The dynamic nature of chromatin, the complex of DNA and histones, is not fully understood and continues to be studied. During infection, viruses utilize host cell machinery, including chromatin, to promote viral proliferation. Adenovirus protein VII is a small histone-like core protein that tightly binds DNA and disrupts host chromatin in the infected cell . By studying protein VII, we aim to uncover how chromatin manipulation affects the health of the cell. To this end, our lab is using the model eukaryote, Saccharomyces cerevisiae or budding yeast, to understand the interaction between protein VII and chromatin. Yeast is an excellent model for studying chromatin due to the high conservation of histone proteins across all eukaryotes. By inducibly expressing protein VII in yeast, we tested how the expression of protein VII affects yeast growth and viability. Our preliminary findings show that protein VII expression is toxic to yeast—cell growth is slowed resulting in the formation of fewer and smaller colonies. This result suggests that protein VII perturbation of host chromatin is sufficient to inhibit growth in our model organism. Currently, we are generating yeast mutants and expressing different viral protein variants to understand the mechanism of these phenotypes . Through studying the chromatin perturbation caused by adenovirus protein VII, we will further our understanding of fundamental chromatin structure.

Mouse adenoviruses (MAdV), like human adenoviruses (HAdVs), have specific tissue tropisms. MAdV-1 infects macrophages and vascular endothelial cells, which can result in encephalitis, while MAdV-2 infects epithelial cells of the intestine but does not cause overt disease. The viral protein that determines MAdV tropism is unknown; however, for many viral families it is the viral attachment protein that is critical. For MAdVs, fiber is the viral attachment protein, and the receptors used by MAdV-1 and MAdV-2, although unknown, are distinct. To test whether MAdV receptor usage dictates tissue tropism, I constructed a MAdV-2 chimeric virus, replacing its fiber protein with that of MAdV-1 using a gene-editing recombination system. The chimera was used to infect a 3D culture model of the intestinal epithelium called “enteroids.” As expected, MAdV-1 does not replicate in enteroids and MAdV-2 does, consistent with their in vivo tropisms. Remarkably, the chimera replicated efficiently, indicating that the fiber protein is not the sole determinant of MAdV-2 intestinal tropism. Although fiber is not the main contributor to tropism, its interactions with host factors are still likely important for productive infection. A recent study identified N-acetylglucosamine (GlcNAc) as a specific ligand for MAdV-2 fiber. We have shown that GlcNAc is not the primary receptor for MAdV-2; however, binding to GlcNAc may aid in adhesion of MAdV-2 and penetration through the mouse intestinal mucus layer. To test this hypothesis, I mutated the GlcNAc interacting residues in MAdV-2 fiber to prevent GlcNAc binding. I am currently comparing the infectivity of this mutant virus to wild type MAdV-2 in both epithelial tumor cells and enteroids. Unlike tumor cell cultures, enteroids contain mucus-secreting goblet cells which will recreate the in vivo context more accurately. Together, these studies of MAdV may help us to understand why different HAdVs infect specific tissues.

Human alpha defensins, a component of the innate immune system, are small cationic peptides that possess antiviral activity against non-enveloped viruses. The effect of defensins on human adenoviruses (HAdV) is serotype-dependent, infection by some serotypes is enhanced while for others it is neutralized. Enhanced infection correlates with increased cell binding; however, the mechanism of increased binding is unclear. One hypothesis is that defensins mediate receptor-independent binding. Inhibitor studies support this hypothesis, although formal proof is still needed. To test this hypothesis, we used CRISPR/Cas9 lentivirus to knockout the primary receptor, coxsackie adenovirus receptor(CAR), in A549 lung cells. In order to vet these cell lines, they were infected with different HAdV serotypes that use either CAR or an unrelated molecule, sialic acid, as their primary receptors. As expected, the sialic acid-utilizing but not the CAR-utilizing serotype was able to infect the CAR KO A549 cells. We have used these cell lines in combination with integrin co-receptor inhibitors to measure binding and infection of wildtype and mutant adenoviruses in the presence and absence of defensins. These experiments allowed us to determine the extent to which defensin-mediated attachment and entry is receptor-independent.
 

For many years, concerted efforts to combat malaria through the use of antimalarial drugs, bed nets, and other public health measures led to marked reductions in morbidity and mortality. Unfortunately, progress has stalled. Reductions in malaria have leveled off and even reversed in certain areas (WHO, 2017). As of 2016, there were 216 million cases and 445,000 deaths annually due to Plasmodium infections (WHO, 2017). To regain momentum and accelerate malaria eradication efforts, an effective and durable vaccine is needed. The Murphy Laboratory focuses on developing novel pre-erythrocytic (PE) malaria vaccines that can effectively stop the Plasmodium sporozoite (spz) before the clinically symptomatic blood stage begins. Identification and inclusion of multiple different protective Plasmodium antigens is thought to be crucial to developing a broad immune response and durable protection against this intracellular parasite. To test and define protective antigens, the Laboratory developed an “Acute Challenge” (AC) model in order to sensitively measure T-cell responses that are completely or partially protective. In this model, DNA vaccines encoding Plasmodium yoelii proteins are delivered by gene gun to induce CD8+ T-cell responses in BALB/c mice. At the peak of the immune response, we challenge the mice with luciferase-expressing P. yoelli sporozoites and measure the parasite burden and protection using IVIS imaging. A known protective epitope derived from P. yoelii circumsporozoite protein (CSP) induces a potent and protective response in this system. My project is to utilize the AC model to assess P. yoelli candidate antigens, of unknown protective potential, that are putatively exported or secreted from the parasite-containing vacuole into the host cell cytoplasm. Confirmed protective antigens will then be assessed for their localization and defined T-cell epitopes. The results will be used to create vaccines designed to maximize such responses and target the responding T-cells to the liver.

Mouse adenoviruses (MAdVs) are non-enveloped double stranded DNA viruses. There are two different types of MAdVs with different tropisms or ability to infect particular cells or tissues. MAdV-1 infects macrophages and endothelial cells and causes encephalitis. MAdV-2 infects intestinal cells but causes no disease. Although, the MAdV fiber capsid proteins are important for attachment of the virus to host cells, it is not known if they are the major determinant of tissue tropism in the mouse. To address this question, I use recombination-mediated genetic engineering to make chimeric MAdVs, wherein I keep most of the genome of one strain but replace the fiber protein with that of the other strain. I then use transfection to introduce the DNA of the chimeric virus into a mouse cell line to allow the virus to replicate. I am currently designing and testing the proper chimeric fiber construct that will result in a replication-competent virus. Ultimately, I compare infection of the chimeric virus to that of the parent viruses in intestinal organoids, a tissue culture model that allows us to faithfully test tropism without the need for mouse studies. These experiments may reveal general principles of AdV tropism that will allow us to understand why different human AdVs cause disease in specific organs.

In cystic fibrosis (CF), a genetic defect in the CFTR anion channel compromises host defenses and causes chronic lung infections with organisms like Staphylococcus aureus. Our lab has been studying the effects of combining ivacaftor, a CFTR modulator which increases CFTR channel activity, with a period of intensive antibiotic treatment. We found that S. aureus lung infections generally persisted despite this aggressive treatment. However, most subjects undergoing treatment were found to be infected by different S. aureus strains one year after treatment than were present before treatment was initiated. Understanding the dynamics of strain switching provide new knowledge about the natural history of chronic CF infections, help define the effects of CFTR modulators and antibiotics, and inform new approaches that might produce infection eradication. We hypothesize that (1) strain switching is most likely to occur during the period of combined ivacaftor and antibiotic treatment, as sputum bacterial burdens were lowest during combined treatment; and that (2) strain switching is rare in the absence of combined treatment. To test this, we used a new population-based multilocus sequence typing (PopMLST) method we developed to perform strain-level genotyping on S. aureus. PopMLST uses PCR amplification and next generation sequencing of housekeeping genes from bacterial isolate pools cultured from sputum. Sequencing determines the number and relative abundance of unique sequence types present, and the data can be used to infer the number of strains present. This analysis was performed on samples obtained before treatment, during treatment with ivacaftor alone, and during combined treatment. We also examined a cohort of subjects receiving usual care. These data improve understanding of strain dynamics during CF infections and suggest new strategies to eliminate infection.

Bacteria inhabit a world filled with threats, including antagonism from other species throughout different environmental conditions. One mechanism microbes employ as protection from these hazards is the type VI secretion system (T6SS) - a system bacteria utilize to inject toxic proteins into neighboring cells, leading to cell death. The H1-T6SS of Pseudomonas aeruginosa comprises 7 pairs of toxins and cognate immunity proteins (which prevent self-intoxication). I hypothesized that each of these toxins could be maximally effective against specific kinds of competing bacteria, however the function of many effector proteins is unknown. My research focuses on characterizing the protein Tse7, encoded by the gene PA0099, and elucidating its potential role in species-specific antagonism. To begin, I identified its key functional amino acids and the gene encoding its immunity protein. I designed and created mutant strains with several genes adjacent to PA0099 deleted and co-cultured these with wild-type Pseudomonas aeruginosa to identify any mutants with a loss in competitive fitness. This lead to the discovery that the gene PA0100 encodes the cognate immunity protein. To determine key functional residues, I identified potential candidates using conserved motifs in the toxin’s amino acid sequence. By creating mutants of these residues and analyzing their change in competitive fitness compared to wild type, I recognized histidine 230 as the residue vital for Tse7 function. Going forward, I will attempt to determine whether Tse7 improves fitness of P. aeruginosa against any specific families of bacteria, indicating the toxin targets that particular bacteria. Almost one-third of Gram-negative bacteria have T6SSs. These systems largely dictate the ability of bacterial species to compete with one another, dramatically affecting bacterial community structure and the landscape of human infections. Therefore, this deepened understanding of its function could further our knowledge of and ability to manipulate bacterial interactions’ impact on environmental and human health.

Modification of biomolecules is responsible for the regulation of cellular activities in all organisms. A type of enzyme, named ADP-ribosyltransferase (ART), plays a crucial role in such modifications by transferring one or multiple ADP-ribose moieties onto its target molecules. Some bacterial ART proteins are toxins that serve as virulence factors that enable pathogens to disrupt host cell functions during persistent infection. However, it was unclear if ARTs play roles in interbacterial interactions. Here, I report the discovery of the first interbacterial ART toxin encoded by a Serratia proteamaculans strain, a commensal bacterium isolated from plant root. Growth competition assays showed that the toxin is capable of conferring a higher fitness for S. proteamaculans. Subsequent analysis by microscope revealed that target bacterial cells became elongated, leading up to cell lysis. Together, my results offered new insights into the complex question of how bacteria compete against each other. The finding expanded our knowledge of the diverse roles of ART proteins and their cellular activities.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi’s Sarcoma (KS), a highly vascularized tumor composed of cells of endothelial origin. KSHV, while possessing both lytic and latent replication programs, predominantly exists in the latent form during infection. While KSHV infects both blood (BECs) and lymphatic (LECs) endothelial cells, LECs are more susceptible to infection and express fewer antiviral genes during infection compared to BECs. Recent experiments have shown that LECs, but not BECs, have a defect in STING, a critical signaling protein that is activated during herpesvirus infections and results in the production of antiviral signaling molecules such as IFN-β. It remains unknown whether the defect in STING plays a direct role in increasing susceptibility to KSHV infection and if the defect impacts the ability of STING to suppress lytic reactivation in LECs. Accordingly, I propose to construct a constitutively active (CA)-STING and express it in LECs. Because CA-STING results in the continuous induction of IFN-β, I hypothesize that CA-STING-LECS will show decreased susceptibility to the establishment of latency by KSHV and have increased ability to suppress lytic reactivation compared to empty vector-expressing (EV)-LECs. First, I will infect EV-LECs and CA-STING-LECs with KSHV and measure the infection rates 48 hours post infection (hpi). I expect the number of infected cells in the CA-STING-LECs to be decreased relative to the EV-LECs. Next, I will infect EV-LECs and CA-STING-LECs with KSHV, and at 48 hpi, I will induce lytic reactivation in the two cell types and quantify the virus produced. If CA-STING suppresses lytic reactivation in LECs, I expect less virion production from CA-STING-LECs than from EV-LECs. The results from these experiments will further elucidate how KSHV exploits defects in innate-immunity to infect and transform host cells.

During HIV infection, CD8+T-cells are crucial for the control of viral replication. Increased CD8+T-cell polyfunctionality, which is the ability for one cell to perform more than one function within the immune response, was associated with improved clinical outcomes, is now the focus of experimental curative therapies for HIV. One therapy that showed promise is a Conserved Elements DNA (CE) vaccine, which is designed to prime the immune system with essential regions of the virus, thereby eliciting polyfunctionality and circumventing escape mutants. A novel immunotherapeutic combinatorial approach was investigated for the ability to reduce or eliminate viral burden in Simian Human Immunodeficiency Virus (SHIV) infected macaques receiving antiretroviral drug therapy. We evaluated effects of this regimen on CD8+T-cell polyfunctionality and its correlation to viral control after analytical antiretroviral treatment interruption (ATI). Our combinatorial immunotherapy regimen uses the CE vaccine to target highly conserved viral sequences, latency reversal agent GS986, exhaustion reversal agent anti-PD-1, and CCR5 gene editing to delete the co-receptor for viral entry in CD4+T-cells. Analysis of CD8+T-cell polyfunctionality was performed using intracellular cytokine staining and flow cytometry to measure the frequency of CD8+T-cells secreting one or more of the following effector functions: TNFα, IFNγ, IL-2, and both CD107a and Granzyme B. Polyfunctionality induced by the combinatorial regimen was algorithmically quantified and compared to a control group receiving no interventions and a group receiving CE vaccine and CCR5 gene editing alone. Our analysis showed neither significant differences in polyfunctionality between treated and control groups, nor any changes in polyfunctionality within the subset of CD8 targeting conserved regions of the virus. However, we observed diverse viral control post-ATI among all animals, and additional experiments are in progress to determine if CD8 polyfunctionality played a role in improved viral control post-ATI.

Microbial toxins are a molecular weapon involved in pathogenesis, immune evasion, and bacterial competition. A prime example of such microbial toolkits are polymorphic toxin systems, which consist of multi-domain proteins and are widespread in all major bacterial lineages. A polymorphic toxin system called MuF has been newly identified and is the first to be discovered in temperate phages and their bacterial hosts. Though it is highly abundant in the human gut microbiome, its biological role has not been defined. To better understand the toxin system, our team is studying a model species Enterococcus faecalis, a commensal bacterium encoding a two-domain MuF toxin protein on one of its phages, consisting of an N-terminal MuF domain and a C-terminal toxin domain. The toxin domain is predicted to be an ADP-ribosyltransferase (ART), which post-translationally attaches ADP-ribose moieties to its target molecules and can profoundly impair cell processes, leading up to cell death. Using genetic approaches to generate phages with malfunctional ART activity, I have found that the mutations change phage infectivity and the morphology of the plaques formed (clear zones in a cell layer formed due to lysis by phage). Moreover, heterologous expression of the toxin domain in E. faecalis results in cell aggregation. From this, I hypothesize that the MuF toxin is delivered by phages to help infection and ensure phage DNA incorporation into host genomes. To further dissect the mechanism by which the MuF toxin system operates, our team is currently developing a fluorescent protein reporter system to investigate and track the detailed process of phage infection. In addition, by applying X-ray crystallography and electron microscopy, I aim to uncover structural information on the toxin, which may lend insight into the mechanism of MuF toxicity and its larger role in the human microbiome.

Secondary nucleotide messengers are used by all domains of life to sense and respond to the changes in their environment. In bacteria these secondary nucleotide messengers play a role in regulating several signaling pathways such as cell wall homeostasis, motility, and the expression of virulence genes. The nucleotide cyclic di- 3, 5’ adenosine monophosphate (c-di-AMP) was recently added to the list of secondary nucleotides. C-di-AMP is found in many bacteria such as S. aureus, S. pneumoniae, B. subtilis, and L. monocytogenes (Lm). C-di-AMP has been best characterized in Lm, a well-studied intracellular pathogen. Lm has adapted to survive and replicate in the host cell cytosol by evading host cell defenses through use of key virulence factors. In Lm, synthesis of c-di-AMP is catalyzed by the diadenylate cyclase dacA and degradation is coordinated by the phosphodiesterases, pdeA and pgpH. Studies using Lm mutants that lack both pdeA and pgpH contain abnormal c-di-AMP levels that cause growth and virulence defects of about four logs compared to wild type Lm. This highlights the importance of c-di-AMP regulation for bacterial virulence and growth, but we still know very little about c-di-AMP regulation and toxicity. Our goal is to further understand the toxicity of high levels of c-di-AMP during bacterial infection. We aim to create a transposon library in the double phosphodiesterase KO (ΔΔ Pde) background to identify suppressor mutations. Previous approaches to analyzing suppressor mutations in the ΔΔ Pde strain has not been thorough or cannot be utilized in vivo. Therefore, we have created an amenable phosphodiesterase mutant that knocks out the phosphodiesterases in Lm (pdeA and pgpH) to grow in vivo successfully to investigate c-di-AMP regulation. Understanding the regulation of c-di-AMP could result in targets for novel treatments against Lm and allow for ways to investigate regulation methods of c-di-AMP in other organisms.

Molecular interactions that occur between surface components of pathogens, termed adhesins, and complementary receptors presented on host cells are critical for establishment of infection. The adhesion-receptor binding event may trigger diverse signaling cascades in the host cell that result in activation of innate host defenses or the subversion of cellular processes facilitating bacterial colonization or invasion. Hence, microbial adhesins are important targets for the development of antimicrobial therapeutics and vaccines. One of the interests of the Sokurenko Lab at UW is understanding the structure and function of the ubiquitous Escherichia coli adhesin, FimH, and its potential as a vaccine candidate against urinary tract infections. FimH is expressed on top of filamentous hair-like appendages called type 1 fimbriae that confer bacterial binding to glycoproteins with terminally exposed mannose. In the laboratory, we use a variety of assays to study FimH-mediated bacterial adhesion and test different mannose-containing compounds and glycoproteins for optimal inhibition of FimH-mediated interactions. We also elicited a great number of FimH-specific monoclonal antibodies that are tested for their potency to inhibit binding properties of the FimH, and to understand the structural mechanisms of antibody-mediated inhibition. By generating and analyzing various FimH mutants, we seek to elucidate how the function of FimH protein can be efficiently regulated. The ultimate goal of these experiments is to gain knowledge on the optimal structure (conformation) of the FimH protein that can be used as an antigen for efficient induction of protective antibodies during immunization.

Interferons are signal proteins released by cells in response to infections which induce neighboring cells to synthesize anti-viral genes like Mx2, which encodes the MxB protein. MxB was thought to only restrict lentiviruses but recent studies have shown that MxB also restricts herpesviruses. It is hypothesized that herpesvirus and MxB have co-evolved in an ‘arms race’ to compete against each other. This suggests that regions of MxB that are under positive selection may have interfaces for its anti-herpesvirus activity. In order to test this hypothesis, I plan to determine whether different homologs of MxB can restrict human herpesviruses. I created MxB inducible cell lines using MxB homologs from three different primates - humans, rhesus monkeys and owl monkeys. Subsequently, I infected these cells with the following GFP labeled viruses: herpes simplex virus 1 (HSV-1), Kaposi’s sarcoma-associated herpesvirus (KSHV), and human cytomegalovirus (HCMV). I assessed the effect of these MxB alleles on viral replication by measuring viral titer and viral protein production (including GFP). I expect that the MxB restriction of herpesviruses will be species-specific, with only human MxB showing meaningful reduction in human herpesvirus replication. These results will guide future research aiming to understand the physical interactions and mechanisms of suppression between MxB and herpesviruses.

Cystic Fibrosis (CF) is a genetic disorder associated with chronic, polymicrobial lung infections. One of the most common treatments for these infections involves inhalation of the antibiotic Tobramycin, used to treat Pseudomonas aeruginosa infections. Tobramycin’s effect on other members of the CF respiratory microbial community is unclear. The Tobramycin inhaled powder (TIP) study assessed the effect of Tobramycin on the entire respiratory microbial community before, during and after one month of therapy and demonstrated that, on average, Staphylococcus aureus viable counts dropped during the first week of therapy before returning to pre-therapy levels. However, there was variability in the response in viable counts to antibiotics in different patients with some that did not change at all throughout the course of drug therapy and no individual clearing their S. aureus infection. The purpose of this study is to better understand why Tobramycin did not clear S. aureus in people with CF as well as determining why patients responded differently. We initially hypothesized that the infection was not cleared due to antibacterial resistance to Tobramycin. I answer this question using standard susceptibility tests on Tobramycin, Cefoxitin, Sulfamethoxazole and Levofloxacin. The use of four different antibiotics help determine if the bacteria are resistant to Tobramycin and if there is a viable alternative to Tobramycin. We anticipate that results will show a shift from susceptibility before therapy to resistance after week one due to the proportion of resistant bacteria increasing. CF lung infections can be a model for many other diseases as well and we hope that this study may provide more insight into how to treat these infections better.

There is a critical lack of knowledge regarding the effects of human immunodeficiency virus (HIV) and zika virus (ZIKV) coinfection in respect to ZIKV pathogenesis, vertical transmission and current vaccine strategies. ZIKV has received global attention because of its clinical complications including congenital malformations during vertical transmission and Guillain-Barré syndrome, a neurocognitive disorder in adults. Recently, ZIKV outbreaks have occurred in tropical and subtropical regions endemic with HIV, therefore it is imperative to understand the impact HIV-ZIKV coinfection may have when moving forward with ZIKV vaccine design. Furthermore, in humans and non-human primates (NHPs) frequencies of blood monocytes increase during HIV and simian immunodeficiency virus (SIV) infection and monocytes are early targets of ZIKV infection. Therefore, we hypothesize that cells from HIV infected individuals have the capacity to harbor increased ZIKV replication and could lead to enhanced ZIKV viremia and pathogenesis. For this study, we use a NHP model to determine the impact of acute SIV infection on ZIKV pathogenesis. We determine ZIKV cellular targets in the blood and preliminary results show an increase in NHP blood monocyte within the first six weeks of SIV infection. We evaluate whether SIV infection increases the susceptibility of cells to ZIKV infection by isolating cells from SIV- and SIV+ animals, infecting them in vitro with ZIKV, and assessing viral replication by plaque assay. Our preliminary findings suggest that in vitro ZIKV replication may increase in cells from SIV+ NHP blood when compared to SIV- blood. Future studies will look at the impact of SIV infection on in vivo ZIKV replication and whether ZIKV replication is enhanced in cells from HIV-infected individuals.