Seunghwa Lee

The α-barrel tip region of Escherichia coli TolC homologs of Vibrio vulnificus interacts with the MacA protein to form the functional macrolide-specific efflux pump MacAB-TolC

TolC and its homologous family of proteins are outer membrane factors that are essential for exporting small molecules and toxins across the outer membrane in Gram-negative bacteria. Two open reading frames in the Vibrio vulnificus genome that encode proteins homologous to Escherichia coli TolC, designated TolCV1 and TolCV2, have 51.3% and 29.6% amino acid identity to TolC, respectively. In this study, we show that TolCV1 and TolCV2 functionally and physically interacted with the membrane fusion protein, MacA, a component of the macrolide-specific MacAB-TolC pump of E. coli. We further show that the conserved residues located at the aperture tip region of the α-hairpin of TolCV1 and TolCV2 played an essential role in the formation of the functional MacAB-TolC pump using site-directed mutational analyses. Our findings suggest that these outer membrane factors have conserved tip-to-tip interaction with the MacA membrane fusion protein for action of the drug efflux pump in Gramnegative bacteria.

Interaction Mediated by the Putative Tip Regions of MdsA and MdsC in the Formation of a Salmonella-Specific Tripartite Efflux Pump

To survive in the presence of a wide range of toxic compounds, gram-negative bacteria expel such compounds via tripartite efflux pumps that span both the inner and outer membranes. The Salmonella-specific MdsAB pump consists of MdsB, a resistance-nodulation-division (RND)-type inner membrane transporter (IMT) that requires the membrane fusion protein (MFP) MdsA, and an outer membrane protein (OMP; MdsC or TolC) to form a tripartite efflux complex. In this study, we investigated the role of the putative tip regions of MdsA and its OMPs, MdsC and TolC, in the formation of a functional MdsAB-mediated efflux pump. Comparative analysis indicated that although sequence homologies of MdsA and MdsC with other MFPs and OMPs, respectively, are extremely low, key residues in the putative tip regions of these proteins are well conserved. Mutagenesis studies on these conserved sites demonstrated their importance for the physical and functional interactions required to form an MdsAB-mediated pump. Our studies suggest that, despite differences in the primary amino acid sequences and functions of various OMPs and MFPs, interactions mediated by the conserved tip regions of OMP and MFP are required for the formation of functional tripartite efflux pumps in gram-negative bacteria.

Functional analysis of Vibrio vulnificus RND efflux pumps homologous to Vibrio cholerae VexAB and VexCD, and to Escherichia coli AcrAB

Resistance-nodulation-division (RND) efflux pumps are associated with multidrug resistance in many gram-negative pathogens. The genome of Vibrio vulnificus encodes 11 putative RND pumps homologous to those of Vibrio cholerae and Escherichia coli. In this study, we analyzed three putative RND efflux pumps, showing homology to V. cholerae VexAB and VexCD and to E. coli AcrAB, for their functional roles in multidrug resistance of V. vulnificus. Deletion of the vexAB homolog resulted in increased susceptibility of V. vulnificus to bile acid, acriflavine, ethidium bromide, and erythromycin, whereas deletion of acrAB homologs rendered V. vulnificus more susceptible to acriflavine only. Deletion of vexCD had no effect on susceptibility of V. vulnificus to these chemicals. Upon exposure to these antibacterial chemicals, expression of tolCV1 and tolCV2, which are putative outer membrane factors of RND efflux pumps, was induced, whereas expression levels of vexAB, vexCD, and acrAB homologs were not significantly changed. Our results show that the V. vulnificus homologs of VexAB largely contributed to in vitro antimicrobial resistance with a broad substrate specificity that was partially redundant with the AcrAB pump homologs.

Crystal Structure of a Soluble Fragment of the Membrane Fusion Protein HlyD in a Type I Secretion System of Gram-Negative Bacteria

The protein toxin HlyA of Escherichia coli is exported without a periplasmic intermediate by the type I secretion system (T1SS). The T1SS is composed of an inner membrane ABC transporter HlyB, an outer-membrane channel protein TolC, and a membrane fusion protein HlyD. However, the assembly of the T1SS remains to be elucidated. In this study, we determine the crystal structure of a part of the C-terminal periplasmic domain of HlyD. The long α-helical domain consisting of three α helices and a lipoyl domain was identified in the crystal structure. Based on the HlyD structure, we modeled the hexameric assembly of HlyD with a long α-helical barrel, which formed a complex with TolC in an intermeshing cogwheel-to-cogwheel manner, as observed in tripartite RND-type drug efflux pumps. These observations provide a structural blueprint for understanding the type I secretion system in pathogenic Gram-negative bacteria.

Interaction between the α-barrel tip of Vibrio vulnificus TolC homologs and AcrA implies the adapter bridging model.

The AcrAB-TolC multidrug efflux pump confers resistance to Escherichia coli against many antibiotics and toxic compounds. The TolC protein is an outer membrane factor that participates in the formation of type I secretion systems. The genome of Vibrio vulnificus encodes two proteins homologous to the E. coli TolC, designated TolCV1 and TolCV2. Here, we show that both TolCV1 and TolCV2 partially complement the E. coli TolC function and physically interact with the membrane fusion protein AcrA, a component of the E. coli AcrAB-TolC efflux pump. Using site-directed mutational analyses and an in vivo cross-linking assay, we demonstrated that the α-barrel tip region of TolC homologs plays a critical role in the formation of functional AcrAB-TolC efflux pumps. Our findings suggest the adapter bridging model as a general assembly mechanism for tripartite drug efflux pumps in Gram-negative bacteria.

FOXL2 Is an Essential Activator of SF-1-Induced Transcriptional Regulation of Anti-Müllerian Hormone in Human Granulosa Cells.

Anti-Müllerian hormone (AMH) is required for proper sexual differentiation by regulating the regression of the Müllerian ducts in males. Recent studies indicate that AMH could be an important factor for maintaining the ovarian reserve. However, the mechanisms of AMH regulation in the ovary are largely unknown. Here, we provide evidence that AMH is an ovarian target gene of steroidogenic factor-1 (SF-1), an orphan nuclear receptor required for proper follicle development. FOXL2 is an evolutionally conserved transcription factor, and its mutations cause blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES), wherein affected females display eyelid defects and premature ovarian failure (POF). Notably, we found that functional FOXL2 is essential for SF-1-induced AMH regulation, via protein–protein interactions between FOXL2 and SF-1. A BPES-inducing mutant of FOXL2 (290–291delCA) was unable to interact with SF-1 and failed to mediate the association between SF-1 and the AMH promoter. Therefore, this study identified a novel regulatory circuit for ovarian AMH production; specifically, through the coordinated interplay between FOXL2 and SF-1 that could control ovarian follicle development.

Corrected QT interval on the electrocardiogram after liver transplantation: Surrogate marker of poor clinical outcomes?

Background Prolongation of corrected QT interval (QTc) on the electrocardiogram is associated with cardiac arrhythmia and sudden death. Changes in the QTc (corrected QT) interval before and after liver transplantation (LT) for the treatment of liver cirrhosis (LC) and its association with clinical outcomes have not been fully evaluated. Methods From January 2011 to May 2016, consecutive 516 consecutive recipients were enrolled into LT registry and the median follow-up was 31 months (IQR 12–52). Patients with an available electrocardiogram before LT and 1 month after from LT were analyzed. Patients were divided into 2 groups according to prolonged QTc interval. The patient groups were analyzed separately according whether the electrocardiogram was preoperative or postoperative. The primary outcome was all-cause death during the follow-up period. Results A total of 283 patients were enrolled in the study. In the preoperative QTc prolongation group, there was not a significant rate difference in all-cause mortality in multivariate analysis (hazard ratio [HR], 0.94; 95% confidence interval [CI], 0.53–1.66; P = 0.26). However, in the postoperative QTc prolongation group, mortality was significantly increased (HR, 1.78; 95%CI, 1.05–3.03; P = 0.03) in patients who underwent LT. Conclusion In patients who underwent LT for LC, postoperative QTc prolongation on ECG, rather than preoperative, is associated with mortality. Larger clinical trials are needed to support this finding.