bacterial toxins

الرحمان الله الرحمان بسم الله بسمالرحيمالرحيم

Bacterial toxinsBacterial toxins

Bacterial toxinsBacterial toxins

Endotoxins Endotoxins ExotoxinsExotoxins

EndotoxinsEndotoxins

cell-associatedcell-associated toxinstoxins

lipopolysaccharide lipopolysaccharide ((LPSLPS) ) or or lipooligosaccharide lipooligosaccharide ((LOSLOS) ) locatedlocated in in the the

outer membrane of Gramouter membrane of Gram--negative bacterianegative bacteria..

ExotoxinsExotoxinsBACTERIAL PROTEIN TOXINSBACTERIAL PROTEIN TOXINS

Bacterial protein toxins are the most powerful human Bacterial protein toxins are the most powerful human poisons known and retain high activity at very high poisons known and retain high activity at very high dilutionsdilutions

GramGram--positive and Grampositive and Gram--negative bacteria produce negative bacteria produce soluble protein toxins secreted by bacteriasoluble protein toxins secreted by bacteria

**Clostridium tetaniClostridium tetani produces tetanus toxin produces tetanus toxinCorynebacterium diphtheriaeCorynebacterium diphtheriae produces the diphtheria toxin produces the diphtheria toxin

Exotoxin ‘s target sitesExotoxin ‘s target sites

Intestinal tract Intestinal tract enterotoxinenterotoxinNervous systemNervous system neurotoxinneurotoxin

Leukocytes Leukocytes leukocidinleukocidin Red blood cells Red blood cells hemolysinhemolysin

Entry of toxins into target cellsEntry of toxins into target cells

Attachment Pore Attachment Pore formationformation

((pore forming toxinspore forming toxins ) )Direct entryDirect entry

ReceptorReceptor- - mediatedmediatedEndocytosis (RME)Endocytosis (RME)

11--Pore formation (pore Pore formation (pore forming toxins)forming toxins)

Some bacterial toxins Some bacterial toxins act locally to promote act locally to promote bacterial invasin bacterial invasin into mammalian cellsinto mammalian cells . .

**Examples are extracellular enzymes that Examples are extracellular enzymes that degrade tissue matrices or fibrin, allowing the degrade tissue matrices or fibrin, allowing the bacteria to spreadbacteria to spread collagenase, hyaluronidase collagenase, hyaluronidase and streptokinaseand streptokinase

*Other toxins, also considered invasins, degrade membrane components, such as phospholipases and lecithinases

22--AttachmenAttachmen

A-Direct entryA-Direct entry the B subunit of the native (A+B) toxin the B subunit of the native (A+B) toxin

binds to a specific receptor on the target binds to a specific receptor on the target cell and induces the formation of a pore in cell and induces the formation of a pore in the membrane through which the A the membrane through which the A subunit is transferred into the cell subunit is transferred into the cell cytoplasmcytoplasm . .

B-ReceptorB-Receptor- - mediated endocytosis mediated endocytosis (RME)(RME)

The toxin is internalized in the cell in a membraneThe toxin is internalized in the cell in a membrane--enclosed vesicle called an endosomenclosed vesicle called an endosom..

+ + H ions enter the endosome lowering the internal pH H ions enter the endosome lowering the internal pH

which causes the Awhich causes the A++B subunits to separateB subunits to separate. . The B The B subunit affects the release of the A subunit from the subunit affects the release of the A subunit from the endosome so that it will reach its target in the cell endosome so that it will reach its target in the cell cytoplasmcytoplasm. . The B subunit remains in the endosome The B subunit remains in the endosome and is recycled to the cell surfaceand is recycled to the cell surface.. Specific receptor for the B subunit of toxins on Specific receptor for the B subunit of toxins on target cells or tissues are usually sialogangliosides target cells or tissues are usually sialogangliosides ((glycoproteinsglycoproteins) ) calledcalled--G G proteins on the cell proteins on the cell

membranemembrane. . ..

Toxin damage on target Toxin damage on target cellscells 11--Pore forming toxinsPore forming toxins

11--Toxin protomers bind to target membranes by either Toxin protomers bind to target membranes by either unidentified highunidentified high--affinity receptors or through nonspecific affinity receptors or through nonspecific absorption to substances such as phosphotidylcholine or absorption to substances such as phosphotidylcholine or cholesterol on the lipidcholesterol on the lipid

bilayerbilayer 2- membrane2- membrane--bound protomers must oligomerize bound protomers must oligomerize into a nonlytic prepore heptamer complexinto a nonlytic prepore heptamer complex..

33 - -the heptamer must undergo a series of conformational the heptamer must undergo a series of conformational

changes that create the stem domain of the toxin, which is changes that create the stem domain of the toxin, which is then inserted into the membranethen inserted into the membrane

Clostridium perfringens, Escherichia coli, S. aureusClostridium perfringens, Escherichia coli, S. aureus

22--Inhibit protein SynthesisInhibit protein Synthesis

A-Substrates for toxins in this groupA-Substrates for toxins in this group are elongation are elongation factors( EF2) and ribosomal RNAfactors( EF2) and ribosomal RNA . .

**The modified EF2 is no longer able to function in protein The modified EF2 is no longer able to function in protein synthesissynthesis

* * Inactivate ribosomal RNA so that the affected ribosome Inactivate ribosomal RNA so that the affected ribosome interactinteract

This group of toxins ultimately results in death of the This group of toxins ultimately results in death of the

target celltarget cell

33--ACTIVATION SECANDRY MASSENGER PATHACTIVATION SECANDRY MASSENGER PATH WAYWAY

**Bacterial toxins can also target and alterBacterial toxins can also target and alter the function of a the function of a variety of cellular proteins without directly killing the variety of cellular proteins without directly killing the intoxicated cellintoxicated cell..

** Toxin activation or modification of secondary messengers Toxin activation or modification of secondary messengers can cause dramatic alterations to signal transduction can cause dramatic alterations to signal transduction pathways critical in maintaining a variety of cellular pathways critical in maintaining a variety of cellular

functionsfunctions

MethodologyMethodology

Test PrincipleTest Principle

Antibody – Antibody – Antigen reactionAntigen reaction

Methods for bacterial Methods for bacterial toxins detectiontoxins detection

11--ELISA (EnzymeELISA (Enzyme--Linked ImmunoSorbent AssayLinked ImmunoSorbent Assay))

5-Rapid Methods: antibody- and DNA-based tests,6-Gen detection

2-Latex Agglutination Method 3-Radioimmunoassay

4-Micro slid technique

((EnzymeEnzyme--Linked ImmunoSorbent Linked ImmunoSorbent AssayAssay))

Can be used both qualitatively and Can be used both qualitatively and quantitatively to measure antigenquantitatively to measure antigen--antibody bindingantibody binding. . Depending on Depending on what variation you use, it will detect what variation you use, it will detect antigen antigen ((hormones, enzymes, hormones, enzymes, microbial antigens, illicit drugsmicrobial antigens, illicit drugs) ) or or antibody antibody ((antianti--HIV in the screening HIV in the screening test for HIV infectiontest for HIV infection) ) in body fluids in body fluids or tissue culture supernatantsor tissue culture supernatants . .

ELISAELISA

ELISAELISA Can be used both qualitatively andCan be used both qualitatively and quantitatively quantitatively

to measure antigento measure antigen--antibody bindingantibody binding.. Depending on what variation you use, it will detect Depending on what variation you use, it will detect

antigen antigen ((hormones, enzymes, microbial antigens, hormones, enzymes, microbial antigens, illicit drugsillicit drugs) ) or antibody or antibody ((antianti--HIV in the HIV in the screening test for HIV infectionscreening test for HIV infection) ) in body fluids or in body fluids or tissue culture super natantstissue culture super natants

What you need to do the assayWhat you need to do the assay

Purified antigen Purified antigen ((if you want to detect or quantify if you want to detect or quantify antibody)antibody) . .

Purified antibody Purified antibody ((if you want to detect or quantify if you want to detect or quantify antigen)antigen) . .

Standard solutions Standard solutions ((positive and negative controls)positive and negative controls) . .Sample to be testedSample to be tested . .

Microtiter dishesMicrotiter dishes: : plastic trays with small wells in plastic trays with small wells in which the assay is donewhich the assay is done . .Wash fluid Wash fluid ((buffer)buffer) . .

EnzymeEnzyme--labeled antibody and enzyme substratelabeled antibody and enzyme substrate . .ELISA reader ELISA reader ((spectrophotometerspectrophotometer) ) for quantitative for quantitative

measurementsmeasurements

ELISAELISA

ELISAELISA11--Coat the microtiter plate with purified antigen by letting an Coat the microtiter plate with purified antigen by letting an

antigen solution sit in the wells for 30-60 minutesantigen solution sit in the wells for 30-60 minutes . . Wash away Wash away unbound antigen with buffer and cover any sites that might unbound antigen with buffer and cover any sites that might nonspecifically bind antibody with unrelated protein nonspecifically bind antibody with unrelated protein ((such as such as solution of powdered milksolution of powdered milk)), again washing away unbound , again washing away unbound proteinprotein . .

22--Add serum sample to be tested for specific antibody to plate Add serum sample to be tested for specific antibody to plate and allow specific antibody to bind to the antigenand allow specific antibody to bind to the antigen . . Wash off Wash off unbound antibodyunbound antibody . .

33--Add antiAdd anti--Ig that will bind to Fc region of specific antibody Ig that will bind to Fc region of specific antibody ((for example, antifor example, anti--human gamma chain that will bind human human gamma chain that will bind human IgGIgG). ). The Fc region of the antiThe Fc region of the anti--Ig is covalently linked with Ig is covalently linked with enzymeenzyme. . Wash off unbound antibodyWash off unbound antibody--enzyme complexenzyme complex . .

AddAdd chromogenicchromogenic 4-substrate4-substrate: : colorless substrate that the colorless substrate that the enzyme will convert to a colored productenzyme will convert to a colored product . . Incubate until color Incubate until color develops; measure color in a spectrophotometerdevelops; measure color in a spectrophotometer . . The more The more color that is detected, the more specific antibody is present in color that is detected, the more specific antibody is present in the unknown samplethe unknown sample . .

ELISAELISA . .Negative controls includeNegative controls include

omit the antigenomit the antigen omit the test antiserum or substitute with omit the test antiserum or substitute with

an antibody that will not bind the antigenan antibody that will not bind the antigen . .Positive control substitutes known Positive control substitutes known

positive serum for unknown serumpositive serum for unknown serum

Sandwich ELISASandwich ELISA

Sandwich ELISA Sandwich ELISA To detectTo detect antigenantigen

11--Coat the microtiter plate with purified antibody Coat the microtiter plate with purified antibody to the antigento the antigen. . Wash away unbound antibody and Wash away unbound antibody and cover any sites that might nonspecifically bind cover any sites that might nonspecifically bind with unrelated proteinwith unrelated protein . .

22--Add sample to be tested for antigen to plate Add sample to be tested for antigen to plate and allow antigen to bind antibodyand allow antigen to bind antibody. . Wash off Wash off unbound antigenunbound antigen . .

33--Add enzymeAdd enzyme--labeled specific antibody to a labeled specific antibody to a different epitope of the antigen to make a different epitope of the antigen to make a ""sandwichsandwich""; wash away unbound antibody; wash away unbound antibody . .

44--Add chromogenic substrate for enzyme that Add chromogenic substrate for enzyme that will be converted to a colored productwill be converted to a colored product . .

55--Negative control omits unknown antigen; Negative control omits unknown antigen; positive control uses known antigenpositive control uses known antigen . .

Latex TestLatex Test

LatexLatex

The latex agglutinationThe latex agglutination

11--Mixing 10 Mixing 10 μμl of a toxin culture with 10 l of a toxin culture with 10 μμl of l of latex reagent on a microscope slide, and latex reagent on a microscope slide, and thereafter the slide was manually rocked for 5 thereafter the slide was manually rocked for 5 to 8to 8

22-- . .A positive reaction A positive reaction ((the mixture became the mixture became fluffy, ifluffy, i..ee.., an agglutination, an agglutination) ) could be read could be read with the naked eye within 5 to 10 s if the latex with the naked eye within 5 to 10 s if the latex reagent contained antiserum homologous to reagent contained antiserum homologous to the capsule of the pneumococcal culturethe capsule of the pneumococcal culture. . Agglutinations observed after more than 30 s Agglutinations observed after more than 30 s were nonspecificwere nonspecific . .

RadioimmunoassayRadioimmunoassay A mixture is prepared ofA mixture is prepared of

radioactive antigenradioactive antigen Because of the ease with which iodine atoms can be Because of the ease with which iodine atoms can be

introduced intointroduced into tyrosinetyrosine residues in a protein, the residues in a protein, the radioactiveradioactive isotopesisotopes 125 125I orI or 131 131I are often usedI are often used..

antibodies against that antigenantibodies against that antigen..Known amounts of unlabeled Known amounts of unlabeled ("("coldcold") ") antigen are added to antigen are added to

samples of the mixturesamples of the mixture. . These compete for theThese compete for the binding sitesbinding sites of of the antibodiesthe antibodies . .

At increasing concentrations of unlabeled antigen, an At increasing concentrations of unlabeled antigen, an increasing amount of radioactive antigen is displaced from the increasing amount of radioactive antigen is displaced from the antibody moleculesantibody molecules . .

The antibodyThe antibody--bound antigen is separated from the free antigen bound antigen is separated from the free antigen in the supernatant fluid, andin the supernatant fluid, and

the radioactivity of each is measuredthe radioactivity of each is measured . . . .

FromFrom these data, a standard binding curve, like this these data, a standard binding curve, like this one shown in red, can beone shown in red, can be drawn The samples to be drawn The samples to be assayed assayed ((the unknownsthe unknowns) ) are run in parallelare run in parallel . .

After determining the ratio of bound to free After determining the ratio of bound to free antigen in each unknown, the antigen antigen in each unknown, the antigen concentrations can be read directly from the concentrations can be read directly from the standard curvestandard curve

There are several ways of doing thisThere are several ways of doing this . .Precipitate the antigenPrecipitate the antigen--antibody complexes by antibody complexes by

adding a adding a ""secondsecond" " antibody directed against the antibody directed against the firstfirst. . For example, if a rabbit IgG is used to bind For example, if a rabbit IgG is used to bind the antigen, the complex can be precipitated by the antigen, the complex can be precipitated by adding an antirabbitadding an antirabbit--IgG antiserum IgG antiserum ((ee..gg.., raised by , raised by immunizing a goat with rabbit IgGimmunizing a goat with rabbit IgG)) . .

Separating Bound from Free AntigenSeparating Bound from Free Antigen

. .The antigenThe antigen--specific antibodies can be coupled to the inner walls of specific antibodies can be coupled to the inner walls of

a test tubea test tube]. ]. After incubationAfter incubation , ,the contents the contents ("("freefree") ") are removedare removed ; ;the tube is washed the tube is washed ("("boundbound")"), and, and

the radioactive of both is measuredthe radioactive of both is measured..The antigenThe antigen--specific antibodies can be coupled to particles, likespecific antibodies can be coupled to particles, like

SephadexSephadex. . Centrifugation of the reaction mixture separatesCentrifugation of the reaction mixture separates the bound counts the bound counts ((in the pelletin the pellet) ) fromfrom

the free counts in the supernatant fluidthe free counts in the supernatant fluid . .Radioimmunoassay is widelyRadioimmunoassay is widely--used because of its greatused because of its great sensitivitysensitivity. .

Using antibodies of high affinityUsing antibodies of high affinity ( (KK00 = 108–1011 = 108–1011 MM −1), −1), it is possible it is possible to detect a few picograms to detect a few picograms ((1010−12 −12 gg) ) of antigen in the tubeof antigen in the tube. [. [Link to Link to page that discussespage that discusses antibody affinityantibody affinity]]The greater the specificity of The greater the specificity of the antiserum, the greater the specificity of the assaythe antiserum, the greater the specificity of the assay. . Link to an Link to an illustration ofillustration of antibody specificityantibody specificity demonstrated by demonstrated by radioimmunoassayradioimmunoassay..The main drawbacks to radioimmunoassay are The main drawbacks to radioimmunoassay are the expense and hazards if preparing and handling the radioactive the expense and hazards if preparing and handling the radioactive antigenantigen

RadioimmunoassayRadioimmunoassay

Micro slid techniqueMicro slid technique

Food extractionFood extraction