2011. target. Sequence alignments were performed to spotlight the altered sequences, and the structural effects of the mutations were evaluated in structural models. Overexpression of the gene in SCVs or introducing the mutation found in the gene of one of the high-level TO-resistant mutants into the gene provided resistance to TO and further validated the identity of the cellular target. FC04-100, a TO derivative which also possesses activity against non-SCV strains, prevents high-level resistance development in prototypic strains and limits the level of resistance observed in SCVs. An ATP synthesis assay allowed the observation of a correlation between antibiotic potency and ATP synthase inhibition. The selectivity index (inhibition of ATP production by mitochondria versus that of bacterial ATP synthase) is usually estimated to be 105-fold for FC04-100. spp. (11,C13). and methicillin-resistant (MRSA) are often associated with recurrent and difficult-to-treat hospital- or community-acquired infections (14, 15) and are among the bacterial threats identified by the CDC that require immediate attention (16). MRSA strains are also found in livestock and domestic pets, and transmissions from animals to humans have often been reported (17,C19). In addition, staphylococcus infections are also associated with food poisoning (20), comparable to several pathogens that belong to the order and can cause gastrointestinal infections as well as food poisoning (21, 22). Though infections are relatively rare, they can lead to invasive Rocaglamide listeriosis, cause severe symptoms, and be fatal. Persistence of this pathogen in food is a major problem and is associated with transmission to humans (23). spp. are well known for their ability to form endospores that can persist in the environment (24). Among pathogenic species, can cause anthrax by contact with infected food-producing animals, as well as by direct contact with endospores when it is used as a biological weapon (25). However, remains the most clinically prominent pathogen of this group. Among the mechanisms that allow to cause prolonged infection is usually its ability to adopt a slow-growth phenotype, called small-colony variant (SCV). SCVs have been associated with chronic and prolonged infections and are often recovered from lungs of patients with cystic fibrosis (CF) or from osteomyelitis, septic arthritis, bovine mastitis, and colonized orthopedic devices (26,C29). SCVs possess an impaired respiratory chain, which affects their oxidative metabolism, causes slow growth, and changes the expression of virulence factors (28, 30). The reduced proton motive pressure (PMF) of SCVs reduces their susceptibility to aminoglycosides, which rely Rocaglamide on an active PMF to cross the cell membrane barrier. Respiratory deficiency is usually often caused by mutation in genes involved in hemin or menadione biosynthesis, which are important components of the respiratory chain (28). SCVs have the ability to produce large quantities of biofilm (31,C33) and also persist within nonphagocytic host cells (12, 34, 35), enhancing their ability to survive in the presence of antibiotics and host immune factors. Switching between the normal and SCV phenotypes seems to be a part of pathogenesis and is a phenomenon that may promote recurrence and chronicity of infections (36). Our laboratory extensively documented the very potent (in the nanomolar range) and selective antibacterial activities of TO against SCVs of spp. (11,C13, 37). We also investigated several structural analogs of TO in order to better understand its structure-activity relationship (37) and recognized a encouraging TO derivative possessing a diamino group replacing the 3-hydroxyl group of TO (FC04-100) (Fig. 1). In addition to its anti-SCV activity, FC04-100 interestingly gained antibacterial activity against prototypical strains (13). The mode of action of TO and its analogs was clearly Rocaglamide associated with the respiratory chain, but their molecular target was unknown until now. Open in a separate windows FIG 1 Structures of TO and analogs used in Rabbit Polyclonal to RPS6KB2 this study. (A) TO is usually characterized by 6 rings, 12 stereogenic centers, a 3 -hydroxyl group, and spiro-fused rings in the form.