Bacterial cell wall and applied importance

Dr.Raghu prakash

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 our

most important antibiotics.

Primary function of the bacterial cell wall

• To prevent rupture or osmoticlysis of the cell protoplast

Lysis of a pair of dividing E. coli cells

Chemical nature of bacterial cell walls

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

• Chemical nature of murein accounts for the function of the cell wall

• Murein is only found in the cell walls of bacteria

r r rrr

r

Gram Positive Cell EnvelopeGram Positive Cell Envelope

CytoplasmCytoplasm

rrrr

Lipoteichoic acid

Peptidoglycan-teichoic acid

Cytoplasmic membrane

M-Protein

Chemical nature of bacterial cell walls

Peptidoglycan is made up of

• 2 amino sugars N-acetyl-glucosamine = G N- acetylmuramic acid = M

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

G—M

L-ala

D-glu

DAP

D-ala

Peptidoglycan Synthesis

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-Ala

Cycloserine inhibits

9MurF

+ L-R3

+D-Glu

+L-Ala

+NADPH

+PEP

- PP1

Phosphonomycininhibits

D-ALaD-Ala transport

Undecaprenyl-PP-Mur NAc- pentapeptide

Undecaprenyl-P

Cytoplasm Membrane Wall

Undecaprenyl-PPDisaccaride-pentapeptide

Bacitracininhibits

Disaccaride-pentapeptide

Nascent Peptidoglycan

Expanding cross linked wallPeptidoglycan

3 LactumAntibioticinhibits

Vancomycininhibits

Surface Proteins

• Five penicillin binding proteins (PBPs)

• Two neuraminidases

• IgA protease

• Twelve choline-binding proteins (CBPs) - include important determinants 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 interstitial cement

• IgA protease - cleaves and inactivates secretory IgA

• Bacterial Determinants of Virulence

• Cell Wall Components

• Phosphorylcholine decorating the teichoic acid and the lipoteichoic acids act both as adhesins and as docking sites for the choline-binding proteins (CBPs).

• The peptidoglycan/teichoic acid complex is highly inflammatory. The cell wall directly activates the alternative pathway of the complement cascade, and the coagulation cascade.

• Peptidoglycan binds to CD14, a cell surface receptor known to initiate the inflammatory response for endotoxin. This induces a cytokine cascade resulting in production of interleukin-1, interleukin-6 and tumor necrosis factor from human cells.

Peptidoglycan hydrolases

• Three types –Glycan-strands hydrolyzing

Endo-N-acetylmuramidasesEndo-N-acetylgucosaminidases

Endopeptidase hydrolyzing Peptide bonds in the interior of the peptide

bridges Bonds involving the C-terminal D-alanine residue

N-acetylmuramoyl –L-alanine amidaseActing at the junction b/w glycan strands and the peptide units

This enzymes appear to play an imp role in number of cellular activities septum and wall extension during cell growth ,cell separation,turn over of wall components,sporulation competency for transformation, excretion of toxins and exoenzymes

Gram-negative cell walls include

an outer membrane

Lipopolysaccharide

Lipid A• Glucosamine disaccharide

• Beta hydroxy fatty acids

Core • Heptoses• Ketodeoxyoctonic acid

O-antigenHighly variablen

PorinsThree Types • Type I :- Nonspecific subrstate on 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.

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.

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 group a colicins

Ferrichrome siderophore uptake;receptor for phages T1,T5,80,&COLICIN M.

Dependent proteins . transport vit B12 &Siderophores

Other characteristics of bacterial cell walls

Outer membrane of Gram-negatives has two importantproperties1. It protects the cells from permeability by many

substances including penicillin and lysozyme.2. It is the location of lipopolysaccharide (endotoxin) which

is toxic for animals.

• 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 layer

or 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

Bacterial Surface Appendages

• Flagella - organelles for swimming motility

• Pili (or fimbriae) - for attachment or adherence to surfaces; sex pilus used during some genetic exchange processes

Salmonella enterica, like most enteric bacteria, is capable of swimming movement by means of flagella.

Flagella

Flagella are long whiplike filaments composed of protein that originate in the cell membrane.

Flagella rotate and impart swimming movement on the cells

Proteus mirabilis swims by means of peritrichous flagella

Vibrio cholerae has a single polar flagellum

Flagella are for swimming movement

Peritrichous flagella are distributed all over the cell surface

Polar flagella originate at the pole of a cell

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

Detecting Motility in Bacteria

• By inoculation of the bacteria into motility test medium (SIM).

Staphylococcusepidermidis

Non motile

E. coli

motile

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

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 membrane allowing 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 certain chemicals 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

Functions of Pili and Fimbriae

Attachment to a surface or substrate

Shigella dysenteriae uses its fimbriae to attach to the intestineand then produces a toxin that causes diarrhea.

Neissera gonorrhoeae, 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-like structures composed of protein on the cell surface.

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

Functions of Pili and Fimbriae

• A special type of pilus called the sex pilus is used in mating between bacteria

E. coli uses its sex pilus (called the F-pilus) to transfer DNA between mating bacteria during conjugation.

How Flagella Work

• Filament is rotated by a protein “motor” in the cell membrane

• Motor is powered by proton motive force (pmf) on the outside of membrane

motor

basalbody

filament

pmf on this side ofmembrane

Endospores are produced as intracellular structures within the cytoplasm of certain bacteria, most notably Bacillus and Clostridium species.

Endospore forming bacteria left to right: Clostridium botulinum, Bacillus brevis, Bacillus thuringiensis

Properties of Endospores• Resting (dormant) cells -

“cryptobiotic” i.e.,show no signs of life..primarily due to lack of water in the spore

• Several unique surface layers

not found in vegetative cells :

exosporium, spore coat,

cortex, and core wall

• Highly resistant to heat (boiling), acids, bases, dyes ( don’t stain) irradiation, disinfectants, antibiotics, etc.

Properties of Endospores

• Spores and parasporal crystals produced by some bacteria are toxic to insects

Parasporal crystalEndospore

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

Under favorable nutritional and environmental conditions, an endospore germinates into a vegetative cell.

A germinating spore

Medically-important Endospore-forming Bacteria

• Bacillus anthracis causes anthrax• Bacillus cereus causes food poisoning• Clostridium tetani causes tetanus• Clostridium botulinum causes botulism• Clostridium perfringens causes food poisoning

and gas gangrene• Clostridium difficile causes antibiotic-induced

diarrhea and pseudomembranous colitis

Bacterial Cell Envelope

• Capsules - for adherence, resistance to engulfment, storage

• Cell wall - protection against lysis or rupture of the cell

• Cytoplasmic membrane - transport of nutrients, energy generation, ATP production, special functions

Capsules• Capsules are composed of polysaccharides

(occasionally polypeptides) deposited outside the cell wall.

Using special staining techniques, some capsules can be demonstrated as a halo surrounding the bacterial cells.

Bacterial cell

Capsular material

Types of Capsules• True capsules are discrete layers enclosing a cell or group

of cells that can be readily visualized microscopically.

Negative stain of Streptococcus pneumoniae outlining its notorious polysaccharide capsule

Usually, if a bacterium forms a capsule, it will grow on certain media with a gummy or mucoidtype of colony, such as these colonies of Bacillus anthracis.

Types of Capsules• Microcapsules, or glycocalyx, are a web of carbohydrate

molecules that envelops the cell. Microcapsules cannot be seen 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

Types of Capsules A slime layer or biofilm is a diffuse matrix of

polysaccharide which imbeds one or more types of bacteria.

or Various bacteria growing in a slime layer biofilm

Functions of Capsules

• Protection against phagotrophic engulfment

• Mediate adherence to surfaces

• Protection against drying

• Reserve of nutrients

• Biofilms for protection and metabolic communication among microbes

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.

Functions of Capsules

• Mediate adherence to surfaces

Oral streptococci use their capsular slime to adhere to the the surfaces of the teeth and gums.

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.

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

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

In medical situations as determinants of virulence

(1) Colonize tissues

(2) Resist phagocytosis, antibiotics and host immune responses

(3) Induce inflammation, complement activation and immune responses .

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 SurfaceSpecific :

Reversible or permanent ,specific covalent bonds between adhesion and receptor molecules.

Species specific tropism

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

(CFA-1 and CFA-2)

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

Non specific:

Reversible attachment ,Attractions, Brownian movement, bacterial cell wall

traping by biofilm

Endotoxin

• Cell envelop component shed as a membrane blebs or vesicles .

• They are exemplified by LOS and LPS .• When bacterial endotoxins releases • Fever change in wbc count DIC ,

hypotension shock death follows

Bacterial Determinants of Virulence

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

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

• PspA inhibits complement-mediated opsonization.• Autolysin LytA is responsible for pneumococcal lysis in

stationary phase as well as in the presence of antibiotics. • Autolysin LytB is a glucosaminidase involved in cell

separation. • LytC exhibits lysozyme-like activity.• CbpA is a major pneumococcal adhesin. It interacts with

carbohydrates on the pulmonary epithelial surface carbohydrates.

• CbpA also has been reported to bind secretory IgA and complement component C3.

THANK U