Disparate outer membrane exclusionary properties underlie intrinsic resistance to hydrophobic substances in Pseudomonas spp. isolated from surface waters under triclosan selection.

Representative members of surface water microbiota were obtained from three unrelated municipal sites in Oklahoma by direct plating under selection by the hydrophobic biocide triclosan. Multiple methods were employed to determine if intrinsic triclosan resistance reflected resistance to hydrophobic molecules by virtue of outer membrane impermeability. While all but one organism isolated in the absence of triclosan were able to initiate growth on MacConkey agar, only one was able to initiate significant growth with triclosan present. In contrast, all bacteria selected with triclosan were identified as Pseudomonas spp. using 16S RNA gene sequencing and exhibited growth comparable to Pseudomonas aeruginosa controls in the presence of hydrophobic antibacterial agents to include triclosan. Two representative bacteria isolated in the absence of triclosan allowed for greater outer membrane association with the fluorescent hydrophobic probe 1-N-phenylnapthylamine than did two triclosan-resistant isolates. Compound 48/80 disruption of outer membrane impermeability properties for hydrophobic substances either partially or fully sensitized nine of twelve intrinsically resistant isolates to triclosan. These data suggest that outer membrane exclusion underlies intrinsic resistance to triclosan in some, but not all Pseudomonas spp. isolated by selection from municipal surface waters and implicates the involvement of concomitant triclosan resistance mechanisms.

Xenobiotic Metabolism in Mice Lacking the UDP-Glucuronosyltransferase 2 Family.

UDP-Glucuronosyltransferases (UGTs) conjugate a glucuronyl group from glucuronic acid to a wide range of lipophilic substrates to form a hydrophilic glucuronide conjugate. The glucuronide generally has decreased bioactivity and increased water solubility to facilitate excretion. Glucuronidation represents an important detoxification pathway for both endogenous waste products and xenobiotics, including drugs and harmful industrial chemicals. Two clinically significant families of UGT enzymes are present in mammals: UGT1s and UGT2s. Although the two families are distinct in gene structure, studies using recombinant enzymes have shown considerable overlap in their ability to glucuronidate many substrates, often obscuring the relative importance of the two families in the clearance of particular substrates in vivo. To address this limitation, we have generated a mouse line, termed ΔUgt2, in which the entire Ugt2 gene family, extending over 609 kilobase pairs, is excised. This mouse line provides a means to determine the contributions of the two UGT families in vivo. We demonstrate the utility of these animals by defining for the first time the in vivo contributions of the UGT1 and UGT2 families to glucuronidation of the environmental estrogenic agent bisphenol A (BPA). The highest activity toward this chemical is reported for human and rodent UGT2 enzymes. Surprisingly, our studies using the ΔUgt2 mice demonstrate that, while both UGT1 and UGT2 isoforms can conjugate BPA, clearance is largely dependent on UGT1s.

An NLRP3 Mutation Causes Arthropathy and Osteoporosis in Humanized Mice.

The NLRP3 inflammasome plays a critical role in host defense by facilitating caspase I activation and maturation of IL-1ß and IL-18, whereas dysregulation of inflammasome activity results in autoinflammatory disease. Factors regulating human NLRP3 activity that contribute to the phenotypic heterogeneity of NLRP3-related diseases have largely been inferred from the study of Nlrp3 mutant mice. By generating a mouse line in which the NLRP3 locus is humanized by syntenic replacement, we show the functioning of the human NLRP3 proteins in vivo, demonstrating the ability of the human inflammasome to orchestrate immune reactions in response to innate stimuli. Humanized mice expressing disease-associated mutations develop normally but display acute sensitivity to endotoxin and develop progressive and debilitating arthritis characterized by granulocytic infiltrates, elevated cytokines, erosion of bones, and osteoporosis. This NLRP3-dependent arthritis model provides a platform for testing therapeutic reagents targeting the human inflammasome.