Microbiology

Permanent URI for this collectionhttps://digital.lib.washington.edu/handle/1773/4942

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Understanding the Role of Delta-catenin in Virus-Positive Merkel Cell Carcinoma
(2025-08-01) Landazuri Vinueza, Joselyn; Galloway, Denise
Merkel Cell Carcinoma (MCC) is a highly aggressive neuroendocrine skin cancer often driven by the integration of Merkel cell polyomavirus (MCPyV) into the host genome and the persistent expression of its viral oncoproteins, small tumor (ST) antigen and truncated large tumor (t-LT) antigen. However, the MCC cell of origin remains unknown. Identifying the cell of origin could provide critical insights into MCC pathogenesis and lead to more effective therapeutic strategies for this deadly cancer.To date, only human skin fibroblasts (HFFs) have been shown to support the complete MCPyV life cycle. Given that fibroblasts are permissive for viral replication but unlikely to be the cell of origin for MCC, we hypothesized that MCPyV initially replicates in fibroblasts but, in rare cases, infects Merkel cell progenitors or their descendants, Merkel cells, contributing to MCC development. In Chapter 2, we identified delta-catenin as a novel ST interactor in HFFs. While ST bound delta-catenin in HFFs, this interaction was absent in virus-positive (VP)-MCC cell lines. We found that while HFFs predominantly express isoform 1, a mesenchymal marker, MCC cells primarily express isoform 3, an epithelial marker. However, overexpression of delta-catenin isoform 1 in VP-MCC cells failed to restore ST binding. These results suggest that the MCC cell of origin may possess epithelial characteristics and that the host cell environment shapes delta-catenin’s function. In Chapter 3, we found that delta-catenin is essential for VP-MCC cell proliferation but was not essential for HFFs proliferation. Delta-catenin promotes proliferation in VP-MCC cells by regulating the expression of cell cycle genes through its interaction with Kaiso, a transcriptional repressor. Additionally, we found that lysine-specific histone demethylase 1A (LSD1/KDM1A) regulates delta-catenin isoform 3 expression by modulating Epithelial Splicing Regulatory Protein (ESRP1), a delta-catenin splicing factor and epithelial marker. Taken together, this work reveals novel host factors involved in MCPyV infection and MCC tumorigenesis, supporting the idea that the cells permissive for viral replication and those giving rise to MCC are distinct.
Investigating Listeria monocytogenes colonization of the gallbladder
(2025-01-23) Schwardt, Nicole Helen; Reniere, Michelle
Listeriosis, caused by the bacterium Listeria monocytogenes, is the third leading cause of death among foodborne illnesses in the United States. Following oral inoculation of the host, L. monocytogenes transverses the intestinal epithelium and rapidly disseminates to the mesenteric lymph nodes, liver, and spleen via the lymph and bloodstream. Bacteria colonizing the liver replicate intracellularly in hepatocytes, and as infected cells lyse some bacteria migrate to the gallbladder via hepatic ducts. Once in the gallbladder, L. monocytogenes replicates extracellularly to high bacterial densities. The gallbladder then becomes the primary bacterial reservoir and the source of fecally excreted bacteria. Despite its importance in L. monocytogenes pathogenesis, little is known about how L. monocytogenes survives and replicates in the gallbladder. The goal of this dissertation is to characterize the gallbladder as an infectious niche for L. monocytogenes, which will lead to a more complete understanding of its multi-organ infection cycle and the determinants of intra- vs. extracellular infection. In this work, I investigated both bacterial and host factors that contribute to gallbladder colonization. First, I developed a novel genetic screen using an ex vivo primate gallbladder infection model to reveal L. monocytogenes genes that are required for replication. Further characterization candidates from the ex vivo screen showed that many are also required for intracellular infection and virulence in an oral murine model of listeriosis. Of note, this study identified sugar transport by phosphoenolpyruvate-dependent phosphotransferase systems (PTS) as being important for replication in the gallbladder. These findings represent novel insights into L. monocytogenes carbon metabolism during infection and suggests a role for these transporters for infection in extracellular niches. Next, using a L. monocytogenes mutant deficient in gallbladder colonization, I found that STING-mediated innate immunity plays a role in gallbladder colonization following oral infection. Finally, I investigated the roles peroxide resistance mechanisms and iron transport systems during intracellular replication, which revealed extensive redundancy in the stress responses employed by L. monocytogenes during infection. Overall, my dissertation research highlights the gallbladder as a replicative niche for L. monocytogenes and genetic determinants required for L. monocytogenes replication, which will lend further insight into the gallbladder’s role in its multi-organ pathogenic life cycle.
Mechanisms of Staphylococcus aureus survival of trimethoprim-sulfamethoxazole-induced thymineless death
(2025-01-23) Gonsalves, Lauren Josephine Elizabeth; Hoffman, Lucas R.
Trimethoprim-sulfamethoxazole (SXT) is commonly used to treat diverse Staphylococcus aureus infections, including those associated with cystic fibrosis (CF) pulmonary disease. Studies with Escherichia coli found that SXT impairs tetrahydrofolate production, leading to DNA damage, stress response induction, and accumulation of reactive oxygen species (ROS) in a process known as thymineless death (TLD). TLD survival can occur through uptake of exogenous thymidine, countering the effects of SXT; however, a growing body of research has implicated central metabolism as another potentially important determinant of bacterial survival of SXT and other antibiotics. Here, we conducted studies to better understand the mechanisms of TLD survival in S. aureus. We found that thymidine abundances in CF sputum were insufficient to prevent TLD of S. aureus, highlighting the importance of alternative survival mechanisms in vivo. In S. aureus cultured in vitro with SXT and low thymidine, we frequently identified adaptive mutations in genes encoding carbohydrate, nucleotide, and amino acid metabolism, supporting reduced metabolism as a common survival mechanism. Although intracellular ROS levels rose with SXT treatment in vitro, survival was not improved in the presence of ROS scavengers, unlike in E. coli. SXT challenge induced the SOS response, which was alleviated by added thymidine. Lastly, an inactivating mutation in the phosphotransferase gene ptsI conferred both limitation in cellular ATP and improved survival against TLD. Collectively, these results suggest that alterations in core metabolic functions, particularly those that reduce ATP levels, predominantly confer S. aureus survival and persistence during SXT treatment, potentially identifying novel targets for co- treatment.
RNA structures within Venezuelan equine encephalitis virus E1 alter macrophage replication fitness and contribute to viral emergence
(2025-01-23) Hickson, Sarah; Hyde, Jennifer
Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne positive-sense single-stranded RNA genome virus belonging to the Togaviridae. Present throughout Central and South America, VEEV is responsible for significant outbreaks of epidemic/epizootic VEEV causing febrile disease and encephalitis in both equids and humans. While endemic/enzootic VEEV persists in nature and circulates between reservoir host rodents and mosquitoes, periodic mutation of enzootic VEEV gives rise to the emergence of epizootic VEEV. Using equines as amplification hosts, epizootic VEEV can have devastating outcomes for equine populations as well as cause large spill over events and disease in humans. The main mutations linked to epizootic VEEV emergence involve amino acid mutations within the E2 glycoprotein, which enhance viral entry and equine amplification. Interestingly, the majority of mutations found within epizootic strains are synonymous, indicating that other viral factors, such as RNA secondary structure, may play a critical role in their emergence. Understanding these mechanisms is crucial for predicting and mitigating future outbreaks.In this study, we discovered novel RNA structures within the E1 coding sequence that specifically affect VEEV replication in macrophages, which are critical early targets during infection. Using mass spectrometry and targeted gene knockdown, we identified several RNA-binding proteins essential for the altered macrophage phenotype, none of which had previously been associated with VEEV replication. Our findings also revealed the conservation of single-nucleotide polymorphisms (SNPs) within epizootic VEEV lineages, as well as the preservation of RNA structures across all lineages. Taken together, these findings suggest a previously unrecognized role for RNA secondary structure in the emergence of epizootic VEEV.
A CRISPR-screening approach to increase HIV latency reversal to improve an HIV cure strategy
(2025-01-23) Gray, Carley; Emerman, Michael
Despite the availability of effective antivirals, HIV remains a global burden. Although virus replication can be suppressed, people living with HIV (PLWH) need to continue life-long antiviral therapy as there is currently no way to eradicate the long-lived and stable HIV reservoir that evades the immune system but retains the ability to reactivate and spread virus. Understanding mechanisms that govern HIV latency within these reservoirs, such as host and viral factors that influence transcription initiation, transcription elongation, and chromatin dynamics, has led to the discovery of a number of small molecule drugs called latency reversal agents (LRAs) capable of stimulating HIV transcription. One strategy being explored to target the HIV reservoir is called “shock-and-kill” where the silent provirus in latently infected cells is transcriptionally stimulated such that the immune system can recognize the subsequently synthesized viral proteins and clear infected cells. However, LRAs used to date in this strategy have not been potent or specific enough on their own to sufficiently activate and lead to clearing of infected cells. While pairing together LRAs that function through differing mechanisms of action have proven synergistic in their ability to activate HIV transcripts, these combinations in relevant model systems, such as ex vivo systems from cells from people living with HIV (PLWH), have demonstrated there are more blocks to latency reversal present in primary cells that prevent full virus reactivation. These results highlight a need to find ways to uncover these blocks and improve reactivation. In my thesis, I used a CRISPR-based screening approach with the LRA combination AZD5582 and I-BET151. This combination synergizes on HIV RNA synthesis by targeting the noncanonical NF-kB pathway to increase transcription initiation and targeting BET proteins to increase available cellular PTEF-b to aid in HIV specific transcription elongation in conjunction with HIV Tat. This combination was effective in in vitro models, but not in an ex vivo system. With this screening approach, I was able to identify a gene target, INTS12, that is a part of a larger complex called the Integrator Complex that could be targeted to improve reactivation in an ex vivo system. These studies provide a framework for predicting in vivo blocks to HIV latency reversal and strategically improving LRAs and identifying novel targets for which small molecule drugs should be developed.
Deciphering the functions of protein VII during adenovirus infection.
(2024-10-16) Arnold, Edward Arthur; Avgousti, Daphne C
Adenoviruses are double-stranded DNA viruses that cause various common illnesses. Although usually considered self-limiting infections, adenoviruses pose significant risks for immunocompromised individuals. Thus, a comprehensive understanding of adenovirus pathogenesis is crucial for advancing strategies aimed at mitigating adenovirus-related diseases. As adenoviruses are nuclear replicating viruses, they necessarily interact with host chromatin. One way in which adenovirus controls host chromatin function is through the histone-like protein, protein VII. Protein VII is an essential viral protein that packages with and condenses the viral genome within the core of the virion. Much like histone proteins, protein VII contains several post-translational modifications (PTMs) that impact its localization within the nucleus. However, the specific impacts of these PTMs on protein VII function during infection remain unexplored. During late infection when protein VII is abundantly expressed, it localizes to and distorts host chromatin and interacts with several host chromatin-associated proteins, including high mobility group box one (HMGB1). In the nucleus HMGB1 binds to and bends DNA to reorganize chromatin. During times of stress or infection, HMGB1 is released from the cell as an alarmin, where it stimulates an inflammatory response. Protein VII retains HMGB1 on chromatin, yet the mechanism of this interaction and its implications during infection are poorly understood. In human adenoviruses, protein VII (hVII) exhibits high conservation, whereas in different vertebrate adenoviruses, such as murine adenovirus-1 (MAdV-1), protein VII (mVII) shows significant divergence with only 39% similarity to hVII. Despite mVII's known role in viral genome packaging within virions, its specific functions remain largely unexplored. MAdVs serve as a valuable in vivo model for investigating adenovirus pathogenesis, thus understanding how mVII contributes to MAdV infection can elucidate how adenoviruses cause disease.In this thesis, I addressed these gaps in our understanding of protein VII function. I investigated how protein VII interacts with HMGB1 and found that protein VII interacts with HMGB1's A-box and anchors it to chromatin using bacterial two-hybrid and human cell culture assays. Furthermore, I showed protein VII suppresses interferon β induction and exploits HMGB1 for this purpose. To determine how PTMs on protein VII impact adenovirus infection, I created mutant viruses that abrogated or mimicked PTMs on protein VII. I determined that acetylation on protein VII may impact expression of early viral genes, but the PTMs did not appear to impact later stages of infection or localization of protein VII. However, I also discovered that protein VII is likely modified at alternative residues, which may compensate for the mutations we introduced. Finally, I used a chimeric HAdV-5 with a protein VII from MAdV-1, to show that mVII does not directly interact with HMGB1, but HMGB1 is still retained on chromatin during infection. I also discovered that in this chimeric virus, mVII was expressed at lower levels than hVII during infection, resulting in a reduction in viral titers. Additionally, mVII frequently localized to the nucleolus, suggesting a potential divergence in function from hVII. These investigations significantly contributed to our knowledge of the interplay between protein VII and the host nuclear environment and its evolutionary adaptations across diverse adenoviral species.
Host and Bacterial Functions in Bacterial Vaginosis Elucidated by Metaproteomics
(2024-02-12) Lee, Elliot Michael; Fredricks, David N
Bacterial vaginosis (BV) is a highly prevalent dysbiosis of the vaginal microbiota which causes a variety of unpleasant symptoms and places patients at higher risk of adverse sequelae. BV is a complex condition, and many of the host and bacterial functions which contribute to its development and recurrence are unknown. This dissertation describes an optimized metaproteomic analysis of cervicovaginal lavage samples from women with and without BV to identify host and bacterial proteins that may contribute to BV. Using this approach, we uncovered new potential synergistic interactions between BV-associated bacteria (BVAB) based on glutamate and identified a possible host response to increased concentrations of free heme in BV. We also demonstrated a novel syntrophic interaction between Dialister micraerophilus and Fannyhessea vaginae to increase putrescine biosynthesis, likely through cross-feeding of the arginine metabolite ornithine. Despite past reports that human amylase is primarily responsible for breaking down vaginal glycogen into fermentable carbohydrates, we identified glycogen-degrading enzymes from L. crispatus and G. vaginalis in samples from both BV- and BV+ study participants. This observation led to our discovery that a wide range of BVAB, but only L. crispatus and L. iners among the commensal Lactobacillus spp., can directly metabolize glycogen. Finally, we described the construction of an E. coli-Gardnerella shuttle vector that can be applied for genetic manipulation of this genus. This work contains novel insights into BV and opens new avenues to study the biology of vaginal bacteria, with implications for treatment and prevention of this condition.
Regulation of coronavirus nsp15 cleavage specificity by RNA structure
(2024-02-12) Salukhe, Indraneel A.; Hyde, Jennifer
SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, has had a significantimpact on global public health. However, SARS-CoV-2 is only one of multiple pathogenic human coronaviruses (CoVs) to have emerged since the turn of the century. CoVs encode for several nonstructural proteins (nsps) that are essential for viral replication and pathogenesis. Among them is nsp15, a uridine-specific viral endonuclease that is important in evading the host immune response and promoting viral replication. Despite the established endonuclease function of nsp15, other determinants of its cleavage specificity have only recently begun to be investigated. In this study we investigate the role of RNA secondary structure in SARS-CoV-2 nsp15 endonuclease activity. We identified regions of differing predicted RNA secondary structure across the SARS-CoV-2 genome. Using a series of in vitro endonuclease assays, we observed that thermodynamically stable RNA structures were protected from nsp15 cleavage relative to RNAs lacking stable structure. We leveraged the s2m RNA from the SARS-CoV-1 3’UTR as a model RNA structure for our studies as it adopts a well-defined structure with several uridines, two of which are unpaired and thus highly probable targets for nsp15 cleavage. We found that SARS-CoV-2 nsp15 specifically cleaves s2m at the unpaired uridine within the pentaloop of the RNA. Further investigation revealed that the position of this uridine also impacted nsp15 cleavage efficiency suggesting that positioning within the pentaloop is necessary for optimal presentation of the scissile uridine and alignment within the nsp15 catalytic pocket. Our findings indicate that RNA secondary structure is an important determinant of nsp15 cleavage and provides insight into the molecular mechanisms of RNA recognition by nsp15. Understanding the broader implications of nsp15 activity will provide further insight not only into CoV biology but also into drug development against nsp15.
Higher HIV-1 Env gp120-Specific Antibody-Dependent Cellular Cytotoxicity (ADCC) Activity Is Associated with Lower Levels of Defective HIV-1 Provirus
(2024-02-12) Yucha, Ryan; Emerman, Michael; Overbaugh, Julie
During HIV-1 (HIV) infection, continued administration of antiretroviral therapy (ART) can decrease a person’s viral load to undetectable levels and allow for a near-normal lifespan, though ART is not a cure due to a reservoir of cells that harbor latent, integrated HIV provirus capable of host immune evasion and stochastic reactivation. While the vast majority of HIV proviruses are defective due to large internal deletions or hypermutations, a fraction of proviruses are intact and replication competent which contribute to the rebound of viremia that occurs upon cessation of ART, necessitating its life-long administration. The majority of archived proviral sequences that persist on ART are genetically similar to those of circulating viruses at the time of ART initiation which suggests that factors present at this time can impact HIV reservoir dynamics, and that additional influences at this critical time for reservoir establishment may also be identified. Host immune responses such as Antibody-Dependent Cellular Cytotoxicity (ADCC), which act to clear infected cells, have been suggested to have the potential to impact reservoir size and characteristics. Children living with HIV represent a significant fraction of people living with HIV, yet studies of the pediatric HIV reservoir, especially those focused on its establishment during chronic HIV infection, are relatively few compared to those in adults. The work detailed in this thesis tested the hypothesis that the ability of autologous plasma antibodies to mediate ADCC against HIV Env at the time of ART initiation inversely correlates with the size of the established HIV reservoir. This was done using samples from the Pediatric Adherence Diary study, the rapid and fluorometric ADCC (RFADCC) assay, and the Cross-Subtype Intact Proviral DNA assay (CS-IPDA). The results demonstrated a moderate, inverse correlation between HIV Env gp120-specific ADCC activity in plasma at the time of ART initiation and the level of defective (r = -0.285, p-value = 0.0214), but not intact (r = -0.0321, p-value = 0.800), HIV proviral copies that persist during ART. These findings suggest that host immune factors prior to ART initiation may impact the proviruses that persist during ART. Additionally, it adds to the mounting evidence that cells harboring defective HIV provirus may face different immune selection pressures than cells harboring intact provirus.
Bcl-xL is required to protect endothelial cells latently infected with KSHV from virus induced intrinsic apoptosis
(2024-02-12) Moore, Lyndsey; Lagunoff, Michael
Kaposi’s Sarcoma Herpesvirus (KSHV) is an oncogenic gammaherpesvirus that, like other herpesviruses, can establish both lytic and latent replication programs. It is the etiologic agent of Kaposi’s Sarcoma (KS), Multicentric Castleman’s Disease (MCD), and Primary Effusion Lymphoma (PEL). Within Kaposi’s Sarcoma tumors, cells are predominantly latently infected. This poses a significant problem, as all current antivirals for herpesviruses target cells that are lytically infected, KSHV included. It is necessary to develop therapeutics that can target latently infected cells to effectively eradicate herpesvirus infections. Unfortunately, latent viruses are difficult to target since they do not produce infectious virions and they dramatically repress expression of viral genes. Targeting host cell requirement for latency, though, is a potentially viable course of action. Previously our lab performed a CRISPR/Cas9 essentiality screen in endothelial cells latently infected with KSHV. In this thesis work, I follow up on the most promising hit from that screen, Bcl-xL. Bcl-xL is an anti-apoptotic protein in the Bcl-2 protein family. The Bcl-2 family proteins are the master regulators of intrinsic apoptosis. I found that Bcl-xL is required for the survival of KSHV latently infected endothelial cells. Bcl-xL is needed during KSHV infection to inhibit virus induced apoptosis by sequestering the pro-apoptotic pore-former Bax. Bcl-xL, but not other anti-apoptotic Bcl-2 family proteins, is uniquely required because endothelial cells do not express additional anti-apoptotic proteins, such as Mcl-1 and Bcl-2, like other cell types. No other cell types were found to require Bcl-xL for survival during KSHV latent infection, likely due to the fact that they express several Bcl-2 family anti-apoptotic proteins. It is unlikely that the virus has evolved to induce apoptosis itself, rather it is more likely that the virus is inducing some host cell change that results in the activation of apoptosis. I found that the KSHV latent locus alone is sufficient to induce cell death in the absence of Bcl-xL. This led me to investigate if there was a specific KSHV latent gene that was inducing apoptosis during KSHV infection. I found that the kaposins, but no other KSHV latent genes or microRNAs (miRNAs), are necessary to render Bcl-xL necessary for survival. In addition to Bcl-xL, I sought to validate other top hits from the previous CRISPR/Cas9 screen. I examined the requirement of several genes for the survival and proliferation of KSHV latently infected cells, and determined that CYP27A1, a sterol hydroxylase, is needed for the proliferation of KSHV infected endothelial cells. Following the introduction and background (chapter 1) and materials and methods (chapter 2), chapter 3 details the validation of the top hit from our CRISPR/Cas9 screen, Bcl-xL. Chapter 4 describes how KSHV induces apoptosis during latent infection. In chapter 5, I validate the necessity of three other genes, Cyp27a1, Ylpm1, and Cmklr1, and determine that CYP27A1 is required for the proliferation of KSHV latently infected cells. Together this work identifies promising potential therapeutics for the specific treatment of Kaposi’s Sarcoma, improves our understanding of how KSHV changes the host cell during latent infection, and provides insight into the accuracy of global CRISPR/Cas9 screens.
Investigating the host determinants of Listeria monocytogenes cytosolic infection
(2023-09-27) Glover, Rochelle; Reniere, Michelle L
Listeria monocytogenes (Lm) is an intracellular foodborne pathogen which causes the severe disease listeriosis in immunocompromised, elderly, and pregnant individuals. Lm infects host cells using a well-defined intracellular lifecycle involving invasion, cytosolic replication, and cell-to-cell spread. While the bacterial virulence factors required for this lifecycle have been extensively studied, less is known about the host determinants of intracellular infection. The goal of this dissertation is to identify and characterize host proteins required for two stages of the Lm intracellular lifecycle: invasion of phagocytic cells through phagocytosis, and subsequent activation of virulence gene expression in the host cytosol. In this work, I developed and performed unbiased biochemical and genetic screens to identify host factors involved in these processes. First, the role of the host cytosol in activating the master virulence regulator PrfA was investigated using cytosolic cell-free extracts derived from Xenopus tropicalis eggs. These studies revealed that a host protein present in eukaryotic cytosol is required for activation of PrfA and expression of virulence factors. Fast protein liquid chromatography was used to separate Xenopus extracts and mass spectrometry identified candidate proteins involved in PrfA activation. As a complementary approach, a genome-wide CRISPR/Cas9 screen was performed and mutant macrophages with reduced levels of virulence factor expression were isolated. This screen also identified genes important for phagocytosis of Lm by macrophages. The role of one candidate gene, Pten, was extensively characterized. I discovered that the tumor suppressor PTEN promotes macrophage phagocytosis of Lm and L. ivanovii, but not other Gram-positive bacteria. Additionally, I found that PTEN enhances phagocytosis of Lm via its lipid phosphatase activity by promoting adherence to macrophages. Using conditional knockout mice lacking Pten in myeloid cells, PTEN-dependent phagocytosis was found to be important for host protection during oral Lm infection. Overall, this thesis provides a comprehensive identification of macrophage factors involved in regulating Lm virulence at the molecular level. In addition, the in vivo studies presented here demonstrate for the first time that macrophage phagocytosis is an important immune defense against invasive Lm during the foodborne route of infection.
Insights into alpha-defensin modulation of non-enveloped viral infection
(2023-09-27) Hu, Ciara Tipping; Smith, Jason G
Alpha-defensins are small antimicrobial peptides that play a crucial role in innate immunity across various mammalian species. Possessing broad antimicrobial properties, they have neutralizing activity against bacteria and both enveloped and non-enveloped viruses. These peptides are either constitutively secreted into the intestinal lumen (enteric) or stored inside granules in neutrophils which subsequently fuse to pathogen-carrying vesicles (myeloid). Despite the presence of defensins, some pathogens can resist their antimicrobial effects or even exploit these antimicrobial peptides to enhance their infection. Given the observed trend of many enteric pathogens being resistant to or enhanced by enteric alpha-defensins, we hypothesized that alpha-defensins can assert selective pressure on pathogens. To investigate this hypothesis, I focused on two non-enveloped viruses: adenovirus and rotavirus. Prior studies on adenoviruses identified the vertex proteins, fiber and penton base, as being important determinants of defensin antiviral activity. Using a directed evolution approach, a different major capsid protein, hexon, was identified as being an additional determinant of defensin activity and a driver of defensin-mediated enhancement. My infection and biochemical assays suggest that a balance between increased cell binding and a downstream block in intracellular trafficking mediated by defensin interactions with all the major capsid proteins dictates the outcome of infection. Investigation into defensins’ effects and mechanisms of action on rotavirus are in their infancy. We examined how defensins impact the infection of various rotavirus strains, and we observed that rotaviruses were resistant to or enhanced by their host’s enteric defensins. We also interrogated the potential role of myeloid and non-host alpha-defensins as cross-species barriers and found that the outcome varied depending on the specific rotavirus strain tested. To elucidate the proteins involved, we used reassortant viruses, and identified the rotaviral spike protein, VP4, as a determinant for defensin-mediated modulation of rotavirus infection. Subsequently, I investigated if using a directed evolution approach, similar to that employed with adenovirus, would provide greater insight into defensins’ ability to drive viral evolution. Moreover, I sought to identify specific regions of the rotavirus virion that are targeted by defensin. I found that rotavirus was able to evolve resistance to a previously neutralizing myeloid defensin and that most of those mutations were in the spike protein. This reinforces the significance of VP4 in determining defensins’ effect on rotavirus. Interestingly, despite the emergence of defensin resistance mutations in the receptor binding pocket, I found that defensin does not inhibit rotavirus binding to cells. Future investigations into entry and trafficking may reveal that rotavirus neutralization by defensins follows a similar overarching mechanism observed for other non-enveloped viruses. This mechanism involves α-defensins disrupting proper trafficking of the virus by altering capsid dynamics.
Understanding Chronic Lung Infections after Correcting the Basic Cystic Fibrosis Defect
(2023-08-14) Durfey, Samantha; Singh, Pradeep K
Cystic fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In 2012, the first drug that can correct the physiologic defect was approved. These drugs, called “CFTR modulators”, offer great hope to people with CF, as many clinical measures of health improve, including lung function, nutritional status, and frequency of disease flares. Unfortunately, despite marked improvements in overall health, there are hints that modulators cannot restore patients to full health. Early studies indicate chronic bacterial infections persist in many patients, and these patients’ lungs remain inflamed. This is important because before modulators, chronic infection and inflammation were the main drivers of lung disease progression and death. The failure of modulators to resolve these pathologies suggests that lung disease may continue to progress in persistently infected patients taking CFTR modulators. The goal of my thesis work was to better understand persistent post-modulator lung infections. In chapter 2, I describe a method we developed to enumerate and quantify the bacterial strains present in a pool of thousands of cultured isolates or directly from the sputum of people with CF. This method enables new questions to be addressed regarding the frequency of multi-strain infections and the degree of strain turnover during infections before and after modulator therapy. In chapter 3, we test an eradication protocol that combined intensive antibiotic treatment with modulator therapy. While antibiotics have not previously been able to clear chronic infections, we hypothesized modulator-induced changes in the lung environment could make antibiotics more effective. Finding that infections generally persisted after combined treatment inspired me to develop a deeper understanding of the causes of persistent infection after modulators. In chapter 4, we used bronchoscopy to sample many lung regions within each subject to identify characteristics of regions which remain persistently infected compared to those that clear infection. Results from this study suggest that structural lung damage may allow bacterial pathogens to persist, and show persistent infection is strongly associated with levels of inflammation after modulators. The work presented in this thesis reveals difficulties in eradicating chronic infection after modulators, and sheds light on the underlying causes of this difficulty.
The transcriptional regulator SpxA1 impacts the virulence, morphology, and redox response of Listeria monocytogenes
(2023-01-21) Cesinger, Monica R; Reniere, Michelle L
Bacterial pathogens require precise transcriptional regulation to effectively cause disease. Using a complex regulatory strategy, the model intracellular pathogen Listeria monocytogenes grows successfully as a saprophyte and can rapidly initiate an infectious lifecycle upon ingestion by a host. This dissertation presents a comprehensive analysis of the redox responsive transcriptional regulator SpxA1, which is required for both L. monocytogenes virulence and environmental growth. Whole-cell proteomics and transcriptomics provide evidence that SpxA1 regulates peroxidase abundance, heme biosynthesis, and motility. Subsequent experiments reveal that SpxA1 directly regulates genes via a conserved promoter motif and this interaction is necessary for aerobic growth. Our data further suggest that SpxA1-regulated heme biosynthesis and catalase are essential for aerobic growth in rich broth but dispensable for virulence. While investigating the role of SpxA1 in virulence, we discovered that the ∆spxA1 mutant forms elongated cells with inappropriate frequencies and distributions of division septa. However, critical proteins involved in cell wall biosynthesis, cell division, and DNA damage-regulated elongation were unchanged between wt and ∆spxA1, a result that diverges from previously described homologs. Finally, we demonstrated that both elongation and decreased motility impact the phagocytosis of ∆spxA1. Together, these results describe a novel role for the highly conserved SpxA1 regulator in L. monocytogenes.
Effect of Exopolysaccharides on Pseudomonas aeruginosa Physiology
(2023-01-21) Dreifus, Julia Ellen; Parsek, Matthew R
Bacteria form multicellular aggregates called biofilms. Cells in biofilms are encased by aself-produced matrix composed of proteins, exopolysaccharides (EPS), nucleic acids, and lipids. Biofilm matrix composition mediates how bacteria interact with one another and their environments. The biofilm matrix of the opportunistic pathogen P. aeruginosa contains up to three chemically distinct EPS: Pel, Psl, and alginate. EPS production is necessary for robust P. aeruginosa biofilms formation, however, the amount and number of EPS produced varies between strains and growth conditions. Differences in EPS production alter how P. aeruginosa interacts with its environment, from promoting initial attachment to enhancing antimicrobial tolerance. The goal of my doctoral thesis work has been to determine whether the EPS Pel and Psl are produced and relevant during infection, and to understand how their production is regulated. In this dissertation, I provide evidence that Pel and Psl are produced and co-localize with P. aeruginosa in cystic fibrosis (CF) lung infections. I also demonstrate that ionic interactions between Pel and extracellular DNA (eDNA) increase tolerance to aminoglycoside treatment. Additionally, I determine that cell-association of Psl is promoted post-translationally by the diguanylate cyclase SiaD, which stimulates initial attachment to surfaces. This work provides valuable insight into how and when P. aeruginosa EPS are produced. These and future studies will inform practices to prevent patient colonization and improve the efficacy of treatments.
A Nascent Peptide Code for Translational Control of mRNA Stability in Human Cells
(2023-01-21) Burke, Phillip Cannon; Bloom, Jesse
Stability of eukaryotic mRNAs is associated with their codon, amino acid, and GC content. Yet, coding sequence motifs that predictably alter mRNA stability in human cells remain poorly defined. Here, we develop a massively parallel assay to measure mRNA effects of thousands of synthetic and endogenous coding sequence motifs in human cells. We identify several families of simple dipeptide repeats whose translation triggers mRNA destabilization. Rather than individual amino acids, specific combinations of bulky and positively charged amino acids are critical for the destabilizing effects of dipeptide repeats. Remarkably, dipeptide sequences that form extended β strands in silico and in vitro slowdown ribosomes and reduce mRNA levels in vivo. The resulting nascent peptide code underlies the mRNA effects of hundreds of endogenous peptide sequences in the human proteome. Our work suggests an intrinsic role for the ribosome as a selectivity filter against the synthesis of bulky and aggregation-prone peptides.
STING is required in conventional dendritic cells for DNA vaccine induction of type I T helper cell-dependent antibody responses
(2022-07-14) Ulrich-Lewis, Justin Theophilus; Fuller, Deborah H
DNA vaccines elicit antibody, T helper cell, CD8+ T cell, and cytotoxic T lymphocyte (CTL) responses. Currently, little is known about the mechanism that DNA vaccines employ to induce adaptive immune responses. Studies have demonstrated that stimulator of interferon genes (STING) and conventional dendritic cells (cDCs) play critical roles in DNA vaccine-induced antibody and T cell responses. STING activation by double stranded DNA (dsDNA)-sensing proteins initiates the production of type I interferons (IFNs); however, the link between STING and cDCs remains unclear. In this study, I investigated the role of STING within cDCs on DNA vaccine induction of antibody and T cell responses. STING knockout (STING-/-) and conditional knockout mice that lacked STING in cDCs (cDC STING cKO) were immunized intramuscularly with a DNA vaccine that expressed influenza A nucleoprotein (pNP). Both STING-/- and cDC STING cKO mice had significantly lower type I T helper (Th1) antibody (anti-NP IgG2C) responses as well as lower frequencies of Th1-associated T cells (NP-specific IFN-γ+CD4+ T cells) post-immunization than wild-type (WT) and cDC STING littermate control mice. By contrast, all mice had similar Th2-type (NP-specific IgG1) antibody concentrations. Moreover, STING-/- mice developed significantly lower polyfunctional effector CD8+ T cells than WT, cDC STING cKO, and cDC STING littermate control mice. These findings suggest that STING within cDCs mediates DNA vaccine induction of Th1 responses, including IFN-γ+CD4+ T cell and Th1-type IgG2C antibody responses. The induction of CD8+ effector cell responses also requires STING, but not within cDCs. These findings provide new insight into the mechanism through which DNA vaccines induce Th1 responses.
Dissecting the Multifaceted Antibody Response to Influenza A Virus and Severe Acute Respiratory Syndrome Coronavirus 2
(2022-04-19) Gentles, Lauren; Bloom, Jesse
Antibodies are the first line of defense against invading respiratory pathogens, and their effectiveness at preventing infection and disease depends heavily on what antigens are targeted. For both influenza A virus and severe acute respiratory coronavirus 2 (SARS-CoV-2), antibodies directed at the receptor-binding surface proteins, hemagglutinin (HA) and spike, respectively, are known to neutralize virus infection and are associated with protection [1–5]. Antibodies targeting non-neutralizing epitopes on many different viral proteins also play important roles in reducing the length and severity of disease and tend to target more conserved regions than most neutralizing antibodies [1,5–11]. Here, I present my work aimed at understanding the development and interaction of neutralizing and non-neutralizing antibodies with two respiratory viruses, influenza A virus and SARS-CoV-2.First, I test the durability of antibody binding to the stem domain of HA after the accumulation of mutations naturally over 17 years in both the H1 and H3 lineages. To accomplish this, I created chimeric HA proteins combining the head domain of an avian influenza virus to which humans have not been exposed with the HA stem domain of seasonal influenza strains spanning 17 years of sequence evolution [12–16]. This allowed me to measure serum antibody binding specifically to the HA stem domain. I measured the binding of human sera collected concurrent to when the first seasonal stem strain I tested was circulating and compared that to the binding of the same sera to the stem from a seasonal strain circulating 17 years later. Based on these measurements for a number of human serum samples, we find that HA stem antibody binding wanes at a similar rate for both H1 and H3 HAs despite a faster rate of sequence evolution in the H3 lineage. In a separate set of experiments, I describe a new assay for measuring the potency of antibodies targeting the influenza A virus receptor-cleaving protein neuraminidase (NA). Our method allows for the testing of anti-NA antibodies in a format similar to traditional neutralization assays for antibodies targeting (HA). Briefly, we paired a modified HA that can perform membrane fusion but cannot bind to cellular receptors and NA containing the mutation G147R which confers the receptor-binding function to NA [17,18]. This creates a virus that is dependent on NA for attachment to cells and can thus be neutralized by anti-NA antibodies. We found that this method works best for antibodies targeting sites near the catalytic active site of NA. We were able to select for an escape mutation with one such monoclonal antibody demonstrating the utility of our methods for mapping anti-NA antibody epitopes. Finally, I address the pressing issue of antibody durability in the context of the ongoing SARS-CoV-2 pandemic by measuring neutralizing and non-neutralizing antibody responses in children for up to 52 weeks following infection. In collaboration with Seattle Children’s hospital, we performed pseudoneutralization [3,19,20] and the Abbott Laboratories SARS-CoV-2 IgG assay on samples collected from 32 children at approximately 4- and 24- weeks post-symptom onset. For both neutralizing anti-spike antibody titers and anti-nucleocapsid (N) antibodies, we observed a high degree of variability from participant to participant. Overall, neutralization titers changed very little over the observation period, while anti-N levels decrease substantially. When compared to a separate, previously characterized, adult cohort [3], we find age-related differences in the antibody responses. Specifically, children tend to have lower neutralizing antibody titers than adults early following infection that become similar to adults in the following six months. Strikingly, anti-nucleocapsid antibodies are much lower in children than adults and wane faster in children over time.
Characterizing the immunogenicity of a therapeutic DNA vaccine and determinants of viral control in SIV-infected rhesus macaques with variable responses to antiretroviral therapy
(2021-10-29) Tunggal, Hillary; Fuller, Deborah
A therapeutic vaccine that induces lasting control of HIV infection could eliminate the need for lifelong adherence to antiretroviral therapy. This study investigated a therapeutic DNA vaccine delivered with a single adjuvant or a novel combination of adjuvants to augment T cell immunity in the blood and gut-associated lymphoid tissue in SIV-infected rhesus macaques. Animals that received DNA vaccines expressing SIV proteins, combined with plasmids expressing adjuvants designed to increase peripheral and mucosal T cell responses, including the catalytic subunit of the E. coli heat-labile enterotoxin, IL-12, IL-33, retinaldehyde dehydrogenase 2, soluble PD-1 and soluble CD80, were compared to mock-vaccinated controls. Following treatment interruption, macaques exhibited variable levels of viral rebound, with four animals from the vaccinated groups and one animal from the control group controlling virus at median levels of 103 RNA copies/ml or lower (controllers) and nine animals, among all groups, exhibiting immediate viral rebound and median viral loads greater than 103 RNA copies/ml (non-controllers). Although there was no significant difference between the vaccinated and control groups in protection from viral rebound, the variable virological outcomes during treatment interruption enabled an examination of immune correlates of viral replication in controllers versus non-controllers regardless of vaccination status. Lower viral burden in controllers correlated with increased polyfunctional SIV-specific CD8+ T cells in mesenteric lymph nodes and blood prior to and during treatment interruption. Notably, higher frequencies of colonic CD4+ T cells and lower Th17/Treg ratios prior to infection in controllers correlated with improved responses to ART and control of viral rebound. These results indicate that mucosal immune responses, present prior to infection, can influence efficacy of antiretroviral therapy and the outcome of immunotherapeutic vaccination, suggesting that therapies capable of modulating host mucosal responses may be needed to achieve HIV cure.
Within-Host Diversity of Kaposi Sarcoma-associated Herpesvirus
(2021-10-29) Santiago, Jan Clement Ang; Mullins, James I
Kaposi sarcoma (KS) is a progressive, incurable soft tissue disease that is the most common cancer of men in regions of sub-Saharan African and the second most common among people with HIV. Kaposi Sarcoma-associated Herpesvirus (KSHV) is the infectious cause of KS, but why only a small fraction of those infected develop KS is not understood. Immune status, viral co-infection, host genetics and environmental factors all are likely to play a role. My thesis sought to identify a possible viral genetic component of KS, through an analysis of the diversity of whole KSHV genomes within individuals afflicted by KS. Strain variation or de novo mutations occurring in other oncoviruses have been associated with variations in disease risk, disease manifestation and clinical course. These variations have the potential to become diagnostic biomarkers and help reveal insights into the pathogenicity of the virus. To identify potential tumor-associated mutations in KSHV I analyzed the ~131 kb unique sequence regions of 43 whole KSHV genomes from tumors and oral swabs from 22 individuals and then screened additional tumors by targeted sequencing and ddPCR to better assess the frequency of identified mutations. In total, 65 KS tumors and 18 oral swabs were evaluated from a cohort of 30 study participants with KS, all from the Uganda Cancer Institute. In addition, the major internal repeat regions of the KSHV genome were examined from 16 individuals. The newly developed technologies of duplex Unique Molecular Identifier (dUMI) and single molecule real time sequencing with UMI (SMRT-UMI) were employed to provide an unprecedented accuracy and depth of study of herpesvirus populations within individuals. These studies revealed recurring tumor-associated mutations, specifically an overrepresentation of a specific region encompassing the K5 and K6 genes and inactivating mutations in the K8.1 gene, both of which were found in at least a third of the cohort and had associations with late-stage tumor characteristics. In contrast, the translation potential of full-length Kaposin proteins from IR2 was lost in a majority of individuals, but no tumor association or clinical correlate was identified. Rearrangement breakpoints were sometimes shared between different tumors from the same person, indicating spread by metastases, helper viruses or residual infectivity. In summary, these studies revealed the presence of selection for tumor- and clinical-stage specific changes the KSHV genome. Hypotheses on whether these changes play a role in KS tumor formation, immune evasion and driving persistence in the host are put forth along with proposed experiments to test them. Among them, findings from my thesis suggest that targeted screening of the K5-K6 and K8.1 gene regions can be helpful in tumor classification.

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