Oren S. Rosenberg, Ph.D. - US grants

Dr. Oren Rosenberg is an infectious disease physician with a background in structural biology. His objective in this current project is to combine his clinical and basic science interests by studying the structure and function of key virulence determinants in Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. Through a series of specific aims that use a combined structural, biochemical and genetic approach, Dr. Rosenberg will examine the role of essential components in the type VII secretion system, which is known to be critical for the virulence of Mtb. The goal is to understand mycobacterial infection better so as to aid in the design of faster, cheaper, less toxic and more effective ways to prevent and treat tuberculosis. Additionally this proposal is designed to complement Dr. Rosenberg's prior laboratory experience and provide him with the requisite technical and intellectual background in microbial pathogenesis and structural biology to function as an independent investigator. A committee of physicians and scientists will oversee Dr. Rosenberg's progression towards independence. His scientific development will also be enriched through attendance at UCSF courses in pertinent topics, several seminar series, departmental retreats and national meetings. Dr. Rosenberg will develop a clinical specialization in tuberculosis and other respiratory infections through work at the tuberculosis clinic at San Francisco General Hospital. At the end of the granting period Dr. Rosenberg will be prepared to develop his own research and clinical agenda as an independent investigator in the basic biology of microbial pathogenesis.

PROJECT SUMMARY: This proposal addresses a major challenge that radiologists and other physicians encounter frequently, namely distinguishing active infection from other processes in the human body. Existing clinical techniques target the host immune response, for example 111In SPECT white blood cell scanning or 18F-FDG PET. Although these modalities can sometimes be useful, they lack the specificity required to distinguish living bacteria from sterile inflammation, cancer, and other highly metabolic tissues. Therefore we are proposing a PET imaging technique that exploits metabolic pathways specific to bacteria, targeting both gram-positive and gram- negative organisms. We believe a technique that detects ALL or at least a majority of pathogenic bacteria will be most useful in clinical practice. Once an imaging abnormality has been identified as infection, tissue sampling, staining and culture may still be required. An imaging method that could distinguish active infection from frequent mimics, would instantly become the standard of care in a variety of inpatient and outpatient settings. In this proposal, we develop 18F and 11C tracers for positron emission tomography (PET) using D-Met derived tracers, and study them for the first time in human patients with spinal infections. We have identified 11C D-met as a radiotracer with (1) a simple, high-yield radiosynthesis (2) good in vivo stability (3) appropriate mimicry of the endogenous substrate (4) high rate of incorporation into bacterial peptidoglycan and (5) low uptake in background tissues. We will start by refining the synthesis of enantiopure 11C D-met, and investigating close structural relatives of 11C D-met for enhanced bacterial uptake (Specific Aim 1). We will then study our lead D-met tracer in compelling preclinical infection models, including models of vertebral osteomyelitis-discitis (Specific Aim 2). In Specific Aim 3, we will take all steps needed for an investigational new drug (IND) approval for our lead tracer, and study its performance in patients suffering from spinal infection.

Project Summary This project brings together structural, biochemical, proteomic and genetic expertise at UCSF to provide a detailed understanding of the structure and post-translational regulation of a key virulence factor in Mycobacterium tuberculosis. A hallmark of virulent, intracellular bacteria is their use of specialized secretion systems to inject proteins into mammalian cells. Multiple such systems are known in Gram-negative bacteria (termed the Type I-VI and VIII-IX) systems), but only one is found exclusively in Gram-positive bacteria: the type VII secretion (T7S) system. Mycobacterium tuberculosis uses multiple T7S systems to promote infection through secretion of effectors. In fact, the main genetic difference between the harmless vaccine strain BCG and pathogenic M. tuberculosis is the loss of the ESX-1 T7S system. T7S is also essential for abscess formation in Staphylococcus aureus and other pathogens may require T7S for virulence, as these systems are broadly conserved in Gram-positive bacteria. In this proposal we aim to build on our preliminary data regarding the basic mechanisms of T7S and unravel the biochemical and structural foundations of this complex biological machine.

PROJECT SUMMARY: This application addresses a major challenge that radiologists and other physicians encounter frequently, namely distinguishing active infection from other processes in the human body. Specifically, the proposed work is motivated by the difficulty in diagnosing and treating pneumonias in cystic fibrosis patients, especially those caused by P. aeruginosa. Clinically available imaging tools either (1) are limited by their background accumulation in the lungs or (2) image the host response to infection rather than the living bacteria themselves. To address this challenge, we have developed several PET radiotracers that exploit metabolic pathways specific to bacteria, including D-amino acid derived probes most recently D-[3-11C]alanine. When D-[3-11C]alanine was applied to several compelling preclinical models of infection, we found that it was exquisitely sensitive to P. aeruginosa, which is not the case for the vast majority of reported radiotracers. We further showed that D-[3-11C]alanine is a radiotracer with (1) a simple, high-yield radiosynthesis (2) good in vivo stability (3) appropriate mimicry of the endogenous substrate (4) high rate of incorporation into both gram- negative and gram-positive bacteria and (5) low uptake in background tissues. These studies demonstrate the outstanding potential of D-[3-11C]alanine, with the proposed work necessary to further validate and understand this tracer. We will first expand our radiochemical methods, synthesizing the D-[1-11C]alanine isotopomer and structurally related 18F probes (Specific Aim 1). We will then further investigate the lead 11C isotopomer in vitro, analyzing its performance in clinical P. aeruginosa strains and validated biofilm models (Specific Aim 2). In Specific Aim 3, we will extend these concepts in vivo employing both acute and chronic models of P. aeruginosa infection.