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Bacterial cell wall and applied

importance

Dr.Raghu prakash

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Cell Walls

Why study bacterial cell walls?

They are essential structures in bacteria.

They are made of chemical components

found nowhere else in nature.

They may cause symptoms of disease in

animals.

They are the site of action of some of ourmost important antibiotics.

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Primary function of the bacterial cell wall

To prevent

rupture or

osmoticlysis

of the cell

protoplast

Lysis of a pair of dividing E. colicells

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Chemical nature of bacterial cell walls

Bacterial cell walls always contain murein,which is a type of peptidoglycan

Chemical nature of murein accounts for thefunction of the cell wall

Murein is only found in the cell walls ofbacteria

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r r rrr

r

Gram Pos it ive Cel l Envelope

Cytoplasm

r

rrr

Lipoteichoicacid

Peptidoglycan-teichoic acid

Cytoplasmic membrane

M-Protein

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Chemical nature of bacterial cell walls

Peptidoglycan is made upof

2 amino sugars

N-acetyl-glucosamine = GN- acetylmuramic acid = M

4 amino acidsL-alanine = L-alaD-glutamic acid = D-gludiaminopimelic acid = DAPD-alanine = D-ala

GM

L-ala

D-glu

DAP

D-ala

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Peptidoglycan Synthesis

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UMP

UDP

UTP

UTP + Glc NAc-1-p

UDP-Glc NAc

UDP-Glc NAcEnolpyruvate

UDP Mur NAc

UDP Mur NAc-L-Ala

UDP Mur NAc-L-Ala-D-Glu

UDP Mur NAc-L-Ala-D-Glu-L-R3

UDP Mur NAc-L-Ala

-D-Glu-L-R3-(DAla)2

D-Ala-DAla

2-L-AlaCycloserineinhibits

9MurF

+ L-R3

+D-Glu

+L-Ala

+NADPH

+PEP

- PP1

Phosphonomycin

inhibits

D-ALaD-Ala transport

Undecaprenyl-PP-Mur NAc-

pentapeptide

Undecaprenyl-P

Cytoplasm Membrane Wall

Undecaprenyl-PP

Disaccaride

-pentapeptide

Bacitracin

inhibits

Disaccaride

-pentapeptide

Nascent

Peptidoglycan

Expanding cross

linked wall

Peptidoglycan3 Lactum

Antibiotic

inhibits

Vancomycin

inhibits

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Surface Proteins

Five penicillin binding proteins (PBPs)

Two neuraminidases

IgA protease

Twelve choline-binding proteins (CBPs) - include importantdeterminants of virulence such as PspA (protective antigen), LytA,

B, and C (three autolysins), and CbpA (an adhesin)

Neuraminidase - cleaves sialic (neuraminic) acid,

possibly to expose receptors or to dissolve interstitialcement

IgA protease - cleaves and inactivates secretory IgA

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Bacterial Determinants of Virulence

Cell Wall Components

Phosphorylcholine decorating the teichoic acid and thelipoteichoic acids act both as adhesins and as docking sitesfor the choline-binding proteins (CBPs).

The peptidoglycan/teichoic acid complex is highlyinflammatory. The cell wall directly activates the alternativepathway of the complement cascade, and the coagulationcascade.

Peptidoglycan binds to CD14, a cell surface receptor knownto initiate the inflammatory response for endotoxin. Thisinduces a cytokine cascade resulting in production ofinterleukin-1, interleukin-6 and tumor necrosis factor from

human cells.

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Peptidoglycan hydrolases

Three typ es

Glycan-strands hydrolyzing

Endo-N-acetylmuramidases

Endo-N-acetylgucosaminidases

Endopeptidase hydrolyzing

Peptide bonds in the interior of the peptide bridgesBonds involving the C-terminal D-alanine residue

N-acetylmuramoylL-alanine amidase

Acting at the junction b/w glycan strands and the

peptide unitsThis enzymes appear to play an imp role in number of cellularactivities septum and wall extension during cell growth ,cellseparation,turn over of wall components,sporulationcompetency for transformation, excretion of toxins andexoenzymes

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Gram-negative cell walls include

an outer membrane

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Lipopolysaccharide

Lipid A Glucosamine disaccharide

Beta hydroxy fatty acids

Core

Heptoses Ketodeoxyoctonic acid

O-antigen

Highly variablen

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Porins

Three Types Type I :- Nonspeci f ic subrstateon the

basis of size.

ex .Omp.F, Omp.C of E.coli. Type II :- Trasport small subrates , but

preferentially transport certain substrates ex,.

LamB transport maltose & maltodextrins ,

binding sites for lambda phage.

Type III :-Ton.B-dependent proteins . transport vit

B12 & Siderophores. Transport is energy

dependent.

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PROTEINS FUNCTIONS

Omp.A

Murien

lipoprotein

OmpB

Lam.B

Omp.C &

Omp.F

Omp.T

Stabilization of outer membrane &mating

aggregates in F-dependent conjugation;receptor for

phage Tu11.

Most abundant surface protein in E.coli &S.enterica

;major structural protein ;stabilizes cell surface.

Diffusion channel for various metabolies

ex: maltose .

LamB transport maltose&maltodextrins,binding

sites for lambda phages .

Diffusion channel for small molecules ;receptors for

Tulb&T3,T4.

Protease.

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PhoE

Protein P

TolA

TonA

Ton.B

Anion-selective diffusion channel under

phosphate limitation

Anion-selective diffusion channel in

P.aeruginosainduced under phosphate .

Maintenance of OM integrity;activity of groupa colicins

Ferrichrome siderophore uptake;receptor for

phages T1,T5,80,&COLICIN M.

Dependent proteins . transport vit B12

&Siderophores

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Other characteristics of bacterial cell

wallsOuter membrane of Gram-negatives has two important

properties

1. It protects the cells from permeability by manysubstances including penicillin and lysozyme.

2. It is the location of lipopolysaccharide (endotoxin) which

is toxic for animals.

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S-layer composed of identical proteins or

glycoproteins sub units

s-layer is quiet different in many organisms

In Gram + sub units it is linked to peptidoglycan layeror secondary cell wall polymers

In Gram it is linked to lipopolysaccharides of outer

membrane S-layer contain acidic and hydrophobic A.A

Functions / attachment to surfaces and to host tissues

Campylobacter and Aeromonas S-layer serves as

virulence factors

In archaea S-layer is outer most layer ,next to cell

membrane it must contribute to the shape of the cell

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Bacterial Surface Appendages

Flagella -organelles forswimmingmotility

Pili (or fimbriae)- for attachmentor adherence tosurfaces; sexpilus usedduring somegeneticexchangeprocesses

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Salmon ella enterica, like

most enteric bacteria, is

capable of swimming

movement by means of

flagella.

Flagella

Flagella are longwhiplike filamentscomposed of proteinthat originate in thecell membrane.

Flagella rotate andimpart swimmingmovement on the cells

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Proteus mirabi l isswims by means of peritrichous

flagella

Vibr io cho leraehas a single polar

flagellum

Flagella are for swimming movement

Peritrichous flagellaare distributed allover the cellsurface

Polar flagella

originate at the poleof a cell

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Detecting Motility in Bacteria

By using flagellar stains to detect the presence

and distribution of flagella

Bacillus cereus Vibrio cholerae Bacillus brevis

Polar flagellumPeritrichous flagella

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Detecting Motility in Bacteria

By inoculation of the bacteria into motility test

medium (SIM).

Staphylococcus

epidermidis

Non motile

E. coli

motile

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Ecological Advantages to Swimming

1. Survival: escape predatory protozoa and white blood cells

(phagocytes)

2. Swim towards nutrients or away from harmful substances(chemotaxis)

3. Swim towards or away from O2 (aerotaxis)

4. Swim towards light (phototaxis)

5. Swim toward the North Pole or the South Pole(magnetotaxis)

The Structure of the Bacterial Surface: Flagella Summary

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The Structure of the Bacterial Surface: Flagella Summary

Flagella filamentous protein structures attached to the cell surface that

provide swimming movement for most motile bacterial cells.

The flagellar filament is rotated by a motor apparatus in the plasma membraneallowing the cell to swim in fluid environments.

tactic behavior or motility is the ability to move (swim) in response to

environmental stimuli.

Chemotaxis: a bacterium can sense the quality and quantity of certainchemicals in its environment and swim towards them (if they are useful

nutrients) or away from them (if they are harmful substances).

Aerotaxis: bacteria swim toward or away from O2

motility as a determinant of virulence: e.g. Vibrio cholerae, Campylobacter,

Helicobacter, Pseudomonas

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Functions of Pili and Fimbriae

Attachment to a surface

or substrateShigella dysenteriaeuses its

fimbriae to attach to the intestine

and then produces a toxin that

causes diarrhea.

Neissera g ono rrhoeae, the cause of the

gonorrhea, uses pili to attach to the urogenital

and cervical epithelium when it causes disease

Pili (also called fimbriae)

are short hair-likestructures composed ofprotein on the cellsurface.

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Functions of Pili and Fimbriae

Resistance to Phagocytic engulfment

Phagocytosis of streptococci by a macrophage

Chain of streptococci protected from engulfment by fimbrial (M)protein

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Functions of Pili and Fimbriae

A special type of pilus called the sex pilus

is used in mating between bacteria

E. coliuses its sex pilus (called the F-pilus)

to transfer DNA between mating bacteria

during conjugation.

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How Flagella Work

Filament is rotatedby a protein motorin the cellmembrane

Motor is poweredby proton motiveforce (pmf) on the

outside ofmembrane

motor

basal

body

filament

pmf

on this

side of

membrane

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Endospores are produced as intracellular structures within the cytoplasm

of certain bacteria, most notably Bacillus and Clostridium species.

Endospore forming bacteria left to right: Clostridiumbotulinum, Bacillus brevis, Bacillus thuringiensis

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Properties of Endospores

Resting (dormant) cells -cryptobiotic i.e.,show no signs oflife..primarily due to lack of waterin the spore

Several unique surface layersnot found in vegetative cells :

exosporium, spore coat,

cortex, and core wall

Highly resistant to heat (boiling),

acids, bases, dyes ( dont stain)

irradiation, disinfectants,

antibiotics, etc.

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Properties of Endospores

Spores and parasporal crystals produced by

some bacteria are toxic to insects

Parasporal crystalEndospore

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Endospore formation is NOT a mechanism of reproduction. Rather it is a

mechanism for survival in deleterious environments. During the process of

spore formation, one vegetative cell develops into one endospore.

The sequential steps of endospore formation in a Bacillus species. The process of

endospore formation takes about six hours. Eventually the mature endospore is

released from its mother cell as a free spore

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Under favorable nutritional and environmental conditions, an

endospore germinates into a vegetative cell.

A germinating spore

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Medically-important Endospore-

forming Bacteria Bacillus anthracis causes anthrax

Bacillus cereus causes food poisoning

Clostridium tetanicauses tetanus

Clostridium botulinum causes botulism

Clostridium perfringens causes food poisoning

and gas gangrene

Clostridium difficile causes antibiotic-induceddiarrhea and pseudomembranous colitis

B t i l C ll E l

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Bacterial Cell Envelope

Capsules - foradherence,resistance toengulfment, storage

Cell wall - protection

against lysis orrupture of the cell

Cytoplasmicmembrane -transport of nutrients,

energy generation,ATP production,special functions

C l

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Capsules

Capsules are composed of polysaccharides

(occasionally polypeptides) deposited outside thecell wall.

Using special staining

techniques,

some capsules can be

demonstrated as a halo

surrounding the bacterial cells.

Bacterial

cell

Capsular material

T f C l

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Types of Capsules

True capsules are discrete layers enclosing a cell or groupof cells that can be readily visualized microscopically.

Negative stain ofStreptococcus pneumoniae

outlining its notorious

polysaccharide capsule

Usually, if a bacterium forms a capsule, it willgrow on certain media with a gummy or mucoi

type of colony, such as these colonies of

Bacillus anthracis.

T f C l

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Types of Capsules

Microcapsules, or glycocalyx, are a web of carbohydrate

molecules that envelops the cell. Microcapsules cannot beseen with light microscope.

Microcapsules can be detected by chemical means or by

carefully-prepared electron micrographs.

The hyaluronic acid capsue of

Streptoccus pyogenes is a

microcapsule

T f C l

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Types of Capsules

A slime layer or biofilm is a diffuse matrix ofpolysaccharide which imbeds one or more types ofbacteria.

or Various bacteria growing in a slime layer biofilm

F ti f C l

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Functions of Capsules

Protection against phagotrophicengulfment

Mediate adherence to surfaces

Protection against drying Reserve of nutrients

Biofilms for protection and metabolic

communication among microbes

F ti f C l

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Functions of Capsules

Protection against phagotrophic

engulfment

Three bacteria that use capsules to protect themselves from attack by

phagocytesduring infections. L to R. Streptococcus pneumoniae - pneumonia; Bacillus

anthracis -

anthrax; Streptococcus pyogenes - strep throat.

F ti f C l

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Functions of Capsules

Mediate adherence to surfaces

Oral streptococci use their capsular slime to adhere to the the surfaces of the

teeth and gums.

F ti f C l

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Functions of Capsules

Reserve of nutrients

Colonies of oral streptococci growing on mitis-salivarius agar. The

medium contains 5% sucrose. Streptococcus salivarius (left)stores excess sugar as levan polymer; Streptococcus mutans

(right) stores the carbohydrate as a dextran polymer. The

polysaccharide polymers give the colonies there glistening, sugary

appearance.

F ti f C l

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Functions of Capsules

Biofilms for protection and metabolic

communication among microbes

Biofilm development by Pseudomonas aeruginosa. Figure from: Kolter, R. and

R. Losick. 1998. One for all and all for one. Science 280:226-227. After the

bacteria form the biofilm, they are protected from antibiotics, detergents,

disinfectants, etc., which cannot penetrate the slime.

The Importance of the Bacterial Surface

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Possible natural functions of bacterial surface components(1) Permeability barriers that allow selective passage of nutrients and exclusion of harmful

substances (e.g. antimicrobial agents)

(2) Adhesins used to attach or adhere to specific surfaces or tissues

(3) Enzymes to mediate specific reactions on the cell surface important in the survival of the

organism

(4) Protective structures against phagocytic engulfment or killing

(5) Antigenic disguises

(6) Sensing proteins" that can respond to temperature, osmolarity, salinity, light, oxygen,

nutrients, cell density (quorum sensing), etc.

Cell surface of a Bacillus

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In medical situations as determinants of virulence

(1) Colonize tissues

(2) Resist phagocytosis, antibiotics and host immuneresponses

(3) Induce inflammation, complement activation andimmune responses .

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Colonization

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Colonization Colonization is a firs step of infection. Establishment of

pathogen at a specific body site frequently followed

after entry to the host tissue,. Colonization occurs in body systems intact with

external environment,eg:- urogenital tract , digestive

tract, respiratory tract and peritoneum in females

through the fallopian tubes.

Adherence to Surface :

1) Specific

2) Non specific

Adherence to Surface

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Adherence to Surface

Specific :

Reversible or permanent ,specific covalent bonds between

adhesion and receptor molecules.

Species specific tropism

E.g:-N.gonorehea ,N meningitis,group A

Eg:-E.coli,uropathogenic pattern are

determined by binding specificity of the

PapG adhesion

Pap G alleles of E.coli exists in three

typesClass 1, 2, 3

Tissue host specific

complementery

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Non specific:

Reversible attachment ,Attractions,

Brownian movement, bacterial cell wall

traping by biofilm

Endotoxin

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Endotoxin

Cell envelop component shed as amembrane blebs or vesicles .

They are exemplified by LOS and LPS .

When bacterial endotoxins releases

Fever change in wbc count DIC ,hypotension shock death follows

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Bacterial Determinants of Virulence

Choline Binding Proteins (CBPs) Includes such important determinants as PspA (protective

antigen), LytA, B, and C (three autolysins), and CbpA (anadhesin).

PspA inhibits complement-mediated opsonization.

Autolysin LytA is responsible for pneumococcal lysis instationary phase as well as in the presence of antibiotics.

Autolysin LytB is a glucosaminidase involved in cellseparation.

LytC exhibits lysozyme-like activity.

CbpA is a major pneumococcal adhesin. It interacts withcarbohydrates on the pulmonary epithelial surfacecarbohydrates.

CbpA also has been reported to bind secretory IgA andcomplement component C3.

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THANK U