efflux pumps

Presenter:Mahsa jaliliMSc in Medical Microbiologyefflux pumps1

Abstract: Infectious diseases remain one of the principal causes of morbidity and mortality in the world.

Efflux pumps are elements that antibiotics are effective in resistance and are involved in the pathogenesis of bacteria .

efflux pumps are important for processes of detoxification of intracellularmetabolites, bacterial virulence in both animal an Man hosts, and etc .

So today we're in a selection pressure for antibiotic use and for the treatment of bacterial infections are a new development .

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Efflux as a mechanism for antibiotic resistance was firstdescribed in 1980 .Multidrug efflux pumps cause serious problems in cancer chemotherapy and the treatment of bacterial infections.In Gram negative bacteria, the pumps belonging to the resistance-nodulation-division (RND) family are especially effective in generating resistance. 3

Antimicrobial resistance has been considered the new challenge of the 21st century for example : Salmonella enterica , Escherichia coli ,Different studies have demonstrated that MDR pumps are capable of extruding not only antibiotics but also antiseptics.4

Functional role of MDR pumps in clinical and nonclinical environments.5

The importance of efflux pumps :In general it can be said:Promotes the excretion of drugs and chemicals are.Metabolites and toxins that repel bacteriaMicroorganisms against antibiotics and chemicals, toxins, stress and protectThe survival of microorganisms in different environments.The supply of materials for the synthesis of bacterial surface structures involvedIn balancing solutions, ionic homeostasis and balance are important.

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Classification :Efflux pump that was first detected : Efflux pumps was for a family of tetracyclineBacterial efflux transporters are classified into five major superfamilies, based on the amino acid sequence and the energy source used to export their substrates.In the prokaryotic kingdom there are five major families of efflux transporters.

(ATP)-binding cassette (ABC) superfamilythe resistance-nodulation-division (RND)family , the small multidrug resistance (SMR) familythe major facilitator superfamily(MFS) the multidrug and toxic compound extrusion (MATE) family

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ABC: membrane proteins that translocate variety of substrates across extra- and intra-cellular membranes48 known ABC transporters; divided into 7 subfamilies of proteins

RND: Fall into 7 phylogenetic families 3 Restricted to Gram ive Bacteria; other 4 either restricted to Gram + ive or represented in bothAlso in bacteria, archaea and eukarya

SMR The smaller size . is unusual bacteria. Its pumps contain only 100-120 amino acid

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MFS: One of two largest families17 MFS FamiliesPresent in bacteria, archaea and eukarya

MATE :14 Phylogenetic FamiliesAlso in bacteria, archaea and eukarya . It has the same size mfs

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Classification :

Schematic representation of the main types of bacterial efflux systems10

Gram-positive bacteriaGram-negativebacteria

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Energy used in the efflux pump : The energy source :ABC transporters are dependent on ATP hydrolysis;)The primary active transport( MFS, RND and SMR are proton-driven efflux pumps. MATE transporters consist of a Na/H drug antiporter system .)Secondary active transport(12

Gram-negative species with known efflux systems:Haemophilus influenzae Campylobacter jejuniHelicobacter pyloriVibrio parahaemolyticusVibrio choleraeNeisseria gonorrhoeae... Salmonella TyphimuriumShigella dysenteriaeKlebsiella pneumoniaeEnterobacter aerogenesSerratia marcescensProteus vulgarisCitrobacter freundii... Bacteroides fragilis...And .

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The most important mechanisms of drug resistance:InactivatingChange the form of the drugThe aim of antibiotic succession and delegationIncrease the amount of targetReduced affinity:RecombinationModification enzymesEfflux pumps

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Important note :The resistance who efflux pump caused by antibiotic depends on the nature of microorganisms and substrate

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Structure of drug efflux systems:In Gram-positive bacteria:A cytoplasmic membrane transport proteins that recognize specific substrate . and the resistance.In Gram-negative bacteria:3 component in a system:Two transport proteins within the membrane.A periplasmic lipoprotein adapter17

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ABC tranceporters :In contrast with prokaryotes,the major mechanism of efflux in eukaryotes is dependent on proteins that derive their transport energy from the hydrolysis of ATP.

Has a membrane-binding region of the cytoplasmic membrane is in addition to the Alpha Helix to allow them The energy of ATP hydrolysis to transport the drug, unlike other gradient used for this purpose are proton exchange membrane of the electrochemical gradient.

Is an arsenic/antimony pump that is responsible for resistance to the antimonial drug Pentostam in leishmania

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ABC tranceporters :ABC 200 amino acid, domains specific areas ABC, which is the second hydrophobic combination of 6 alfa hlyksmy.The presence of walkers areas A, B sequence LSGGQ on all members of the family.

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E.coli E.coliS.TyphimuriumBacillus subtilisEukaryotes21

The SMR family :The unusual bacteria foundOfthe 100 amino acidsthat madethefour-helixFor example : EmrE is in E.coliQacH-2 resistance in Staphylococcus aureus causes a number of disinfectants.

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The MFS family :In eukaryotes, bacteria and Archaea are available.MFS are made of 400 amino acids arranged in 12 Helix membrane and a cytoplasmic loop between helices 6 and 7 are large.

Mfs transport of drugs in two classes be:1- Class B: Transport tetracycline in E Coli2-class k: Transport tetracycline in Staphylococcus aureus

MFS characterized by multi-drug pumps that to be anti ports drug / proton acts and the transmission becomes very large sugar and drugs.

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RND transporters :Typical emerged were larger than 1,000 amino acids. 12-helix structure. They have a great second or subsequent intracytoplasmic Periplasmic between helices 1 and 2, 7 and 8.There are more gram-negative bacteria.Pump compounds: toxic metal ions, lipophilic drugs such as tetracycline, quinolones, and beta-lactamase and chloramphenicol factors chemotherapy.

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Structure :DNA structure is composed of 3 components:1-AcrA: inner membrane.2-AcrB: inner membrane.3-TolC: in the outer membrane.The third monomer forming the inner membrane creates an area that extends Periplasmic..27

TOLC PROTEIN;Is a multifunctional protein. Moving in small drugs and toxins polypeptide involved.for example: hemolysin .

TolC is divided into two domains:1-domain beta : in the outer membrane.2-domain alpha : in the periplasmic space.

TolC several actions have to cross the outer membrane periplasmic substrates.28

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-barrel :Opens the outer membrane.Beta consists of 12 strings and are arranged in a right-handed barrel.30

-helical :12 left-handed filament is composed of two types of grape that areShortTall

Alpha Helix Coils-Coiled arranged in the area and the composition and structure of alpha and beta binding steadily alpha helix, like a belt around it were shorter.31

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This substrate system:AminoglycosidesTetracyclineFluoroquinolonesChloramphenicolTrimethoprim33

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MATE transporters :Of 450 amino acids that have been arranged in 12 Helix These trans Porter Na or protons act as anti ports.NorM resistance in Vibrio parahaemolyticus color, aminoglycosides and fluoroquinolones cause.YdhE in E.Coli is a cationic resistance to antibiotics causeMepA in the transfer tetracycline Staphylococcus aureus, detergents and paints as well.PepM in Pseudomonas aeruginosa substrate fluoroquinolones, is ethidium bromide and disinfectants.

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The pump substrates include:GentamicinCiprofloxacinErythromycinTrimethoprim

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Genetic of the efflux pump E.coli :Efflux system in E Coli:ChromosomeTransferable elements (plasmids, transposons, Integron)

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There are various regulators in E. Coli :Including : SdiA ,Sox ,Rob ,Mar A

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Efflux pump genes in Salmonella :

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Salmonella has a specific regulator, RamA, which controls the expression ofacrABin response to environmental signals. We suggest thatRamA is a master regulator ofacrABand that the AcrAB induction pathway inSalmonellais different from that inE. coli. In one pathway, bile binds to the RamA protein, which is thenconverted from a low to a high activity state. In the other pathway, indole may activateramAtranscription to induceacrAB.41

Inhibitors :Inhibitors of efflux pumps have great potential as pharmacological agents that restore the drug suseptibility of multidrug resistant bacterial pathogens

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High-potential pharmacological agents that can be multi-drug resistant pathogens return sensitivity to the drug.Example:Phenylalanine, arginine beta - naphthyl amide

Phenyl-Arginyl N-naphtylamide43

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