Gram-negative bacteria produce extracellular outer membrane vesicles (OMVs) that interact with host cells. Unlike Gram-negative bacteria, less is known about the production and role of extracellular membrane vesicles (MVs) in Gram-positive bacteria. The food-borne pathogen Listeria monocytogenes can survive under extreme environmental and energy stress conditions and the transcription factor σB is involved in this survival ability. Here, we first determined the production of MVs from L. monocytogenes and evaluated whether general stress transcription factor σB affected production of MVs in L. monocytogenes. L. monocytogenes secreted MVs during in vitro broth culture. The wild-type strain actively produced MVs approximately nine times more and also produced more intact shapes of MVs than those of the isogenic ΔsigB mutant. A proteomic analysis showed that 130 and 89 MV proteins were identified in the wild-type and ΔsigB mutant strains, respectively. Wild-type strain-derived MVs contained proteins regulated by σB such as transporters (OpuCA and OpuCC), stress response (Kat), metabolism (LacD), translation (InfC), and cell division protein (FtsZ). Gene Ontology (GO) enrichment analysis showed that wild-type-derived MV proteins corresponded to several GO terms, including response to stress (heat, acid, and bile resistance) and extracellular polysaccharide biosynthetic process, but not the ΔsigB mutant. Internalin B (InlB) was almost three times more contained in MVs derived from the wild-type strain than in MVs derived from the ΔsigB mutant. Taken together, these results suggest that σB plays a pivotal role in the production of MVs and protein profiles contained in MVs. L. monocytogenes MVs may contribute to host infection and survival ability under various stressful conditions.

Acinetobacter baumannii secretes outer membrane vesicles (OMVs) during both in vitro and in vivo growth, but the biogenesis mechanism by which A. baumannii produces OMVs remains undefined. Outer membrane protein A of A. baumannii (AbOmpA) is a major protein in the outer membrane and the C-terminus of AbOmpA interacts with diaminopimelate of peptidoglycan. This study investigated the role of AbOmpA in the biogenesis of A. baumannii OMVs. Quantitative and qualitative approaches were used to analyze OMV biogenesis in A. baumannii ATCC 19606T and an isogenic ΔAbOmpA mutant. OMV production was significantly increased in the ΔAbOmpA mutant compared to wild-type bacteria as demonstrated by quantitation of proteins and lipopolysaccharides (LPS) packaged in OMVs. LPS profiles prepared from OMVs from wild-type bacteria and the ΔAbOmpA mutant had identical patterns, but proteomic analysis showed different protein constituents in OMVs from wild-type bacteria compared to the ΔAbOmpA mutant. In conclusion, AbOmpA influences OMV biogenesis by controlling OMV production and protein composition.

ABSTRACT:Ebastine undergoes extensive metabolism to form desalkylebastine and hydroxyebastine. Hydroxyebastine is subsequently metabolized to carebastine. Although CYP3A4 and CYP2J2 have been implicated in ebastine N-dealkylation and hydroxylation, the enzyme catalyzing the subsequent metabolic steps (conversion of hydroxyebastine to desalkylebastine and carebastine) have not been identified. Therefore, we used human liver microsomes (HLMs) and expressed cytochromes P450 (P450s) to characterize the metabolism of ebastine and that of its metabolites, hydroxyebastine and carebastine. In HLMs, ebastine was metabolized to desalkyl-, hydroxy-, and carebastine; hydroxyebastine to desalkyland carebastine; and carebastine to desalkylebastine. Of the 11 cDNA-expressed P450s, CYP3A4 was the main enzyme catalyzing the N-dealkylation of ebastine, hydroxyebastine, and carebastine to desalkylebastine [intrinsic clearance (CL int ) ‫؍‬ 0.44, 1.05, and 0.16 l/min/pmol P450, respectively]. Ebastine and hydroxyebastine were also dealkylated to desalkylebastine to some extent by CYP3A5. Ebastine hydroxylation to hydroxyebastine is mainly mediated by CYP2J2 (0.45 l/min/pmol P450; 22.5-and 7.5-fold higher than that for CYP3A4 and CYP3A5, respectively), whereas CYP2J2 and CYP3A4 contributed to the formation of carebastine from hydroxyebastine. These findings were supported by chemical inhibition and kinetic analysis studies in human liver microsomes. The CL int of hydroxyebastine was much higher than that of ebastine and carebastine, and carebastine was metabolically more stable than ebastine and hydroxyebastine. In conclusion, our data for the first time, to our knowledge, suggest that both CYP2J2 and CYP3A play important roles in ebastine sequential metabolism: dealkylation of ebastine and its metabolites is mainly catalyzed by CYP3A4, whereas the hydroxylation reactions are preferentially catalyzed by CYP2J2. The present data will be very useful to understand the pharmacokinetics and drug interaction of ebastine in vivo.Ebastine, a potent and selective histamine H 1 -receptor antagonist, belongs to a second generation of nonsedating antihistamines but with negligible anticholinergic and antiserotonergic properties (Llupia et al., 2003). Ebastine undergoes extensively sequential metabolism in the liver (Hashizume et al., 1998(Hashizume et al., , 2001). The major primary metabolites identified in humans are hydroxy-and desalkylebastine, and hydroxyebastine is further metabolized to carebastine. In vitro studies indicate that the formation of desalkyl-and hydroxyebastine from ebastine is catalyzed by CYP3A4 and CYP2J2, respectively (Hashizume et al., 2002). The specific hepatic cytochrome P450 (P450) enzymes involved in hydroxy-and carebastine metabolism have not been identified so far, despite some information which could be obtained from the previously published pharmacokinetics of ebastine. After oral administration to experimental animals and humans, ebastine is almost completely metabolized to the pharmacologically active princi...

In patients with PTC, MEE does not impact RFS. Thus, appropriate surgical intervention and postoperative follow up are mandatory in PTC, regardless of its extent.

Acinetobacter baumannii is a Gram-negative, nonmotile aerobic bacterium that has emerged as an important nosocomial pathogen. Multidrug-resistant (MDR) A. baumannii is difficult to treat with antibiotics, and treatment failure in infected patients is of great concern in clinical settings. To investigate proteome regulation in A. baumannii under antibiotic stress conditions, quantitative membrane proteomic analyses of a clinical MDR A. baumannii strain cultured in subminimal inhibitory concentrations of tetracycline and imipenem were performed using a combination of label-free (one-dimensional electrophoresis-liquid chromatography-tandem mass spectrometry) and label (isobaric tag for relative and absolute quantitation) approaches. In total, 484 proteins were identified, and 302 were classified as outer membrane, periplasmic, or plasma membrane proteins. The clinical A. baumannii strain DU202 responded specifically and induced different cell wall and membrane protein sets that provided resistance to the antibiotics. The induction of resistance-nodulation-cell division transporters and protein kinases, and the repression of outer membrane proteins were common responses in the presence of tetracycline and imipenem. Induction of a tetracycline resistant pump, ribosomal proteins, and iron-uptake transporters appeared to be dependent on tetracycline conditions, whereas β-lactamase and penicillin-binding proteins appeared to be dependent on imipenem conditions. These results suggest that combined liquid chromatography-based proteomic approaches can be used to identify cell wall and membrane proteins involved in the antibiotic resistance of A. baumannii.