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

Gram Positive Cell Envelope

Lipoteichoic acid
r r

M-Protein

Peptidoglycan-teichoic acid
r r r r r r r

Cytoplasmic membrane

Cytoplasm

Chemical nature of bacterial cell walls

Peptidoglycan is made up of 2 amino sugars N-acetyl-glucosamine = G N- acetylmuramic acid = M 4 amino acids L-alanine = L-ala D-glutamic acid = D-glu diaminopimelic acid = DAP D-alanine = D-ala
GM

L-ala
D-glu DAP D-ala

Peptidoglycan Synthesis

Cytoplasm
UMP UDP UTP
D-ALa

Membrane
D-Ala transport Vancomycin inhibits

Wall
Expanding cross linked wall Peptidoglycan
3 Lactum Antibiotic inhibits

UTP + Glc NAc-1-p

- PP1

UDP-Glc NAc
+PEP

UDP-Glc NAc
Enolpyruvate

Phosphonomycin inhibits

Nascent Peptidoglycan

UDP Mur NAc

+L-Ala +D-Glu + L-R3

+NADPH

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

D-Ala-DAla
9MurF

Undecaprenyl-PP Disaccaride -pentapeptide

Undecaprenyl-PP -Mur NAcpentapeptide

Disaccaride -pentapeptide

UDP Mur NAc-L-Ala -D-Glu-L-R3-(DAla)2 Cycloserine inhibits

2-L-Ala

Bacitracin inhibits

Undecaprenyl-P

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-acetylmuramidases Endo-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 amidase Acting 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
O-antigen Highly variable Core Heptoses Ketodeoxyoctonic acid

Lipid A Glucosamine disaccharide Beta hydroxy fatty acids

Porins
Three 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
Omp.A

FUNCTIONS
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 .

Murien lipoprotein OmpB

Lam.B

LamB transport maltose&maltodextrins,binding sites for lambda phages .

Diffusion channel for small molecules ;receptors for Tulb&T3,T4. Protease.

Omp.C & Omp.F Omp.T

PhoE

Anion-selective diffusion channel under phosphate limitation

Protein P

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

TolA

TonA

Ton.B

Other characteristics of bacterial cell walls

Outer membrane of Gram-negatives has two important properties 1. 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

Flagella
Flagella are long whiplike filaments composed of protein that originate in the cell membrane.
Salmonella enterica, like most enteric bacteria, is capable of swimming movement by means of flagella.

Flagella rotate and impart swimming movement on the cells

Flagella are for swimming movement

Peritrichous flagella are distributed all over the cell surface
Proteus mirabilis swims by means of peritrichous flagella

Polar flagella originate at the pole of a cell

Vibrio cholerae has a single polar flagellum

Detecting Motility in Bacteria

Peritrichous flagella Polar flagellum

Bacillus cereus

Vibrio cholerae

Bacillus brevis

By using flagellar stains to detect the presence and distribution of flagella

Detecting Motility in Bacteria

Staphylococcus epidermidis Non motile

E. coli motile

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

Ecological Advantages to Swimming

1. 2. 3. 4. 5. Survival: escape predatory protozoa and white blood cells (phagocytes) Swim towards nutrients or away from harmful substances (chemotaxis) Swim towards or away from O2 (aerotaxis) Swim towards light (phototaxis) 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

Pili (also called fimbriae) are short hair-like structures composed of protein on the cell surface. Attachment to a surface or substrate
Shigella dysenteriae uses its fimbriae to attach to the intestine and 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

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

basal body

Filament is rotated by a protein motor in the cell membrane

motor filament

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

pmf on this side of membrane

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 ( dont stain) irradiation, disinfectants, antibiotics, etc.

Properties of Endospores

Parasporal crystal

Endospore

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

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 Endosporeforming 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.
Bacterial cell

Capsular material

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

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 mucoid type 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 phagocytes during 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 Surface
Specific :
Reversible or permanent ,specific covalent bonds between complementery adhesion and receptor molecules.

Tissue host specific

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

Species specific tropism

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

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