SpvC is a Salmonella effector with phosphothreonine lyase activity on host mitogen-activated protein kinases.

SpvC is encoded by the Salmonella virulence plasmid. We have investigated the biochemical function of SpvC and the mechanism by which it is secreted by bacteria and translocated into infected macrophages. We constructed a strain carrying a deletion in spvC and showed that the strain is attenuated for systemic virulence in mice. SpvC can be secreted in vitro by either the SPI-1 or SPI-2 type III secretion systems. Cell biological and genetic experiments showed that translocation of the protein into the cytosol of macrophages by intracellular bacteria is dependent on the SPI-2 T3SS. Using antibodies specific to phospho-amino acids and mass spectrometry we demonstrate that SpvC has phosphothreonine lyase activity on full-length phospho-Erk (pErk) and a synthetic 13-amino-acid phospho-peptide containing the TXY motif. A Salmonella strain expressing spvC from a plasmid downregulated cytokine release from infected cells.

Dynamin is required for F-actin assembly and pedestal formation by enteropathogenic Escherichia coli (EPEC).

After attaching to human intestinal epithelial cells, enteropathogenic Escherichia coli (EPEC) induces the formation of an actin-rich pedestal-like structure. The signalling pathway leading to pedestal formation is initiated by the bacterial protein Tir, which is inserted into the host cell plasma membrane. The domain exposed on the cell surface binds to another bacterial protein, intimin, while one of the cytoplasmic domains binds the adaptor protein Nck. This leads to recruitment of other cytoskeletal proteins including neural Wiskott-Aldrich syndrome protein (N-WASP) and Arp2/3, resulting in focused actin polymerization at the site of bacterial attachment. In this study we investigated the role of the large GTPase dynamin 2 (Dyn2) in pedestal formation. We found that in HeLa cells, both endogenous and overexpressed Dyn2 were recruited to sites of EPEC attachment. Recruitment of endogenous Dyn2 required the presence of Tir, Nck and N-WASP but was independent of cortactin and Arp2/3. Knock-down of Dyn2 expression by RNA interference reduced actin polymerization and pedestal formation. Overexpression of dominant-negative mutants of Dyn2 also reduced pedestal formation and prevented recruitment of N-WASP, Arp3 and cortactin, but not Nck. Together, our results indicate that Dyn2 is an integral component of the signalling cascade leading to actin polymerization in EPEC pedestals.

Signature-tagged mutagenesis: barcoding mutants for genome-wide screens.

DNA signature tags (molecular barcodes) facilitate functional screens by identifying mutants in mixed populations that have a reduced or increased adaptation to a particular environment. Many innovative adaptations and refinements in the technology have been described since its original use with Salmonella; they have yielded a wealth of information on a broad range of biological processes--mainly in bacteria, but also in yeast and other fungi, viruses, parasites and, most recently, in mammalian cells. By combining whole-genome microarrays and comprehensive ordered libraries of mutants, high-throughput functional screens can now be achieved on a genomic scale.

Proton motive force-dependent Hoechst 33342 transport by the ABC transporter LmrA of Lactococcus lactis.

The fluorescent compound Hoechst 33342 is a substrate for many multidrug resistance (MDR) transporters and is widely used to characterize their transport activity. We have constructed mutants of the adenosine triphosphate (ATP) binding cassette (ABC)-type MDR transporter LmrA of Lactococcus lactis that are defective in ATP hydrolysis. These mutants and wild-type LmrA exhibited an atypical behavior in the Hoechst 33342 transport assay. In membrane vesicles, Hoechst 33342 transport was shown to be independent of the ATPase activity of LmrA, and it was not inhibited by orthovanadate but sensitive to uncouplers that collapse the proton gradient and to N,N'-dicyclohexylcarbodiimide, an inhibitor of the F0F1-ATPase. In contrast, transport of Hoechst 33342 by the homologous, heterodimeric MDR transporter LmrCD showed a normal ATP dependence and was insensitive to uncouplers of the proton gradient. With intact cells, expression of LmrA resulted in an increased rate of Hoechst 33342 influx while LmrCD caused a decrease in the rate of Hoechst 33342 influx. Cellular toxicity assays using a triple knockout strain, i.e., L. lactis delta lmrA delta lmrCD, demonstrate that expression of LmrCD protects cells against the growth inhibitory effects of Hoechst 33342, while in the presence of LmrA, cells are more susceptible to Hoechst 33342. Our data demonstrate that the LmrA-mediated Hoechst 33342 transport in membrane vesicles is influenced by the transmembrane pH gradient due to a pH-dependent partitioning of Hoechst 33342 into the membrane.

What do proton motive force driven multidrug resistance transporters have in common?

The extensive progress of genome sequencing projects in recent years has demonstrated that multidrug resistance (MDR) transporters are widely spread among all domains of life. This indicates that they play crucial roles in the survival of organisms. Moreover, antibiotic and chemotherapeutic treatments have revealed that microorganisms and cancer cells may use MDR transporters to fight the cytotoxic action of drugs. Currently, several MDR extrusion systems are being investigated in detail. It is expected that understanding of the molecular basis of multidrug recognition and the transport mechanisms will allow a more rational design of new drugs which either will not be recognized and expelled by or will efficiently inhibit the activity of the MDR transporters. MDR transporters either utilize ATP hydrolysis or an ion motive force as an energy source to drive drugs out of the cell. This review summarizes the recent progress in the field of bacterial proton motive force driven MDR transporters.

Energetics of wild-type and mutant multidrug resistance secondary transporter LmrP of Lactococcus lactis.

LmrP, a proton/multidrug antiporter of Lactococcus lactis, transports a variety of cationic substrates. Previously, two membrane-embedded acidic residues, Asp142 and Glu327, have been reported to be important for multidrug transport activity of LmrP. Here we show that neither Glu327 nor Asp142 is essential for ethidium binding but that Glu327 is a critical residue for the high affinity binding of Hoechst 33342. Substitution of these two residues, however, negatively influences the transport activity. The energetics of transport was studied of two closely related cationic substrates ethidium and propidium that carry one and two positive charges, respectively. Extrusion of monovalent ethidium is dependent on both the electrical membrane potential (Deltapsi) and transmembrane proton gradient (DeltapH), while extrusion of propidium predominantly depends on the DeltapH only. The LmrP mutants D142C and E327C, however, mediate electroneutral ethidium extrusion, but are unable to mediate DeltapH-dependent extrusion of propidium. These data indicate that Asp142 and Glu327 are involved in proton translocation.

ydaG and ydbA of Lactococcus lactis encode a heterodimeric ATP-binding cassette-type multidrug transporter.

Multidrug resistance (MDR)-type transporters mediate the active extrusion of structurally and functionally dissimilar compounds from the cells, thereby rendering cells resistant to a range of drugs. The ydaG and ydbA genes of Lactococcus lactis encode two ATP-binding cassette half-transporters, which both share homology with MDR proteins such as LmrA from L. lactis or the mammalian P-glycoprotein. The ydaG/ydbA genes were cloned and expressed separately and jointly in L. lactis using the nisin-inducible system. When both proteins are co-expressed, several structurally dissimilar drugs such as ethidium, daunomycin, and BCECF-AM are extruded from the cell. YdaG and YdbA could be co-purified as a stable heterodimer. ATPase activity was found to be associated with the YdaG/YdbA heterodimer only and not with the individual subunits. Both the ydaG and ydbA genes are up-regulated in multidrug-resistant L. lactis strains selected for growth in the presence of a variety of toxic compounds. This is the first demonstration of a functional heterodimeric ATP-binding cassette-type MDR transporter.

Facilitated drug influx by an energy-uncoupled secondary multidrug transporter.

The majority of bacterial multidrug resistance transporters belong to the class of secondary transporters. LmrP is a proton/drug antiporter of Lactococcus lactis that extrudes positively charged lipophilic substrates from the inner leaflet of the membrane to the external medium. This study shows that LmrP is a true secondary transporter. In the absence of a proton motive force, LmrP facilitates downhill fluxes of ethidium in both directions. These fluxes are inhibited by other substrates of LmrP. The cysteine-reactive agent p-chloromercuri-benzene sulfonate inhibits these fluxes in wild type LmrP but not in the cysteine-less LmrP C270A mutant. Cysteine mutagenesis of LmrP resulted in three mutants, D68C/C270A, D128C/C270A, and E327C/C270A, with an energy-uncoupled phenotype. Asp68 is located in the conserved motif GXXX(D/E)(R/K)XGRK for the major facilitator superfamily of secondary transporters and was found to play an important role in energy coupling, whereas the negatively charged residues Asp128 and Glu327 have indirect effects on the transport process. L. lactis strains expressing these uncoupled mutants of LmrP show an increased rate of ethidium influx and an increased drug susceptibility compared with cells harboring an empty vector. The rate of influx in these mutants is enhanced by a transmembrane electrical potential, inside negative. These observations suggest a new strategy for eliminating drug-resistant microbial pathogens, i.e. the design and use of modulators of secondary multidrug resistance transporters that uncouple drug efflux from proton influx, thereby allowing transmembrane electrical potential-driven influx of cationic drugs.

Non-radiative deactivation pathways of subphthalocyanine and subnaphthalocyanine dyes and of their mixture.

Subphthalocyanine and subnaphthalocyanine dyes and their mixture were investigated by means of the spectroscopic and photoelectric methods. Absorption, fluorescence, steady-state and time-resolved photothermal measurements for the dyes and their mixture were done in order to get information about the radiative and non-radiative deactivation processes as competetive processes to charge separation. It was shown that energy transfer between the dyes improved the photocurrent generation in photoelectrochemical cells (PEC) based on In(2)O(3) and SnO(2) as an electrode. The possible participation of the dye triplet states in non-radiative processes was discussed.

The Escherichia coli multidrug transporter MdfA catalyzes both electrogenic and electroneutral transport reactions.

The resistance of cells to many drugs simultaneously (multidrug resistance) often involves the expression of membrane transporters (Mdrs); each recognizes and expels a broad spectrum of chemically unrelated drugs from the cell. The Escherichia coli Mdr transporter MdfA is able to transport differentially charged substrates in exchange for protons. This includes neutral compounds, namely chloramphenicol and thiamphenicol, and lipophilic cations such as tetraphenylphosphonium and ethidium. Here we show that the chloramphenicol and thiamphenicol transport reactions are electrogenic, whereas the transport of several monovalent cationic substrates is electroneutral. Therefore, unlike with positively charged substrates, the transmembrane electrical potential (negative inside) constitutes a major part of the driving force for the transport of electroneutral substrates by MdfA. These results demonstrate an unprecedented ability of a single secondary transporter to catalyze discrete transport reactions that differ in their electrogenicity and are governed by different components of the proton motive force.

Comparison of planning and using free time among students of the Faculty of Pharmacy and the Faculty of Nursing and Health Sciences.

The work presents a research of the group of third year students from two faculties of the Medical University in Lublin. The aim of the research was find out the correlation and differences in the ways of managing and using the so-called 'leisure time'; in relation to stable factors, as well as the system and the direction of studies and environmental factors, dependent on the influences of the psychosociological surrounding. 114 students of the Faculty of Pharmacy and also 55 daily and 51 extramural students of the Faculty of Nursing and Health Education were examined. It has been stated that students of the Faculty of Nursing and Health Education devoted most of their time to house work, reading books and magazines and watching TV, which differentiated them from pharmacy students. Daily students of both faculties in comparison with extramural students spent more time on listening to the radio, going to the cinema or taking part in social meetings. Daily students of the Faculty of Nursing and Health Education devoted more time to sport, walking, reading books and magazines, hobbies and going to the theatre, opera, museum, concerts and exhibitions, not only in comparison with the extramural students, but also with the pharmacy students. A few students showed interest in part-time work during the academic year, although their number rose during the period of holidays, especially among daily students of the Faculty of Nursing and Health Sciences. On the basis of the research it was stated that the way of managing and actual spending of leisure time was influenced by the direction of studies, family situation and sex of the examined students.

[Hygiene during leisure time among third year students from the Department of Nursing and Health Sciences]. / Higiena wypoczynku studentów III roku Wydzialu Pielegniarskiego i Nauk o Zdrowiu.

The aim of the study was the assessment of hygiene of leisure time among third year students from Faculty of Nursing and Health Science of Lublin Medical Academy. It analysed passive and active ways of spending free time. The study involved 106 students (55 stationary and 51 extramural) and it was conducted by means of questionnaire. The study revealed that students prefer passive types of spending their leisure time. The most popular activity was listening to the radio, to which they devoted average 2.9 hours a day (listening to music mainly). Extramural students listened to the radio shorter than stationary ones (the difference was statistically significant). Students spent also a lot of their time watching television (average 1.5 hours a day), reading books and newspapers (average 1.85 hours a day) and doing housework, which is an active way of rest (average 2.7 hours a day), mainly preparing meals and shopping. Students devoted the least of their free time to sleep during the day in spite of the fact it is an excellent way of rest. The study found also that physical activity was not a favourite type of spending free time. Every third student did not do any sport. Stationary students did sport 4 times longer than extramural (the difference was statistically significant). Only 31% practiced taking a daily walk and only 44% of students made tourist trips. 81.9% of them went away during summer holidays, but only 31% of them during the winter break. Undoubtedly, the way of spending free time by the students under examination was not hygienic as it did not give them a sense of relaxation and rest; also the students themselves were not satisfied with it.

Multidrug transporters and antibiotic resistance in Lactococcus lactis.

The Gram-positive bacterium Lactococcus lactis produces two distinct multidrug transporters, designated LmrA and LmrP, that both confer resistance to a wide variety of cationic lipophilic cytotoxic compounds as well as to many clinically relevant antibiotics. While LmrP is a proton/drug antiporter that belongs to the major facilitator superfamily of secondary transporters, LmrA is an ATP-dependent primary transporter that belongs to the ATP-binding cassette superfamily of transport proteins. Both LmrA and LmrP function as "hydrophobic vacuum cleaners" by excreting lipophilic cationic compounds from the inner leaflet of the membrane directly into the external water phase. LmrA is both functionally and structurally homologous to the human multidrug transporter P-glycoprotein. LmrA is a half ABC transporter that is functional as a homodimer, consistent with the general four-domain organization of ABC transporters, and is proposed to mediate drug transport by an alternating two-site transport mechanism.

Acidic residues in the lactococcal multidrug efflux pump LmrP play critical roles in transport of lipophilic cationic compounds.

The proton motive force-driven efflux pump LmrP confers multidrug resistance on Lactococcus lactis cells by extruding a wide variety of lipophilic cationic compounds from the inner leaflet of the cytoplasmic membrane to the exterior of the cell. LmrP contains one cysteine (Cys(270)), which was replaced by alanine. This cysteine-less variant was used in a cysteine scanning accessibility approach. All 19 acidic residues in LmrP were replaced one by one by cysteine and subsequently challenged with the large thiol reagent fluorescein maleimide. The labeling pattern strongly indicates that only three acidic residues (Asp(142), Glu(327), and Glu(388)) are membrane-embedded. The roles of these residues in drug recognition were evaluated based on transport experiments with two cationic substrates, ethidium and Hoechst 33342, after replacing each of these residues with cysteine, alanine, lysine, glutamate, or aspartate. The obtained results suggest that the negative charges at positions 142 and 327 are not critical for the transport function but are important for drug recognition by LmrP. Surprisingly, the residues Cys(142) and Cys(327) become accessible for fluorescein maleimide upon binding of substrates, indicating a movement of these residues from a nonpolar to a polar environment. Substrate binding apparently results in a conformational change in this region of the protein and a reorientation of a lipid-embedded, hydrophobic substrate-binding site to an aqueous substrate translocation pathway.

Ways of spending leisure time by the third year-students of the Faculty of Pharmacy, Medical University of Lublin.

The aim of the study was the assessment of leisure time among third-year students from the Faculty of Pharmacy of the Medical University of Lublin. It analysed quantity of time devoted to school activity and ways of spending free time. The study involved 114 students (82 women and 32 men). The study revealed that women had less free time than men, who, most probably did not attend some lectures. The most popular activities among the questioned students were: sleeping (average 6.8 hours a day), studying (average 3.6 hours a day), listening to the radio (average 2.9 hours a day), talking with friends (average 1.9 hours a day), personal hygiene (average 1.1 hours a day), watching TV (average 1.1 hours a day), housework. Students devoted the least of their free time on active rest, for example walking (women did it more often than men) or practising sport (more popular among men). Cultural life of the students consisted only of meetings with friends and going to the cinema (women did it more often). The least popular way of spending free time was going to the theatre, opera, concerts and exhibitions. Few students spent their time working. Their number increased significantly during holidays. The way of spending free time by third-year students from the Faculty of Pharmacy (both men and women) during the day was similar, differences related only to the amount of time devoted to each activity.

An ABC-type multidrug transporter of Lactococcus lactis possesses an exceptionally broad substrate specificity.

LmrA is a 590-amino acid membrane protein which confers multidrug resistance on Lactococcus lactis cells by extruding amphiphilic compounds from the inner leaflet of the cytoplasmic membrane at the expense of ATP hydrolysis. Its structural and functional characteristics place it in the P-glycoprotein cluster of the ATP-binding cassette transporter superfamily, making it the first prokaryotic multidrug transporter of this cluster. The number of compounds recognized and transported by LmrA is remarkably vast and includes many lipophilic cations as well as a record of eight classes of clinically relevant broad-spectrum antibiotics. Homologs of LmrA have been found in pathogenic bacteria, suggesting that these putative efflux pumps may play a crucial role in antibiotic resistance of human pathogens. Recent evidence indicates that LmrA is functional as a homodimer, consistent with the overall structure of P-glycoprotein, and mediates drug transport by an alternating two-site transport mechanism. Copyright 2000 Harcourt Publishers Ltd.