Chlamydia, caused by the bacteria Chlamydia trachomatis, is one of the most common bacterial sexually transmitted infections of the urogenital and gastrointestinal tracts. Preliminary whole genome sequencing of C. trachomatis isolates from women and men who have sex with men (MSM) identified the gene pmpE. This gene encodes for a putative membrane protein that is a key locus of genetic variation between these two populations of isolates. Here, I performed deeper sequencing and structural analysis of natural pmpE variants to better understand how these genetically distinct strains exhibit their tissue tropism differences. PCR-based sequencing of C. trachomatis isolates was conducted against pmpE and ompA to initially type the strain, and then followed by whole genome sequencing to derive complete genomic information. Bioinformatic analysis showed that the cervical and rectal strains are sorted into two distinct clades based on pmpE. To predict possible functional differences, structures of pmpE sequence variants were computationally modeled using Alphafold v3, and analysis demonstrated that variations were confined to predicted surface-exposed loops. Based on the collective data, we hypothesize that two loops may provide functional differences in the ability of cervical versus rectal C. trachomatis strains to interact with tissue-specific host attachment proteins.

Mycobacterium abscessus is a non-tuberculous mycobacterial (NTM) species that causes severe pulmonary infections, particularly in immunocompromised patients and those with preexisting lung diseases such as cystic fibrosis. Treating M. abscessus infections is challenging due to its intrinsic antibiotic tolerance and capacity to develop multidrug resistance. To identify novel molecules that can target this pathogen and enhance current treatments, we screened a library of FDA-approved drugs (n = 2,400). Our data shows that Netupitant, a drug commonly used to prevent chemotherapy-induced nausea and vomiting, exhibits potent antibacterial activity against a broad range of M. abscessus clinical isolates, including multidrug-resistant strains, with a minimum inhibitory concentration (MIC) ranging from 4 to 16 µg/mL. Furthermore, in combination with amikacin, a standard treatment for M. abscessus infections, Netupitant demonstrated strong synergistic interactions, as confirmed by checkerboard microdilution and time-kill assays. These findings highlight Netupitant’s potential as a novel therapeutic option for M. abscessus, particularly in combination with existing antibiotics. Future studies exploring its mechanism of action and in vivo efficacy could further advance antibacterial drug discovery for difficult-to-treat NTM infections.

The obligate intracellular bacterium Chlamydia trachomatis is one of the leading causes of sexually transmitted bacterial infections. Despite its great prevalence and systemic impact on women's reproductive health, no vaccine has been developed. As an obligate intracellular parasite, Chlamydia trachomatis infection occurs within a host-derived vacuole, and Chlamydia have evolved numerous pathways for evading cellular immune defenses. Recent work has highlighted a mechanism through which intracellular Chlamydia interferes with a cell-intrinsic, interferon-gamma (IFN_γ) activated pathway. Our lab discovered a secreted CT virulence factor (GarD) that is important of preventing the IFN_γ -dependent recruitment of ubiquitin to CT-containing vacuoles, and we hypothesize that a gene of unknown function (ct134) immediately upstream of garD may also play a role in this immune evasion pathway. In this project, we used a new genetic system to produce a double knockout mutant for these genes, and we investigated in a cell culture model the fitness and susceptibility of mutants in cell stimulated with human IFN_γ. We expect this work to either define a broader role of this gene locus in mediating chlamydial anti-IFN_γ defenses. In addition, we generated a homologous double knockout lesion in the mouse-tropic pathogen Chlamydia muridarum, and we hypothesize that the C. muridarum homologs of ct134 and garD fulfill this phenotype will support future studies focused on investigating the in vivo role of these genes in Chlamydial infection and virulence in the upper genital tracts of infected female mice.

Members of the gut microbiota produce an array of bioactive metabolites that impact many aspects of host metabolism, immunity, and behavior. However, the mechanisms by which these metabolites are generated remain poorly understood and the biosynthetic enzymes are largely understudied. Recently, our laboratory discovered a family of gut microbial amidases that were found to affect hunger-related biological pathways in malnourished children. These amidases hydrolyze N-acyl ethanolamines (NAEs), lipid messengers with known roles in satiety, visceral pain, and inflammation. Using one of these family members as a model amidase, I am exploring and defining the catalytic mechanisms that are responsible for NAE hydrolysis and the production of a new class of gut microbial metabolites, N-acyl amino acids (NAAAs). I used computational models to predict specific residues that might be important for the amidase activity. I then cloned mutant enzymes into an E. coli expression vector, induced recombinant protein expression, and tested the ability of the purified mutant enzymes to hydrolyze labeled NAEs using liquid chromatography - tandem mass spectrometry (LC-MS/MS). These analyses have pinpointed residues that are important for substrate recognition and binding. My work is advancing our understanding of the selectivity of these intriguing gut microbial enzymes and the regulation of NAAAs in the gut lumen. These efforts are expected to generate the knowledge required to engineer more selective enzymes that produce metabolites of known bioactivity.

Pulling apart DNA during replication induces DNA strands to wrap around each other, producing positive supercoils ahead of the replication fork. Positive supercoils hinder further DNA replication, and are removed by Type II Topoisomerases (Top2s), a group of essential enzymes that cleave positive supercoils to relax DNA for easy separation. Errors in supercoil resolution are linked to diseases like cancer and autoimmune disorders. A key question in the field is the mechanism by which Top2s locate positive supercoils. We recently discovered that GapR, an essential DNA binding protein conserved across α-proteobacteria, binds positive supercoils and stimulates the activity of bacterial Top2s DNA Gyrase and Topoisomerase IV. We hypothesized that GapR recruits Top2s to positive supercoils by direct interaction. We investigated this mechanism by using a Bacterial Two-Hybrid assay to screen for GapR interaction with Top2 subunit and identified an interaction between GapR and the A subunits of DNA Gyrase and Topoisomerase IV. Additionally, we discovered that GapR interacts with Top2 A subunits, and not with Top2 B subunits, in a gel shift assay. In collaboration with the David Baker lab, we generated predictions of the GapR-Top2 interaction which together support a model of interaction between GapR and the Top2 A subunit that is mutually exclusive to the Top2 B subunit. In our current work, we aim to identify the mechanism of direct interaction between GapR and Top2s aided by mass photometry and biochemical experiments to reveal a previously unknown mechanism of Top2 recruitment. Because GapR is conserved by alphaproteobacteria, our research could reveal a target for inhibition by antibiotics. If such Top2 recruiters are more broadly conserved, our work provides a novel pathway to target with anticancer therapeutics as human Top2 inhibitors are important chemotherapy drugs.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus known to cause Kaposi sarcoma (KS), a cancer of the soft tissues, and several other diseases. KSHV has two distinct replication cycles: a latent and lytic cycle. During latent infection, only a small section of the viral genome, the KSHV latency-associated region (KLAR), is expressed. Spindle cells, the main proliferating cell type in KS tumors, are thought to be of endothelial origin and are primarily latently infected. Due to lowered viral gene expression during latency, these cells have few viral factors to target therapeutically. Cellular factors required for infected cell survival, like altered metabolic pathways, are potential therapeutic targets for latently infected cells. One metabolic pathway altered by latent infection is fatty acid synthesis (FAS). My research focuses on understanding how KSHV infection induces FAS in cells. Since previous research has shown that expression of KLAR is sufficient to increase lipid droplet formation, a measure of FAS induction, I hypothesize that expression of one of the four genes present in KLAR is likely what upregulates this metabolic pathway. To test this hypothesis, I infected telomerase-immortalized microvascular endothelial (TIME) cells with lentivirus containing a viral plasmid overexpressing one of the four KLAR genes. I then measured lipid droplet formation across each transduced cell population using a flow cytometer. This project is still in progress; however, if the increase in lipid droplet production in transduced cells is similar to the increase observed in cells latently infected with wild-type KSHV, then I will conclude that the over-expressed gene was sufficient to up-regulate FAS. Identifying which viral gene induces FAS in infected cells will provide a new direction for future mechanistic studies and aid in identifying potential therapeutic targets for KSHV-associated diseases.

The activation of inflammasomes is a crucial component of the early immune response to pathogens and initiates a form of inflammatory programmed cell death called pyroptosis. During infection, a cytosolic inflammasome-forming protein sensor detects a pathogen to assemble the inflammasome complex, which subsequently activates the protease Caspase-1 (CASP1). CASP1 processes Gasdermin D (GSDMD), inducing pyroptosis through pore formation in the plasma membrane, while also facilitating the release of proinflammatory cytokines, such as IL-1β and IL-18. Adenovirus (AdV) is a common pathogen that causes inflammatory symptoms by infecting multiple mucosal epithelial tissues in the respiratory tract and intestinal tract such as the nose, mouth, and eyes. We wanted to test whether AdV infection could activate one of the main inflammasome sensors in human conjunctival epithelial cells (hCjE cells), which is NLRP1. However, we found that upon AdV infection, NLRP1-mediated cytokine release is absent, suggesting that CASP1 signaling is suppressed. Interestingly, despite the loss of IL-1β and IL-18, pyroptosis remains unaffected. Recent studies indicate that in the absence of CASP1, inflammasomes can activate Caspase-8 (CASP8), leading to the cleavage of Caspase-3 (CASP3) and Gasdermin E (GSDME), resulting in an alternative, incomplete form of pyroptosis. Thus, I hypothesize that during AdV infection, host cells are still able to induce pyroptosis by activating the alternative CASP8-GSDME pathway. To test this hypothesis, we generated and validated genetic knockouts of CASP8, GSDMD, GSDME, and CASP3 in hCjE cells to assess their roles in pyroptosis during AdV infection. These findings will provide new insights into viral immune evasion strategies and inflammasome regulation in epithelial cells.

​​​​Bacteria evolve primarily through horizontal gene transfer, the movement of genetic material between organisms that are not directly related. This allows the rapid acquisition of traits like virulence and antibiotic resistance, an increasing public health concern. The mechanisms by which bacteria integrate and control these new traits is incompletely understood. Acquired virulence genes have allowed uropathogenic Escherichia coli (UPEC) to become the predominant cause of urinary tract infections (UTI) throughout the world. A critical step in the UPEC infectious process is the transition from a free-swimming, unicellular, “planktonic” form in the urinary tract to an stationary multi-cellular community, or “biofilm,” when it invades the bladder epithelium. The Fang lab has shown that the transcription factor, MprA, promotes the expression of a horizontally-acquired gene cluster encoding the enzymes required for the biosynthesis of polysaccharide capsule, an important UPEC virulence factor. We hypothesize that this capsule is associated with planktonic growth, and that by regulating capsule, MprA controls the switch between planktonic and biofilm-associated growth. We will test this by growing biofilms of wildtype, mprA, and capsule-deficient mutant strains in the laboratory. Additionally, we will assess their impact on virulence by infecting larvae of Galleria mellonella, the wax moth, with each strain. Understanding how factors like MprA control horizontally-acquired genes can inform the development of future antibacterial therapies.

It is estimated that ~15% of all cancers are caused by oncogenic virus infections. Two of the top seven cancer-causing human viruses are members of the gammaherpesvirus family: Epstein Barr Virus (EBV) and Kaposi’s Sarcoma Herpesvirus (KSHV). Our lab uses Murine Herpesvirus 68 (MHV-68), a mouse gammaherpesvirus with shares significant genetic homology to KSHV and EBV, as a model system to understand how gammaherpesviruses alter the metabolism of their host during lytic infection to promote their replication. We recently metabolically profiled MHV-68 infected host cells at various time points during the lytic infectious cycle. Our data showed nucleotide metabolism is significantly induced in MHV-68 infected NIH/3T3 cells, revealing a potential antiviral target. This study investigates the antiviral efficacy of Methotrexate (MTX), an FDA-approved nucleotide biosynthesis inhibitor currently used to treat cancer, rheumatoid arthritis, and psoriasis. MTX inhibits dihydrofolate reductase (DHFR), an enzyme crucial for producing thymidylate and purine nucleotides, which are essential for de novo nucleotide synthesis. We hypothesized that MTX can block MHV-68 production and be repurposed as an antiviral drug. To test our hypothesis, we first determined a safe concentration of MTX in NIH/3T3 cells using both qualitative (microscopy) and quantitative (trypan blue exclusion) cell viability assays. Next, we infected NIH/3T3 cells with MHV-68 and treated them with a safe level of MTX or solvent control. After 48 hours, we assessed viral production in control vs MTX treated cellular supernatants via viral plaque assays. Our results revealed that MTX significantly suppressed MHV-68 virion production by ~50-fold. These findings suggest that targeting host metabolic pathways could be an effective antiviral strategy against gammaherpesviruses in humans. Further research is needed to explore the use of MTX as a broad viral therapy against other viruses.

In 2023, the World Health Organization (WHO) estimated that there were 263 million malaria cases and 597,000 deaths globally. Parasites of the genus Plasmodium are the causative agent of malaria, deposited into the dermis of a human host through the bite of a female Anopheles mosquito carrying infected sporozoites (spz). From the dermis, spz migrate through the bloodstream and into the liver where they infect hepatocytes, producing potentially thousands of merozoites from a single hepatocyte which then enter the symptomatic erythrocytic stage of the disease. Higher numbers of CD8+ T cells per infected hepatocyte have been associated with Plasmodium clearance and because eliminating all infected hepatocytes during the pre-erythrocytic stage prevents malaria onset, identifying causes of CD8+ T cell recruitment provides critical insights for malaria prevention. The liver is one of the most sexually-dimorphic organs in both mice and humans, leading us to utilize immunohistochemical light microscopy to observe CD8+ cells in inflammatory foci, defined as abnormal concentrations of hepatic nuclei including at least one CD8+ cell. Using digital pathology software, we quantified these in female, male, and orchiectomized male (ORX) BALB/cJ mice that were either unvaccinated or repeatedly vaccinated with radiation-attenuated spz allowing us to assess the role of androgens in this recruitment. We found that following challenge with the rodent malaria wild-type parasite Plasmodium yoelii spz, vaccinated mice had more inflammatory foci and CD8+ cells than unvaccinated mice while intact male mice had fewer CD8+ cell and inflammatory foci than ORX or females of similar vaccination status. These findings suggest that androgens reduce recruitment of CD8+ T cells to inflammatory foci, providing a potential explanation for the reduced parasite clearance in male mice compared to their female counterparts. Further studies should explore the mechanism behind this reduced recruitment to inform important decisions in malaria vaccinology and translational medicine.

Inflammasomes are cytosolic innate immune complexes that initiate pyroptotic cell death and the release of inflammatory cytokines. Inflammasomes are a critical component of the host innate immune response to viral pathogens. The inflammasome-forming sensor NLRP1 functions in barrier defense against a diversity of viral and bacterial pathogens, necessitating multiple modes of pathogen recognition. For instance, NLRP1 directly senses viral infection by detecting viral protease activity. NLRP1 is also activated indirectly by the ribotoxic stress response caused by radiation or toxins. Moreover, NLRP1 has been proposed to directly bind dsRNA. However, it is now understood that dsRNA-induced NLRP1 activation also requires p38-mediated phosphorylation. Thus, it is unclear whether NLRP1 directly or indirectly senses dsRNA. To address how dsRNA activates NLRP1, we reconstituted the NLRP1 inflammasome in inflammasome-deficient 293T cells. We found that reconstitution of the minimal NLRP1 inflammasome responds to viral proteases and other activating stimuli but not to dsRNA. This suggests that NLRP1 is insufficient to respond to dsRNA and instead requires uncharacterized host cofactors. We then hypothesized the NLRP1 response to dsRNA is an indirect event that requires upstream sensing events by canonical dsRNA receptors, and we found that co-expression of RIG-I or MDA5 restores NLRP1 responsiveness to dsRNA in 293T cells. We further investigated this pathway in the context of pathogen infection. During viral replication, dsRNA is generated, and the host has evolved mechanisms to detect it. Since viral dsRNA sensing is detrimental to viral replication, viruses have evolved strategies to evade detection. Notably, influenza A virus (IAV) encodes NS1, a protein that limits dsRNA accumulation. To investigate how IAV potentially counteracts NLRP1 activation by dsRNA, we transfected NS1 into 293T cells reconstituted with the NLRP1 inflammasome system and observed that NS1 significantly attenuated dsRNA-induced NLRP1 activation.

Multidrug resistant Gram-negative bacteria are an emerging threat to public health, continuously evolving to survive under an increasing number of antibiotics and evade the immune system. A major feature of these bacteria is a polysaccharide capsule, which prevents their immune detection. Thus, there is a need to therapeutically restore an effective immune response against them. The Woodward Lab verified that bacteriophage tail spike proteins (TSPs) act as opsonins, which coat and increase phagocytosis of bacteria by macrophages as part of a novel phagocytic pathway. To expand on these data, I am assessing how the adaptive immune system is influenced by the TSP opsonization pathway, analyzing markers of T cell activation and macrophage polarization as starting points. I hypothesize that this pathway has distinct effects on antigen presentation, costimulation, and cytokine expression, compared to better known opsonization pathways like complement and immunoglobulins, and that some of these effects are conserved across bacterial species. To first assess this, I infected macrophages in tissue culture with bacteria, with or without TSP, and measured MHC-II and costimulatory marker expression, an increase which would be associated with enhanced ability to induce T cell responses. I did not observe any differences when TSP was added to the infection. To characterize macrophage cytokine expression, I am treating cultured macrophages with TSP and bacteria-specific antibodies, with the latter serving as a point of comparison between the TSP and antibody opsonization pathways, and quantifying proinflammatory and anti-inflammatory cytokines resulting from this treatment. These studies will reveal whether the TSP opsonization pathway promotes or inhibits adaptive immune responses, which would implicate their utility as a therapeutic and contribute to our understanding of the interaction between bacteriophages, bacteria, and the immune system.

Pseudomonas aeruginosa, a rod-shaped, Gram-negative bacteria, can cause various opportunistic infections, and it is a common pathogen in hospitals because of its antibiotic resistance and virulence. In P. aeruginosa, virulence is primarily regulated by cyclic adenosine monophosphate(cAMP), which binds to two effector proteins: virulence factor regulator(Vfr) and cAMP-binding protein A(CbpA). As cAMP binds to Vfr, this secondary signal promotes transcription of genes involved in virulence, such as the type IV pili system, which mediates twitching motility, and the Type III secretion system, which releases toxins into the host cell cytoplasm. However, regarding CbpA, all that is known so far is that its expression is strongly regulated by cAMP-Vfr signaling, and cAMP-CbpA binding localizes this protein to the P.aeruginosa cell pole. My project aims to determine the function of CbpA and how this effector protein regulates the cAMP-related processes of P.aeruginosa. To meet these goals, I have generated a construct that overproduces CbpA and am making mutant strains lacking cbpA. Using these constructs, I will evaluate how CbpA influences cAMP levels using a fluorescence reporter and assess its function in twitching and swimming motility using macroscopic assays.  Given that cbpA is regulated by cAMP-Vfr signaling, I will perform these experiments in strains of the wild-type (normal cAMP levels), ∆cyaAB(lacking cAMP synthesis, low cAMP levels), and ctx::araBAD-cyaB(inducible cAMP synthesis, high cAMP levels).  These experiments will provide insights into the roles of CbpA in P.aeruginosa virulence and motility. A deeper understanding of cAMP signaling and its effectors will enhance our understanding of the pathogenesis of P. aeruginosa, facilitating the development of therapeutic strategies against its infections.

Pseudomonas aeruginosa (Pa) is an opportunistic pathogen that infects the airways of people with cystic fibrosis, a genetic disease that increases susceptibility to lung infections. Pa uses an intercellular communication system called quorum sensing (QS) that allows bacteria to sense cell density and coordinate behaviors among the population, including regulation of virulence. In the laboratory strain PAO1, there are three complete QS systems in Pa that are regulated by the transcription factors LasR, RhlR, and PqsR. PAO1 QS is organized hierarchically with LasR regulating RhlR, and the hierarchy is influenced by the transcription factor MexT that delays RhlR activity. However, it is unknown if QS hierarchy is found widely in Pa strains. My project tested whether the QS hierarchy exists in clinical isolates of Pa. We obtained 3 clinical isolates with intact lasR, rhlR, and mexT genes and created lasR and mexT knockout mutants for each strain to test the effects on RhlR activity compared to wild-type. To measure RhlR activity, we transformed each strain with a RhlR reporter plasmid. We found that a PAO1 mexT mutant shows greater RhlR activity compared to wild-type, while each clinical isolate showed similar RhlR activity between wild-type and the mexT mutant. We observed lower RhlR activity in clinical-isolate lasR mutants compared to wild-type, demonstrating LasR-dependent QS like PAO1. In PAO1, a ∆lasR∆mexT double knockout mutant restored RhlR activity. Interestingly, in clinical isolates, we observed no change in RhlR activity in these ∆lasR∆mexT double knockout mutants as compared to the lasR mutant, indicating MexT is not regulating QS hierarchy in these clinical isolates. Altogether, the clinical isolates displayed a LasR-dependent QS architecture similar to PAO1, but this was not dependent on MexT. Thus, my work points to undiscovered factors that influence QS architecture and highlight the diversity of QS regulation in strains of Pa.

In Escherichia coli, the Cpx system is understood to be a two-component cell envelope stress response system. In Pseudomonas aeruginosa however, the Cpx system is largely unstudied. Based on predictive modeling, the Cpx two-component system in P. aeruginosa is thought to involve interactions with two novel accessory proteins, PA3203 and PA3207. Previous genetic analysis in our lab has indicated that PA3207 acts as a negative regulator of Cpx signaling, while PA3203 promotes activity of the system. I evaluated biochemical interactions between these two proteins using the Bacterial Two-Hybrid assay. I generated N- and C-terminal fusions to two functional domains (T18 and T25) of an adenylate cyclase enzymatic reporter. Adenylate cyclase activity, occurring when T18 and T25 were brought into proximity by fusion protein interactions, was measured by a qualitative color assay on MacConkey agar. By this method, I confirmed functional interactions between PA3207 and cytoplasmic signaling domains of both CpxS and CpxR. Interactions between PA3203 and CpxSR were also detected, but were more dependent on the orientation of protein fusions. These findings indicate that CpxSR signaling is regulated through protein-protein interactions with multiple accessory proteins, a unique mechanism among bacterial two-component systems.

Kaposi’s sarcoma (KS) is a cancer caused by Kaposi’s sarcoma-associated herpesvirus (KSHV). While most KS tumor cells are latently infected, where KSHV is inactive, all current treatments for herpesviruses target lytic infection. The Lagunoff lab has shown that latent KSHV infection, similarly to cancer cells, induces the Warburg effect, in which glycolysis is used as an energy source rather than oxidative phosphorylation. Inhibition of lactate dehydrogenase (LDH), an enzyme that catalyzes the last step of glycolysis, increases cell death specifically in latently infected cells. This indicated that the KSHV-induced upregulation of glycolysis was necessary for the survival of these cells; however, it is unknown how KSHV induces this requirement. The goal of my proposal is to determine the viral mechanism for the induction of the Warburg effect in latently infected cells. During latent infection, only the KSHV-latency-associated-region (KLAR) of the viral genome is expressed. KLAR encodes 4 genes: vFLIP, vCyc, LANA, the kaposins, and a cluster of 12 microRNAs. I hypothesized that one of the genes or miRNAs is necessary and/or sufficient to induce the requirement for glycolysis in latently infected cells. To test for necessity, I am using KSHV recombinant viruses that have a deletion in vFLIP, vCyc, the kaposins, or the entire miRNA locus to infect endothelial cells. To test sufficiency, our lab has created lentiviral vectors that contain one of the KLAR genes or the miRNA locus to overexpress these genes in endothelial cells. I anticipate that vCyc and/or the miRNA locus might exhibit necessity/sufficiency, since prior studies have identified these as important for the regulation of other metabolic pathways. Understanding KSHV’s alteration of specific metabolic pathways in latently infected endothelial cells provides novel therapeutic targets for the inhibition of latent KSHV infection and ultimately KS tumors.

Malaria, caused by the Plasmodium parasite, remains a relentless and destructive infectious disease, disproportionately affecting children in Sub-Saharan Africa, due in part to the absence of a highly effective, widely deployable malaria vaccine. Lipid nanoparticle (LNP) vaccines are a promising approach for vaccine development, especially against pathogens such as Plasmodium, which have proven historically difficult to vaccinate against. When coupled with the glycolipid adjuvant 7DW8-5 at a 5ug LNP to 0.5ug adjuvant ratio, malaria-targeting LNP formulations confer protection in mouse models. However, the optimal vaccine-to-adjuvant ratio and the mechanisms underlying 7DW8-5-mediated protection remain unclear. Here, we present a study that aims to refine dosing strategies and elucidate the role of CD8+ T and NKT cells in adjuvant-induced protection in a human-translatable mouse model. Different groups of mice will be vaccinated with varying LNP-to-adjuvant ratios, and immune response will be assessed via ELISPOT 28 and 56 days post-vaccination. Furthermore, we will use ELISA to reveal variations in innate immune response between groups 3 hours after vaccine administration. In parallel, we will investigate the necessity of CD8+ T cells and/or NKT cells in protecting from malaria challenge. Mice will be vaccinated using the standardized LNP-to-adjuvant ratio and treated with depletion antibodies targeting CD8+T or NKT cells 24 hours before challenge with Plasmodium sporozoites. Protection will be assessed via blood smear analysis. Our findings will reveal optimal dosing strategies for malaria-specific LNP vaccines and provide insight into the immunological mechanisms behind 7DW8-5-driven protection. This research will contribute to the development of effective nanoparticle-based malaria vaccines — a necessary innovation to help relieve the global malaria burden.

There is a crucial need for a vaccine that produces a robust immune response against Human Immunodeficiency Virus (HIV), particularly for those without access to effective treatments. We investigated the immunogenicity of a novel self-amplifying RNA (RepRNA) vaccine for HIV in non-human primates (NHPs). RepRNA vaccines encode subgenomic sequences that enable the self-amplification of additional copies of RNA, inducing strong immune responses with lower doses of RNA. The repRNA was formulated with a lipid nanocarrier called LION (HDT Bio), which protects the RNA from degradation and enables its delivery into the cell. This platform has shown success in a licensed SARS-CoV-2 vaccine, suggesting it may be similarly promising as an HIV vaccine. I aim to evaluate whether the RepRNA/LION vaccine can elicit robust systemic and mucosal responses in NHPs. I hypothesized that the vaccine would increase HIV-specific T-cell responses in PBMCs and induce HIV Env-specific antibody production in nasal and rectal secretions. To investigate the immunogenicity of this vaccine, we vaccinated twelve cynomolgus macaques, divided into three groups, with HIV Env and/or HIV Gag-Env. To determine vaccine efficacy, I measured the frequency of antigen-specific T-cells in blood using interferon-gamma (IFN-γ) Enzyme-Linked ImmunoSpot (ELISpot) assays because activated T-cells secreting IFN-γ help eliminate infected cells. I also assessed HIV Env-specific Immunoglobulin A (IgA) levels in nasal and rectal secretions using Enzyme-Linked Immunosorbent Assays (ELISAs) because IgA is key in neutralizing pathogens at mucosal surfaces. My preliminary results show an increase in IFN-γ production after the first vaccination, which indicates a systemic antigen-specific T-cell response. We will continue to run assays to see if further vaccination doses can induce more robust immune responses. Results from this study indicate that the RepRNA/LION HIV vaccine may be a promising approach to induce mucosal and systemic immune responses against HIV.

Vibrio parahaemolyticus is a gram-negative marine bacterium that causes acute gastroenteritis in humans generally following the consumption of raw or undercooked shellfish. Mice are highly resistant to many human gut pathogens, including Vibrio, Salmonella, and Shigella spp., which has hindered our understanding of bacterial pathogenesis, immunity, and the development of therapeutics. Inflammasomes are cytosolic innate immune complexes that assemble in response to pathogen infection or harmful stimuli. Once the inflammasome is assembled, inflammatory caspases like caspase 1 are activated, driving a lytic cell death termed pyroptosis and the maturation and release of pro-inflammatory cytokines (i.e., IL-1β, IL-18). Inflammasomes have recently emerged as a necessary mediator of mouse resistance to Shigella and Salmonella, suggesting that inflammasomes may also be the cause of mouse resistance to V. parahaemolyticus. Consistent with that possibility, our preliminary data suggest that inflammasomes prevent intestinal inflammation in mice infected with V. parahaemolyticus, although the mechanism of protection is unknown. To identify the inflammasome(s) responsible for mouse resistance, I reconstitute specific murine inflammasomes in HEK293T cells, which lack most components of the inflammasome pathway. Then, I assess their activation in response to V. parahaemolyticus infection. Our previous findings demonstrated that V. parahaemolyticus robustly activates the mouse NAIP-NLRC4 inflammasome. However, we unexpectedly observed that V. parahaemolyticus infection also induces inflammasome activation in HEK293T cells even in the absence of NAIP-NLRC4 inflammasome reconstitution. This suggests the presence of an inflammasome-sensor in 293T cells that is responsive to V. parahaemolyticus infection. I am currently using inflammasome inhibitors and gene knockouts to identify this unknown inflammasome, which will ultimately aid in our understanding of host factors that mediate host defense against V. parahaemolyticus.

The bacterium Pseudomonas aeruginosa (PA) is an opportunistic pathogen that regulates certain virulence traits via quorum sensing (QS). In PA the QS signaling molecules are acyl-homoserine lactones (AHL). In the well-studied laboratory strain PAO1, there are two complete QS systems: Las and Rhl. Here we study the Rhl system, which has two QS genes that have coevolved and regulate QS activity— rhlI, which codes for an enzyme that produces the signaling molecule N-butyryl-L-homoserine lactone (C4-HSL) and rhlR, which codes for the C4-HSL receptor. In clinical isolates of PA, there are variant rhlR genes, which we hypothesize are important for receptor specificity to C4-HSL and therefore QS activity. We created rhlR genes coding for single amino acid variants of PAO1 RhlR to replicate genotypes found in the clinical isolates. To measure how each variant affects QS activity, we we will use rhlA-gfp as a reporter. The rhlA gene is directly activated by RhlR, and we will compare GFP fluorescence of variants to wild type PAO1 rhlR. QS is a tightly regulated system in PA, and receptor specificity is vital for ensuring this metabolically taxing system is turned on at the right time and properly regulates subsequent protein activity.

Malaria is a mosquito-borne infectious disease caused by Plasmodium parasites and in 2023 caused an estimated 597,000 deaths. Although two currently approved malaria vaccines are available, they offer insufficient protection in endemic populations, which prompts the need for new vaccines. Here we tested several lipid nanoparticle (LNP) vaccines and quantified the number of surviving parasites in vaccinated mice challenged with Plasmodium yoelii sporozoites. To quantify surviving parasites, we utilized the Plasmodium 18S rRNA reverse transcription PCR assay, which is a highly sensitive assay that can quantify the amount of Plasmodium parasites in liver or blood samples. The assay works by amplifying and detecting parasite 18S rRNA in a sample through specific primers, probes and quenchers for mouse GAPDH mRNA and pan-Plasmodium 18S rRNA and can be used to quantify the burden of Plasmodium in a sample. Through the 18S assay, we identified LNP formulations that most effectively protected against rodent malaria. Notably, these LNPs required the adjuvant 7DW85 to be protective.  In the absence of the adjuvant, fewer mice vaccinated with LNPs were protected against rodent malaria. Together, we identified our leading LNP vaccines, which we continue to optimize with the goal of attaining sterile protection against rodent malaria. 

Antibiotic resistant bacteria are an issue of significant global concern and contribute to the increased health burden caused by pathogens. Ongoing research has found that a pandemic urinary tract pathogenic strain of E. coli, ST1193, acquired a high level of fluoroquinolone resistance (FQ-R ) due to two point mutations in gyrA and one in parC, which encode subunits of the topoisomerases DNA gyrase and topoisomerase IV. Rather than a drug sensitive strain acquiring these mutational changes in a sequential, stepwise process, research shows these mutations were acquired by ST1193 during a single horizontal gene transfer event via Hfr conjugational transfer of chromosomal sequences from a distantly related FQ-R commensal E. coli strain. In order to model the evolution of ST1193, we are studying the transfer of the gyrA and parC genes to drug sensitive recipients related to the drug resistant ST1193 strain. I have created a donor derived from an E. coli K12 laboratory strain that can simultaneously transfer mutated gyrA and parC genes to a FQ sensitive recipient. With this I have been able to recover diverse isolates where gyrA and/or parC genes from a FQ-R donor have recombined into the recipient cell chromosomes. Comparisons from whole genome sequencing of these recombinants show a vast diversity in lengths and chromosomal sequences recombined into the recipient chromosomes, ranging from 33kb to as much as 558.5kb. As part of my analysis, I am examining whether some fitness cost are associated in transferring one or two gyrA mutations with or without parC. My comparisons of transfer and recombination events between different branches of the E. coli evolutionary tree are illuminating diverse ways in which bacterial pathogens with high resistance to antibiotics arise.