Tunable force transduction through the Escherichia coli cell envelope.

The outer membrane (OM) of Gram-negative bacteria is not energised and so processes requiring a driving force must connect to energy-transduction systems in the inner membrane (IM). Tol (Tol-Pal) and Ton are related, proton motive force- (PMF-) coupled assemblies that stabilise the OM and import essential nutrients, respectively. Both rely on proton-harvesting IM motor (stator) complexes, which are homologues of the flagellar stator unit Mot, to transduce force to the OM through elongated IM force transducer proteins, TolA and TonB, respectively. How PMF-driven motors in the IM generate mechanical work at the OM via force transducers is unknown. Here, using cryoelectron microscopy, we report the 4.3Å structure of the Escherichia coli TolQR motor complex. The structure reaffirms the 5:2 stoichiometry seen in Ton and Mot and, with motor subunits related to each other by 10 to 16° rotation, supports rotary motion as the default for these complexes. We probed the mechanism of force transduction to the OM through in vivo assays of chimeric TolA/TonB proteins where sections of their structurally divergent, periplasm-spanning domains were swapped or replaced by an intrinsically disordered sequence. We find that TolA mutants exhibit a spectrum of force output, which is reflected in their respective abilities to both stabilise the OM and import cytotoxic colicins across the OM. Our studies demonstrate that structural rigidity of force transducer proteins, rather than any particular structural form, drives the efficient conversion of PMF-driven rotary motions of 5:2 motor complexes into physiologically relevant force at the OM.

The quantitative basis for the redistribution of immobile bacterial lipoproteins to division septa.

The spatial localisation of proteins is critical for most cellular function. In bacteria, this is typically achieved through capture by established landmark proteins. However, this requires that the protein is diffusive on the appropriate timescale. It is therefore unknown how the localisation of effectively immobile proteins is achieved. Here, we investigate the localisation to the division site of the slowly diffusing lipoprotein Pal, which anchors the outer membrane to the cell wall of Gram-negative bacteria. While the proton motive force-linked TolQRAB system is known to be required for this repositioning, the underlying mechanism is unresolved, especially given the very low mobility of Pal. We present a quantitative, mathematical model for Pal relocalisation in which dissociation of TolB-Pal complexes, powered by the proton motive force across the inner membrane, leads to the net transport of Pal along the outer membrane and its deposition at the division septum. We fit the model to experimental measurements of protein mobility and successfully test its predictions experimentally against mutant phenotypes. Our model not only explains a key aspect of cell division in Gram-negative bacteria, but also presents a physical mechanism for the transport of low-mobility proteins that may be applicable to multi-membrane organelles, such as mitochondria and chloroplasts.

Antifungal Styryloquinolines as Candida albicans Efflux Pump Inhibitors: Styryloquinolines are ABC Transporter Inhibitors.

Styrylquinolines are heterocyclic compounds that are known for their antifungal and antimicrobial activity. Metal complexation through hydroxyl groups has been claimed to be a plausible mechanism of action for these types of compounds. A series of novel structures with protected hydroxyl groups have been designed and synthesized to verify the literature data. Their antifungal activity against wild-type Candida albicans strain and mutants with silenced efflux pumps activity has been determined. Combinations with fluconazole revealed synergistic interactions that were dependent on the substitution pattern. These results open a new route for designing active antifungal agents on a styrylquinoline scaffold.

The multifarious roles of Tol-Pal in Gram-negative bacteria.

In the 1960s several groups reported the isolation and preliminary genetic mapping of Escherichia coli strains tolerant towards the action of colicins. These pioneering studies kick-started two new fields in bacteriology; one centred on how bacteriocins like colicins exploit the Tol (or more commonly Tol-Pal) system to kill bacteria, the other on the physiological role of this cell envelope-spanning assembly. The following half century has seen significant advances in the first of these fields whereas the second has remained elusive, until recently. Here, we review work that begins to shed light on Tol-Pal function in Gram-negative bacteria. What emerges from these studies is that Tol-Pal is an energised system with fundamental, interlinked roles in cell division - coordinating the re-structuring of peptidoglycan at division sites and stabilising the connection between the outer membrane and underlying cell wall. This latter role is achieved by Tol-Pal exploiting the proton motive force to catalyse the accumulation of the outer membrane peptidoglycan associated lipoprotein Pal at division sites while simultaneously mobilising Pal molecules from around the cell. These studies begin to explain the diverse phenotypic outcomes of tol-pal mutations, point to other cell envelope roles Tol-Pal may have and raise many new questions.

The lipoprotein Pal stabilises the bacterial outer membrane during constriction by a mobilisation-and-capture mechanism.

Coordination of outer membrane constriction with septation is critical to faithful division in Gram-negative bacteria and vital to the barrier function of the membrane. This coordination requires the recruitment of the peptidoglycan-binding outer-membrane lipoprotein Pal at division sites by the Tol system. Here, we show that Pal accumulation at Escherichia coli division sites is a consequence of three key functions of the Tol system. First, Tol mobilises Pal molecules in dividing cells, which otherwise diffuse very slowly due to their binding of the cell wall. Second, Tol actively captures mobilised Pal molecules and deposits them at the division septum. Third, the active capture mechanism is analogous to that used by the inner membrane protein TonB to dislodge the plug domains of outer membrane TonB-dependent nutrient transporters. We conclude that outer membrane constriction is coordinated with cell division by active mobilisation-and-capture of Pal at division septa by the Tol system.

Blocking and dislocation of Candida albicans Cdr1p transporter by styrylquinolines.

Styrylquinolines are a novel group of quinoline drugs that are known to have p53-independent antiproliferative activity and antiviral properties. This study evaluated the antifungal activity of these drugs more deeply, particularly their activity modulation towards Cdr1p, the main multidrug transporter of Candida albicans. Styrylquinolines were found to have antifungal activity and to work synergistically with fluconazole. Additionally, they decreased the extracellular concentration of rhodamine 6G in ABC-transporter-expressing cells. The cellular localization of GFP-tagged Cdr1p was assessed by epifluorescent microscopy. Styrylquinolines induce expression of Cdr1p, as confirmed by Western blotting. Three of four drugs tested caused the partial delocalization of transport protein to the cytoplasm. These results show the first evidence that styrylquinolines decrease the activity of ABC multidrug transporters in C. albicans.

Estimation of Candida albicans ABC Transporter Behavior in Real-Time via Fluorescence.

We present a fluorometric method for determining ABC transporter activity in the pathogenic fungus C. albicans during different growth phases and in response to glucose. The carbocyanine dye diS-C3(3) was previously used to monitor plasma membrane potentials and test the influence of surface-active compounds in membrane polarization. We used diS-C3(3) to show changes in fluorescence kinetics that reflect changes in the activity of ABC transporters in C. albicans growth. Cdr1-GFP fluorescence, revealed that Cdr1p relocates to the inside of the cell after the early-log growth phase. Addition of glucose to the cell suspension resulted in Cdr1p transporter expression in the CDR2-knockout strain. We confirmed the diS-C3(3) results by standard RT-PCR and Western blotting.

Detection of inhibitors of Candida albicans Cdr transporters using a diS-C3(3) fluorescence.

Candida albicans is a major cause of opportunistic and life-threatening, systemic fungal infections. Hence new antifungal agents, as well as new methods to treat fungal infections, are still needed. The application of inhibitors of drug-efflux pumps may increase the susceptibility of C. albicans to drugs. We developed a new fluorescence method that allows the in vivo activity evaluation of compounds inhibiting of C. albicans transporters. We show that the potentiometric dye 3,3'-dipropylthiacarbocyanine iodide diS-C3(3) is pumped out by both Cdr1 and Cdr2 transporters. The fluorescence labeling with diS-C3(3) enables a real-time observation of the activity of C. albicans Cdr1 and Cdr2 transporters. We demonstrate that enniatin A and beauvericin show different specificities toward these transporters. Enniatin A inhibits diS-C3(3) efflux by Cdr1 while beauvericin inhibits both Cdr1p and Cdr2p.

Value of ultrasonography in assessment of recent injury of anterior talofibular ligament in children.

INTRODUCTION: Sprained ankle is a very common injury in children. Proper treatment of ligament injuries enables full recovery. X-ray and US examinations are commonly available diagnostic methods. MATERIAL AND METHODS: Two hundred and six children (113 girls and 93 boys, mean age 10.6) with recent ankle joint sprain (up to 7 days of injury) were subject to a retrospective analysis. All patients underwent an X-ray and US examination of the ankle joint within 7 days of injury. In 19 patients, anterior talofibular ligament reconstruction was conducted. RESULTS: X-ray failed to visualize a pathology in 129 children (63%); in 24 patients (12%), avulsion fracture of the lateral malleolus was found, and in 36 cases (17%), effusion in the talocrural joint was detected. Ultrasonography failed to visualize a pathology in 19 children (9%); in 60 patients (29%), it showed avulsion fracture of the lateral malleolus involving the attachment of the anterior talofibular ligament (ATFL); in 34 cases (17%), complete ATFL tear was detected, and in 51 patients (25%), partial ATFL injury was found. Other injuries constituted 19%. The surgeries conducted to repair the anterior talofibular ligament (19) confirmed the US/X-ray diagnoses in 100% of cases. Avulsion ATFL injury, i.e. the one that involves the ligament attachment site, is usually found in younger children (median: 8 years of age). Complete ATFL tears (not involving the attachment site) concern older children (median: 14 years of age). CONCLUSIONS: Since X-ray is of limited value in diagnosing ankle joint pathologies in recent sprain injuries in children, soft tissue imaging, i.e. ultrasonography, is the basic examination to assess the ligament complex. Avulsion fractures, which involve the ATFL attachment site and are usually found in younger children, are a consequence of the incomplete ossification and require urgent diagnosis and orthopedic consultation.