First Year CBID Trainees
Alexandra Abu-ShmaisResearch: Gaining a better understanding of the fundamental rules of antibody-antigen interactions
Alexandra’s project will focus on gaining a better understanding of the fundamental rules of antibody-antigen interactions. Recently our laboratory developed a technology termed Linking B cell Receptor to Antigen Specificity through Sequencing (LIBRA-seq) that enables the rapid identification of antigen-specific B cells. Using this technology, we have successfully identified B cells capable of recognizing antigens encoded within human immunodeficiency virus (HIV), hepatitis C virus (HCV), and influenza virus. LIBRA-seq turns BCR-antigen interactions into sequence-able events using DNA barcoding, antigen-specific B cell sorting and single-cell sequencing. As her primary project, Alexandra will leverage the LIBRA-seq technology to construct a human antibody-antigen atlas with specificity towards antigens from a number of common viral pathogens, such as influenza, respiratory syncytial virus, human metapneumovirus, and others. Due to the continuous exposure every person experiences to infection by or vaccination against these common pathogens, antigen-specific B cells can be identified in virtually anyone. Yet, there is still extremely limited information about the repertoires of B cells that different individuals use to recognize these common pathogens and vaccines. With the application of the LIBRA-seq technology, Alexandra will aim to create a high-resolution “atlas” of antibodies and their antigen specificity, resulting in unparalleled depth of information about antibody-antigen interactions. Identified B cells will be produced as recombinant monoclonal antibodies and tested in various assays to define their structural, functional, and other characteristics. Aligned with the CBID goals, this project will entail a variety of biochemical methods, such as protein purification and binding assays, oligonucleotideprotein conjugation, structural biology, and others, in order to gain novel insights into the fundamental rules of antibody-antigen interactions for a variety of pathogens. Furthermore, the outcomes of this proposal may lead to the discovery of novel antibodies of potential therapeutic or vaccine template interest against biomedically relevant agents of infectious disease.
Mentor: Ivelin Georgiev, Ph.D.
Jamisha FrancisResearch: The human innate immune protein S100A12 mitigates zinc stress and promotes Group B Streptococcus pathogenesis
Group B streptococcal (GBS) or Streptococcus agalactiae infections are one of the top five leading causes of neonatal mortality, causing chorioamnionitis, fetal infection, neonatal sepsis and preterm birth. GBS colonizes the urogenital and/or the gastro-intestinal tract of about 40-50% of healthy women in the United States. Innate immune cells like neutrophils respond to GBS and deposit antimicrobial molecules like S100A-family proteins. S100A12 or Calgranulin C is a calcium binding proinflammatory protein that is secreted by granulocytes. Under conditions of low metal availability, S100A12 inhibits GBS growth via zinc chelation, a result that was reversed by the addition of exogenous zinc. In conditions of high zinc availability, S100A12 mitigates zinc stress in GBS and promotes biofilm formation on abiotic and biotic surfaces. Conversely, the addition of zinc represses bacterial biofilm formation on polystyrene, gestational membranes and the instrumented fetal membrane on a chip (IFMOC). GBS also mitigates zinc stress via deployment of the CadD efflux determinant, which aids in zinc detoxification. Our proposed Aims will seek to determine the role that cadD plays in promoting biofilm formation on gestational tissues, IFMOC, and in vivo under differing conditions of dietary zinc intake. These aims fit within the mission of the training grant and will promote investigation at the interface of chemistry and biology.
Mentor: Jennifer Angeline Gaddy, Ph.D.
Christopher GoodResearch: IMS of Staphylococcus aureus Osteomyelitis
Uncovering the complex biological processes which drive pathogenesis requires multimodal technologies that consolidate multi-omics data with spatial and temporal analysis in tissue. Matrix assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is one such technology that offers spatially correlated molecular detection with high sensitivity and label-free chemical specificity. In our laboratory, MALDI IMS has been successfully applied to infectious disease models in order to understand the dynamic molecular interactions at the host-pathogen interface.
We are interested in exploring Staphylococcus aureus infection in situ to discover bacterial and host factors fundamental to abscess pathology. Currently, I have piloted MALDI IMS lipidomic studies of undecalcified cortical bone, marrow, and surrounding tissue. Using a murine osteomyelitis model, lipid alterations localized to inflammatory lesions will be identified after proper registration of complementary histological techniques. Proteins involved at the foci of infection will be defined and correlated to lipid signatures by using additional spatial proteomic methods. Molecular heterogeneity between abscesses and differences attributed to a host comorbidity like hyperglycemia are unique research directions obtainable by MALDI IMS.
Regarding the mission of the CBID Training Program, elucidating the factors involved in pathogenesis with MALDI IMS is a valid approach to combating the growing threat of infectious diseases. Classic industrial approaches to antibiotic and drug discovery are insufficient; a more advanced technical investigation of the processes involved is necessary to further drug development and improve human health.
Mentor: Richard M. Caprioli, Ph.D.
Samantha GrimesResearch: Biochemical and genetic determinants of small molecule inhibitors of coronavirus replication
Samantha’s research project will focus on the biochemical and genetic determinants of small molecule inhibitors of coronavirus replication. The pandemic SARS-CoV-1 (COVID-19) pandemic has with incredible force demonstrated the capacity of CoVs to emerge to cause profound health consequences and societal disruption, perhaps unprecedented in human history. The need for effective countermeasures (antivirals, monoclonal antibodies, vaccines) is both immediate and long-term. The Denison lab has been a world leader over the past 10 years in preparing for this eventuality, preforming pre-clinical development on two drugs already in human testing in COVID- 19 patients: Remdesivir and EIDD-1931. Both are nucleoside analogs that target the viral RNA dependent RNA polymerase to interfere with viral RNA synthesis by chain termination (remdesivir) and lethal mutagenesis (EIDD-1931). An important feature of both compounds is their ability to evade the unique CoV proofreading exonuclease to inhibit the viral polymerase. Samantha’s project will focus on the use of remdesivir and EIDD-1931 in combination to test their impact on efficacy, prevention of resistance, mechanism of action against the proofreading exonuclease, and testing additional nucleoside analog inhibitors as well as potential direct inhibitors of the exonuclease. She will initiate work with the model CoV, murine hepatitis virus, which has been directly applicable in the results to MERS and SARS-CoV. This will allow rapid progress and testing at BSL2. Concurrently she will initiate training for work at BSL3 with SARSCoV- 2 and will be able to apply her results on inhibitor efficacy, combination Rx, exonuclease mutations and other findings directly to SARS-CoV-2. Based on our previous studies, student outcomes and her project, I believe this work will be of high impact from both research productivity and training perspectives.
Relevance to CBID: The project has high relevance to the CBID in that the project will use small molecule inhibitors to directly attack CoV replication and determine the virological response to identify new targets. Additionally, this project will work to directly support global efforts to control the COVID-19 pandemic and address future zoonotic CoVs. Further, the project is integrated across multiple disciplines – genetics, biochemistry, pathogenesis, drug development - and is based on established collaborations with a pharmaceutical company and UNC for animal model development. Samantha is very interested in this interface of fundamental biology, biochemistry, and throughput to translational potential. Samantha’s background and training are in microbiology and immunology with demonstrated success in these areas. Now with training and direct application to viral inhibition the project is likely to lead to important discoveries and prepare her for a career at the interface of Chemistry and Biology.
Mentor: Mark R. Denison, M.D.
Second Year CBID Trainees
Grace MoralesResearch: Defining the role of CDT binary toxin in the context of Clostridium difficile infection
Clostridium difficile is a gram-positive, spore-forming anaerobe, and a leading cause of nosocomial infection in the United States. The infection is dependent on the secretion of one or more AB-type toxins: toxin A (TcdA), toxin B (TcdB), and the C. difficile transferase toxin (CDT, or binary toxin). While TcdA and TcdB are considered the primary virulence factors, multiple studies suggest that CDT increases the severity of CDI. While the mechanism of how CDT contributes to disease is unclear, there are reports that suggest roles for CDT in epithelial cell remodeling and inflammasome activation. This work will define the cell types targeted by CDT and the consequences of CDT intoxication in the context of infection. Training in chemical biology will drive the generation of innovative reagents (ex. toxins with site-specific crosslinkers, fluorescent reporter constructs, cellular probes, and nanobodies) for addressing fundamental questions about CDT function. A better understanding of how CDT contributes to disease is expected to guide vaccine development and therapeutic strategies.
Mentor: Borden Lacy, Ph.D.
Catherine SheltonResearch: Propionate utilization by Salmonella provides growth advantage during infection
Propionate utilization by Salmonella provides growth advantage during infection Salmonella enterica is an enteric bacterial pathogen that causes 1.2 million infections every year. During infection, Salmonella induces intestinal inflammation through virulence factors deployed by two type III secretion systems. This pathogen-induced gut inflammation leads to the production of new compounds, such as tetrathionate and nitrate, by the host. Salmonella is capable of using these compounds as alternative electron acceptors to undergo anaerobic respiration. By performing anaerobic respiration, Salmonella is able to outgrow the resident microbiota which lack the ability to respire and instead rely on fermentation for energy production. In order to effectively respire, Salmonella must find carbon sources within the nutrient-limited environment of the gut. A potential carbon source within the gut is propionate, a short chain fatty acid produced by the resident microbiota. Importantly, Salmonella possesses machinery that enables utilization of propionate as a carbon source through the prpBCDE operon. This project will investigate the hypothesis that propionate can be used by Salmonella to grow during infection. To do so, Salmonella mutants will be generated in the prpBCDE operon and growth will be monitored in differing concentrations of propionate under fermentation and anaerobic respiration conditions. We will use gene expression profiling and epithelial invasion assays to determine the interactions between propionate metabolic pathways and expression and function of virulence genes. Additionally, Salmonella prpBCDE mutants will be used for in vivo infections in a mouse model to determine if propionate utilization confers a growth advantage to Salmonella during intestinal inflammation. If successful, this research will provide a deeper understanding into a novel mechanism used by this bacterial pathogen to outsmart the intestinal microbiota and establish infection.
Sydni Caet SmithResearch: Mechanisms by which viruses of the Reoviridae family acquire genetic diversity and interact with the host to mediate infection
Extracellular vesicles, including exosomes and ectosomes, are used for intercellular communication and cargo transfer, including modulation of inflammation and immunity. Apoptotic bodies are used to systematically clear dead cell debris. A small but growing body of research suggests several viruses are transmitted in extracellular vesicles, which may enable immune evasion and multi-particle transmission. In the Ogden lab, we study mechanisms by which viruses of the Reoviridae family acquire genetic diversity and interact with the host to mediate infection. We have previously observed reovirus release in extracellular vesicles similar in size to ectosomes or apoptotic bodies. We hypothesize that reovirus transmission in extracellular vesicles promotes genetic complementation by enabling simultaneous multi-particle infection of target cells. For my thesis project, I will determine (1) the origin of reovirus-containing vesicles released from infected cells and (2) effects of vesicle-contained virus release on genetic complementation in vitro.
Mentor: Kristen M. Ogden, Ph.D.
Sirena TranResearch: Identifying and characterizing proteins that accelerate evolution (“evolvability factors”)
One of the most pressing clinical challenges that we are currently facing today is the antimicrobial resistance (AMR) problem. Historically we have been trying to battle this problem by creating more potent antibiotics. However, it has become evident that this approach is failing. Part of the efforts in the Cover lab are focused on the development of an alternate strategy to resolve this problem: inhibiting evolution. As the first step towards this goal, the lab is identifying and characterizing proteins that accelerate evolution (“evolvability factors”). One of these proteins is the DNA translocase and RNA polymerase interacting protein, Mfd. The lab has recently found that Mfd is required for the rapid development of AMR, to multiple classes of antibiotics in highly divergent bacteria. However, it is unclear 1) how Mfd increases mutagenesis and 2) whether the microbiome impacts Mfd-dependent AMR development (or AMR development in general). To investigate these questions, Salmonella typhimurium will be used as the model system with various biochemical, molecular biology, and genetic techniques. Additionally, a mouse model of evolution will be developed where we can both monitor the kinetics and degree of AMR development in bacterial pathogens in “regular” versus germ-free mice with and without Mfd.
Mentor: Timothy L. Cover, M.D.
Michelle WiebeResearch: Investigating how the BtsSR and YpdAB systems interact to regulate serine and pyruvate metabolism
Two-component signaling (TCS) systems are an important way bacteria sense and respond to changes in their environment. TCSs are typically composed of a sensor histidine kinase and its cognate response regulator. Recent work has revealed that noncognate components of different TCSs can cross interact with each other, complicating the relatively simple signal transduction of these systems. One example of this cross interaction in Uropathogenic E. coli (UPEC) is BtsSR and YpdAB. Serine-pyruvate homeostasis in UPEC appears to be controlled by two two-component systems, BtsSR and YpdAB, with BtsS and YpdA being the receptors and BtsR and YpdB being the corresponding cognate response regulators. Previous studies by our lab demonstrated that YpdAB and BtsSR are active during acute urinary tract infection, where serine is one of the key amino acids utilized by UPEC. Serine is imported into the cell via the action of the SdaC transporter and is then broken down into pyruvate and ammonia by the actions of the SdaA and SdaB enzymes. My project will investigate how the BtsSR and YpdAB systems interact to regulate serine and pyruvate metabolism.
Mentor: Maria Hadjifrangiskou, Ph.D.
Third Year CBID Trainees
Katherine AlmasyResearch: Probing Host-Pathogen Protein-Protein Interactions During Flavivirus Infection
Flaviviruses, such as Dengue virus, are known to co-opt several host factors within the host cell endoplasmic reticulum (ER) proteostasis network, which is composed of chaperones and other factors important for protein folding, assembly, and quality control. As the virus begins to translate, replicate, and assemble new virions around the ER membrane, the unfolded protein response (UPR) is activated to increase the folding capacity of the organelle. It is composed of three distinct signaling branches, each of which the virus modulates. The specific function of each UPR branch in the context of viral propagation is not well characterized, largely due to a dearth of tools to selectively modulate each branch. We hypothesize that some of the protein-protein interactions between host proteostasis factors and viral proteins are crucial for viral propagation. Using small molecule modulators of the UPR, the project’s aim is to develop a profile of how selective chemical modification of each branch affects specific protein-protein interactions which are critical for viral propagation. Various transcriptomic and proteomic techniques will also be used to determine which steps in the viral life cycle are affected by the modulation. This work may be extended to other flaviviruses such as Yellow Fever and Zika.
Mentor: Lars Plate, Ph.D.
Casey ButricoResearch: Host immunity during Staphylococcus aureus osteomyelitis infections mediates bacterial metabolic adaptations
The molecular mechanisms that facilitate nutrient acquisition in S. aureus in osteomyelitis is of great interest. This project will utilize forward genetic screens to identify nutrient transporters and surface molecules of interest, which will then be validated by mono-infections to assess their essentiality in vivo. Based on previous data and preliminary studies, the driving hypothesis is that fibrinogen-binding proteins that enhance surface adhesion in abscesses as well as nutrient transporters that facilitate the uptake of alanine are crucial for S. aureus growth in bone. In addition, manipulation of immune pathways and populations in mouse models of osteomyelitis will be used to address the role of the innate immune system during infection. The implications of the host immune response on S. aureus nutrient acquisition will be determined using a transposon mutant library. In parallel, creation of a unique S. aureus pooled mutant library with distinct defects in central metabolism marked by a variety of antibiotic resistant cassettes will provide a valuable tool for deciphering which metabolic pathways are crucial for S. aureus survival in a variety of in vivo models. Finally, biochemical strategies will be implemented to modulate the host immune system and assess the metabolome at the host-pathogen interface.
Mentor: James E. Cassat, M.D., Ph.D.
Catherine LeasureResearch: Heme Homeostasis Mechanisms in S. aureus
Staphylococcus aureus is a major human pathogen that can cause devastating disease in almost every site in the body. With antibiotic resistant strains becoming increasingly prevalent, basic research is needed to identify new targets for antimicrobial development. This research project focuses on mechanisms that S. aureus uses to maintain heme homeostasis. Because the bacterium is capable of both acquiring exogenous heme and synthesizing it de novo, it must tightly regulate heme biosynthesis to avoid accumulation of toxic levels. Previous work in the Skaar laboratory has shown that both heme and the membrane protein HemX post-transcriptionally regulate the levels of GtrR, a key heme biosynthetic enzyme. To define the mechanism of this regulation, current experiments are using systematic mutagenesis of GtrR to identify residues important for regulation of heme synthesis. In addition, genetic selection is being leveraged to uncover new players in the regulation of heme synthesis as well as key residues in known regulators or enzymes. A better understanding of how S. aureus regulates the synthesis of heme could lead to the development of new antimicrobials and help combat the development of multi-drug resistance.
Mentor: Eric P. Skaar, Ph.D., M.P.H.
Fourth Year CBID Trainees
Samuel DooyemaResearch: Unearthing microbial mechanisms and host responses to DNA translocation by Helicobacter pylori
Helicobacter pylori is a bacterial pathogen that conveys the highest known risk for gastric cancer, the third leading cause of cancer-related death worldwide. One H. pylori oncogenic determinant is the cag type IV secretion system (T4SS) which translocates pro-inflammatory effectors, such as CagA and peptidoglycan, into epithelial cells. It has also recently been demonstrated by our lab that H. pylori can translocate DNA into host epithelial cells in a cag-dependent manner and subsequently activate the innate immune receptor TLR9, which detects and responds to hypo-methylated CpG DNA motifs commonly found in microbial genomes. TLR9 expression is increased in gastric cancer tissue compared with normal gastric tissue, and H. pylori strains that confer a higher risk for gastric cancer are more potent in their ability to activate TLR9. However, TLR9 activation is known to have both pro- and anti-inflammatory effects in humans. Therefore, my overarching hypothesis is that H. pylori DNA translocation and selective activation of TLR9-dependent pathways contributes to the increase in carcinogenic risk conferred by H. pylori cag+ strains by promoting persistence and activating epithelial responses with carcinogenic potential. My project will test this hypothesis via the following aims: 1) defining the microbial mechanisms that regulate this rare, trans-kingdom DNA translocation resulting in TLR9 activation by H. pylori, and determine the identity of the translocated DNA, 2) determining the host responses to translocated DNA by visualizing translocated H. pylori DNA on its intracellular course and examining its ability to activate additional host nucleic acid sensing pathways, and 3) elucidating the impact of DNA translocation and TLR9 activation on gastric inflammation and carcinogenesis in vivo.
Mentor: Richard Peek, M.D.
Michael DoyleResearch: Characterizing the Human Antibody Response to Henipavirus Infection
Hendra (HeV) and Nipah (NiV) viruses, the prototypic henipaviruses, are emerging zoonotic paramyxoviruses known to cause severe disease across six mammalian orders, including humans. Carried by flying foxes, these viruses infect humans through a variety of avenues, and human-to-human transmission has been observed. Humans infected by Hendra or Nipah viruses display severe respiratory and/or neurological disease, with mortality rates reaching as high as 90%. With such high morbidity and mortality associated with infection, coupled with the potential for global spread and use as agents of bioterrorism, there is significant interest in understanding how the human adaptive immune system neutralizes these viruses and using this information to develop novel therapeutics and vaccines. To this end, this project involves isolating henipavirus-specific monoclonal antibodies from survivors of natural Hendra or Nipah virus infection, and from a subject who was inoculated with the Hendra equine vaccine. By utilizing a highly optimized human hybridoma technique to first isolate antibodies, over 50 antibodies have been isolated to date. A variety of biochemical techniques will be employed to define antigenic sites on the henipavirus glycoproteins recognized by these antibodies. In collaboration with scientists at UTMB and USU, antibodies will also be evaluated for neutralization capacity against fully virulent Hendra and Nipah virus isolates and for therapeutic efficacy in ferret challenge models. The studies will ultimately provide a detailed understanding of how the adaptive arm of the human immune system can combat these deadly viruses, and lead to antibody-based therapeutic candidates against Hendra and Nipah viruses.
Mentor: James E. Crowe, Jr., M.D.
Kelsey PilewskiResearch: Antibody Cross-Reactivity in Chronic HIV/HCV Co-infection
In the fight against highly genetically diverse and immune system-evading pathogens such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV), co-infection only compounds the problem. The roles of humoral-mediated immunity against both HIV and HCV have remained controversial, as natural sterilizing immunity is rarely achieved in HCV infection, and never achieved in HIV infection. Numerous neutralizing antibodies have been isolated from HIV- and HCV-infected donors over the years, leading to many novel vaccine candidates, therapeutics, and clinical trials, yet determinants influencing the development of a protective humoral response remain poorly understood. The most successful antibodies against these viruses recognize not only the strain with which an individual is infected, but also additional disparate viral variants. Therefore, although antigen specificity is almost always in the best interest of the host, antibody polyspecificity seems to provide selective advantage in defense against highly mutable pathogens. As this multi-specificity allows for recognition of multiple variants within a virus family, we wanted to investigate the extent to which antibody cross-reactivity can extend across viral families. In order to examine this question, we isolated a panel of antibodies from the peripheral blood of a HIV/HCV co-infected donor that can bind to both HIV and HCV envelope glycoprotein antigens. Interestingly, these antibodies are encoded by divergent germline variable genes, suggesting multiple potential modes of binding. Using next-generation sequencing techniques at multiple time points, we will characterize the development of these antibodies over the course of chronic co-infection. The goal of this project is to examine how polyreactivity contributes to the development of broadly neutralizing anti-viral antibodies.
Mentor: Ivelin Georgiev, Ph.D.
Jade WilliamsResearch: “Total Synthesis of the Siderophore Coelichelin and Alkylpiperidine Arenosclerin A”
The primary project focuses on the development of efficient and concise syntheses of naturally occurring microbial metabolites in order to enable their biological study. There is a growing need for novel therapeutic agents and identification of bacterial targets in the treatment of infectious disease. Thus far, successful completion of the synthesis of the siderophore coelichelin has enabled a collaboration with the Skaar Lab. To date, it has been demonstrated that synthetic coelichelin as well as an N-acylated analog can be utilized to promote bacterial growth in gram-negative, P. aeruginosa. Preliminary results indicate that coelichelin and its analogs could be used in the development of siderophore-antibiotic conjugates and enable advanced study of metal acquisition pathways. A second project focuses on the development of a synthetic route allowing access to the alkylpiperidine arenosclerin, which has not previously been synthesized. Arenosclerin has demonstrated antibacterial activity against several antibiotic-resistant clinical isolates in both gram-negative and gram-positive bacteria. However, further evaluation of arenosclerin has been limited by its availability. An efficient synthetic strategy to provide sufficient material for biological study has now been developed. Following total synthesis of arenosclerin, preliminary studies will commence to further characterize its antibacterial activity and identify its cellular target.
Mentor: Gary A. Sulikowski, Ph.D.
Fifth Year CBID Trainees
Eric HusemanResearch: Total Synthesis of Arimetamyin A and Biological Evaluation of Its Disaccharide Glycan
Arimetamycin A is a recently isolated anthracycline natural product with potent anticancer activity against various cell lines, including two lines resistant to doxorubicin and daunorubicin, which are among the most commonly employed anthracyclines in the clinic. Structurally, Arimetamycin A features a steffimycin type aglycone decorated with a disaccharide composed of two branched, deoxy amino sugars named brasiliose and lemonose. We have devised a semisynthetic route to Arimetamycin A that will derive the aglycone from Steffimycin, the brasiliose sugar from Vancomysin, and the lemonose sugar from D-threonine. To this point, we have synthesized and united the two sugar units and are currently working to transform the disaccharide into a molecule that can be joined with the aglycone to complete the total synthesis.
Mentor: Steven D. Townsend, Ph.D.
Amyn MurjiResearch: The Development of Therapeutic Antibodies and Vaccines Targeting Viral Pathogens
HIV-1 continues to impose a large global health burden. Candidate vaccines using HIV-derived antigens have not proven effective to date, and efforts toward protection against new infections remain a high priority in HIV-1 research. In recent years, strategies that target the elicitation of broadly neutralizing antibodies (antibodies that are capable of neutralizing a large fraction of circulating HIV-1 variants) have emerged as a potential avenue to a prophylactic HIV-1 vaccine. The sole target of neutralizing antibodies elicited thus far is the envelope protein (Env) of HIV-1. However, due to the extensive global diversity of HIV-1, Env-based vaccine candidates have only led to the elicitation of antibodies with limited neutralization breadth. To address this challenge, we are developing technologies for the simultaneous presentation of multiple diverse Envs to the immune system. We are designing and validating a number of these technologies in animal models and are assessing the elicited antibody response to determine efficacy.
Mentor: Ivelin Georgiev, Ph.D.