Microbiology and Immunology
The following grants/studies are under way in the Department of Microbiology and Immunology
within the School of Medicine.
Structural requirements for sterol 14-alpha-demethylases
Funding source: National Institute of General Medical Sciences (NIGMS) R01 subcontract
Researcher: Fernando Villalta, Ph.D.
Project Summary: Sterol 14α-demethylase (CYP51) is a drug target in lower eukaryotic pathogens such as Trypanosoma cruzi, which causes Chagas disease, because the reaction it catalyzes is essential for membrane formation and therefore loss of this activity is lethal. There are no effective treatments for Chagas disease, which causes significant mortality worldwide and represents a serious global health problem. In this project, in collaboration with Dr. Galina Lepesheva at Vanderbilt University, the team focuses on structure-based development of selective inhibitors for protozoan CYP51s. They also will synthesize derivatives of the three original scaffolds they discovered that bind to the active site of the enzyme and analyze the efficacy of the best of these derivatives in disease models. The results arising from these studies will lead to rational design of pathogen-selective drugs for the treatment of Chagas disease.
Molecular analysis of trypanosome infection
Researcher: Fernando Villalta, Ph.D.
Founding source: National Institute of Allergy and Infectious Diseases (NIAID)
Project Summary: Dr. Villalta’s laboratory studies the molecular interface between the human protozoan Trypanosoma cruzi and its host, including the host and pathogen molecules required for intracellular parasitism, mechanisms of pathogenesis and strategies for control. These studies combine a genetics system approach with protein structural analysis, knockout and transgenic animal and parasite models, electron, video and confocal microscopy, functional genomics, gene network analysis and bioinformatics in acute and chronic models of disease. This research program is aimed to discover the cellular and molecular basis of host cell invasion by T. cruzi and identify the molecular signatures caused by the parasitein host cells leading to pathogenesis and disease. Fundamental questions about the structural basis of molecular interactions involved in the pathogenesis of Chagas disease are being addressed. This work will provide clues to the structure and function of the key virulence factors that are implicated in gastrointestinal and cardiac pathology of Chagas heart disease.
Manipulation of macrophage Alu RNA metabolism by breast cancer cells
Researcher: Gautam Chaudhuri, Ph.D.
Founding source: National Cancer Institute (NCI)
Project Summary: Macrophages are a heterogeneous collection of terminally differentiated mononuclear phagocytes that are distributed all over the mammalian body to perform tissue clearing, tissue remodeling and other functions. These cells act in diverse capacities as the primary responders of our innate and adaptive immune systems. As a part of their tissue clearing roles, macrophages recruited at the site of breast tumor development ingest significant mass of cellular debris formed from the initially dying tumor cells. High levels of phagocytosis induce the production of reactive oxygen and nitrogen species, which in turn elevates the expression of cytotoxic non-coding RNAs (ncRNAs) like Alu RNAs. If not interfered by the tumor cells, high levels of Alu RNA would produce severe inflammatory immune responses through the activation of the inflammasome and accelerated death of the overfed macrophages bringing collateral damage to the breast tumor cells. The preliminary data generated in the PI's laboratory revealed that during the breast tumor formation, inflammatory development of the recruited macrophages is prevented by breast tumor cells through the reduction of Alu RNA levels in the tumor-associated macrophages converting them to accessory cells supporting tumor growth. We postulate that breast tumor cell-induced reduction in the levels of the cytotoxic Alu RNA in tumor-associated macrophages creates a tissue microenvironment that fosters tumor progression. The long-term goal of the proposed research is to understand how breast tumor cells manipulate macrophages to make them docile and supportive to the development of breast tumors. The central hypothesis is that as a part of the regulation of macrophage turnover, oxidative stress generated during intense phagocytosis by the macrophages at the site of tissue damage (e.g. breast tumor) increases the levels of cytotoxic Alu RNA in these cells. This response in breast tumor-associated macrophages (TAMs) is suppressed by the tumor cells so that these longer-living immune-suppressed macrophages in the tumor microenvironment can be utilized for further development and progression of the breast tumor. Specific Aims to test the hypothesis are: (a) Identification of the mediators secreted from breast cancer cells that manipulate Alu RNA metabolism in the macrophages; and, (b) Evaluation of the mechanisms of manipulation of Alu RNA metabolism in tumor-associated macrophages during breast tumor development. This research proposes an innovative basic mechanism to explain macrophage-assisted breast tumor development highlighting the critical importance of Alu RNAs in the maintenance and disposal of macrophages. The proposed research will make a significant contribution because it will not only highlight a new direction in the understanding of macrophage biology and the etiology of macrophage-associated breast tumor development but also will lead us towards the development of rational chemotherapy against such diseases of the breast.
Characterizing a small molecule of Streptococcus cristatus for HIV drug design
Researcher: Bindong Liu, Ph.D.
Founding source: National Institute of General Medical Sciences (NIGMS)
Project Summary: The overall number of people living with HIV-1 has continued to increase in all regions of the world. With no prospect for an effective vaccine, containment of the spread of HIV-1 relies on measures to prevent transmission, and treatment relies on antiretroviral therapy. However, drug resistance is becoming increasingly problematic, with some individuals harboring and transmitting viruses that are resistant to a number of different drugs. Thus, an increasing number of people are left with little or no options for new therapeutics. This highlights the need for the development of new antiretroviral agents. We recently discovered that a small molecule of Streptococcus cristatus CC5A (S. cristatus CC5A) is able to up-regulate APOBEC3G and APOBEC3F expression and inhibit HIV replication. This specific research is aimed to characterize this small molecule and to examine the clinical potential of this small molecule in development of novel anti-HIV drugs. We hypothesize that the small molecule of S. cristatus CC5A enhances innate immunity through up- regulation of APOBEC3G and APOBEC3F expression and could be a potential novel anti-HIV drug candidate. To test the hypothesis, we propose following specific aims. (1) To identify and characterize the small molecule of S. cristatus CC5A that promotes A3Fand A3G expression. (2) To exploit the possibility that S. cristatus upregulates APOBEC3 and inhibits HIV replication or transmission in primary HIV target cells, including CD4+ T-cells, macrophages and dendritic cells. (3) To elucidate the mechanism by which this small molecule activates APOBEC3 gene transcription. The successful completion of this proposal will lay the groundwork for developing the small molecule to a novel anti-HIV drug.
Contribution of cellular factor to HIV-1 assembly
Researcher: Xinhong Dong, Ph.D.
Founding source: National Instituteon Minority Health and Health Disparities (NIMHD)
Project Summary:The global HIV epidemic continues to expand exceeding previous predictions and has become one of the deadliest epidemics in human history. The high prevalence of HIV infection in African-American women points to the need to develop new medical interventions toward eliminating women health disparities in HIV/AIDS. The emergence and transmission of HIV-1 isolates resistant to currently approved drugs makes the discovery of novel anti-HIV drugs with new mechanisms and targets a high research priority. HIV-1 Gag protein directs the highly ordered process of particle assembly and release. Distinct steps involved in these late stages of the HIV-1 replication cycle are being defined, yet significant gaps still need to be filled in our knowledge. By yeast two-hybrid screening of a human cDNA library, we identified a novel Gag-binding partner, filamin A. Filamin A (FLNa) is a non-muscle actin binding protein that plays an important role in cross-linking cortical filaments into a dynamic three-dimensional structure. FLNa interacts with different cellular proteins, and serves as a versatile scaffold required for protein trafficking, signaling transduction, and cell-cell and/or cell-matrix connections. The discovery of the FLNa-Gag interaction in a productive manner in HIV-1 particle assembly and release suggests that FLNa facilitates HIV-1 Gag trafficking to the plasma membrane by regulating the actin cytoskeleton remodeling. The overall goal of this research is to define the molecular basis of the FLNa-Gag interaction and its biological significance. Our studies will provide important new information regarding retrovirus-host interactions, and will impact anti-HIV therapy by discovering and developing novel assembly inhibitors. This research proposal will be accomplished in a series of experiments organized within three integrated specific aims. Specific Aim 1: To define the molecular basis of the FLNa-Gag interaction. Specific Aim 2: To define the mechanism of FLNa-regulated HIV-1 Gag trafficking. Specific Aim 3: To define the role of FLNa in human primary CD4+ T cells and macrophages.
Novel Targets for discovering peptide inhibitors of HIV replication
Researcher: Xinhong Dong, Ph.D.
Founding source: National Institute of Allergy and Infectious Diseases (NIAID)
Project Summary: The continual emergence of HIV strains that are resistant to currently approved anti-HIV drugs is an increasing threat to the effective treatment of HIV infection and control of the HIV/AIDS epidemic. Therefore, the discovery and development of new anti-HIV drugs with novel antiviral mechanisms and targets are urgently needed. The long-term objective of this research is to develop a novel class of anti-HIV drugs representing novel chemical entities targeting late stages of the HIV-1 replication cycle. Our recent studies demonstrate a novel protein- protein interaction between HIV-1 Gag and host filamin A, which is involved in late stages of the HIV-1 replication cycle in a productive manner. Disruption of the interaction redistributes Gag subcellular localization and inhibits particle release. These data suggest that the Gag-filamin A interaction could be developed as targets for HIV therapeutics. We hypothesize that small synthetic peptides, containing the binding site required for the interaction, might block specifically the interaction resulting in the impaired virus assembly and release. Experiments will be performed to test this central hypothesis. In specific aim 1, the binding site for Gag and filamin A will be defined by mutagenesis, as well as in vitro and in vivo binding studies. Surface plasmon resonance (SPR) will be used to evaluate the binding kinetics of the interaction. Experiments in specific aim 2 will be designed to identify peptide candidates to specifically block the interaction. Gag- and filamin A-based libraries with overlapping peptide sequences covering the binding site and its surrounding region will be generated. Screening for peptide candidates against libraries will be performed using direct binding and competitive binding inhibition assays. The binding dynamics of peptide candidates with target proteins will be characterized. In specific aim 3, the role of peptide candidates on virus assembly and release will be examined. Cellular uptake and targeting validation of peptide candidates, which are mediated by Tat peptide, will be evaluated by fluorescence microscopy and flow cytometry. The role of peptide candidates on HIV-1 assembly and release will be determined in human T cell lines, and primary human CD4+ T cells and macrophages. Taken together, these studies will not only provide new sight into retrovirus-host interaction, but also impact the HIV/AIDS therapy by developing novel peptide inhibitors targeting the Gag-filamin A interaction.
Novel trypanosome receptor for Thrombospondin-1
Researcher: Pius N. Nde, Ph.D.
Founding Source: National Institute of Allergy and Infectious Diseases (NIAID)
Project Summary: Trypanosoma cruzi, the causative agent of Chagas heart disease affects several million individuals causing significant morbidity and mortality, yet it remains incurable. T. cruzi modulates the gene expression profiles of a few extracellular matrix proteins to facilitate infection. One of the genes up-regulated early during infection by the parasite is host thrombospondin-1 (TSP-1), a matricellular protein. TSP-1 binds specifically to the surface of invasive forms of T. cruzi trypomastigotes and knockdown of host TSP-1 by RNA interference causing significant inhibition of T. cruzi infection. We hypothesize that the trypomastigote form of T. cruzi up- regulates host TSP-1 that interacts with trypanosome surface receptor(s) to enhance the infection of heart cells. The long-term goal of this research is to understand the molecular mechanisms that allow T. cruzi to infect heart cells, so that specific molecular intervention strategies can be developed to prevent infection of heart cells. This hypothesis will be tested by experiments based on these specific aims: 1. To clone and characterize the novel T. cruzi TSP-1 binding molecule. We will use affinity chromatography, MALDITOF-MS, PCR and purification of recombinant proteins approaches to identify, clone and characterize the trypomastigote receptor that is important in the process of T. cruzi infection; 2. To determine the in vivo role of TSP-1 gene in the process of T. cruzi infection using TSP-1 KO mice model.
Human brain-on-a chip: Regional communication, drug and toxin responses; and Inner
blood-retinal barrier-on-a-chip: Implications for ocular disease
Researcher: Donald J. Alcendor, Ph.D.
Funding source: National Center for Advancing Translational Sciences (NCATS)
Project Summary: Physical or pharmacological disruption of chemical signals between the systemic blood flow and the brain impairs normal functioning and responsiveness of the brain. Long-range chemical signaling through dysregulation of cytokines, nutrients, growth factors, hormones, lipids, neurotransmitters, drugs and their metabolites is also important, but these chemical signals are difficult to quantify and cells are usually studied in isolation. The blood-brain barrier (BBB) dynamically controls exchanges between the brain and body, but this cannot be studied directly in the intact human brain or adequately represented by animal models. Most existing in vitro BBB models do not include neurons and glia with other BBB elements and cannot adequately predict drug efficacy and toxicity. This research will develop an in vitro, three-dimensional, multi-compartment, organotypic model of a central nervous system (CNS) neurovascular unit (NVU) and cerebral spinal fluid (CSF) compartment, both coupled to a realistic blood-surrogate supply system that also incorporates circulating immune cells. Primary and stem-cell-derived human cells will interact with a variety of agents to produce critical chemical communications across the BBB and between brain regions, providing a compact device that faithfully reproduces the properties of the human BBB, the CNS, and the CSF. The proposed in vitro BBB/CNS/CSF model will have a small volume, requires a limited number of human cells, can recreate interactions between different brain regions, and will be coupled in real time to advanced electrochemical and mass spectrometry instruments. This transformative technological platform will replicate chemical communication, molecular trafficking, and inflammation in the brain, and will enable targeted and clinically relevant nutritional and pharmacologic interventions or prevention. This platform will be used to examine the role of the BBB in modulating chemical body-brain interactions, characterize glial and neural cell interactions in the brain, and assess the effect of a wide range of drugs, chemicals, infectious agents and xenobiotics on various brain regions. The model’s clinical utility rests on its ability to 1) recreate unique regions by selecting specific combinations of neurons, endothelial cells, astrocytes, other neuroglia, pericytes and systemic leukocytes, 2) use cells and fluids derived from patients with known pathologies to assess drug treatments and physiological stress from chronic diseases such as obesity and acute injury such as stroke, 3) uncover potential adverse effects during drug discovery as well as those that are being used in clinical trials, such as toxic transformation of approved drugs by brain endothelial cells, 4) detect novel and unbiased correlations between large numbers of chemical signals which converge at the BBB, and 5) combine microfluidic devices, state-of-the-art cell culture and organotypic human brain-cell preparations, analytical instruments, bioinformatics, control theory, and neuroscience drug discovery. An integrated approach will provide technologies of widespread applicability and reveal new mechanistic and region-specific insights into how the brain receives, modifies, and is affected by drugs, neurotropic agents and disease. Dr. Alcendor will aid in integration of vascular pericytes of the neurovascular units (NVU) of the blood brain barrier (BBB) and inner blood retinal barriers (IBRB) to develop separate and unique tri-cell culture models. The model of the IBRB will include retinal pericytes, retinal microvascular endothelial cells and Muller cells. The BBB model will include brain vascular pericytes, brain microvascular endothelial cells and astrocytes. These tri-cell culture models will be incorporated into a microfluidic device modeled after the normal brain and retina that will allow the monitoring of molecular traffic for therapeutic analysis and cellular responses after CMV infection (diseased brain and retina). Dr. Alcendor will interact with Dr. John P. Wikswo’s team at Vanderbilt and grant collaborators from the Cleveland Clinic in the development of these model devices.
Capacity Building Assistance (CBA) to improve the Delivery and Effectiveness of Human
Immunodeficiency Virus (HIV) Prevention Services for High-risk and/or Racial/Ethnic
Researcher: Donald J. Alcendor, Ph.D.
Founding source: Center for Disease Control and Prevention (CDC)
Project Summary: The name of this initiative is Project SAVED – Strengthening Access via Empowerment and Diligence. The overall goal of the project is to strengthen the capacity of African American faith leaders, health providers (trained), Historically Black College and University (HBCU) leaders in metropolitan and non-metropolitan US southern states to collaboratively increase access and the use of HIV prevention services for African American high-risk adolescents and adults (predominantly heterosexual) in their communities utilizing the principles of the Centre for Ethnicity and Health Community Engagement Model. Project SAVED will 1) build SCRET’s capacity to utilize the CEH Community Engagement Model to create opportunities for African American high-risk heterosexuals to access and use HIV prevention services, and 2) build SCRET’s capacity to provide trained CBA members who are able to facilitate and empower community stakeholders to be agents of change by identifying and building upon their assets to increase access to HIV prevention and care services. Their roles and responsibilities will be to: 1) provide guidance and input regarding all aspects and activities of the Project SAVED CBA initiative; 2) undergo master trainer education and receive HIV capacity building training regarding the CEH Model, and to in turn diffuse, adapt, and/or adopt the model within their respective communities; 3) mobilize, identify, and engage persons interested in receiving capacity building information and training; and 4) liaison between Project SAVED staff and community stakeholders.
CD556, infection, and race in preterm delivery
Researcher: Stella Nowicki, D.D.S.
Founding source: National Institute of Child Health and Human Development (NICHD)
Project Summary: The rate of preterm delivery among African American (AA) women is 1.5 to 2.4 times higher than Caucasian (CS). The national public health goal is eliminating ethnic disparity in preterm delivery (PTD). Although the mechanism of PTD is unclear, infections are among the main ethnologic factors implicated. We propose a unifying hypothesis by which infection and host factors may increase the risk of PTD among African Americans (AA). The fetus is a semiallogenic antigen that requires efficient protection from the maternal humoral immune system. Decay accelerating factor (DAF) is a protective host factor, which is expressed in the feto-maternal interphase and its main function is protection from cytotoxic effect of autologous complement (c) attack. DAF expression is controlled by progesterone (P) and P was implicated in pregnancy losses. Infection upregulates nitric oxide (NO) and in turn NO downregulates DAF expression. Decreased DAF expression alters the protective quality of the feto-maternal interphase and may trigger complement mediated increases in proinflammatory tumor necrosis factor (TNF), and prostaglandin production resulting in PTL/PTD. We propose that both infection and biologic/genetic factors may contribute to the disturbances in the endocrine-NO-immune axis. Various factors in concert may act to alter the C/DAF ratio in the feto-maternal interface thereby leading to the activation of proinflammatory/prostaglandin pathway. The C cascade and TNF response occurs upon infection or vaginal colonization. Virulent E. coli is capable of displaying synergistic signaling via E.coli adhesin-host receptor and eliciting aggressive cytokine responses. Although these factors may affect all women, AA are at higher risk due to an increased inherent capacity of rejection or hyperresponsiveness of alloantigens. The inherent differences may result in the observed PTD disparities between AA and CS. We propose the following aims: 1. Characterize expression of DAF among AA and CS women undergoing elective pregnancy termination, term and preterm labor. 2. Characterize allelic polymorphism in the DAF Tcb, Cr(a-), and TNFA alleles and analyze possible association with infection, PTD and race. 3. Characterize genotypes of vaginal colonization isolates in patients with term and preterm delivery. The long-term goal of our project is to characterize mechanism of infection associated PTL/PTD and the role of the host and pathogen factors interacting at the urogenital interphase in birth outcome.
Molecular microbial pathogenesis training program
Researcher: Fernando Villalta, Ph.D.
Funding Source: National Institute of Allergy and Infectious Diseases (NIAID)
Project Summary: Highly interactive Meharry Medical College and Vanderbilt University mentors who conduct cutting-edge research in molecular microbial pathogenesis participate in this training. The program, for trainees committed to infectious diseases careers, offers trainees opportunities for new discoveries and breakthroughs in the study of the pathogen-host interactions of microbes causing disease including biodefense agents. The innovative features of this program are grounded on cutting-edge science and molecular approaches to study the pathogenesis of microbe-host cell interactions in the following areas: (i) Microbial attachment to receptors, invasion and replication; (ii) Functional genomics and systems biology of microbial infections; (iii) Cell host signaling evoked by pathogens including toxins; (iv) Unique pathogen target genes required for survival; (v) Structural biology and function of new microbial virulent factors; and (vi) Interactions of novel immune molecules with pathogens.
Research training in cardiovascular biology at Meharry
Researcher: Fernando Villalta, Ph.D.
Funding Source: National Heart, Lung and Blood Institute (NHLBI)
Project Summary: This is the third renewal of a NHLBI-supported Minority Institutional Research Training Program in cardiovascular biology at Meharry Medical College (MMC) that will focus in the considerable strengths and diversity of multidepartmental research in cardiovascular biology at MMC and Vanderbilt University School of Medicine (VUMC) into a unique and coherent framework for specialized training. The proposed program will support five pre-doctoral trainees per year and will involve 28 faculty members at MMC and VUMC. Three departments including Cardiovascular Biology at MMC and six departments at VUMC, participate in the program. The research training will focus in cardiovascular biology using cutting edge science and approaches to elucidate mechanisms causing cardiovascular as well as hematologic diseases.
Mechanism based targeted therapies for membranous nephropathy
Researcher: Dorin Bogdan Borza, Ph.D.
Funding Source: Satellite HealthCare, Inc.
Project Summary: Membranous nephropathy (MN) is a major cause of nephrotic syndrome in adults. Up to 40 percent of patients progress to end-stage kidney disease, and another 30 percent suffer from complications of persistent proteinuria and chronic kidney disease. Current therapies are unsatisfactory. Development of much-needed novel therapies require a better understanding of the pathogenic mechanisms. This study will test the hypothesis that the formation of subepithelial immune complexes promoting complement activation via the alternative pathway plays a central role in the pathogenesis of membranous nephropathy, mediating damage to the glomerular filtration barrier and proteinuria. It aims to determine whether targeted therapies specifically inhibiting the activation of the terminal complement cascade or of the alternative pathway , as well as the ablation of plasma cells producing pathogenic antibodies, are effective strategies for ameliorating glomerular damage and proteinuria in animal models of MN. The new knowledge gained from these studies may eventually translate into new treatments for human disease.
Mechanism of biogenesis of atypical alphaviruses
Researcher: Raju Ramasamy, Ph.D.
Funding Source: National Institute of Allergy/Infectious Diseases (NIAID)
Project Summary: Dr. Raju's long-term goal is to elucidate the pathways of alphavirus RNA genome repair and remodeling leading to the generation of atypical viruses in infected host cells. Alphaviruses are an important cause of emerging viral encephalitides in animals and humans and are significant biodefense agents. Delineating these pathways should lead to strategies to control emergence of outbreaks of alphaviruses and perhaps other mosquito-transmitted RNA viruses. Since alphaviruses are vigorously pursued as gene therapeutic and vaccine delivery vehicles, the team's work will also be useful in the development of improved RNA vectors.
Research Centers in Minority Institutions (RCMI)- Molecular Biology Core Component
Researcher: Robert Holt, Ph.D.
Funding Source: National Institute on Minority Health and Health Disparities (NIMHD)
Project Summary: Dr. Holt is the Scientific Director of the Molecular Biology Core at Meharry Medical College supported by RCMI. This core is a multi-functional facility that provides to faculty, students and staff of the college the primary service of DNA sequencing but also provides access to shared equipment housed in a centralized location and facilitates convenient and rapid access to frequently used molecular biological reagents. This core supports the enhancement of the research enterprise at the College.
Mitochondrial inner membrane protein translocase in trypanosoma brucei
Researcher:Minu Chaudhuri, Ph.D.
Funding Source: National Institute of General Medical Sciences (NIGMS)
Project Summary: African trypanosomiasis, a fatal disease in humans as well as in domestic animals, is caused by the parasitic protozoa, Trypanosoma brucei. As available drugs for this disease are inadequate, it is critical to identify targets to design new drugs. Import of essential mitochondrial proteins is crucial for survival of this parasite in mammalian hosts. Therefore the unique structure and function of mitochondrial protein import molecules could be exploited as novel drug target(s).