Role of internal loop dynamics in antibiotic permeability of outer membrane porins

Gram-negative bacteria pose a serious public health concern due to resistance to many antibiotics, caused by the low permeability of their outer membrane (OM). Effective antibiotics use porins in the OM to reach the interior of the cell; thus, understanding permeation properties of OM porins is instrumental to rationally develop broad-spectrum antibiotics. A functionally important feature of OM porins is undergoing open–closed transitions that modulate their transport properties. To characterize the molecular basis of these transitions, we performed an extensive set of molecular dynamics (MD) simulations of Escherichia coli OM porin OmpF. Markov-state analysis revealed that large-scale motion of an internal loop, L3, underlies the transition between energetically stable open and closed states. The conformation of L3 is controlled by H bonds between highly conserved acidic residues on the loop and basic residues on the OmpF β-barrel. Mutation of key residues important for the loop’s conformation shifts the equilibrium between open and closed states and regulates translocation of permeants (ions and antibiotics), as observed in the simulations and validated by our whole-cell accumulation assay. Notably, one mutant system G119D, which we find to favor the closed state, has been reported in clinically resistant bacterial strains. Overall, our accumulated ∼200 µs of simulation data (the wild type and mutants) along with experimental assays suggest the involvement of internal loop dynamics in permeability of OM porins and antibiotic resistance in Gram-negative bacteria.

Antibiotic resistance is a major concern in the treatment of bacterial infections (1–3). Design of antibiotics targeting Gram-negative bacteria is particularly challenging due to the presence of an outer membrane (OM) containing lipopolysaccharide glycolipids. The OM provides a major permeation barrier against the uptake of various substrates, including antibiotics (2, 4–11). Due to the low permeability of the OM, essential nutrients for the bacteria typically diffuse into the cell through a variety of general diffusion, β-barrel OM porins (6). Several OM porins have been shown to also be the main pathways for penetration of antibiotics into Gram-negative bacteria (6, 12).The general diffusion OM porins are typically organized into trimeric β-barrel structures with multiple loops connecting individual β-strands in each monomer. Of particular functional interest is a long internal loop (L3) that folds into the lumen of the monomeric β-barrels and forms a constriction region (CR) within the porin. The CR significantly narrows the pore (Fig. 1A) and acts as a major permeation barrier (13). Additionally, the CR contains many charged and polar residues, including acidic residues on the L3 loop and a cluster of tyrosine and basic residues on two opposite sides of the barrel wall (termed here as Y and B face, respectively) (Fig. 1B). The nature and organization of the B-face and Y-face residues will determine their interaction with the L3 loop and therefore, are expected to influence the dynamics of L3 and thus, the permeation properties of the porin.Open in a separate windowFig. 1.Structural features of OmpF in E. coli. (A) The monomeric radius profile [calculated using the program HOLE (31)] of the crystal structure of OmpF [PDB code 3POX (26)]. A molecular representation of a single monomer of OmpF is shown highlighting the internal loop L3 (orange) that constricts the pore. (B) A top-down view of OmpF showing that two acidic residues of L3 (D121 and E117) form hydrogen bonds with residues in the Y face. A cluster of basic residues (B face) on the opposite side of the Y face is hypothesized to facilitate the movement of L3 to further narrow the pore.A remarkable feature of many OM porins is their ability to undergo spontaneous conformational transitions between macroscopically distinct “open” and “closed” states. Functionally relevant conformational transitions in OM transport proteins are often active processes, coupled to and driven by an external energy input, such as the transmembrane voltage change (14–18). However, in OM porins, thermal fluctuations seem to be the main source for such conformational changes (19–24). The most abundant OM porin in Escherichia coli, OmpF, is an ideal model to study such conformational changes. OmpF is known for spontaneously fluctuating between highly stable conducting (open) and less stable nonconducting (closed) states, as observed in electrophysiological measurements under a low electric voltage (21, 22, 25). Notably, spontaneous fluctuations between conducting and nonconducting states have also been observed at 0 mV for OmpC, a close homolog of OmpF, where a KCl concentration gradient was used to drive ion diffusion (19). The asymmetric voltage-dependent inactivation of OmpC was not affected by mutations in these experiments, suggesting different molecular mechanisms for the spontaneous and voltage-dependent gating processes. However, the molecular mechanism by which OmpF undergoes these transitions is still not well understood.We expect that the internal loop, L3, might play a direct role in the open–closed transitions in OmpF due to its location within the CR. Several of the acidic residues in L3, including E117 and D121, interact with tyrosine residues of the Y face in the crystal structure of OmpF (Protein Data Bank [PDB] code 3POX) (Fig. 1A) (26). It has been observed in a previous molecular dynamics (MD) study that L3 leaves the Y face to transiently interact with the B face, thereby narrowing the pore (27). These results led to the hypothesis that L3 movement could be responsible for controlling open–closed transitions of the pore. However, due to the short duration of the simulations and the nonphysiological conditions (vacuum) used in that computational study, structural support for this hypothesis remained lacking. The role of L3 conformational dynamics in permeability of OM porins remains an open subject. Notably, it is reported that the movement of L3 may not be critical to “voltage gating” of OM porins, a process occurring at voltages of 100 mV or more (28–31). However, this does not exclude the possibility that L3 can be involved in the open–closed transition at low or zero external voltage.As a support for the latter hypothesis, mutations to disrupt the hydrogen bonds between L3 and Y face in OmpC, a homologous protein to OmpF, have been reported to increase closed-state visiting frequency of the porin under no voltage conditions in electrophysiology experiments (19). Furthermore, mutation of B-face residues to uncharged or negatively charged residues in OmpF showed an increase in substrate permeation (32–34), possibly as a result of reduced attraction of L3 to the B face, shifting the equilibrium toward the open state. Moreover, the crystal structure of a clinically relevant mutant of OmpF, G119D (35), suggested that adding a negative charge to L3 can potentiate its attraction toward the B face, thereby further reducing the pore size (SI Appendix, Fig. S1) as compared with the wild type (WT) and therefore, shifting the porin toward the closed state. This shift resulted in decreased permeability of the mutant to substrates such as carbohydrates and antibiotics (35). Strikingly, this mutant porin was shown to confer colicin N resistance to clinical strains of E. coli (35).The aim of this study is to provide atomistic insight into the mechanisms controlling the equilibrium between open and closed states of OmpF using extended MD simulations, which are used to construct a Markov-state model (MSM) for the process. MSMs are a class of model used to describe the long-timescale dynamics of molecular systems and to obtain the thermodynamic and kinetic information about dynamic processes from MD simulations (36). Using MSMs, we find that large-scale motion of L3 controls the equilibrium between conducting and nonconducting states of OmpF. Further analysis using the transition path theory (TPT) (37) revealed that transitions between open and closed states occur in two steps: 1) movement of E117 to the B face to initiate the transition from the open state and 2) movement of D121 to the B face that drives a large-scale movement of L3 to mediate complete closure of the pore.The significant effect of pore narrowing in the closed state on OmpF permeability is examined by electrophysiology simulations, in which ionic currents are shown to be substantially reduced in the closed state, and by free energy calculations indicating the presence of a higher-energy barrier against permeation of an antibiotic. Furthermore, simulations of charge-reversal mutants of B-face residues key in our proposed mechanism show a significant decrease in the probability of the closed state. This agrees with the increased accumulation observed for several antibiotics in our whole-cell assays, in which we expressed the mutant porins. Furthermore, previous experiments also report an increase in substrate permeability of these mutants (32–34). According to our model, these mutations reduce the attraction of E117 or D121 to the B face, thereby reducing the closed-state probability. Overall, our results provide mechanistic details on how thermal motion of an internal loop controls a dynamic equilibrium between open and closed states, thereby regulating permeability of OM porins.     

Prevalence of Chicken Infectious Anaemia Virus (CIAV) in Commercial Poultry Flocks of Northern India: A Serological Survey

Globally, the chicken infectious anaemia virus (CIAV) has gained much importance as an immunosuppressive and economically important emerging pathogen of poultry. In recent years, the virus has been detected and isolated from poultry flocks of India. The present study reports the first sero‐epidemiological investigation of the presence of CIAV infection in poultry flocks of the country. A total of 404 serum samples were collected from chicken flocks of eleven poultry farms, which contain a total of 0.34 million birds from four Northern states, suspected of having chicken infectious anaemia (CIA). Screening of the sera samples using a commercially available enzyme‐linked immunosorbent assay (ELISA) kit revealed 351 serum samples (86.88%) to be positive for CIAV antibodies. A high CIAV prevalence rate recorded in the present investigation, along with earlier virus detection reports, indicates the widespread distribution of the virus and that CIAV should be considered an economically important poultry pathogen affecting poultry industry of India. Extensive nationwide epidemiological studies are suggested for revealing the economic impact of CIA and to initiate further research along with devising and adapting suitable prevention and control strategies especially the use of suitable vaccines for safeguarding poultry health and production in the country.     

A Novel Metabolite from Aspergillus ochraceus JGI 25 Showing Cytotoxicity to Hela Cells

This study aims at the isolation of filamentous fungi, extraction of metabolites, and evaluation of the cytotoxic properties on HeLa cells and normal human lymphocytes. We isolated fungi from the soil by serial dilution method. One of the isolates was chosen and identified as Aspergillus ochraceus Wilhelm (Trichocomaceae) by standard techniques. The metabolites were extracted using methanol. Different concentrations of the extract were evaluated for their potential anticancer activity on HeLa cells by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide assay and the safety of the extract was checked on normal human lymphocytes. The extract was purified by chromatographic techniques like thin-layer chromatography and high-performance liquid chromatography, and subjected to mass spectrometric analysis. The extract showed significant cytotoxic potential on HeLa cells at low concentrations with a half maximal inhibitory concentration value of <50 μg/ml. The extract gave 10 fractions by thin layer chromatography, and fraction B had higher toxicity than the rest. This fraction gave a single peak by high-performance liquid chromatography and had a mass-to-charge ratio of 905.65, which did not match any of the earlier known fungal metabolites or metabolites from other strains of A. ochraceus. The metabolite from A. ochraceus is alkaloid in nature, cytotoxic to HeLa cells, and appears to be a novel with anticancer potentials, which could be explored further for characterization of the active component.     

Pancreato‐enteric anastomosis: The duct evagination technique

Pancreatic anastomotic failure remains the most frequent and potentially life‐threatening complication following Pancreatoduodenectomy. Numerous modifications in the technique of the pancreatoenteric anastomosis have been reported. We suggest a simple modification which involves “evaginating” the cut end of the pancreatic duct. This technique helps avoid a compromise of the pancreatic ductal patency, and by achieving a wide pancreatic ductal opening can facilitate a safer pancreato‐enteric anastomosis. In addition, by possibly decreasing the likelihood of post‐operative pancreatic ductal stenosis, it has the potential to reduce post‐Pancreatoduodenectomy pancreatic exocrine insufficiency. The modification acts as an adjunct to an already established technique yielding good results. J. Surg. Oncol. 2009;100:277–278. © 2009 Wiley‐Liss, Inc.     

Altered protein kinase a in brain of learned helpless rats: effects of acute and repeated stress.

BACKGROUND: Stress-induced learned helplessness (LH) in animals serves as a model of behavioral depression and some aspects of posttraumatic stress disorder. We examined whether LH behavior is associated with alterations in protein kinase A (PKA), a critical phosphorylating enzyme, how long these alterations persist after inescapable shock (IS), and whether repetition of IS prolongs the duration of LH behavior and changes in PKA. METHODS: Rats were exposed to IS either on day 1 or twice, on day 1 and day 7. Rats were tested for escape latency on days 2 and 4 after day 1 IS or days 2, 8, and 14 after day 1 and day 7 IS. [(3)H]cAMP (cyclic adenosine monophosphate) binding, catalytic activity and expression of PKA subunits were determined in frontal cortex and hippocampus. RESULTS: Higher escape latencies were observed in rats tested on day 2 after single IS and on day 14 after repeated IS. Concurrently, reduced [(3)H]cAMP binding, PKA activity, and expression of selective PKA RIIbeta and Calpha and Cbeta subunits were observed in the brains of these rats. CONCLUSIONS: Repeated IS prolongs the duration of LH behavior, and LH behavior is associated with reductions in apparent activity and expression of PKA. These reductions in PKA may be critical in the pathophysiology of depression and other stress-related disorders.     

Pre-cancerous lesions of stomach

Total 39 cases of carcinoma stomach were noticed out of 142 malignant tumours of GIT (27.46 percent). Histologically maximum cases were of diffuse type (56.41 percent) followed by intestinal type (35.89 percent) and indolent mucoid carcinoma (7.69 percent) of the stomach. The surrounding epithelium showed lot of changes in the intestinal type of carcinoma stomach. About 78.57 percent showed intestinal metaplasia, 14.28 percent of these cases showed chronic gastric ulcer and severe dysplasia (carcinoma in situ) and another 14.28 percent revealed villous adenoma with carcinoma in situ. In contrast to this, in diffuse variety, only 13.63 percent cases revealed intestinal metaplasia, 27.27 percent showed basal cell hyperplasia, stratification of the epithelium of crypts and diffuse infiltration of mucosa by malignant cells and 4.54 percent showed atrophic gastritis also. In mucoid carcinoma all cases had basal cell hyperplasia and stratification of crypts. Hence these conditions should be taken as premalignant lesions of stomach and should be cured in proper time.     

A missing denture''s misadventure!

We report a late onset, benign, tracheoesophageal fistula in a 51-year-old man, due to an accidentally swallowed denture. In view of the extensive peri-esophageal sepsis and fibrosis, he was managed by a subtotal esophagectomy and a cervical esophagogastric anastomosis. The tracheal defect was closed with the help of an intercostal muscle flap. This report also highlights the difficulty in identifying swallowed prosthetic dental material radiologically, when no metallic component is present. This fact was also responsible for the delay in diagnosis, eventually leading to the rare complication of a tracheoesophageal fistula.