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.     

National Rural Health Mission--"rhetoric or reality"

National Rural Health Mission (NRHM) launched by Government of India holds great hopes and promises to serve the deprived and underserved communities of rural areas. The backbone of the programme is Accredited Social Health Activist (ASHA), which will play major role in the implementation of the programme. The invariable existence of socio-cultural clusters in the community has always been a major challenge to the health care efforts made by the government. Though ASHA is a novel concept to melt the ice in the culture of silence among the various cluster community groups, it is important to emphasize that inter-cluster communication may still pose a problem, which ASHA may be unable to address. Considering the constraints of ASHA and success of cluster community approach in Unicef supported community based Maternal Child Health & Nutrition (MCHN) Project, it is quite reasonable to state that inclusion of community mobilisers (Bal Parivar Mitra) from within the cluster community group might well be an asset to the programme, who may actually bring about the task of spreading the spirit of NRHM. These set of functionaries may work in coordination to bring about the desired behaviour changes and decrease the social delays responsible for maternal and childhood mortality. It will also bring about the feeling of community participation and ownership. The programme is in its initial phase but has years ahead of it to bring visible changes at community level to make it a reality.     

Clinical modifiers for heart failure following myocardial infarction

Heart failure (HF) is a clinical syndrome that occurs when the ability of the heart to meet the requirements of the body fails. Myocardial infarction (MI) is a common antecedent event that predisposes a patient to HF. Loss of cardiac function following MI occurs in the context of myocyte death and ventricular remodeling. The clinical significance of HF following MI is underscored by the fact that among MI survivors, the risk of death is markedly elevated in those who develop HF compared with those who do not. Various modifying factors associated with the development of HF following MI have been identified. Use of multimodality therapy with improved clinical outcomes for HF has increased the need to specifically identify the failing heart at an earlier stage. The ability to identify heart failure early in its pathogenesis will enable finer risk stratification following MI. This article reviews various risk predictors for the development of HF following MI.     

A comparison of beta adrenergic blocking drugs in the treatment of hypertension

Beta adrenergic blocking drugs were found to be effective hypotensive agents in the long-term treatment of patients with hypertension. In 40% of patients they appeared to be an extremely satisfactory antihypertensive agent. The fall in blood pressure was confirmed in a double blind study which also indicated that propranolol, prindolol, alprenolol and MK 950 (timolol) had similar antihypertensive properties. Propranolol and timolol reduced the pulse rate more than prindolol and alprenolol but the fall in blood pressure induced by the four drugs was not significantly different. The incidence of volunteered side effects was low, but when questioned specifically a large number of patients has disturbance of their dream pattern. This was more frequent and severe with prindolol and alprenolol. In this study bronchospasm was associated with the use of propranolol and timolol but not with that of prindolol and alprenolol.     

An improved in vitro pyrogen test: to detect picograms of endotoxin contamination in intravenous fluids using limulus amoebocyte lysate.

A method for in vitro pyrogen testing using Limulus amoebocyte lysate (LAL) has been described. The method is based upon the measurement of endotoxin-precipitable protein and can be used to measure picogram quantities equivalent to E. coli endotoxin in unknown solutions. When increasing concentrations of E. coli endotoxin are added to a constant amount of LAL and the reaction is allowed to proceed to completion, there is a proportional increase in the protein precipitated by endotoxin. Therefore, by measuring the amount of protein precipitated from LAL, it is possible to determine the equivalent E. coli endotoxin concentration in unknown solutions, when samples of the unknowns are run simultaneously with E. coli endotoxin standards and negative controls. The endotoxin proportional precipitation of protein occurs in reaction mixture showing gelation as well as in reaction mixture where the levels of endotoxin are lower than required for gelation. Determination of precipitated protein provides greater sensitivity for endotoxin detection than the gelation methods currently in use.