Therapeutic intervention for Brucella

SUMMARY 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 (http://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 a nimals. 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 ana lysis 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 i s 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 resista nce 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 permi tted 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 a ct 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 bacte riocins (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 ami no acid sequencing will be used to identify protein(s) that interact with TFX. Also, Brucella engineered to express the resistance-associated TFX G gene, follow