R01AI154524

Molecular Elucidation of the Francisella Tularensis Virulence Mechanism

Francisella Tularensis (FT), the causative agent of tularemia, is one of the most infectious bacterial pathogens known. Due to its high infectivity and ease of aerosolization, it has been classified as a Category A bioweapon by the US government. The morbidity and mortality of tularemia are substantial, and given its extreme infectivity, a significant outbreak of tularemia would readily overwhelm the capabilities of even the largest US medical centers.

FT virulence requires genes expressed from the chromosomally encoded FT Pathogenicity Island (FPI). The expression of these genes is activated by a combination of FT regulators: the stringent starvation protein A (SSPA), the macrophage growth locus protein A (MGLA), and the pathogenicity island gene regulator (PIGR), which are expressed during FT infection. MGLA and PIGR are unique to FT, whereas SSPA proteins are found in multiple bacteria. The FT SSPA, however, is unusual as it does not homodimerize but rather heterodimerizes with MGLA. PIGR is a DNA-binding protein with a predicted winged-helix-turn-helix DNA-binding motif. Intriguingly, the "alarmone," guanosine tetraphosphate (ppGpp), is also necessary for FT virulence. Recently, we showed that this alarmone binds directly to the MGLA-SSPA complex. We further showed that ppGpp binding to MGLA-SSPA mediates high-affinity binding of PIGR to this heterodimer. Strikingly, our data also revealed that MGLA-SSPA interacts constitutively with the FT RNAPs70 holoenzyme, suggesting it represents a virulence specialized RNAP.

Given the extreme virulence and potential use of FT as a bioweapon, there is an urgent need to decipher the molecular mechanisms driving its virulence. The overarching goal of this proposal is the molecular dissection of these mechanisms. Our central hypothesis is that FT employs a conceptually novel form of pathogenesis requiring a virulence-specialized RNAP containing MGLA-SSPA. We shall test our central hypothesis and delineate the molecular mechanisms controlling the activation of FT virulence genes through the completion of three specific aims:

Specific Aim 1: To fully characterize MGLA-SSPA interaction with FT RNAP. Structural, biochemical, and cellular studies will dissect the mechanism of virulence regulation by MGLA-SSPA.

Specific Aim 2: To carry out structure and function analyses of FT RNAP(MGLA-SSPA)-ppGpp-PIGR complexes. The mechanism behind PIGR-mediated activation of the FPI will be analyzed structurally, biochemically, and in vivo.

Specific Aim 3: To determine a high-resolution (MGLA-SSPA)-ppGpp-PIGR crystal structure, identify inhibitors of ppGpp binding to MGLA-SSPA, and obtain structures of MGLA-SSPA inhibitor complexes.

The successful completion of these aims will reveal new paradigms in transcription regulation and enable the rational design of novel anti-Francisella-virulence therapeutics.

Duke University

TO ASSIST PUBLIC AND PRIVATE NONPROFIT INSTITUTIONS AND INDIVIDUALS TO ESTABLISH, EXPAND AND IMPROVE BIOMEDICAL RESEARCH AND RESEARCH TRAINING IN INFECTIOUS DISEASES AND RELATED AREAS, TO CONDUCT DEVELOPMENTAL RESEARCH, TO PRODUCE AND TEST RESEARCH MATERIALS. TO ASSIST PUBLIC, PRIVATE AND COMMERCIAL INSTITUTIONS TO CONDUCT DEVELOPMENTAL RESEARCH, TO PRODUCE AND TEST RESEARCH MATERIALS, TO PROVIDE RESEARCH SERVICES AS REQUIRED BY THE AGENCY FOR PROGRAMS IN INFECTIOUS DISEASES, AND CONTROLLING DISEASE CAUSED BY INFECTIOUS OR PARASITIC AGENTS, ALLERGIC AND IMMUNOLOGIC DISEASES AND RELATED AREAS. PROJECTS RANGE FROM STUDIES OF MICROBIAL PHYSIOLOGY AND ANTIGENIC STRUCTURE TO COLLABORATIVE TRIALS OF EXPERIMENTAL DRUGS AND VACCINES, MECHANISMS OF RESISTANCE TO ANTIBIOTICS AS WELL AS RESEARCH DEALING WITH EPIDEMIOLOGICAL OBSERVATIONS IN HOSPITALIZED PATIENTS OR COMMUNITY POPULATIONS AND PROGRESS IN ALLERGIC AND IMMUNOLOGIC DISEASES. BECAUSE OF THIS DUAL FOCUS, THE PROGRAM ENCOMPASSES BOTH BASIC RESEARCH AND CLINICAL RESEARCH. SMALL BUSINESS INNOVATION RESEARCH (SBIR) PROGRAM EXPANDS AND IMPROVES PRIVATE SECTOR PARTICIPATION IN BIOMEDICAL RESEARCH. THE SBIR PROGRAM INTENDS TO INCREASE AND FACILITATE PRIVATE SECTOR COMMERCIALIZATION OF INNOVATIONS DERIVED FROM FEDERAL RESEARCH AND DEVELOPMENT, TO INCREASE SMALL BUSINESS PARTICIPATION IN FEDERAL RESEARCH AND DEVELOPMENT, AND TO FOSTER AND ENCOURAGE PARTICIPATION OF SOCIALLY AND ECONOMICALLY DISADVANTAGED SMALL BUSINESS CONCERNS AND WOMEN-OWNED SMALL BUSINESS CONCERNS IN TECHNOLOGICAL INNOVATION. THE SMALL BUSINESS TECHNOLOGY TRANSFER (STTR) PROGRAM STIMULATES AND FOSTERS SCIENTIFIC AND TECHNOLOGICAL INNOVATION THROUGH COOPERATIVE RESEARCH AND DEVELOPMENT CARRIED OUT BETWEEN SMALL BUSINESS CONCERNS AND RESEARCH INSTITUTIONS, TO FOSTER TECHNOLOGY TRANSFER BETWEEN SMALL BUSINESS CONCERNS AND RESEARCH INSTITUTIONS, TO INCREASE PRIVATE SECTOR COMMERCIALIZATION OF INNOVATIONS DERIVED FROM FEDERAL RESEARCH AND DEVELOPMENT, AND TO FOSTER AND ENCOURAGE PARTICIPATION OF SOCIALLY AND ECONOMICALLY DISADVANTAGED SMALL BUSINESS CONCERNS AND WOMEN-OWNED SMALL BUSINESS CONCERNS IN TECHNOLOGICAL INNOVATION. RESEARCH CAREER DEVELOPMENT AWARDS SUPPORT THE DEVELOPMENT OF SCIENTISTS DURING THE FORMATIVE STAGES OF THEIR CAREERS. INDIVIDUAL NATIONAL RESEARCH SERVICE AWARDS (NRSAS) ARE MADE DIRECTLY TO APPROVE APPLICANTS FOR RESEARCH TRAINING IN SPECIFIED BIOMEDICAL SHORTAGE AREAS. IN ADDITION, INSTITUTIONAL NATIONAL RESEARCH SERVICE AWARDS ARE MADE TO ENABLE INSTITUTIONS TO SELECT AND MAKE AWARDS TO INDIVIDUALS TO RECEIVE TRAINING UNDER THE AEGIS OF THEIR INSTITUTIONAL PROGRAM.

93.855 - Allergy and Infectious Diseases Research

National Institute of Allergy and Infectious Diseases (NIAID) [HHS - NIH]

Durham, North Carolina 27710 United States

Single Zip Code

NIH Research Project Grant (Parent R01 Clinical Trial Not Allowed) (PA-20-185)

Amendment Since initial award the total obligations have increased 373% from $855,904 to $4,049,782.