I am interested in producing Human Papillomavirus (HPV) type specific monoclonal antibodies (mAbs) from B cells derived from adolescent females and young women vaccinated with the licensed human papillomavirus vaccine, Gardasil 9, which provides protection against HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58. Fluorescently labeled pseudoviruses for these nine HPV types, as well as phenotypic markers of memory B cells are used to isolate HPV specific memory B cells via fluorescence-activated cell sorting (FACs). Gene transcripts for Immunoglobulin (Ig) heavy and corresponding light chain variable regions are amplified through Reverse Transcription Polymerase Chain Reaction (RT-PCR), cloned into their respective IgG1 backbone vectors, and subsequently transfected into eukaryotic cells to produce antibodies. HPV types [6, 11, 16, and 18] have been previously produced by our lab. I aim to expand on this work by developing antibodies against types 31, 33, 45, 52, and 58. I will characterize these mAbs for HPV binding and neutralization. Production of these well-characterized, type specific antibodies are useful for HPV studies, because they provide standards in HPV serologic assays. 

Pulling apart DNA during replication induces DNA strands to wrap around each other, producing “positive supercoils” ahead of the replication fork. These positive supercoils present a significant obstacle for further DNA replication. Type II topoisomerases (Top2s), a group of essential DNA replication enzymes, cleave these positive supercoils and relax DNA to a state that is easy to separate. Errors in the resolution of supercoils are implicated in many health conditions including autoimmune diseases and cancer. However, the mechanism by which Top2s localize to positive supercoils is unknown. We recently discovered that GapR, an essential DNA binding protein conserved 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 may recruit Top2s. We hypothesize that GapR recruits Top2s to positive supercoils by direct interaction. We investigated this mechanism by using a Bacterial Two-Hybrid assay to screen for GapR interaction with Top2 subunits, and I formed GapR truncations to probe for interaction in biochemical and genetic assays. We identified an interaction between GapR and the homologous Top2 subunits GyrA of DNA Gyrase and ParC of Topoisomerase IV. I discovered that this interaction terminates with the truncation of the last 13 amino acids of GapR. In the future, we aim to identify the surface that mediates direct interaction between GapR and Top2s aided by structure prediction. This work will reveal a previously unknown mechanism of Top2 recruitment. Because GapR is broadly conserved by alphaproteobacteria, our research could reveal a novel mechanism to inhibit with antibiotics. As GapR is the first identified Top2 recruiter, our work could reveal a novel pathway to target with anticancer therapeutics if conserved, as human Top2 inhibitors are important chemotherapy drugs.

Biofilm is a community of bacteria enclosed in an extracellular polymeric substance (EPS) attached to a surface. Inside the biofilm, bacteria can collaborate to increase their survival. The EPS also protects bacteria from drug penetration, leading to increased antibiotic resistance. Therefore, biofilm formation is often linked with chronic bacterial infections. Pseudomonas aeruginosa is an opportunistic pathogen that often causes chronic lung infections in cystic fibrosis patients. It is also a common model for studying biofilm formation. The initial step for biofilm formation is bacteria attaching to and sensing a surface. Upon surface contact, P. aeruginosa may produce cyclic adenosine monophosphate (cAMP), which is a universal second messenger that regulates cellular functions in both eukaryotes and prokaryotes. In P. aeruginosa, cAMP is synthesized by two adenylate cyclases, CyaA & CyaB, and degraded by a cAMP phosphodiesterase, CpdA. cAMP is a key regulator for P. aeruginosa virulence by upregulating the production of the type III secretion system, the type II secretion system, and the type IV pili. However, recent observations in our lab suggest that cAMP may also contribute to the homeostasis of the cell envelope. To investigate this phenomenon, I used microscopy to characterize the cell morphology of strains with different cAMP levels and found that increased cAMP levels lead to longer cells. I also found that high cAMP strains are more sensitive to êžµ-lactam antibiotics specifically, while low cAMP strains become more resistant. Ongoing work includes characterizing the genetic factors that connect cAMP and êžµ-lactam sensitivity, as well as using microscopy to determine changes in cell envelop induced by cAMP. Overall, this work reveals an important role of cAMP in bacterial physiology and provides insight into the complex relationship between virulence and antimicrobial resistance.

Coccidioidomycosis, also known as Valley Fever (VF) is caused by the fungus Coccidioides. Pigtail macaques (PTMs) bred at the Washington National Primate Research Center (WaNPRC) in Mesa, AZ are naturally infected with Coccidioides and are similar to humans in their physiology, symptoms, and immune responses. Populations with a weakened immune system, notably older individuals, are at risk for severe complications from infection. Additionally, there is evidence that males have a higher incidence of VF than females in endemic areas. I characterized the immune responses in a PTM model across age and sex to better understand how VF affects the immune response of these populations. Forty-two PTMs (2.25-19.24 years, 3.66-18.29 kg, 37 female, 5 male) at the WaNPRC were sampled for blood. The frequencies of immune cell subsets in whole blood were characterized by flow cytometry and compared for significant differences based on age and sex. I analyzed sex-based differences with Brown-Forsythe and Welch ANOVA t-tests and found no statistically significant differences. For age-based differences, we used a simple linear regression to analyze differences by age in immune cell subsets. We found that old PTMs (10.07-19.24 years) have higher activation of CD8+ T cells, myeloid dendritic cells, intermediate monocytes, and higher frequency of γΔ T cells and CD4+ γΔ T cells than young PTMs (2.25-9.69 years). Young PTMs have a higher frequency of CD45+ granulocytes, PD-1 High CD8+ T cells, plasmacytoid dendritic cells, and NK cells. By correlating older PTMs with higher immune cell activation, and younger PTMs with higher immune cell frequency, we have a better understanding of how a vaccine or treatment could be developed to support older individuals, who are at greater risk of severe infection.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an opportunistic pathogen of wounds, cornea, and the Cystic Fibrosis lung. P. aeruginosa is also a model organism for the study of bacterial biofilm formation. Biofilms are multicellular communities that form from bacterial growth concomitant with the production of extracellular polymeric substances (EPS). EPS includes polymers such as polysaccharides, DNA, and proteins; these polymers provide structure and protection to the biofilm cells. Proteomics experiments by the Parsek Lab and others have demonstrated that a notable component of the biofilm matrix are the secreted proteases. Secreted proteases have defined roles in virulence and nutrient acquisition, but their role in the biofilm matrix of P. aeruginosa has not been explored. I hypothesize that these secreted proteases recycle nutrients, remove cell waste, and protect cells from host immunity. To test my hypothesis, I generated a mutant strain of P. aeruginosa that lacks the six major secreted proteases. While we see that loss of the proteases does not impact planktonic growth, preliminary data suggests that loss of proteolytic activity results in moderately increased biofilm formation. Using a general proteolysis assay relying on casein hydrolysis, I have determined the relative contribution of each of the six proteases to the total proteolytic capacity of P. aeruginosa in planktonic growth. I will further test the impact of the proteases on biofilm growth in different growth environments, including under flow conditions and in artificial sputum medium. I will also assess which proteases contribute the most to proteolysis during biofilm growth. My work fits into a growing body of literature that suggests that the biofilm matrix is not an inert scaffold, but is instead a dynamic and active network.

People living with untreated HIV have a compromised immune system, which increases the risk for enhanced inflammation and disease severity in those co-infected with SARS-CoV-2. This emphasizes the importance of understanding the underlying mechanisms during immunosuppression that impact SARS-CoV-2 pathogenesis and disease outcomes. Since the microbiome plays an important role in immunity, microbial dysbiosis during HIV infection could contribute to prolonged SARS-CoV-2 pathogenesis. Microbial dysbiosis can be determined through the loss of diversity and changes to the composition of the microbiome. There is an established link between the increase in HIV disease progression and gastrointestinal microbial dysbiosis, however, the understanding of HIV-induced microbial dysbiosis during COVID-19 progression is unknown. In this project, we will investigate the gastrointestinal microbiome diversity and composition during SIV infection, to serve as a basis for understanding this undefined association. Utilizing the SIV macaque model for AIDS, we will test the hypothesis that the microbiome diversity and composition during SIV infection will be dissimilar between different gastrointestinal areas (stool, rectal swabs). Seven female rhesus macaques were intravaginally infected with SIVmac251. Rectal swabs and stool samples from the macaques were collected at baseline: 17~34 weeks post-SIV infection, and 7 days pre-SARS-CoV-2 infection. We extracted genomic DNA using a QIAgen PowerFecal Pro DNA kit and sequenced the ribosomal RNA after 16s amplification. We use the bioinformatics platform, QIIME2, to analyze the sequencing data. I am probing the data for relative microbial abundance, and diversity of microbial communities through metrics of richness, evenness, and specific indexes. Preliminary findings report that during SIV infection, the overall diversity of the gut microbiome is similar in stool and rectal swabs, and the microbiota composition is different between them. The results from these studies will then be used to understand the role of SIV-induced microbial dysbiosis on SARS-CoV-2 virological and disease outcomes.
 

Human Immunodeficiency Virus 1 (HIV-1) is a lentivirus and the causative agent of Acquired Immunodeficiency Syndrome (AIDS). HIV encodes a viral protease, the function of which is required for viral replication. The host innate immune sensor CARD8 detects HIV protease activity, leading to inflammasome activation during HIV infection. Inflammasomes are cytosolic innate immune complexes that recruit Caspase-1 and lead to secretion of pro-inflammatory cytokines and lytic cell death. Humans encode a single CARD8 gene; however Old World Monkeys (OWMs), the hosts of Simian Immunodeficiency Viruses (SIVs) encode two copies of CARD8. The function of the OWM CARD8 is unknown. To characterize the function of OWM CARD8s, I cloned CARD8 from representative OWMs and tested their responses to HIV and SIV protease in two ways. First, I determined if OWM CARD8s are capable of forming an inflammasome in response to HIV-1, a panel of SIVs, or the broad CARD8 activator VbP. I found that most, but not all, OWM CARD8s are functional but not responsive to HIV-1/SIVs. Human CARD8 senses HIV-1 through viral protease cleavage of its N-terminus. To determine if this lack of response of OWM CARD8s is due to the absence of viral protease cleavage, I will next perform western blots comparing human and OWM CARD8 proteolysis in the presence of absence of HIV/SIVs. My data suggests that the species-specific differences in CARD8 alters its capacity to detect viral proteases. We speculate that the absence of HIV-like pathogenesis in OWMs with endemic SIV may in part be due to the absence of CARD8 inflammasome activation to SIV protease.

Vaccines have successfully reduced global infectious disease burden, but there is room to improve vaccination technologies. Because many pathogens infect at mucosal sites, a goal of new vaccines is to promote strong mucosal and systemic antibody and T-cell responses. Integrated fiber microneedle devices (iFMN) are a novel oral vaccination method that may achieve this goal. These devices are patches with a polymer backfill matrix and multiple >1 mm pyramidal needles that penetrate immune cell-rich mucosal tissue in the mouth, inducing immune responses at draining lymph nodes. To test the hypothesis that priming with iFMN delivery of a DNA vaccine increases mucosal and systemic antibody responses after systemic booster immunization with the same vaccine, male rhesus macaques (n=6) were primed with an iFMN delivery of a DNA vaccine encoding Influenza A Virus (IAV) Nucleoprotein (NP) at weeks (0) and (6). The macaques then received a single boost of the same NP DNA vaccine at week (12) using the proven delivery modality of Gene Gun epidermal delivery (GG). Mucosal secretions (including bronchoalveolar lavage, saliva, and nasal/tracheal swabs) and serum were collected 2-4 weeks before and after each immunization. I conducted enzyme-linked immunosorbent assays (ELISAs) to quantify antigen-specific IgG and IgA binding antibody at each timepoint. To characterize the priming effect of iFMN oral delivery on systemic and mucosal antibody responses, I compared these animals’ responses to macaques (n=8) previously immunized with a single GG dose of the same NP DNA vaccine. The iFMN-primed animals had robust post-GG boost NP-specific IgG responses in serum but these responses were not significantly higher than for macaques boosted solely with GG DNA. These results demonstrate that iFMN delivery did not effectively prime for robust systemic and mucosal antibody responses. Additional experiments will be done to confirm these findings.

Mycobacterium abscessus are non-motile bacilli that cause soft-tissue and pulmonary infections, commonly in healthcare settings or patients with cystic fibrosis. Though it is considered an opportunistic pathogen, its many virulence factors signal its potential for evolution into a true pathogen. Upon infection, the bacilli are internalized by macrophages, forming granulomas to contain the infection. Macrophages can harbor bacilli during infection stages and induce drug resistance by expelling toxins through ABC transporters. Treatment is often challenging as M. abscessus is intrinsically resistant to many antibiotics. Current treatment uses a combination of two or more intravenous drugs and one or more oral antibiotics over several months. Treatment success is challenged by patient adherence and may also be impacted by drug efflux by macrophage ABC transporters. Transporters identify certain drugs as toxic to the body and try to flush them out of the cell. Since Mycobacteria infect macrophages, these channels pose a significant disadvantage to treatment since the cell will actively efflux the drug, preventing the drug's intracellular concentration from increasing to an effective level against the bacilli inside. Certain drugs are known to inhibit ABC transporters, and the addition of these inhibitors in treatment could increase bacteriocidal activity and reduce the development of drug tolerance. First, we will determine the drug's minimum inhibitory concentrations to each inhibitor to see if there is an intrinsic activity on M. abscessus. Next, we will use the Human THP-1 cell line infect with Mycobacterium abscessus and treat with known ABC transport inhibitors in concert with a current therapeutic drug, Clarithromycin. They will then be plated at various time points to determine the colony-forming units. If efflux by macrophage transporters reduces the efficacy of Clarithromycin, bacteriocidal activity will increase between the combination therapy and the clarithromycin-only treatment. These results may improve the current treatment regimens for M. abscessus.

Cytomegalovirus (CMV) is a nuclear-replicating DNA herpesvirus that rearranges the nucleus to form a kidney bean shape during infection. CMV-induced cellular rearrangement is disrupted by knockout of a histone variant, macroH2A1. This disruption leads to significantly decreased infectious progeny for CMV. We examined how different transcriptional profiles during CMV infection of macroH2A1 knockout cells and found several host genes were misregulated in the absence of macroH2A1. One such gene is KIF1A, a kinesin-3 motor protein found in neurons. I previously found that KIF1A is induced in primary human foreskin fibroblasts (HFFs) during infection, which is unusual since KIF1A is not normally expressed in fibroblasts. Interestingly, KIF1A is not induced during CMV infection in the macroH2A1 knockout cell line. This led me to hypothesize that macroH2A1 is required for induction of KIF1A expression. To test this hypothesis, I overexpressed KIF1A in HFFs to establish if I can rescue the defect in infectious progeny. Wild-type and macroH2A1 knockout HFFs transduced with a plasmid containing the KIF1A gene are analyzed using Western blotting and plaque assays to determine KIF1A expression and viral titers. I anticipate that the defect is rescued by overexpressing KIF1A in macroH2A1 knockout cells. CMV is the leading infectious cause of birth defects in the United States, making its mechanisms of infection a key area of study for development of antiviral therapies.

Bacterial CRISPR immune systems defend against foreign genetic material, such as bacteriophage viruses. CRISPR systems are classified into six types with diverse protein components and mechanisms of interference. Among these, our research investigates the function of CRISPR-Cas13 systems, which uniquely target RNA rather than DNA. To overcome immunity, bacteriophages have evolved anti-CRISPR mechanisms that are designed to inhibit specific CRISPR types, restoring infection and proliferation of the viral invader. We recently discovered a novel anti-CRISPR mechanism, in which a noncoding RNA provides inhibition of CRISPR-Cas13 function. The central questions surrounding this RNA anti-CRISPR (rnAcr) are how it associates with CRISPR-Cas13 in order to inhibit its function, as well as the boundaries of its length and anticipated mechanism of inhibition. rnAcr is predicted to have three vital stem loops, which have been experimentally deleted and structurally disrupted by performing site directed mutagenesis to mutate select regions of nucleotides in each stem’s structure. We did this in order to determine if the stem loops’ structures were necessary for rnAcr’s anti-CRISPR function. We found that these were all essential for its function, which gives rise to the hypothesis that its structure is interacting with the bacterial host’s CRISPR-Cas13 system to effectuate its inhibitory mechanism. In order to test anti-CRISPR function, we conjugated a target and nontarget plasmid, in which the target plasmid would be recognized by Cas13, and cellular RNA would be cleaved, leaving no growth if no anti-CRISPR mechanism is present. We have shown that rnAcr is sufficient for anti-CRISPR function, allowing for tolerance of these target plasmids and cellular growth. rnAcr suggests a novel anti-CRISPR mechanism, as until now, the majority of reported anti-CRISPRs have been composed of small proteins produced during phage infection, suggesting rnAcr’s significant implications when considering new players in the host-bacteriophage evolutionary competition.

Kaposi’s Sarcoma (KS) is among the most common tumors in central Africa and is a prevalent AIDS-associated malignancy. 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. ACADS and CHD1 are two genes identified as some of the top hits from the screen. ACADS encodes a tetrameric mitochondrial flavoprotein, which catalyzes the first step of mitochondrial beta-oxidation. CHD1, or chromodomain helicase DNA binding protein 1 alters gene expression by chromatin modification. I hypothesize that ACADS and CHD1 are required for survival of latently infected KSHV cells but not uninfected cells. To test this hypothesis, I created knockout tert-immortalized microvascular endothelial (TIME) cells of each gene with CRISPR-Cas9 and plasmids containing guide RNAs used in the original screen. Then, I infected control and knockout cells with either KSHV or mock, and conducted trypan blue assay at 72 hours post infection to measure percent of live cells. Preliminary data suggests an increase in cell death for KSHV infected ACADS knockout cells compared to the control cells. In future experiments, I expect a significant decrease in the percentage of live cells in the ACADs and CHD1 knockout cells compared to the control and uninfected cells.

Science is rapidly evolving, yet its advances do not enter classrooms at the same rate. Systems Education Experiences (SEE) is a program in the Baliga Lab at the Institute for Systems Biology (ISB) that accelerates the transfer of scientific knowledge and practices to classrooms. One active area of Baliga Lab research is elucidating the level of resilience organisms have, when faced with complex environmental changes. My role is to design laboratory experiments that investigate this with the model organism Halobacterium salinarum (Halo) and to connect this to broader rules governing natural systems for use in high school classrooms. My first experiment probes the resiliency of Halo with the introduction of a combination of stressors (salt and hydrogen peroxide) and its recovery after population collapse. The second measures the long term phenotypic changes in the population. I wanted to see if after being exposed to a new environment if there was an advantage to having gas vesicles and if it is an irreversible trait that allows Halo to be resilient across a variety of environmental conditions. This relates to broadly applicable rules governing resilience across many systems. This project serves as a model for how all organisms respond to stress. Combinations of stressors in human lives can make us less resilient. However, strategies to quickly prepare, respond, and react can improve outcomes for individuals and the overall population. This project connects to a goal of K-12 science education which is to not just teach academic concepts but to equip students with knowledge that can be applied to all parts of life. Our knowledge on the mechanisms that control how organisms respond to stress is extremely limited. By understanding the biological stress response we can promote resilience in the earth's most vulnerable systems in the wake of climate change.

Bacteria face a variety of threats, including antagonistic killing by other bacteria in competition for space and resources. In response to this antagonism, many bacteria have evolved specific defense systems. One pertinent example is the Pseudomonas aeruginosa Response to Antagonism (PARA) in P. aeruginosa, which provides defense against various antagonists by activating a suite of genes, mediated by two-component pathway Gac/Rsm, in response to kin cell lysis. The Gac/Rsm machinery is conserved across the Pseudomonas genus, but its function in defense has not been studied outside of P. aeruginosa. Here, we investigate whether two divergent Pseudomonas species, P. putida (KT2440) and P. protegens (Pf-5), similarly use Gac/Rsm in defense. To do this, we performed competitive growth assays against an antagonistic competitor, Enterobacter cloacae, comparing Gac/Rsm deletion mutants against wild-type, and quantified relative survival as an indicator of competitive fitness. Preliminary data indicate that the deletion of the core Gac/Rsm gene gacS results in dramatically decreased competitive fitness for Pf-5, but not for KT2440. This indicates that Pf-5 uses the Gac/Rsm system in a similar manner to P. aeruginosa and that, while Gac/Rsm is conserved, it may differ in function between species. To identify additional specific genes involved in defense systems, we set up a genome-wide screen. The screen indicated that genes related to the flagellum and lipopolysaccharide biosynthesis may be involved in defense against antagonism, which was surprising because these well-characterized structures have never before been implicated in defense. Work is currently underway to validate these genes as true defense factors and determine the mechanism by which they confer survival. Our findings advance the understanding of defense systems among Pseudomonas species by shedding light on their conservation and complexity, thus providing a foundation for future work on defense systems across bacterial phyla.

My research explores the metabolic flexibility and longevity of Rhodopseudomonas palustris (R. palustris). This alpha-proteobacterium has become a model organism for studying bacterial survival in non-growing states. R. palustris can endure long-term starvation in a growth-arrested state without forming dormant structures, prompting a comprehensive investigation of the molecular basis of growth-arrest and metabolic modes under these conditions. Recent studies have demonstrated that R. palustris can enter the growth-arrested state due to nutrient limitation but not energy limitation. R. palustris utilizes cyclic phosphorylation to generate ATP, allowing it to sustain viability for an extended period, even in the absence of nutrients including carbon and nitrogen. Earlier research examined the molecular response of R. palustris to growth arrest induced by carbon starvation under light and dark anaerobic conditions. Results indicated that light-incubated cells remained viable for months while dark-incubated cells exhibited a significant decrease in viability following growth arrest. The decline in viability was associated with ATP depletion, which underscores the critical role of ATP in R. palustris’s survival during growth arrest. To further investigate the versatile metabolism of R. palustris, we conducted anaerobic growth experiments using wild-type strain CGA009. We manipulated casamino acids concentrations in nitrogen-rich medium (PM) and nitrogen-free medium (NFM). Results revealed that R. palustris CGA009 utilizes casamino acids as both carbon and nitrogen sources. Our experiments also confirmed that R. palustris CGA009 can grow in the amino acid L-Leucine. Currently, we are researching the capacity of R. palustris CGA009 to utilize diverse carbon substrates through aerobic and anaerobic cultivation on Gelrite medium. Distinct growth patterns provided insights into specific concentrations of carbon substrates tolerated by R. palustris. This ongoing research aims to identify additional carbon substrates supporting R. palustris’s growth, with implications for harnessing its unique metabolic capabilities and expanding our understanding of R. palustris’s metabolic versatility.