Mapping the locations where transcription factors interact with DNA is central to uncovering genome regulation mechanisms and gives key insights as to how organisms respond to intracellular or extracellular signals. The current gold standard for characterizing transcription factor binding sites is Chromatin Immunoprecipitation Sequencing (ChIP-Seq). Despite the ubiquity of ChIP-Seq, significant caveats remain. This method requires high-quality antibodies that may be laborious and costly to acquire, and large amounts of material are needed to obtain reliable data. We propose an alternative to ChIP-Seq: exploiting the interbacterial toxin double-stranded DNA deaminase (DddA). DddA catalyzes the deamination of cytosine to uracil, which is replaced by thymine via DNA replication. We harness this activity by designing translational fusions of DddA to the protein of interest. We hypothesize that the target protein carries DddA to its DNA binding region, introducing local C→T mutations that deep-sequencing detects. As a proof of concept, we tested this system with known bacterial transcription factors GacA, GcsR, and FleQ. Our results confirmed that DddA fusions have higher localized C→T mutations where the characterized transcription factors bind. We also tested different conditions and construct designs to maximize the efficiency of this method. Our results show that the use of DddA as a method of analyzing protein–DNA interactions is a broad and powerful tool with advantages over current methods like ChIp-Seq. Through this, we can better understand how organisms control gene expression when infecting a host, competing for resources, or surviving stressors.

Enteric alpha defensins, such as human defensin 5 (HD5), are antimicrobial peptides secreted by Paneth cells in the lumen of the small intestine as part of the innate immune response. Defensin activity effectively inhibits many bacterial and viral pathogens during infection. However, not all pathogenic human viruses are neutralized in the presence of defensins. For example, among the seven human adenovirus (HAdV) species, infections of certain serotypes are enhanced by HD5, while other serotypes are inhibited. Our goal is to identify the molecular determinants that confer HAdV neutralization or enhancement by HD5. We have previously identified regions of the three major capsid proteins that form the outside of the virus, hexon, penton base, and fiber, as key determinants. We identified these determinants through rational design and the creation of chimeric viruses in which we swapped portions of the three major capsid proteins between two serotypes with opposite HD5-dependent phenotypes. From these data, we have created a model of the enhancement and neutralization mechanisms. To further test these models, I am creating a panel of chimeric HAdVs by swapping capsid components from additional HAdV species and serotypes. I will study the concentration-dependent effect of HD5 on each chimera’s infectivity compared to the wild type viruses, which will uncover new mechanistic detail and allow us to create a more generalized model.

Pseudomonas aeruginosa, an opportunistic pathogen that commonly infects cystic fibrosis patients, uses quorum sensing (QS), a form of cell-cell communication, to regulate the expression of virulence factors and public goods based on population density. P. aeruginosa QS consists in part of N-acyl homoserine lactone signal molecules that activate two separate regulatory proteins, LasR and RhlR, which in turn activates the transcription of other target genes in their respective regulons. The las and rhl regulons are hierarchical in lab strains, with LasR activating the transcription of rhlR; however, many pathogenic variants carry nonfunctional alleles of lasR and rely on rhlR as the dominant QS regulator. Two anti-activator proteins, QteE and QslA, restrict the expression of these two QS regulons; however, it is not clear how P. aeruginosa anti-activators function in many pathogenic strains. Identifying how anti-activators regulate QS in pathogenic variants could be crucial in developing therapies that do not rely on antibiotics. To investigate how QteE and QslA modulate QS, we overexpressed each anti-activator in P. aeruginosa and used transcriptional reporters to monitor the activity of rhlA, a RhlR regulated gene. Expression of both genes is reduced significantly in strains with over-expressed anti-activators. However, in a pathogenic variant, only over-expressing qteE delayed QS induction while over-expressing qslA had no effect. These results indicate that QteE can modulate QS by affecting LasR and RhlR levels, while QslA only modulates LasR levels. These experiments lay the foundation for therapeutic strategies centered on inhibiting QS rather than relying on conventional antibiotics.

Venezuelan Equine Encephalitis Virus (VEEV) is an arthropod-borne virus spread by mosquitos. It causes a range of acute disease from mild flu-like symptoms to more severe illnesses such as encephalitis. Certain species of rodents serve as a reservoir host for VEEV and display no symptoms, whereas horses, as amplification hosts, often exhibit lethal symptoms, suggesting that differential immune responses in these hosts contribute to differences in pathogenesis. Because reservoir and amplification hosts exhibit distinct pathogenesis profiles, this suggests VEEV replicates differently in these hosts and may be able to better evade the equine innate immune system. We have shown that changes in 3’ untranslated region (UTR) sequence and structure affect the ability of VEEV to evade host innate immune sensors such as IFIT2 and RIG-I. We generated mutant viruses which encode 3’-UTR sequences from either pathogenic or attenuated strains of VEEV and are using these to investigate the role of RNA structures in the 3’ UTR in limiting VEEV replication in equine cells. By stimulating the interferon response in equine fibroblast cells (NBL-6 cells) using a synthetic double-stranded RNA analog, Poly I:C, we predict equine antiviral responses will inhibit VEEV replication. Furthermore, we hypothesize pathogenic chimeras of VEEV will replicate more efficiently in Poly I:C-treated cells than attenuated chimeras. These studies will expand our fundamental understanding of the molecular mechanism of how RNA structure in the VEEV genome contributes to host innate immune evasion and has implications in future antiviral therapeutics and vaccination strategies.

Kaposi’s Sarcoma (KS) is a highly vascularized tumor, which affects AIDS patients worldwide and remains endemic to sub-Saharan Africa. Kaposi’s Sarcoma-associated Herpesvirus (KSHV) is the etiological agent of KS and its latent infection is involved in tumor formation and the induction of angiogenesis in the spindle cell, a cell of endothelial origin and the main proliferating cell type in a KS tumor. Previous RNA-Seq data obtained by our group showed that osteopontin (opn), a secreted protein known to act as a ligand for integrin receptors that activate signaling cascades that promote angiogenesis, is highly upregulated at the transcript level during KSHV latent infection of endothelial cells. To determine whether opn is required for the activation of angiogenesis in KSHV latently-infected endothelial cells, we used CRISPR-lentiviral constructs to knock out opn and evaluate changes in angiogenic phenotypes upon KSHV infection via cell proliferation, tubule formation, and cell migration assays. Preliminary results reveal a significant reduction in tubule formation in opn knockout KSHV-infected endothelial cells. This finding suggests that opn upregulation in such cells is responsible for the activation of this angiogenic phenotype. Future experimentation will include evaluating how KSHV induces the upregulation of opn by infecting wild-type endothelial cells with mutant viruses lacking certain latency protein genes and evaluating differences in opn transcriptional and translation, as well as evaluating the mechanisms by which opn activates tubule formation in KSHV latently-infected endothelial cells. Identifying  the drivers of angiogenesis in KSHV-infected endothelial cells will aid the characterization of therapeutic targets for KS progression in such cells, given that KS tumors are highly angiogenic from its early stages.

Human adenoviruses (AdVs) infect and cause disease in multiple organ systems, and certain human AdV serotypes are associated with particular diseases; however, the basis for AdV tissue tropism is unknown. To better understand this problem, I am using mouse adenoviruses (MAdV). Like human AdVs, MAdV serotypes are associated with distinct tissue tropisms: MAdV-1 infects macrophages whereas MAdV-2 infects intestinal epithelial cells. By swapping genes between MAdV-1 and MAdV-2, I hope to uncover the genetic basis of tissue tropism in MAdVs. Importantly, these studies will be aided by the availability of a cell culture system that recapitulates the cellularity of the intestinal epithelium and supports MAdV-2 replication, but not MAdV-1 replication. Due to genetic conservation, principles of MAdV tissue tropism are likely to apply to HAdVs. A major determinant of viral cell tropism is receptor usage, which may also play a primary role in tissue tropism. For both MAdV species, the trimeric fiber protein that extends from the icosahedral capsid is thought to be the viral attachment protein that binds to a host receptor. Although the MAdV receptors are not completely known, they are distinct for MAdV-1 and MAdV-2. To gain insight into tissue tropism, I have created a chimeric MAdV-1-M2f virus, where the fiber gene of MAdV-2 has been inserted in place of the native fiber gene in the MAdV-1 genome. A prior student in the Smith lab created the inverse chimera. If replication is fiber dependent, the chimeric MAdV-1-M2f virus should be capable of infection. By studying these chimeric viruses, I will uncover whether the fiber/receptor interaction plays a central role in determining tissue tropism.

Cystic fibrosis (CF) is a genetic disorder characterized by chronic lung infections involving various organisms, including the gram-positive pathogen Staphylococcus aureus. Antibiotics, such as trimethoprim-sulfamethoxazole (SMX), play key roles in treating CF infections; these drugs inhibit bacterial growth by disrupting important bacterial metabolic processes. SMX specifically inhibits folate metabolism, causing DNA damage that results in bacterial cell death. However, S. aureus is able to persist in CF pulmonary infections despite treatment with antibiotics, and evidence suggests that S. aureus does so through adaptive mutations. Our goal is to identify the adaptive mutations of S. aureus during SMX exposure in vitro, to understand how this pathogen persists and to prevent the emergence of resistance. We grew S. aureus in the presence of super-inhibitory SMX concentrations for 24 hours in Luria Bertani broth. We sampled the culture at specific timepoints and measured viable bacterial counts on chocolate agar; we evaluated all resulting colonies for SMX susceptibility and associated genetic changes. Surprisingly, we identified mutants that survived SMX treatment carrying diverse adaptive changes not associated with folate metabolism or DNA repair, suggesting previously-unknown lethal effects of SMX against S. aureus. These mutants carried mutations predicted to decrease production of reactive oxygen species (ROS) - toxic compounds produced by all cells during aerobic respiration and in response to stress. Our results indicate ROS may play a role in SMX-mediated S. aureus cell death, suggesting that treatments that augment the effects of ROS could improve antibiotic efficacy. We are now exploring the involvement of ROS in S. aureus killing by SMX using engineered S. aureus strains with knockout and overproducing mutations in ROS detoxification genes. This study will help us better understand SMX’s mechanism of action and S. aureus’ response to this drug, in order to improve the treatment of diverse infections caused by this pathogen.

CD8+ T-cell exhaustion during chronic human immunodeficiency virus (HIV) infection is characterized by increasing expression of inhibitory markers on the cell surface that lead to decreased effector function and dampened immune responses that are essential to achieving therapeutic vaccine efficacy. Inhibitory marker PD-1 is upregulated on exhausted CD8+ T-cells, and blockade with a monoclonal antibody (aPD-1) can help reverse exhaustion. We hypothesized that dosing with aPD-1 would boost the immune system and decrease expression of other exhaustion markers throughout infection to improve therapeutic vaccine responses. To investigate this hypothesis, we studied how exhaustion progresses over time in simian-human immunodeficiency virus (SHIV)-infected Rhesus macaques treated with a novel combinatorial therapeutic regimen consisting of a conserved-elements vaccine to circumvent viral mutants, GS986 to reverse latency, CCR5 gene editing to prevent viral entry, and aPD-1 to reverse CD8+ T-cell exhaustion. We characterized exhaustion in peripheral blood mononuclear cells and mesenteric lymph nodes with surface staining and flow cytometry, with a focus on exhaustion markers PD-1, TIGIT, CTLA-4, LAG-3, and TIM-3 at various timepoints throughout infection and vaccination. Although we observed no differences in viral burden between treatment groups, we observed higher CD8+ T-cell vaccine responses in animals receiving aPD-1 compared to vaccinated animals that did not receive aPD-1, suggesting aPD-1 improved vaccine responses. Although we did not find a correlation between PD-1 signaling and any exhaustion markers, we observed a significant negative correlation between CD8+ T-cell vaccine responses and pre-vaccination TIGIT levels (Spearman r= -0.75, p=0.007). We also found that PD-1 and TIGIT are largely independently expressed on CD8+ T-cells. Taken together, these data suggest a role for dual blockade of PD-1 and TIGIT to improve vaccine efficacy in future studies. Defining the impacts of CD8+ T-cell exhaustion on therapeutic vaccine immunogenicity is crucial to improving combinatorial immunotherapy towards a cure for HIV.

Nucleic acid vaccines introduce DNA or mRNA into cells in vivo, instructing them to express antigens from a pathogen resulting in the induction of immune responses that can provide long term protection from that pathogen. They provide many advantages over traditional vaccines including lower cost, improved safety, and the possibility to rapidly update the vaccine since only the genetic sequence of a new variant is required. One drawback of DNA vaccines has been their relatively poor immunogenicity compared to traditional vaccines which has been overcome, to some extent, by using improved delivery methods and co-formulation with plasmids expressing cytokines as adjuvants. Previous studies have established IL-12, as the “gold standard” genetic adjuvant due to its ability to support differentiation of antigen specific CD4+ T cells to produce Th1 cytokines as well as expansion of antigen specific CD8+ T cells to be more cytolytic in vivo. There is growing interest in identifying other adjuvants that not only increase immunogenicity of DNA vaccines but also modulate the types of responses they induce. In this study, we sought to determine if co-administration of an adjuvant cocktail including IL-18, a pro-inflammatory cytokine, and IRF7, a transcriptional activator of type I interferons, along with IL-12 would enhance antibody responses to DNA vaccines expressing SIV and Influenza antigens in a preclinical nonhuman primate model. Plasma samples were collected at different times post vaccination and the effect of the adjuvants on immunogenicity was measured via IgG ELISA and analyzed. After 2 vaccinations, we observed a significant increase (P=0.0272) in antibody responses against SIV gp130 in the adjuvant cocktail group compared to the IL-12 group. These results indicate that combining adjuvants could provide further improvement in DNA vaccine immunogenicity. Additional studies to determine the impact of this adjuvant cocktail on T cell responses are in progress.

Antibiotic treatment often fails in chronic bacterial infections; many resistant bacteria no longer respond to antibiotic treatments once designed to eliminate them. The chronic infections of bacteria such as Pseudomonas aeruginosa (Pa) that afflict people with cystic fibrosis (CF) are a prime example. In CF, standard antibiotic susceptibility testing does not accurately predict treatment efficacy, and genetically unstable resistance is one potential explanation. Genetically unstable resistance occurs when resistance-producing mutations are rapidly lost or compensated for in the absence of antibiotics. Clinical susceptibility testing likely fails to detect unstable resistance as many bacterial growth generations (in the absence of antibiotics) are used in preparing isolates for testing. We hypothesized that Pa cultured from the lungs of tobramycin-treated CF patients exhibit unstable antibiotic resistance that rapidly reverts upon growth in the absence of antibiotic pressure. To test this, we cultured Pa isolates from patients who are being treated with antibiotics in a manner that maintains antibiotic selection during growth steps. We then measured their resistance levels before and after growth in the absence of antibiotics to identify isolates with unstable resistance. Whole genome sequencing of sensitive and resistant isolate pairs identified the genetic mechanisms causing unstable resistance. Our data shows that individual isolates from many patients exhibit a wide range of instability. Some isolates exhibited as much as an over a hundred-fold decrease in inhibitory concentration after antibiotic-free growth, while other isolates from the same sample had stable resistance. This work could lead to new sequenced-based methods to detect unstable resistance in patients, new approaches to select antibiotics for treatment and a greater understanding of antibiotic efficacy in chronic infections.