,000 people worldwide. CF is caused by mutations in the CFTR gene, which encodes for a channel that regulates the flow of water and ions into and out of the cell. CFTR deficiency results in mucus buildup and other defects that lead to increased bacterial burden and inflammation. However, whether or not CFTR deficiency itself is sufficient to initiate or potentiate epithelial inflammatory responses is unknown. Inflammasomes are multiprotein complexes that upon activation initiates a lytic form of cell death called pyroptosis and the release of pro-inflammatory cytokines including IL-1B, a hallmark of CF. Previous studies have shown that inflammasomes are expressed in the epithelia of mice and humans. Although CF is largely a disease that affects airway epithelium, other tissues such as the skin and gut are also affected by CFTR dysfunction. Moreover, we and others recently found that inflammasomes play an important role in host defense of epithelial barriers, including the intestinal epithelium. Thus, we hypothesize that inflammation in the gut of CF patients may be caused or enhanced by inflammasome activation. To test this possibility I used both human and mouse intestinal organoids as models and ran a Forskolin-induced Swelling (FIS) Assay to assess CFTR function in presence and absence of a CFTR inhibitor (CFTRinh-172). Having established this model system, I am now evaluating the impact of CFTR function on inflammasome activation. We anticipate that our work may reveal a link between CFTR dysfunction and inflammasome activation which may provide further insight into how inflammasomes affect inflammation in the gut epithelia of CF patients.