The role of TMS 12 in the staphylococcal multidrug efflux protein QacA.

OBJECTIVES: To elucidate the importance of a region in QacA predicted to be important in antimicrobial substrate recognition. METHODS: A total of 38 amino acid residues within or flanking putative transmembrane helix segment (TMS) 12 of QacA were individually replaced with cysteine using site-directed mutagenesis. The impact of these mutations on protein expression, drug resistance, transport activity and interaction with sulphhydryl-binding compounds was determined. RESULTS: Accessibility analysis of cysteine-substituted mutants identified the extents of TMS 12, which allowed for refinement of the QacA topology model. Mutation of Gly-361, Gly-379 and Ser-387 in QacA resulted in reduced resistance to at least one bivalent substrate. Interaction with sulphhydryl-binding compounds in efflux and binding assays demonstrated the role of Gly-361 and Ser-387 in the binding and transport pathway of specific substrates. The highly conserved residue Gly-379 was found to be important for the transport of bivalent substrates, commensurate with the role of glycine residues in helical flexibility and interhelical interactions. CONCLUSIONS: TMS 12 and its external flanking loop is required for the structural and functional integrity of QacA and contains amino acids directly involved in the interaction with substrates.

A Unique Sequence Is Essential for Efficient Multidrug Efflux Function of the MtrD Protein of Neisseria gonorrhoeae.

Antimicrobial resistance in Neisseria gonorrhoeae has reached an alarming level, severely impacting the effective treatment of gonorrhea. Belonging to the resistance-nodulation-cell division (RND) superfamily of efflux transporters, the MtrD membrane protein of N. gonorrhoeae provides resistance to a broad range of antimicrobial compounds. A unique feature of MtrD is an 11-residue sequence (from N917 to P927 [N917-P927]) that connects transmembrane helices (TMS) 9 and 10; this sequence is not present in homologous RND proteins. This study explores the structural and functional roles of the N917-P927 region by means of mutant analysis and molecular dynamics simulations. We show that N917-P927 plays a key role in modulating substrate access to the binding cleft and influences the overall orientation of the protein within the inner membrane necessary for optimal functioning. Removal of N917-P927 significantly reduced MtrD-mediated resistance to a range of antimicrobials and mutations of three single amino acids impacted MtrD-mediated multidrug resistance. Furthermore, molecular dynamics simulations showed deletion of N917-P927 in MtrD may dysregulate access of the substrate to the binding cleft and closure of the substrate-binding pocket during the transport cycle. These findings indicate that N917-P927 is a key region for interacting with the inner membrane, conceivably influencing substrate capture from the membrane-periplasm interface and thus is essential for full multidrug resistance capacity of MtrD. IMPORTANCE The historical sexually transmitted infection gonorrhea continues to be a major public health concern with an estimated global annual incidence of 86.9 million cases. N. gonorrhoeae has been identified by the World Health Organization as one of the 12 antimicrobial-resistant bacterial species that poses the greatest risk to human health. As the major efflux pump in gonococci, the MtrD transporter contributes to the cell envelope barrier in this organism and pumps antimicrobials from the periplasm and inner membrane, resulting in resistance. This study demonstrates that a unique region of the MtrD protein that connects TMS 9 and TMS 10 forms a structure that may interact with the inner membrane positioning TMS 9 and stabilizing the protein facilitating substrate capture from the inner membrane-periplasm interface. Analysis of mutants of this region identified that it was essential for MtrD-mediated multidrug resistance. Characterization of the structure and function of this unique local region of MtrD has implications for drug efflux mechanisms used by related proteins and is important knowledge for development of antibiotics that bypass efflux.

Coordination of Substrate Binding and Protonation in the N. gonorrhoeae MtrD Efflux Pump Controls the Functionally Rotating Transport Mechanism.

Multidrug resistance is a serious problem that threatens the effective treatment of the widespread sexually transmitted disease gonorrhea, caused by the Gram-negative bacterium Neisseria gonorrhoeae. The drug efflux pump primarily implicated in N. gonorrhoeae antimicrobial resistance is the inner membrane transporter MtrD, which forms part of the tripartite multiple transferable resistance (Mtr) CDE efflux system. A structure of MtrD was first solved in 2014 as a symmetrical homotrimer, and then, recently, as an asymmetrical homotrimer. Through a series of molecular dynamics simulations and mutagenesis experiments, we identify the combination of substrate binding and protonation states of the proton relay network that drives the transition from the symmetric to the asymmetric conformation of MtrD. We characterize the allosteric coupling between the functionally important local regions that control conformational changes between the access, binding, and extrusion states and allow for transition to the asymmetric MtrD conformation. We also highlight a significant rotation of the transmembrane helices caused by protonation of the proton relay network, which widens the intermonomeric gap that is a hallmark of the rotational transporter mechanism. This is the first analysis and description of the transport mechanism for the N. gonorrhoeae MtrD transporter and provides evidence that antimicrobial efflux in MtrD follows the functionally rotating transport mechanism seen in protein homologues from the same transport protein superfamily.

Multidrug Resistance in Neisseria gonorrhoeae: Identification of Functionally Important Residues in the MtrD Efflux Protein.

A key mechanism that Neisseria gonorrhoeae uses to achieve multidrug resistance is the expulsion of structurally different antimicrobials by the MtrD multidrug efflux protein. MtrD resembles the homologous Escherichia coli AcrB efflux protein with several common structural features, including an open cleft containing putative access and deep binding pockets proposed to interact with substrates. A highly discriminating N. gonorrhoeae strain, with the MtrD and NorM multidrug efflux pumps inactivated, was constructed and used to confirm and extend the substrate profile of MtrD to include 14 new compounds. The structural basis of substrate interactions with MtrD was interrogated by a combination of long-timescale molecular dynamics simulations and docking studies together with site-directed mutagenesis of selected residues. Of the MtrD mutants generated, only one (S611A) retained a wild-type (WT) resistance profile, while others (F136A, F176A, I605A, F610A, F612C, and F623C) showed reduced resistance to different antimicrobial compounds. Docking studies of eight MtrD substrates confirmed that many of the mutated residues play important nonspecific roles in binding to these substrates. Long-timescale molecular dynamics simulations of MtrD with its substrate progesterone showed the spontaneous binding of the substrate to the access pocket of the binding cleft and its subsequent penetration into the deep binding pocket, allowing the permeation pathway for a substrate through this important resistance mechanism to be identified. These findings provide a detailed picture of the interaction of MtrD with substrates that can be used as a basis for rational antibiotic and inhibitor design.IMPORTANCE With over 78 million new infections globally each year, gonorrhea remains a frustratingly common infection. Continuous development and spread of antimicrobial-resistant strains of Neisseria gonorrhoeae, the causative agent of gonorrhea, have posed a serious threat to public health. One of the mechanisms in N. gonorrhoeae involved in resistance to multiple drugs is performed by the MtrD multidrug resistance efflux pump. This study demonstrated that the MtrD pump has a broader substrate specificity than previously proposed and identified a cluster of residues important for drug binding and translocation. Additionally, a permeation pathway for the MtrD substrate progesterone actively moving through the protein was determined, revealing key interactions within the putative MtrD drug binding pockets. Identification of functionally important residues and substrate-protein interactions of the MtrD protein is crucial to develop future strategies for the treatment of multidrug-resistant gonorrhea.

The role played by drug efflux pumps in bacterial multidrug resistance.

Antimicrobial resistance is a current major challenge in chemotherapy and infection control. The ability of bacterial and eukaryotic cells to recognize and pump toxic compounds from within the cell to the environment before they reach their targets is one of the important mechanisms contributing to this phenomenon. Drug efflux pumps are membrane transport proteins that require energy to export substrates and can be selective for a specific drug or poly-specific that can export multiple structurally diverse drug compounds. These proteins can be classified into seven groups based on protein sequence homology, energy source and overall structure. Extensive studies on efflux proteins have resulted in a wealth of knowledge that has made possible in-depth understanding of the structures and mechanisms of action, substrate profiles, regulation and possible inhibition of many clinically important efflux pumps. This review focuses on describing known families of drug efflux pumps using examples that are well characterized structurally and/or biochemically.

A genetically encoded and gate for cell-targeted metabolic labeling of proteins.

We describe a genetic AND gate for cell-targeted metabolic labeling and proteomic analysis in complex cellular systems. The centerpiece of the AND gate is a bisected methionyl-tRNA synthetase (MetRS) that charges the Met surrogate azidonorleucine (Anl) to tRNA(Met). Cellular protein labeling occurs only upon activation of two different promoters that drive expression of the N- and C-terminal fragments of the bisected MetRS. Anl-labeled proteins can be tagged with fluorescent dyes or affinity reagents via either copper-catalyzed or strain-promoted azide-alkyne cycloaddition. Protein labeling is apparent within 5 min after addition of Anl to bacterial cells in which the AND gate has been activated. This method allows spatial and temporal control of proteomic labeling and identification of proteins made in specific cellular subpopulations. The approach is demonstrated by selective labeling of proteins in bacterial cells immobilized in the center of a laminar-flow microfluidic channel, where they are exposed to overlapping, opposed gradients of inducers of the N- and C-terminal MetRS fragments. The observed labeling profile is predicted accurately from the strengths of the individual input signals.

GRID: a high-resolution protein structure refinement algorithm.

The energy-based refinement of protein structures generated by fold prediction algorithms to atomic-level accuracy remains a major challenge in structural biology. Energy-based refinement is mainly dependent on two components: (1) sufficiently accurate force fields, and (2) efficient conformational space search algorithms. Focusing on the latter, we developed a high-resolution refinement algorithm called GRID. It takes a three-dimensional protein structure as input and, using an all-atom force field, attempts to improve the energy of the structure by systematically perturbing backbone dihedrals and side-chain rotamer conformations. We compare GRID to Backrub, a stochastic algorithm that has been shown to predict a significant fraction of the conformational changes that occur with point mutations. We applied GRID and Backrub to 10 high-resolution (≤ 2.8 Å) crystal structures from the Protein Data Bank and measured the energy improvements obtained and the computation times required to achieve them. GRID resulted in energy improvements that were significantly better than those attained by Backrub while expending about the same amount of computational resources. GRID resulted in relaxed structures that had slightly higher backbone RMSDs compared to Backrub relative to the starting crystal structures. The average RMSD was 0.25 ± 0.02 Å for GRID versus 0.14 ± 0.04 Å for Backrub. These relatively minor deviations indicate that both algorithms generate structures that retain their original topologies, as expected given the nature of the algorithms.

Treatment of postoperative nausea and vomiting after spinal anesthesia for cesarean delivery: A randomized, double-blinded comparison of midazolam, ondansetron, and a combination.

BACKGROUND: The antiemetic efficacy of midazolam and ondansetron was shown before. The aim of the present study was to compare efficacy of using intravenous midazoalm, ondansetron, and midazolam in combination with ondansetron for treatment of nausea and vomiting after cesarean delivery in parturient underwent spinal anesthesia. MATERIALS AND METHODS: ONE HUNDRED THIRTY TWO PARTURIENTS WERE RANDOMLY ALLOCATED TO ONE OF THREE GROUPS: group M (n = 44) that received intravenous midazoalm 30 µg/kg; group O (n = 44) that received intravenous ondansetron 8 mg; group MO (n = 44) that received intravenous midazoalm 30 µg/kg combined with intravenous ondansetron 8 mg if patients had vomiting or VAS of nausea ≥ 3 during surgery (after umbilical cord clamping) and 24 hours after that. The incidence and severity of vomiting episodes and nausea with visual analog scale (VAS) > 3 were evaluated at 2 hours, 6 hours, and 24 hours after injection of study drugs. RESULTS: The incidence of nausea was significantly less in group MO compared with group M and group O at 6 hours postoperatively (P = 0.01). This variable was not significantly different in three groups at 2 hours and 24 hours after operation. The severity of nausea and vomiting was significantly different in three groups at 6 hours after operation (P < 0.05). CONCLUSION: Our study showed that using intravenous midazolam 30 µg/kg in combination with intravenous ondansetron 8 mg was superior to administering single drug in treatment of emetic symptoms after cesarean delivery under spinal anesthesia.

Prevalence and antimicrobial susceptibility of extended-spectrum beta-lactamase-producing bacteria in intensive care units of Sanandaj general hospitals (Kurdistan, Iran).

This study focused on analyzing the spread of extended-spectrum beta-lactamase (ESBL) enzymes among Gram-negative bacteria at intensive care units (ICUs). Between January 2007 and January 2008, 301 consecutive clinical isolates of Gram-negative type were isolated. Of these, 66 strains were collected from patients in ICUs in two major hospitals in Sanandaj (Kurdistan, Iran). The isolates were identified, tested for antimicrobial susceptibility, and analyzed for the presence of ESBL using the double-disk synergy test. Isolates with a positive ESBL phenotype were subjected to PCR for SHV, TEM, OXA and CTX-M beta-lactamase gene families. Sixty-six Gram-negative bacteria were isolated from clinical samples of 66 ICU patients. These isolates included 16 Escherichia coli, 28 Enterobacter spp., 5 Pseudomonas spp., 10 Klebsiella pneumoniae, 3 Serratia marcescens and 1 Stenotrophomonas maltophilia. Twenty-three (34.85%) of these isolates were ESBL producing. The ESBL genes detected were SHV, TEM, OXA-1, OXA-2 and CTX-M. The results show the presence of ESBL genes among Gram-negative bacteria in the ICU setting of Sanandaj's hospitals. There is a need to institute a strict hospital infection control policy and regular surveillance of bacterial resistance to antimicrobial agents.

Prevalence and significance of a negative extended-spectrum beta-lactamase (ESBL) confirmation test result after a positive ESBL screening test result for isolates of Escherichia coli and Klebsiella pneumoniae: results from the SENTRY Asia-Pacific Surveillance Program.

A negative extended-spectrum beta-lactamase (ESBL) confirmation test result obtained after a positive ESBL screening test result using Clinical and Laboratory Standards Institute methods has been a common occurrence among isolates of Escherichia coli and Klebsiella pneumoniae in the SENTRY Antimicrobial Surveillance Program in the Asia-Pacific region. Among isolates collected between 1998 and 2004 this screen-positive, nonconfirmed profile (failed to show clavulanate synergy) was observed in 8.9% of 4,515 E. coli isolates and 20.3% of 2,303 K. pneumoniae isolates. We then selected 52 E. coli isolates and 68 K. pneumoniae isolates with a negative ESBL confirmation test, as well as comparable number of isolates with confirmed ESBL-positive tests, and examined them for the presence of TEM, SHV, plasmid-borne ampC, and CTX-M genes. We found that 62% of nonconfirming E. coli isolates and 75% of nonconfirming K. pneumoniae harbored a plasmid-borne AmpC enzyme of the CIT or DHA type. The majority of nonconfirming E. coli and K. pneumoniae from the Asia-Pacific region harbor important beta-lactamases, and a positive screening test alone should be sufficient grounds to report resistance to extended-spectrum cephalosporins in this region.