Inhibitors of bacterial virulence identified in a surrogate host model.

Antibiotic resistance continues to reduce the number of available antibiotics, increasing the need for novel antibacterial drugs. Since the seminal work of Sir Alexander Fleming, antibiotic identification has been based exclusively on the inhibition of bacterial growth in vitro. Recently, inhibitors of bacterial virulence which interfere with bacterial pathogenesis mechanisms have been proposed as an alternative to antibiotics, and a few were discovered using assays targeting specific virulence mechanisms. Here we designed a simple surrogate host model for the measurement of virulence and systematic discovery of anti-virulence molecules, based on the interaction of Tetrahymena pyriformis and Klebsiella pneumoniae cells. We screened a library of small molecules and identified several inhibitors of virulence. In a mouse pneumonia model we confirmed that an anti-virulence molecule displayed antibacterial activity against Klebsiella pneumoniae and Pseudomonas aeruginosa, by reducing dramatically the bacterial load in the lungs. This molecule did not inhibit bacterial growth in vitro but prevented biosynthesis of the Klebsiella capsule and lipopolysaccharides, a key requirement for virulence. Our results demonstrate that anti-virulence molecules represent an alternative to antibiotics and those can be discovered using non-animal host models.

Overlapping enhancer/promoter and transcriptional termination signals in the lentiviral long terminal repeat.

Oncoretrovirus, but not lentivirus, displays a high transcriptional readthrough activity in the 3' long terminal repeat (LTR) (Zaiss et al. J. Virol. 76, 7209-7219, 2002). However, the U3-deleted, self-inactivating (SIN) lentiviral LTR also exhibits high transcriptional readthrough activity. Since the canonical "core" polyadenylation signal (AAUAAA) of the lentivirus is located in the R-U5 region, the above finding suggests that additional RNA termination signals must be present in the U3 region. Insertion of alternative termination signals including panhuman T cell leukemia virus type I polyadenylation signal, a 3' end small intron, and a tertiary tRNA motif into the lentiviral SIN LTR did not restore the transcriptional termination function. Functional dissection of the U3 region revealed that 70-80% of the termination signals reside in the transcriptional control region within 124 nt overlapping NFkappaB, Sp1 and TATA binding sites. Serial deletion analysis of the transcriptional control region indicates that the lentiviral enhancer/promoter elements are essential to the RNA termination function. These results characterize the mechanism of lentiviral transcriptional readthrough, which addresses important fundamental and practical issue of RNA readthrough influencing lentiviral gene function and vector safety.