MCBM ELEMENT 6:

INFECTION, IMMUNITY,

INFLAMMATION, CELL INJURY &

REPAIR

Immunity, Infectious Disease

LECTURE 14:

Introduction to Microbiology

&

Bacterial Structure

LECTURE 14: LEARNING

OBJECTIVES

The objectives of this lecture are for students to:

Appreciate the wide prevalence and diversity of microbial forms

Understand the consequences of microbial infectious diseases

Realize the different morphologies of microbes

Realize the difference between prokaryotic and eukaryotic cell structure

Be aware of the medically important microbes

Understand the importance of the Gram stain

LECTURE 14: LEARNING

OBJECTIVES

The objectives of this lecture are for students to:

Understand the structure of a typical bacterial cell

Understand the medical importance of the various appendages of a bacterial cell

Know the detailed structure of both Gram positive and Gram negative cell walls

Realize the difference between Gram positive and Gram negative cells

Understand the action of penicillin and lysozymeenzyme on the cell wall of bacteria

Understand how a bacterial cell enlarges

LECTURE 14: LEARNING

OUTCOMES

At the end of the lecture you should be able to:

Discuss the omnipresence of microorganisms

Understand that microbes are capable of

causing infectious diseases

Appreciate and recognize the various forms

and diversity of microbes

Compare and contrast prokaryotic and

eukaryotic cell structures

Perform the Gram staining procedure during

practicals

LECTURE 14: LEARNING OUTCOMES

At the end of the lecture you should be able to:

Know the structure of a typical bacterial cell and be able relate the medical importance of the various appendages

Discuss the medical importance of the LPS molecule

Structurally distinguish between Gram positive and Gram negative cells

Describe the detailed structure of both types of cell walls

Explain the different mechanisms of actions of penicillin and lysozyme on the cell wall of bacteria

Illustrate the process of bacterial cell enlargement

I. Microorganisms can be found in every

ecosystem and in close association with every type

of multicellular organism

They populate the healthy human body by the

billions as benign passengers and even participate

in bodily functions …… (give examples)

Bacteria, fungi, viruses, protozoa, helminthes

…..their role in the initiation and spread of human

diseases. Such microorganisms are characterized

as Pathogens

Most infectious disease is initiated by colonization

(i.e. the establishment of proliferating microorganisms

on the skin or mucous membranes)

However major exceptions are diseases caused by

introduction of organisms directly into the bloodstream

or internal organs

Microbial colonization may result in 1 ) Elimination of

the microorganism without effecting the host or 2 )

Infection where the organisms multiply and cause the

host to react by making an immune or other type of

response.

Infection can have several consequences ,

including infectious diseases , where the

organism causes tissue damage and

impairment of body function .(Def. of

infection: multiplication of pathogenic

bacteria eg Salmonella species even if the

person is asymptomatic)

I I. PROKARYOTIC PATHOGENS

All prokaryotic organisms are classified as bacteria whereas eukaryotic organisms including fungi, protozoa and helminthes, as well as humans.

Prokaryotic organisms divided into 2 major groups: the eubacteria (which include all medically impt. bacteria) and archae bacteria (a collection of evolutionarily distinct organisms )

A. TYPICAL BACTERIA

Most bacteria have the following shapes : a sphere or coccus , rod shaped or a corkscrew like appearance ( spirochete )

Nearly all bacteria have a rigid cell wall with the exception of the mycoplasma. The cell wall determines the shape of the bacteria. The cell wall also determines whether the bacterium is classified as gram-positive of gram- negative. The cell wall also surrounds the cell membrane. External to the cell-wall may be flagella, piliand/or a capsule.

Bacteria divide by binary fission . Many

bacteria also exchange genetic information

carried on plasmids (small specialized

genetic elements capable of self replication

including transferring antibiotic resistance)

B. ATYPICAL BACTERIA

This group include organisms such as

mycoplasma , Chlamydia and rickettsia .

Although prokaryotic , they lack some

significant structural and metabolic

capabilities

I I I . FUNGI

They are nonphotosynthetic, generally saprophytic, eukaryotic. If filamentous, are called moulds whereas the yeast are unicellular.

Fungal reproduction maybe asexual, sexual or both and all fungi produce spores. Pathogenic fungi can cause diseases such as skin infections (superficial mycoses) to serious, systemic infections (deep mycoses)

I V. PROTOZOA

Protozoa are single – celled , nonphotosynthetic , eukaryotic organisms that have many shapes and sizes . Parasites of humans….they can infect all the major tissues and organs of the body.

Both intracellular as well as extracellular parasites in the blood, urogenital region or the intestine. Transmission is by ingestion of the infective stage of the parasite or by an insect bite.

V. HELMINTHS

Helminths are those group of worms that live as parasites. These are multicellular eukaryotic organisms with complex body organization.

Three major groups: tapeworms (cestodes) flukes (trematodes), and roundworms (nematodes). They are parasitic and all major groups can cause disease.

V I. VIRUSES

Viruses are obligate intracellular parasites that do not have a cellular structure .

A virus consists of molecules of either DNA (DNA Virus) or RNA (RNA Virus) but not both surrounded by a protein coat .

A virus may also have an envelope derived from the plasma membrane of the host cell from which the virus is released .

Viruses contain the genetic information necessary for directing their own replication but require the host’s cellular structure and enzymatic machinery in order to complete the process of their own reproduction

Some Possible Outcomes Following Exposure to

Microorganism

VARIOUS TYPES OF BACTERIA

Comparison of naked and enveloped

virus

BACTERIAL STRUCTURE

Structure of a typical bacteria

Bacterial Capsules

India ink negatively stained preparation of cells of

Streptococcus pneumoniae. Note the extensive capsule

surrounding the cells.

Pili (a) Pili on an Escherichia coli cell. The short pili (fimbriae) mediate adherence;

the F pilus is involved in DNA transfer.(11,980x)

Fimbriae and pili

• Fimbriae and pili are structurally similar to flagella but are not involved in motility. Fimbriae are considerably shorter than flagella and are more numerous (Figure 3.56) but, like flagella, consist of protein. Not all organisms have fimbriae, and the ability to produce them is an inherited trait. The functions of fimbriae are not known for certain in all cases, but there is some evidence that they enable organisms to stick to surfaces including animal tissues in the case of some pathogenic bacteria or to form pellicles or scums on the surfaces of liquids.

• Pili are similar structurally to fimbriae but are generally longer, and only one or a few pili are present on the surface. Pili can be visualized under the electron microscope because they serve as specific receptors for certain types of virus particles, and when coated with virus they can be easily seen (Figure 3.57). There is strong evidence that pili are involved in the process of conjugation in prokaryotes. Pili are also involved in attachment to human tissues by some pathogenic bacteria.

Structure of Gram + Cell Wall

STRUCTURE OF GRAM-NEGATIVE CELL-WALL

Structure of pepitidoglycan

1. It has to add more peptidoglycan

2. It has to produce an enzyme called Autolysin

3. This autolysin breaks cross-linking bonds in the peptidoglycan

4. Bacteria also produces two enzymes called Carboxypeptidases and

Transpeptidases (penicillin binding proteins (PBPs) that reseales the

breaks

5. Penicillin binds to the transpeptidase enzymes that reseal the

peptidoglycan molecule during cell growth

6. For this reason, Carboxypeptidases and transpeptidase enzymes are

called penicillin– binding proteins (PBPs).

7. Binding inactivates these enzymes as a result the cell is unable to close

the breaks in the cell wall that autolysin makes as the cell grows

8. Ultimately the cell is unable to hold the turgor pressure and the cell lyses

resulting in the death of the cell

HOW DOES A BACTERIAL CELL

ENLARGE ?

ANTIBACTERIAL COMPOUNDS THAT

TARGET PEPTIDOGLYCAN

• Compounds that interfere with the synthesis of peptidoglycan or alter its structural integrity weaken the rigid molecule to a point where it is not strong enough to prevent the cell from bursting.

• These compounds include the antibiotic penicillin and the enzyme lysozyme, which is found in many body fluids including tears and saliva.

Penicillin

• Penicillin is the most thoroughly studied of a group of antibiotics that interfere with peptidoglycan synthesis.

• Penicillin binds to proteins involved in cell wall synthesis and, prevents the cross-linking of adjacent glycan chains.

• These proteins are called penicillin-binding proteins, a name that reflects their medical importance rather than their role in peptidoglycan synthesis.

• Generally, penicillin is far more effective against Gram-positive cells than Gram-negative cells.

• This is because the outer membrane of Gram-negative cells prevents the medication from reaching its site of action, the peptidoglycan layer.

• However, the structure of penicillin can be modified to create penicillin derivatives that can pass through porin channels.

• These drugs are effective against a range of Gram-negative bacteria.

Lysozyme

• Lysozyme breaks the bond that links the alternating N-acetylglucosamine and N-acetylmuramic acid molecules and thus destroys the structural integrity of the glycan chain, the backbone of the peptidoglycan molecule.

• Removing that layer from a Gram-positive bacterium creates a protoplast that lacks a cell wall.

• In contrast, removing the peptidoglycan layer from a Gram-negative bacterium creates a spheroplast.

• Spheroplasts retain some portions of the outer membrane.

• Because they lack their rigid cell wall, protoplasts and spheroplasts both become spherical regardless of the original cell shape.

• Due to osmosis, they will burst unless maintained in a solution that has the same relative concentration of ions and small molecules as the cytoplasm.

Penicillin vs. Lysozyme

NAM NAG NAMNAM NAG

NAM NAG NAMNAM NAG

5 5 5

β -1,4 glycosidic linkage

Pentaglycine

cross-linkage

L-ALA

D-I-GLU-N

L-LYS

D-ALA

In cell division,

the pentaglycine

cross-links are

broken in order

to add more

peptidoglycan

strands

PBPs reseal

these breaks.

PB

P

PB

P

PB

P

Penicillins bind

to PBPs

& prevent this!

The broken links

remain broken;

cell wall weakens

Osmolysis!

Lysozymes

lyse (cut) the

β -1,4 glycosidic

links and

result the

same fate but

irrespective of

cell division

Penicillin

Lysozyme

Differences in Cell Wall

Composition and the Gram Stain

• Differences in the cell wall composition of Gram-positive and Gram-negative bacteria account for their staining characteristics.

• It is not the cell wall, however, but the inside of the cell that is stained by the crystal violet-iodine complex.

• The Gram-positive cell wall somehow retains the crystal violet-iodine complex even when subjected to acetone-alcohol treatment, whereas the Gram-negative cell wall cannot.

• The precise mechanism that accounts for the differential aspect of the Gram stain is not entirely understood.

• Presumably, the decolorizing agent dehydrates the thick layer of peptidoglycan and in this dehydrated state the wall acts as a permeability barrier, retaining the dye.

• In contrast, the solvent action of acetone-alcohol easily damages the outer membrane of Gram-negative bacteria; their relatively thin layer of peptidoglycan cannot retain the dye complex.

• These bacteria lose the dye complex more readily than their Gram-positive counterparts.

• Also, as Gram-positive cells age, they often lose their ability to retain the dye.

• This probably results from damage to their peptidoglycan layer that occurs as a consequence of aging.

Bacterial LPS Layer.

Arrangement of Lipopolysaccharide, lipid A, phospholipid, porin and lipoprotein in gram-negative

The Lipopolysaccharide Molecule

Medical importance of the LPS molecule:

1. LPS are found in Gram-negative bacteria.

2. When purified LPS is injected into an animal it

brings about symptoms characteristic of

infection caused by life bacteria.

3. The same symptoms occur regardless of the

bacterial species.

4. Because it is part of the cell wall it is called

Endotoxin

5. Endotoxin is seldom destroyed by heat even through the heat treatment kills the live bacteria.

6. Two parts of the LPS molecule are medically important.

Lipid A is the toxic portion. Because Lipid A is embedded in LPS, its released and is able to exert its toxic effect only if the bacterium is lysed. Lipid A is toxic because it activates complement and stimulates the release of cytokines and these become toxic if produced in very high concentrations.

7. The O-specific polysaccharide side chain is made up of a chain of sugar molecules. The number and composition of which varies among different species of bacteria.

e.g: E.Coli 015:H7

Gram-Negative Cell Wall

The peptidoglycan layer is made up of only one or two sheets of interconnected glycan chains.

The outer membrane is a typical phospholipid bilayer, expect the outer leaflet contain

lipopolysaccharide. Porins span the membrane to allow specific molecular to pass. Periplasm fills

the region between the cytoplasmic and outer membranes

Chemical Structures of Lipopolysaccharide

The Lipid A portion, which anchors the LPS molecule in the bilayer, is responsible for the

symptoms associated with endotoxin. The composition and length of the O-specific polysaccharide

side chain varies among different species of bacteria.