A.
SPECIFIC AIMS Brucella is a Gram-negative facultative intracellular bacterium that induces chronic infectious disease by direct contact or by consumption of animal products. Because of the ease of inserting antibiotic resistance genes, Brucella was one of the first bacteria to be ?weaponized? by the United States for biological warfare and can be readily made resistant to all commercial antibiotics (www.expasy.org/sprot/hamap/BRUSU.html). No human vaccine exists and antibiotic treatment is protracted with combined antibiotics required, eg., Rifampicin and Doxycycline (1). Therefore, a great need exists to find new therapies beyond current antibiotics to treat individuals infected with Brucella. Our long- term research goals are to develop a peptide antibiotic that shows great promise against Brucella, to gain insight into its mechanism of action, and to demonstrate effectiveness in the treatment of antibiotic resistant Brucella. To accomplish this goal, we have developed a peptide antibiotic, trifolitoxin (TFX), with activity against members of the ?-proteobacteria, especially Brucella. Harnessing the clinical ? antibiotic potential of trifolitoxin will define the effective concentration of trifolitoxin to inhibit Brucella under extracellular and intracellular conditions. Now, we will explore the efficacy of trifolitoxin in the following Specific Aims: 1. To determine TFX inhibition of Brucella replication in macrophages. 2. To determine TFX inhibition of Brucella growth and dissemination in animals. 3. To determine the mechanism(s) of antibiotic effect. Trifolitoxin (TFX) is a ribosomally synthesized, posttranslationally modified peptide antibiotic that inhibits a specific group of organisms within the ?-proteobacteria (2, 3). DNA sequence analysis and direct peptide ? sequencing have shown that the sequence of the peptide prior to posttranslational modification but after cleavage of the leader is DIGGSRXGCVA (2, 4, 5). The addition of the seven genes tfxABCDEFG to strains of ?-proteobacteria is sufficient to make active TFX (3). Now, we have exciting data demonstrating ? that TFX can inhibit Brucella in culture. This opportune observation provides the first new therapeutic approach in decades to treat Brucella infected individuals where resistance to current commercial antibiotics may exist.
In Aim 1, we will determine the concentration of TFX required to inhibit Brucella once the bacterium is established within the macrophage, the primary host cell for Brucella residence. The small peptide size coupled with its hydrophobicity and cationic charge suggests rapid penetration of eukaryotic cells. These experiments will give insight into the stage of infection at which TFX is likely to be most active.
In Aim 2 our biophotonic imaging system has permitted us to follow in vivo Brucella infections serially in live mice (6). This extremely valuable tool allows us to image in real time the therapeutic effect of TFX administered to mice over the course of infection. Further, we can rapidly determine if TFX prevents organ dissemination, or if previously infected animals can more rapidly clear a disseminated infection.
In Aim 3 for TFX to be a commercially viable product, the mechanism of TFX action must be understood. Although currently unknown, TFX may not act by a mechanism resembling currently known peptide antibiotics. Since TFX is considered as a bacteriocin (5, 7), electron microscopy of TFX treated Brucella will be performed to examine for distortion of the bacterial membrane as observed for other bacteriocins (8, 9). If TFX targets the bacterial membrane, then bacterial micelles will be produced to examine TFX specificity for the peptidoglycan membrane. If TFX does not interact with the bacterial membrane, a pull- down assay and SDS-PAGE followed by amino acid sequencing will be used to identify protein(s) that interact with TFX. Also, Brucella engineered to express the resistance-associated TFX G gene, followed by a pull-down assay and N-terminal sequencing may identify the target of TFX action. Alternatively, we will attempt to mutate Brucella to produce resistant bacteria. Our working hypothesis is that TFX disrupts an important bacterial process leading to the death of Brucella, and we seek to identify these initial steps. Project Narrative The submitted proposal addresses the request of PA-06-135 using advanced technology to develop a drug to treat an infectious disease. We have a clearly identified product that meets the needs of PA-06-135. The Problem: Brucella, a Gram-negative facultative intracellular bacterium, induces chronic infectious disease. To treat Brucella infected humans, current antibiotic therapy has two problems. First, multiple antibiotics are required combined with long-term therapy of 6 weeks to 6 months to effect clearance of this intracellular bacterium. Current therapy is not ideal because of side effects of present antibiotics , therapeutic failures and relapses. Second, because antibiotic resistance genes are readily inserted, Brucella was one of the first bacteria to be ?weaponized? for biological warfare and can be readily made resistant to all commercial antibiotics. No human vaccine exists and antibiotic treatment requires months. Therefore, a great need exists to find new therapies to treat Brucella infected individuals. The Product: Our long-term research goal is to develop trifolitoxin (TFX), a peptide antibiotic, that shows great promise against Brucella, to gain insight into its mechanism of action, and to demonstrate effectiveness in the treatment of antibiotic resistant Brucella. Now, we have exciting data demonstrating that TFX can inhibit different Brucella species in culture. This opportune observation provides the first new therapeutic approach in decades to treat Brucella infected individuals where resistance to current commercial antibiotics may exist.
| Rambow-Larsen, Amy A; Petersen, Erik M; Gourley, Christopher R et al. (2009) Brucella regulators: self-control in a hostile environment. Trends Microbiol 17:371-7 |
