Objectives: YhiV (MdtF) is an resistance nodulation division (RND) type efflux pump in Escherichia coli with significant homology to AcrB but usually expressed at a low level in clinical isolates. When overexpressed the pump confers decreased susceptibility to a variety of substances including erythromycin and ethidium bromide (EtBr). We characterized two mutants of E. coli E12 (ΔacrB ΔacrF) overexpressing yhiV that showed surprising differences in their spectrum of multidrug resistance (MDR). Methods: The two mutants obtained after repeated exposure of E. coli E12 to levofloxacin were tested for antimicrobial susceptibility to a variety of agents and for intracellular accumulation of selected pump substrates. Gene expression was studied by quantitative RT–PCR, and yhiV was sequenced. Gene inactivation and replacement were done by phage λ-based homologous recombination. Results: Mutant DKO20/1 overexpressed yhiV, showed a wild-type yhiV sequence and had >2-fold increased MICs of fluoroquinolones, novobiocin, macrolides/ketolides, EtBr, oxacillin and Phe-Arg-β-naphthylamide (PAβN, a putative efflux pump inhibitor) compared with the E12 parent. A second mutant, strain DKO1/17 that had the Val-610→Phe point mutation in YhiV differed from DKO20/1 by faster growth, >2-fold increased MICs of linezolid and tetracycline, but >2-fold decreased MICs of PAβN, azithromycin and telithromycin. Inactivation of yhiV in DKO1/17 and reintroduction of the wild-type and mutant yhiV sequence confirmed that the differing MICs of most of the drugs were associated with the observed single point mutation. Intracellular drug accumulation studies with linezolid and PAβN were consistent with the MIC results. Conclusions: The region around amino acid Val-610 in YhiV appears to be involved in determining recognition and efficiency of export of a number of MDR efflux pump substrates. This single point mutation in the periplasmic loop of the pump can increase resistance to a given drug such as a fluoroquinolone while decreasing resistance to another one.
The Escherichia coli AcrB efflux pump is a resistance-nodulation-division (RND) pump that recognizes many unrelated compounds ([9][1], [10][2]). AcrB forms a complex with AcrA and TolC and is the single most important contributor to multidrug resistance in E. coli . Crystallographic models suggest
Arginase 1, an enzyme induced by Th2 cytokines, is a hallmark of alternatively activated macrophages and is responsible for the hydrolysis of L-arginine into ornithine, the building block for the production of polyamines. Upregulation of arginase 1 has been observed in a variety of diseases, but the mechanisms by which arginase contributes to pathology are not well understood. We reveal here a unique role for arginase 1 in the pathogenesis of nonhealing leishmaniasis, a prototype Th2 disease, and demonstrate that the activity of this enzyme promotes pathology and uncontrolled growth of Leishmania parasites in vivo. Inhibition of arginase activity during the course of infection has a clear therapeutic effect, as evidenced by markedly reduced pathology and efficient control of parasite replication. Despite the clear amelioration of the disease, this treatment does not alter the Th2 response. To address the underlying mechanisms, the arginase-induced L-arginine catabolism was investigated and the results demonstrate that arginase regulates parasite growth directly by affecting the polyamine synthesis in macrophages.
The Escherichia coli multidrug efflux pump protein AcrB has recently been cocrystallized with various substrates, suggesting that there is a phenylalanine-rich binding site around F178 and F615. We found that F610A was the point mutation that had the most significant impact on substrate MICs, while other targeted mutations, including conversion of phenylalanines 136, 178, 615, 617, and 628 to alanine, had smaller and more variable effects.
Members of the genus Nocardia are ubiquitous environmental saprophytes capable to cause human pulmonary, disseminated and cutaneous nocardiosis or bovine mastitis. Innate immunity appears to play an important role in early defense against Nocardia species. To elucidate the contribution of antimicrobial peptides (AMPs) in innate defense against Nocardia, the activity of human alpha-defensins human neutrophil peptides (HNPs) 1-3, human beta-defensin (hBD)-3 and cathelicidin LL-37 as well as bovine beta-defensins lingual and tracheal antimicrobial peptides (LAP, TAP) and bovine neutrophil-derived indolicidin against four important Nocardia species was investigated.Whereas N. farcinica ATCC 3318 and N. nova ATCC 33726 were found to be susceptible to all investigated human and bovine AMPs, N. asteroides ATCC 19247 was killed exclusively by neutrophil-derived human alpha-defensins HNP 1-3 and bovine indolicidin. N. brasiliensis ATCC 19296 was found to exhibit complete resistance to investigated human AMPs and to be susceptible only to bovine indolicidin.Selected AMPs are capable to contribute to the first line of defense against Nocardia, yet, susceptibility appears to vary across different Nocardia species. Obtained results of neutrophil-derived AMPs to possess the broadest antinocardial spectrum are remarkable, since nocardiosis is characterized by a neutrophil-rich infiltrate in vivo.
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