Bacteria

A bacterial cell has a nuclear apparatus which is a loose arrangement of DNA This is surrounded cytoplasm which contains ribosomes, mesosomes and inclusion granules. The cytoplasm is enclosed within a cytoplasmic membrane. Bacterium has a rigid cell wall  Fimbriae and flagella are the surface adherents. Some bacteria may have a capsule (or loose slime) around the cell wall.

Shape and Size of Bacteria

The bacteria can be spheroidal (coccus), rod or cylindrical (bacillus) and spirillar (spirochaete). Very short bacilli are called as coccobacilli  Some of the bacilli may be curved or comma shaped (Vibrio cholerae).

Arrangement of Bacterial Cells

Streptococci are present in chains; staphylococci in grape-like clusters Cocci in pairs (diplococci) are suggestive of pneumococci, gonococci or menigococci.
Bacilli do not exhibit typical arrangement pattern except the Chinese letter arrangement shown by Corynebacterium diphtheriae

Surface Adherents and Appendages

CAPSULE The gels formed by the capsule adhere to the cell Capsule can be detected by negative staining ,with specific antiserum and observing the capsular swelling phenomenon called as Quellung reaction
Usually weakly antigenic Capsule production is better in vivo as compared to in vitro environment.
Eg. Capsules seen in Pneumococci,  Klebsiella, Escherichia coli, Haemophilus influenzae

Flagella : provide motility to the bacterium. 
Motile organisms: vibrios, pseudomonas, Esch.coli, salmonellae, spirochaetes and spirilla. 
Pathogenic cocci are nomotile.
Flagella measure in length from 3 to 20 µm and in diameter from 0.01 to 0.0 13 µm.
 
Arrangement

Bacteria with one polar flagellum are known as monotrichous; 
Tuft of several polar flagellae is known as lophotrichous
Presence of  Flagellae at both the ends of organism is amphitrichous 
Flagellae distributed all over the surface of the bacterium, it is called peritrichous.
•    Filament is composed of a protein-flagellin. The flagellar antigen is called as H (Hauch) antigen in contrast to somatic antigen which is called as O (Ohne haunch)

PILI (fimbriae) : hair like structures help in attachment also called sex pilli, transfers genetic material through conjugation , Present in Certain Gram negative bacteria. Only Composed of protein pilin  
Gram positive bacterium that has pili is Cornebacterium renale

The Cell Wall

The cell wall of  bacteria is multilayered structure. The external surface of cell wall is smooth in Gram positive bacteria  Gram negative bacteria have convoluted cell surfaces. The average thickness of cell wall is 0.15 to 0.50 .µm. Chemically composed of mucopeptide scaffolding formed by N acetyl glucosamine and N acetyl muramic acid
The cell wall is a three layered structure in Gram negative bacteria: outer membrane middle layer and plasma membrane. The outer membrane consists of lipoprotein and 1ipoppolysaccaride component

Functions of bacterial cell wall

 Provides shape , Gives rigidity , Protection, Surface has receptor sites for phages, Site of  antibody action,  Provides attachment to complement, Contains components toxic to host
 
Cytoplasmic Structures

The Plasma Membrane: This delicate membrane separates rigid cell wall from cytoplasm. It accounts for 30% of total cell weight. Chemically, it is 60% protein, 20-30% lipids and remaining carbohydrates.

 Mesosomes: 
 
 Principal sites of respiratory enzyme , Seen well in Gram positive bacteria as compared to Gram negative batcteria. Attachement of mesosomes to both DNA chromatin and membrane have been noticed thus help in cell division
 
Ribosomes: 

sites of protein synthesis. These are composed of RNA and proteins and constitute upto 4 of total cell protein and 90% of total cellular RNA.
Cytoplasmic Granules: Glycogen  :  Enteric bacteria
Poly-beta & hydroxy Butyrate : Bacillus & Pseudomonas
Babes-Ernst  :Corynebacterium & Yersinia pestis

Nuclear Apparatus

Bacterial DNA represents 2-3% of the cell weight and 10% of the volume of bacterium. Nucleous can be demonstrated by staining it with DNA specific Fuelgen stain .Consists of a single molecule of  double stranded DNA arranged in a circular form. Bacterial chromosome is haploid and replicates by binary fission, the bacteria may have  plasmid an extrachromosomal genetic material.
 

BACTERIAL GROWTH

The conversion of a parental cell into two daughters constitutes the bacterial life cycle and the time taken to complete cell cycle is known as generation_time. This is around 15 minutes in vegetative bacteria except mycobacteria.

Bacterial Growth Curve

In the presence of fresh growth medium a bacterium shows following four phases;

The Lag phase -> The Log phase -> The Stationary phase  -> The Decline phase

The Lag Phase : short duration , bacteria adapt themselves to new environment 

The Log Phase (Exponential Phase) : Regular growth of bacteria occurs The morphology of bacteria is best developed in this phase and organisms manifest typical biochemical characters. 

- Most of the cidal Abx work best in this phase
•    i.e. Ampicillin
- Best phase for staining bacterial cultures

Chemostat and turbidostat are examples of technique by which this phase can be prolonged.

Stationary Phase : balanced growth and cell division cannot be sustained. The total cell Count remains static till lysis supervenes, but the viable cell count quickly declines.

Decline Phase: death phase. Dyeing bacteria exceed the dividing bacterias.
 

GENETIC VARIATION

Two methods are known for genetic variation in bacteria: mutation and gene transfer.

Mutation : Any change in the sequence of bases of DNA, irrespective of detectable changes in the cell phenotype. Mutations may be spontaneous or induced by various agents which are known as mutagens. 

Spontaneous Mutations: Arise from enzymatic imperfections during DNA replications or with transient insertions of transposable elements.

Induced Mutations: Mutation by physical and chemical mutagens.

Physical mutagens  ultraviolet rays and high-energy ionizing radiations. The primary effect of UV rays on DNA is the production of pyrmidine dimers whereas ionizing radiations cause single_stranded breaks the DNA molecules.

Chemical mutagens :Affecting nucleotide sequence

(i) Agents which cause error in base pairing (e.g. nitrous acid and alkylating agents).
(ii) Agents which cause errors in DNA replication (e.g. acridine dyes such as acridine orange and profiavine).
(iii) Base analogs which are incorporated into DNA and cause replication errors (e.g. 5-bromouracil)

Gene Transfer

Transformation: Uptake of naked DNA

Transduction    : Infection by a nonlethal bacteriophage

Conjugation    : Mating between cells in contact

Protoplast fusion

Transformation: Gene transfer by soluble DNA is called as transformation. it requires that DNA be absorbed by the cell, gain entrance to the cytoplasm and undergo recombination with the host genome. 

Artificial Transformation(transfection) :Some of the bacteria (such as Escherichia coli) resist transformation until they are subjected to some special treatment such as CaCl2 to make the bacterium more permeable to DNA. Such modified cells can also take up intact double stranded DNA extracted from viruses or in the shape of plasmids. Though the process is same as transformation, it is 9 as transfection because it results in infection by an abnormal route

Transduction :The type of gene transfer in which the DNA of one bacterial cell is introduced into another bacterial cell by viral infection is known as transduction. This introduces only a small fragment of DNA. Because the DNA is protected from damage by the surrounding phage coat, transduction is an easier to perform and more reproducible process than transduction. ,

Two types of transduction are known.

- Generalized transduction When a bacteriophage picks up fragments of host DNA at random and can transfer any genes

-  Specialised transduction: phage DNA that has been integrated into the host chromosome is excised along with a few adjacent genes, which the phage can then transfer.

After entry into the host cell, the phage DNA gets incorporated into the host chromosome in such a way that the two genomes are linearly contiguous (lysogeny). The phage genome in this stage is known as prophage, The host cell acquires a significant new property as a consequence of lysogeny because it becomes immune to infection by homologous phage. This is hence called as lysogenic conversion and endow toxigenicity to Corynebacterium diphtheriae

Abortive Transduction :phage DNA fails to integrated into the host chromosome, the process is called as abortive transduction The phage DNA does not replicate and along with binary fission Of the host it goes into one of the daughter cells.

Conjugation :This is defined as the transfer of DNA directly from on bacterial. .cell to another by a mechanism that requires cell-to-cell contact. 

The capacity to donate DNA depends upon the possession of the fertility (F) factor. The F pili  also retard male-male union. Concomitant with effective male-female pair formation, the circular DNA bearing the F factor is converted to a linear form that is transferred to the female cell in a sequential manner. DNA replication occurs in the male cell and the newly synthesized, semiconserved DNA molecule remains in the male. This ensures postmating characters of the male.

Conjugation in Different Bacteria: Unusual form of plasmid transfer, called phase mediated conjugation has  been reported to occur with some strains of Staphylococcus aureus.

Protoplast Fusion: Also called as genetic transfusion. Under osmotically buffered Conditions protoplast fusion takes place by joining of cell membrane and generation of cytoplasmic bridges through which genetic material can be exchanged.

Transposons: Transposons  Tn  are  DNA sequences which are incapable of autonomous existence and which transpose blocks of genetic material back and forth between cell Chromosome and smaller replicons such as plasmids. insertion sequences (IS ) are another similar group of nucleotides which can move from one chromosome to another

Genetic material. IS and  Tn are collectively also known as transposable elements or Jumping genes. These are now recognised to play an important role in bringing about vanous types of mutations.

NITRIC OXIDE-DEPENDENT KILLING

Binding of bacteria to macrophages, particularly binding via Toll-like receptors, results in the production of TNF-alpha, which acts in an autocrine manner to induce the expression of the inducible nitric oxide synthetase gene (i-nos ) resulting in the production of nitric oxide (NO) . If the cell is also exposed to interferon gamma (IFN-gamma) additional nitric oxide will be produced (figure 12). Nitric oxide released by the cell is toxic and can kill microorganism in the vicinity of the macrophage.

NON-SPECIFIC KILLER CELLS

Several different cells including NK and LAK cells, K cells, activated macrophages and eosinophils are capable of killing foreign and altered self target cells in a non-specific manner. These cells play an important role in the innate immune system.

A. NK and LAK cells

Natural killer (NK) cells are also known as large granular lymphocytes (LGL) because they resemble lymphocytes in their morphology, except that they are slightly larger and have numerous granules.

NK cells can be identified by the presence of CD56 and CD16 and a lack of CD3 cell surface markers.

NK cells are capable of killing virus-infected and malignant target cells but they are relatively inefficient in doing so.

However, upon exposure to IL-2 and IFN-gamma, NK cells become lymphokine-activated killer (LAK) cells, which are capable of killing malignant cells.

Continued exposure to IL-2 and IFN-gamma enables the LAK cells to kill transformed as well as malignant cells. LAK cell therapy is one approach for the treatment of malignancies.

NK and LAK cells have two kinds of receptors on their surface – a killer activating receptor (KAR) and a killer inhibiting receptor (KIR). 

When the KAR encounters its ligand, a killer activating ligand (KAL) on the target cell the NK or LAK cells are capable of killing the target. However, if the KIR also binds to its ligand then killing is inhibited even if KAR binds to KAL. 

The ligands for KIR are MHC-class I molecules. Thus, if a target cell expresses class I MHC molecules it will not be killed by NK or LAK cells even if the target also has a KAL which could bind to KAR. 

Normal cells constitutively express MHC class I molecules on their surface, however, virus infected and malignant cells down regulate expression of class I MHC. Thus, NK and LAK cells selectively kill virus-infected and malignant cells while sparing normal cells.

B. K cells 

Killer (K) cells are not a morphologically distinct type of cell. Rather a K cell is any cell that mediates antibody-dependent cellular cytotoxicity (ADCC). 

In ADCC antibody acts as a link to bring the K cell and the target cell together to allow killing to occur. K cells have on their surface an Fc receptor for antibody and thus they can recognize, bind and kill target cells coated with antibody. 

Killer cells which have Fc receptors include NK, LAK, and macrophages which have an Fc receptor for IgG antibodies and eosinophils which have an Fc receptor for IgE antibodies.

ANTIGENS

Immunogen
A substance that induces a specific immune response.

Antigen (Ag)
A substance that reacts with the products of a specific immune response.

Hapten
A substance that is non-immunogenic but which can react with the products of a specific immune response. Haptens are small molecules which could never induce an immune response when administered by themselves but which can when coupled to a carrier molecule. Free haptens, however, can react with products of the immune response after such products have been elicited. Haptens have the property of antigenicity but not immunogenicity.

Epitope or Antigenic Determinant
That portion of an antigen that combines with the products of a specific immune response.

Antibody (Ab)
A specific protein which is produced in response to an immunogen and which reacts with an antigen.

FACTORS INFLUENCING IMMUNOGENICITY

- Larger the molecule the more immunogenic it is likely to be.

- More complex the substance is chemically the more immunogenic it will be.

- Particulate antigens are more immunogenic than soluble ones and denatured antigens more immunogenic than the native form.

- Antigens that are easily phagocytosed are generally more immunogenic. This is because for most antigens (T-dependant antigens, see below) the development of an immune response requires that the antigen be phagocytosed, processed and presented to helper T cells by an antigen presenting cell (APC).

- Some substances are immunogenic in one species but not in another. Similarly, some substances are immunogenic in one individual but not in others (i.e. responders and non-responders). The species or individuals may lack or have altered genes that code for the receptors for antigen on B cells and T cells or they may not have the appropriate genes needed for the APC to present antigen to the helper T cells.

Method of Administration

1. Dose
The dose of administration of an immunogen can influence its immunogenicity. There is a dose of antigen above or below which the immune response will not be optimal.

2. Route
Generally the subcutaneous route is better than the intravenous or intragastric routes. The route of antigen administration can also alter the nature of the response

3. Adjuvants
Substances that can enhance the immune response to an immunogen are called adjuvants. The use of adjuvants, however, is often hampered by undesirable side effects such as fever and inflammation.

TYPES OF ANTIGENS

T-independent Antigens
T-independent antigens are antigens which can directly stimulate the B cells to produce antibody without the requirement for T cell help In general, polysaccharides are T-independent antigens. The responses to these antigens differ from the responses to other antigens.
Properties of T-independent antigens

1. Polymeric structure
These antigens are characterized by the same antigenic determinant .

2. Polyclonal activation of B cells
Many of these antigens can activate B cell clones specific for other antigens (polyclonal activation). T-independent antigens can be subdivided into Type 1 and Type 2 based on their ability to polyclonally activate B cells. Type 1 T-independent antigens are polyclonal activators while Type 2 are not.

3. Resistance to degradation
T-independent antigens are generally more resistant to degradation and thus they persist for longer periods of time and continue to stimulate the immune system.

T-dependent Antigens
T-dependent antigens are those that do not directly stimulate the production of antibody without the help of T cells. Proteins are T-dependent antigens. Structurally these antigens are characterized by a few copies of many different antigenic determinants as illustrated in the Figure 2.

HAPTEN-CARRIER CONJUGATES

Hapten-carrier conjugates are immunogenic molecules to which haptens have been covalently attached. The immunogenic molecule is called the carrier.

Structure
Structurally these conjugates are characterized by having native antigenic determinants of the carrier as well as new determinants created by the hapten (haptenic determinants). The actual determinant created by the hapten consists of the hapten and a few of the adjacent residues, although the antibody produced to the determinant will also react with free hapten. In such conjugates the type of carrier determines whether the response will be T-independent or T-dependent.

SUPERANTIGENS

When the immune system encounters a conventional T-dependent antigen, only a small fraction (1 in 104 -105) of the T cell population is able to recognize the antigen and become activated (monoclonal/oligoclonal response). However, there are some antigens which polyclonally activate a large fraction of the T cells (up to 25%). These antigens are called superantigens .

Examples of superantigens include: Staphylococcal enterotoxins (food poisoning), Staphylococcal toxic shock toxin (toxic shock syndrome), Staphylococcal exfoliating toxins (scalded skin syndrome) and Streptococcal pyrogenic exotoxins (shock).