The FinR-regulated essential gene fprA, encoding ferredoxin NADP+ reductase: Roles in superoxide-mediated stress protection and virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa has two genes encoding ferredoxin NADP(+) reductases, denoted fprA and fprB. We show here that P. aeruginosa fprA is an essential gene. However, the ΔfprA mutant could only be successfully constructed in PAO1 strains containing an extra copy of fprA on a mini-Tn7 vector integrated into the chromosome or carrying it on a temperature-sensitive plasmid. The strain containing an extra copy of the ferredoxin gene (fdx1) could suppress the essentiality of FprA. Other ferredoxin genes could not suppress the requirement for FprA, suggesting that Fdx1 mediates the essentiality of FprA. The expression of fprA was highly induced in response to treatments with a superoxide generator, paraquat, or sodium hypochlorite (NaOCl). The induction of fprA by these treatments depended on FinR, a LysR-family transcription regulator. In vivo and in vitro analysis suggested that oxidized FinR acted as a transcriptional activator of fprA expression by binding to its regulatory box, located 20 bases upstream of the fprA -35 promoter motif. This location of the FinR box also placed it between the -35 and -10 motifs of the finR promoter, where the reduced regulator functions as a repressor. Under uninduced conditions, binding of FinR repressed its own transcription but had no effect on fprA expression. Exposure to paraquat or NaOCl converted FinR to a transcriptional activator, leading to the expression of both fprA and finR. The ΔfinR mutant showed an increased paraquat sensitivity phenotype and attenuated virulence in the Drosophila melanogaster host model. These phenotypes could be complemented by high expression of fprA, indicating that the observed phenotypes of the ΔfinR mutant arose from the inability to up-regulate fprA expression. In addition, increased expression of fprB was unable to rescue essentiality of fprA or the superoxide-sensitive phenotype of the ΔfinR mutant, suggesting distinct mechanisms of the FprA and FprB enzymes.

Inhibition of Plasmodium falciparum proliferation in vitro by double-stranded RNA nanoparticle against malaria topoisomerase II.

The need to develop new effective antimalarial agents is urgent due to the rapid emergence of drug resistance to all current drugs by the most virulent human malaria parasite, Plasmodium falciparum. A promising avenue is in the development of antimalarials based on RNA interference targeting expression of malaria parasite vital genes, viz. DNA topoisomerase II gene (PfTOP2). Biodegradable chitosan nanoparticle system has proven to be effective in delivering DNA and small double-stranded interfering RNA to target cells. We have employed a long double-stranded (dsRNA) targeting the coding region of PfTOP2 that is complexed with chitosan nanoparticles in order to interfere with the cognate mRNA expression and examined its effect on P. falciparum growth in culture. Exposure of ring stage-infected erythrocytes to 10 µg/ml PfTOP2 chitosan/dsRNA nanoparticles for 48 h resulted in 71% growth inhibition as determined by [(3)H] hypoxanthine incorporation and microscopic assays, compared with 41% inhibition using an equivalent amount of free PfTOP2 dsRNA or 12% with unrelated chitosan/dsRNA nanoparticles. This inhibition was shown to occur during maturation of trophozoite to schizont stages. RT-PCR analysis indicated 56% and 38% decrease in PfTOP2 transcript levels in P. falciparum trophozoites treated with PfTOP2 dsRNA nanoparticles and free PfTOP2 dsRNA respectively. These results suggest that chitosan-based nanoparticles might be a useful tool for delivering dsRNA into malaria parasites.