Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi’s Sarcoma (KS), a highly vascularized tumor made up of cells of endothelial origin. KSHV establishes a predominantly latent infection in endothelial cells in culture and in the KS tumor. A previous study has shown that KSHV induction of the Warburg effect is required for the survival of latently infected endothelial cells. The Warburg effect, a common metabolic alteration in cancer cells, refers to an increase in glycolysis and a decrease in oxidative phosphorylation. The mechanism of Warburg induction by KSHV is currently unknown. I proposed to evaluate the role of endothelial cell specific hypoxia-induced factors (HIFs) on KSHV Warburg induction since HIFs have been implicated in Warburg induction in other types of cancer. I hypothesized that HIF2α mediates KSHV Warburg induction through expression of glycolytic genes. To test this, I constructed HIF2α knock-out cells using CRISPR/Cas9 gene editing. I then used RT-qPCR to measure glycolytic gene expression during KSHV infection of wild-type and HIF2α knock-out cells. I found that the transcript levels of certain glycolytic genes remained constant in KSHV-infected HIF2α knock-out cells as compared to KSHV-infected wild-type cells, showing that HIF2α is not responsible for glycolytic gene expression during KSHV infection. I am following up on the role of the KSHV latent gene KapA on the induction of glycolytic gene expression. KapA was previously found to interact with components of the Ras pathway. As the Ras pathway activates glycolytic genes through HIF1α, I hypothesize that exogenous expression of KapA will lead to increased glycolytic gene expression through increased expression of HIF1α. I will construct an endothelial cell line that overexpresses KapA using CRISPR/Cas9 system and then use RT-qPCR to evaluate glycolytic gene expression. These results will aid in the future efforts to develop antiviral drugs by targeting cellular metabolism.

Antibiotic resistance is an intractable clinical challenge that disproportionally affects much of the world’s most impoverished populations. A critical driver of resistance stems from DNA mutations. Therefore, insights into how mutations arise in the genome is critical to understanding the development of antibiotic resistance. Our lab is currently identifying factors and environmental conditions that promote mutagenesis and bacterial evolution. Utilizing mutation rate analysis, we have identified factors and conditions that promote mutagenesis in divergent bacterial species. Additionally, using short-term evolution experiments, we have found that these factors promote the development of antibiotic resistance over time. This work provides novel mechanistic insights into mutagenesis and antibiotic resistance development in bacteria.

Helicobacter pylori is a gastric bacterial pathogen that infects about 50% of the world’s population. Chronic infection with H. pylori causes inflammation and increases the risk for developing gastric cancer. H. pylori colonizes the human stomach where it uses a type IV secretion system to deliver bacterial factors to the gastric epithelial cells. Once infected, gastric epithelial cells initiate different immune responses, one of which is the release of a protein; lipocalin 2 (LCN2). Like nearly all pathogenic bacteria, H. pylori must acquire iron, which is important for colonization, persistence, and virulence. LCN2 behaves like a competitor for iron and limits bacterial growth by depleting intracellular iron stores. LCN2 is highly upregulated in gastric tissue during H. pylori infection, however it is still unclear if LCN2 plays a beneficial role for the host by controlling H. pylori growth. My project tests the hypothesis that if host LCN2 is sequestering iron, then H. pylori survival rates will be decreased. My first aim characterizes the kinetics of LCN2 expression determining when this gene is most highly expressed during H. pylori infection. My second aim uses novel and innovative CRISPR/Cas9 genome editing techniques to engineer LCN2 knockout gastric cells. If my hypothesis is correct, I expect that H. pylori survival rates will increase when co-cultured with LCN2 knockout cells since there will be no sequestering of iron by LCN2. Understanding how the host tries to defend itself through proteins such as LCN2 will provide better insight into the host immune response and may inform vaccine design and novel treatment strategies for populations with a higher predisposition for developing gastric cancer as a result of H. pylori infection.

Klebsiella pneumoniae is a gram-negative bacterium that asymptomatically colonizes the intestinal and upper respiratory tract of healthy humans. It is also an opportunistic pathogen that causes bacteremia, pneumonia, urinary tract infections, and other severe infections in individuals with significant comorbidities. Klebsiella infections are often healthcare-associated and antibiotic-resistance is a major problem for treatment. A K. pneumoniae lineage known as multilocus sequence type 258 (ST258) is resistant to virtually all beta-lactam antibiotics, including the carbapenems, and many clinical isolates are also resistant to fluoroquinolones and aminoglycosides. Additionally, the capsule of ST258 confers resistance to phagocytosis by neutrophils and killing by the complement system. However, it is readily killed if phagocytosed. One possible therapeutic approach is to develop an antibody-based immunotherapy that targets the capsular polysaccharide (CPS) of ST258. As a step toward developing the tools needed for such an approach, we developed an enzyme-linked immunosorbent assay (ELISA) that can be used to detect CPS and titer CPS-specific IgG antibodies. Polystyrene ELISA plates were coated with purified ST258 CPS and then incubated with rabbit antiserum containing antibodies specific for CPS. Anti-CPS IgG was then detected with goat anti-rabbit IgG antibodies and a detection reagent. We anticipate the ELISA will be an important quantitative tool in our efforts to develop an immunotherapy that targets the CPS of K. pneumoniae.

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. 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-mediated signaling, a critical antiviral pathway that is activated during herpesvirus infections. STING induces expression of the antiviral signaling molecule interferon and pro-inflammatory cytokines through the transcription factors IRF3 and NF-kB, respectively. While STING is unable to signal through IRF3 in LECs, it is unknown if NF-kB activation is similarly impaired. Moreover, it is unknown if the defect contributes to the increased susceptibility to KSHV infection in LECs. Accordingly, I hypothesize that in LECs, NF-kB signaling via STING will be impaired and that the STING defect increases susceptibility to infection relative to BECs. To determine if STING activates NF-kB, I will stimulate STING in BECs and LECs and measure expression of NF-kB-activated genes. I expect NF-kB-activated genes to be expressed in BECs but not in LECs, indicating that STING is unable to signal through NF-kB in LECs. To determine if the STING defect renders LECs more susceptible to KSHV infection, I will use CRISPR-Cas9 to knock out STING in BECs. I will then use KSHV to infect the knock-out BECs as well as wild-type BECs and LECs and compare infection rates. I expect the infection rate in the knockouts to be increased relative to wild-type BECs and similar to the infection rate in LECs. The results from these experiments will further elucidate how KSHV exploits defects in innate-immunity to infect and transform host cells.

Human cytomegalovirus (HCMV) is a member of the Herpesvirus family. Most of the US population has been exposed to this virus, but it is only deadly in immunocompromised individuals. When a human cell becomes infected, one of the earliest mechanisms of anti-viral defense is the activation of protein kinase R (PKR). Active PKR phosphorylates the translation initiation factor, eIF2α, shutting down most protein synthesis in the cell. HCMV circumvents cellular shutdown by expressing protein TRS1, which binds to PKR and inhibits it. Another cellular kinase, the heme-regulated inhibitor (HRI), also phosphorylates eIF2α upon activation by reactive oxygen species. Because HRI and PKR are similar in structure, I hypothesize that HRI activation during HCMV infection is inhibited by TRS1. I infected PKR-knockout human fibroblasts with HCMV and then activated HRI expression by inducing production of reactive oxygen species. These cells also have a luciferase gene, expression of which is induced upon eIF2α phosphorylation. I measured the abundance of phosphorylated eIF2α (eIF2α-P) and luciferase activity in the lysate of infected cells in which HRI has been activated. I expect that TRS1 will bind and inhibit HRI, therefore eIF2α-P levels and luciferase activity will be low compared to cells lacking TRS1. These results reveal that HCMV replication is sensitive to HRI activation in reactive oxygen-rich inflammatory tissues and provides insights into the evolutionary history and structural similarities between HRI and PKR.

As an airborne virus, influenza A is a widespread threat to global economies and a consistent danger to public health. Through high reassortment and evolutionary rates, influenza is even able to infect those who have been previously vaccinated against the virus. The innate immune system serves as a key first line of defense against this pathogen, with the signaling components, called interferons, driving the production of a potent cellular antiviral response. Studies have indicated that viral populations replete in defective virus particles, virions with a deletion in a portion of their genome, are less efficient at blocking the antiviral response, as shown by increased interferon in the host. Our project seeks to explore this phenomenon of RNA deletions leading to increased interferon expression in host cells by testing the hypothesis that deletions in the three polymerase genes of influenza alone are sufficient to cause an increase in the interferon response. In addition, we are currently testing if mutational deactivation of one of the other genome segments, or absence of such segments, is capable of producing a more robust immune response when combined with polymerase gene deletions. In order to support this analysis, I began by creating pure populations of PA defective influenza particles grown on PA expressing host cells. Similar to results observed by my mentor Dr. Alistair Russell with PB1 and PB2 defective populations, it was found that these PA defective influenza particles were sufficient to induce the interferon response. Recently, I have assisted in the creation of multiple influenza protein expressing cell lines and influenza populations with simultaneous modifications to the HA, NS, and polymerase genes. It is hoped that immune stimulation data derived from these custom viruses, in combination with previous findings, will improve current antiviral therapies and models of the human immune response to influenza.