Sindbis Virus (SINV) is an alphavirus that is transmitted between birds via mosquitos and causes disease in humans after spillover events. While many alphaviruses have potential to cause severe disease such as Venezuelan Equine Encephalitis Virus (VEEV), SINV is known to cause less life-threatening but still severe and debilitating chronic illness predominantly associated with fever, arthralgia, and myalgia. Given its lower morbidity, SINV often serves as a model system for infectivity and pathogenesis studies of alphavirus. Comparison of the genomes of virulent strains (AR86) and avirulent strains (Girdwood) of SINV help us to define elements in the viral genome of SINV that contribute to pathogenesis. Previous studies have identified key pathogenesis determinants within the E2 glycoprotein and 5’ noncoding regions. Additionally, SINV contains elements in the 3’ UTR that are capable of repressing deadenylation contributing to evasion of host cell mRNA decay machinery. Our recent work with VEEV has identified additional novel RNA structures in the E1 segment that contribute to replication in macrophages and serves as a basis for further exploration of the role of RNA structure in pathogenesis and immune evasion which has been largely unexplored. By exchanging a 4000bp nucleotide segment of the 3’ end in SINV strains Girdwood and AR86, we hope that important conclusions can be made about the importance of differing genomic code and underlying RNA structure between these two strains after observing virus production, replication, and pathogenesis of constructed chimeras. This in turn can be used to further discern the genomic mutations and RNA structure requirements to shift from avirulent to virulent strains and could play an important role in predicting the mutational requirements necessary for SINV and other alphaviruses to make the jump from endemic bird/mosquito hosts to the human population as well as any mutations that contribute to greater pathogenicity.

Human adenoviruses (HAdVs) are known to infect and cause diseases in multiple organ systems. HAdV serotypes are associated with particular diseases, giving us reason to believe there is a genetic link to tissue tropism. To understand this relationship, we are using mouse adenoviruses (MAdVs). Like HAdVs, 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, we hope to uncover the genetic basis of tissue tropism in MAdVs. These studies are 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. Phylogenic analysis indicates that MAdVs are the oldest lineage of Mastadenoviridae, the genus that includes AdVs of mammals. Thus, functions of viral genes that are conserved among viruses within the genus and important for MAdV tissue tropism are likely to apply to HAdVs. A major determinant of viral cellular 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. MAdV-1 has been shown to use specific cell adhesion proteins called integrins as a receptor for viral entry. Although the receptor for MAdV-2 is unknown, it differs from MAdV-1. To gain insight into tissue tropism, I have designed 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. Using these viruses, we can infect intestinal epithelial cells to measure replication. Doing so will help uncover whether the fiber/receptor interaction plays a central role in determining tissue tropism.

Antimicrobial peptide (AMPS) hold promise as novel antibiotics for treatment of human infections. However, in general, they fail to reach clinical application because of issues with human cell toxicity or other factors. The AMP colistin (CST), is a rare AMP in clinical use and despite its nephrotoxic effects, has seen increasing use as a last resort to antibiotics. There’s now widespread CST resistance among bacterial pathogens. Our chemist colleagues recently synthesized a new class of AMP mimics, main chain cationic polyimidazoliums (PIMs). Their lead PIMs are potent broad spectrum antibiotics with a novel mode of action. We are interested in developing PIMs for inhalation therapy to treat respiratory infections in people with the genetic disease cystic fibrosis (CF). The bacterium, Pseudomonas aeruginosa, is the major contributor of disease in patients with CF lung infection. Unlike CST, PIM-resistant P. aeruginosa do not emerge in laboratory evolution experiments. We wanted to assess a particularly potent PIM called PIM1D as a potential CF therapeutic. Many antibiotics are inhibited by the secretions in CF lung and we expected this might be true of PIM1D. We used a special in vitro culture medium designed to mimic CF sputum, synthetic CF medium (SCFM). The medium contains the nutrient composition of CF sputum (SCFM-1) along with mucin and DNA (SCFM-2), polymers present at high concentrations in CF lungs. These polymers are known to inhibit many antibiotics’ activity. PIM1D retains high activity in SCFM-1 but activity is reduced by 90% in SCFM-2 compared to SCFM-1 and standard media. The inhibition might be due to either mucin, DNA, or both together. We aim to answer which of these molecules is responsible for inhibition of PIM1D activity. In collaboration with chemists who synthesized PIMs, they will provide PIM1D derivatives and PIMs packaged in nano-carriers to test for better activity in SCFM-2. 

Cystic fibrosis (CF) is a genetic disease characterized by polymicrobial lung infections. Staphylococcus aureus, a Gram-positive pathogen, is commonly cultured from the secretions of people with CF (PwCF). Treatment for S. aureus infections requires antibiotics, such as the bactericidal antibiotic trimethoprim-sulfamethoxazole (SXT). SXT prohibits growth by targeting folate biosynthesis, a pathway important for DNA replication and maintenance and production of cell metabolites. In many PwCF, S. aureus generally persists despite antibiotic treatment. Our data shows that S. aureus can survive SXT treatment through the accumulation of adaptive mutations. In this project, we examined these adaptive mutations in S. aureus in vitro to better understand the mechanisms of resistance. We grew several S. aureus isolates with adaptive mutations in Luria Bertani (LB) broth. We sampled the culture tubes at several times in a 24-hour period, measuring viable bacterial counts on chocolate agar. We found that isolates with mutations in the sugar transport gene, ptsI, persisted better under SXT selection, relative to wild-type S. aureus. Other S. aureus isolates with mutations in pathways for aerobic respiration, including menaquinone (menB, thiN) and hemin synthesis (hemB), also better survived SXT compared to wild-type. Many of these mutations were also identified in S. aureus infecting PwCF. These results indicate that adaptive mutations in pathways associated with important metabolic processes may allow survival with folate inhibition. We hypothesize that limiting aerobic respiration may assist in S. aureus surviving SXT. As S. aureus can survive without oxygen, we are currently studying whether limiting oxygen and, consequently, aerobic respiration in wild-type S. aureus will improve survival with SXT. This work will help us understand the mechanisms of SXT action and S. aureus’ response, in an effort to improve treatment for S. aureus infections.

In this study we developed a nonhuman primate model of simian immunodeficiency virus (SIV)-Zika virus (ZIKV) co-infection to understand how HIV infection impacts ZIKV pathogenesis and test our hypothesis that ZIKV pathogenesis is enhanced in people living with untreated HIV. Previously, we have found delayed viral clearance, as well as delayed and dampened expansion of whole blood monocytes, the ZIKV cellular targets, during SIV infection. Here, we sought to further characterize the innate immune responses of SIV-ZIKV co-infection, by assessing cytokine and chemokine release. Pigtail macaques (n=7) were infected with SIVmac239M and co-infected with ZIKV at 9 weeks post-SIV infection. Co-infected animals were compared to control animals (n=7) infected with ZIKV only. Longitudinal plasma and cerebral spinal fluid (CSF) were collected at timepoints pre- and post-infection and assayed using a multiplex immunoassay to quantify 24 different cytokine and chemokines ex vivo. SIV and ZIKV both induced pro-inflammatory responses, characterized by transient increases in interleukin-17 (IL-17A) and monocyte chemoattractant protein-1 (MCP-1), with no major differences between experimental groups. Plasma MCP-1 concentrations were also found to be consistent with dampened and delayed whole blood monocyte frequencies. Pro-inflammatory interleukin-8 (IL-8), a chemokine needed for recruitment of neutrophils, increased in the plasma during SIV infection but not following ZIKV infection, a result that is in contrast to our previous findings. Transient increases in IL-8 were detected in a few animals in the CSF after ZIKV infection, which may be evidence for neuroinflammation. Overall, no significant differences between SIV+ vs SIV- groups were found for any analytes detected in plasma or CSF during ZIKV infection. Collectively, our results demonstrate that both SIV and ZIKV infections induce a pro-inflammatory response, that is not enhanced by SIV-ZIKV co-infection. This suggests SIV induced immunosuppression does not impair pro-inflammatory cytokine responses during ZIKV infection.

Nucleic-acid based vaccines, including RNA and DNA, provide protective immunity by eliciting antibody (Ab), and cytotoxic T lymphocyte (CTL) responses. FDA approval of mRNA vaccines against SARS-CoV-2 (COVID-19) provide ample evidence that mRNA vaccines are a viable vaccine platform. Once a mRNA vaccine enters a cells cytoplasm, mRNA encoded antigens are produced rapidly inducing an immune response. The production of mRNA encoded antigens will wane over time as mRNA degrades and transfected cells die. Alpha viruses, a RNA virus, have a unique replication process whereby this virus amplifies its RNA genome upon entering a cell. We, as well as others, have developed RNA vaccines that, like alpha viruses, self-amplify once inside of a cell to create more copies of mRNA than entered cell. This self-replicating RNA vaccine is called a replicon RNA vaccine (repRNA). RepRNA induces robust and sustained antigen production and immune responses. Currently, mRNA vaccines are administered as a homologous prime/boost vaccine regimen where the same mRNA vaccine is given as a priming vaccine and boosting vaccine, but antibody titers wane over time allowing for potential infections. This project aims to evaluate If utilizing a heterologous prime/boost regimen with DNA and repRNA vaccines confers more robust and longer lasting antibody and CTL responses than homologous regimens with DNA or repRNA. Mice will be vaccinated with DNA as a priming vaccine and repRNA as a boosting vaccine that encode SARS-CoV-2 spike protein. Anti-SARS-CoV-2 spike IgG antibody titers will be measured at 3-4 different timepoints, and CTL responses will be evaluated using an interferon gamma (IFN-γ) enzyme-linked immune absorbent spot (ELISpot) assay. The insights gained from this project will help to inform future DNA vaccine formulations and regimens as more DNA vaccines and repRNA vaccines enter clinical trials.

H. pylori is a gram-negative, helical shaped bacteria that infects more than 50% of the world's population. The shape of bacteria is determined by the peptidoglycan cell wall, which is a macromolecule composed of glycan strands cross-linked by short D-amino acid containing peptide stems. The Salama Lab has identified multiple cell-shape-determining proteins that are required for the helical cell shape of H. pylori, including Csd5. Csd5 is a transmembrane protein and loss of Csd5 causes cells to be straight rods instead of helical. The C-terminal bacterial SH3 domain of Csd5 binds directly to the peptidoglycan cell wall. This project aims to identify whether the SH3 domain of Csd5 binds to a specific feature of the peptidoglycan cell wall. We hypothesize that the SH3 domain binds specifically to tetrapeptides in the peptidoglycan cell wall. To investigate whether the SH3 domain binds to tetrapeptides, we performed a pull-down experiment with purified SH3 domain and purified peptidoglycan from mutants that have varying tetrapeptide content. From this we confirmed that peptidoglycan with higher tetrapeptide content pulls down purified SH3 domain from Csd5 at a higher level than peptidoglycan with lower tetrapeptide content, suggesting that the SH3 domain of Csd5 preferentially binds to the tetrapeptides in the sacculus. In addition, we are visualizing where tetrapeptides are localized in H. pylori cells and identifying whether purified SH3 domain incubated with purified cell walls localizes to the same location by 3D microscopy. By investigating how the SH3 domain of Csd5 interacts with the peptidoglycan cell wall, we will learn more about how Csd5 controls the helical cell shape of H. pylori cells.

Mycobacterium abscessus (MABSC) is a species of rapidly growing nontuberculous mycobacteria (NTM) that is most frequently encountered in human NTM infections and is very difficult to treat. Active MABSC disease commonly emerges from pulmonary infections, to which populations with underlying lung disease and depressed immune systems are most susceptible. There is no official standard of care for MABSC infections and current treatment regimens lack efficacy and are met by challenges of intrinsic and acquired resistance mechanisms. Studies evaluating pulmonary disease outcomes report unsatisfactory treatment success rates of approximately 45%. Thus, there is an urgent clinical need for novel antibacterial agents and drug combinations to efficiently and effectively cure MABSC infections. Repurposing FDA-approved drugs can help us economically discover new treatment options with a shorter drug development time, which is critical for antibiotics as the emergence of resistance often outpaces drug development. We approached the repurposing of approved drugs to treat MABSC infections through the whole-cell screening of a drug library of about 2400 FDA-approved compounds and clinical molecules, followed by the selection and characterization of the activity of the most promising hits. Our data revealed a novel antiemetic compound, netupitant, that exhibited a potent anti-MABSC activity (MIC 4-16 µg/ml) and was able to interact synergistically with standard first-line MABSC therapeutics. Netupitant has a good safety profile and accumulates preferentially in lung tissues, making it suitable for treating pulmonary MABSC infections. Additionally, our screen revealed two promising antibiotic drug combinations: eravacycline/clarithromycin and bedaquiline/amikacin that were able to exhibit potent synergistic interactions against clinical MABSC isolates, as determined by checkerboard microdilution assays. Further mechanistic and in vivo studies are needed to evaluate these hits as potential treatment options to improve the current clinical outcomes for MABSC patients.

DNA replication is vital to most every organism, yet key processes in replication are not yet understood. As the replisome moves through a strand of DNA, it naturally induces a state where the DNA strand wraps around itself, termed ‘positive supercoiling.’ Positive supercoiling knots DNA, preventing DNA from being pulled apart further during replication, with ~100 supercoils formed every second during DNA replication in bacteria. These positive supercoils must be resolved for DNA replication to continue, a task performed by topoisomerase enzymes. However, the mechanism for topoisomerase recruitment to positive supercoils is not known. Growth-Associated Protein in Regulation (GapR) is a structuring protein that stimulates topoisomerases in α-proteobacteria: without GapR, α-proteobacteria die off, unable to replicate their DNA, suggesting GapR is likely a missing regulator to topoisomerase recruitment. The focus of my research is the mechanism for how GapR interacts with topoisomerases, and I hypothesize that GapR interacts directly with topoisomerases. I am studying this interaction by analyzing interacting proteins from interaction assays between GapR and proteins from Caulobacter crescentus. In initial assays, I utilized histidine-tagged GapR to interrogate potential interacting proteins and found multiple bands of proteins in the size range of topoisomerases, supplemented with similar findings for 3xFLAG tagged topoisomerase subunits GyrA and ParC identifying GapR-sized proteins, suggesting a possible direct interaction between GapR and topoisomerase enzymes. Because topoisomerase inhibitors are anticancer and antimicrobial therapeutics, understanding the mechanism of how GapR and topoisomerases interact will reveal crucial information regarding the topoisomerase regulation of DNA replication and could have far-reaching implications for both antibacterial drugs and cancer treatment.

Epithelial cells form barriers at mucosal surfaces that protect against pathogen invasion. Recently, epithelial inflammasomes were demonstrated to be a key component of epithelial immunity. Inflammasomes are multiprotein complexes that function as intracellular sensors of pathogens. Upon pathogen detection, the inflammasome-sensor assembles with the adaptor protein Apoptosis-associated speck-like protein containing a CARD (ASC) to activate the pro-inflammatory protease Caspase-1 (CASP1), which then goes on to activate the inflammatory cytokines IL-1β and IL-18 and the pore-forming protein Gasdermin D, which results in a lytic form of cell death called pyroptosis. In epithelia such as the lung and gut, Nucleotide-binding domain, leucine-rich repeat, pyrin domain-containing 1 (NLRP1) is the predominant inflammasome-forming sensor. However, the role of NLRP1 in corneal epithelia is unknown. To characterize the role of inflammasomes in corneal epithelia, we used the human corneal epithelial cell line hTCEpi as a model. We used CRISPR-Cas9 to knock out genes required for NLRP1 inflammasome signaling (NLRP1, CASP1, and ASC) in hTCEpi cells. To induce NLRP1 inflammasome activation, we treated the hTCEpi cells with Val-boroPro (VbP), an inhibitor of the NLRP1 inhibitor dipeptidyl peptidase 9 (DPP9). We then measured IL-1β levels as a readout of inflammasome activation. We found that VbP-induced inflammasome activation was dependent on NLRP1, CASP1, and ASC. Our findings demonstrate that hTCEpi cells have a functional NLRP1 pathway, and suggest that the NLRP1 inflammasome mediates host defense and/or inflammatory pathogenesis in the corneal epithelium. We plan to test if viruses that cause conjunctivitis elicit an NLRP1 response in corneal epithelial cells, which would implicate NLRP1-mediated responses contributing to inflammation during viral conjunctivitis (i.e., pink eye).

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a γ-herpesvirus that is the etiological agent of Kaposi’s sarcoma (KS), a cancer of endothelial cell origin. Like other herpesviruses, KSHV has distinct latent and lytic replication cycles – during latency there is limited viral gene expression and no KSHV virions are produced, while in the lytic life cycle all viral genes are expressed and new virions are assembled. Both lytic and latent genes are implicated in KSHV’s oncogenic properties. During infection, endothelial cells from both blood and lymphatic vessels undergo changes in signaling pathways and morphology. However, differences in the expression of lytic and latent genes have been described for the different sources of endothelium. We have previously observed that blood endothelial cells (BECs) grown in culture are less susceptible to infection as compared to lymphatic endothelial cells (LECs). Other labs have reported higher levels of lytic gene expression in LECs. I aim to determine if there are differential levels of KSHV lytic gene expression in the BEC and LEC lines in our lab. To determine the levels of lytic replication in BECs and LECs, I isolated RNA at different time points post infection and use RT-qPCR to determine the relative levels of viral lytic genes. I tested different infection rates to determine the role of infection rates on levels of lytic replication. I am also testing different cellular growth conditions, including cell medias to determine if the levels of lytic replication depend on cell proliferation levels. The goal is to determine differences seen in the level of lytic replication in different endothelial cell types in different labs. Understanding the conditions for higher lytic replication of KSHV in endothelial cells could help understand KSHV tumorigenesis as lytic replication is a key factor in the way KSHV causes cancer.