NEET MDS Lessons
General Microbiology
Immunoglobulin (Ig)
Immunoglobulins are glycoprotein molecules that are produced by plasma cells in response to an immunogen and which function as antibodies. The immunoglobulins derive their name from the finding that they migrate with globular proteins when antibody-containing serum is placed in an electrical field
FUNCTION
1. Immunoglobulins bind specifically to one or a few closely related antigens. Each immunoglobulin actually binds to a specific antigenic determinant. Antigen binding by antibodies is the primary function of antibodies and can result in protection of the host.
2. The significant biological effects are a consequence of secondary "effector functions" of antibodies.Phagocytic cells, lymphocytes, platelets, mast cells, and basophils have receptors that bind immunoglobulins. This binding can activate the cells to perform some function. Some immunoglobulins also bind to receptors on placental trophoblasts, which results in transfer of the immunoglobulin across the placenta. As a result, the transferred maternal antibodies provide immunity to the fetus and newborn.
STRUCTURE OF IMMUNOGLOBULINS
The basic structure of the immunoglobulins is illustrated in figure 2. Although different immunoglobulins can differ structurally, they all are built from the same basic units.
A. Heavy and Light Chains
All immunoglobulins have a four chain structure as their basic unit. They are composed of two identical light chains (23kD) and two identical heavy chains (50-70kD)
B. Disulfide bonds
1. Inter-chain disulfide bonds - The heavy and light chains and the two heavy chains are held together by inter-chain disulfide bonds and by non-covalent interactions The number of inter-chain disulfide bonds varies among different immunoglobulin molecules.
2. Intra-chain disulfide binds - Within each of the polypeptide chains there are also intra-chain disulfide bonds.
C. Variable (V) and Constant (C) Regions
When the amino acid sequences of many different heavy chains and light chains were compared, it became clear that both the heavy and light chain could be divided into two regions based on variability in the amino acid sequences. These are the:
1. Light Chain - VL (110 amino acids) and CL (110 amino acids)
2. Heavy Chain - VH (110 amino acids) and CH (330-440 amino acids)\(x = {-b \pm \sqrt{b^2-4ac} \over 2a}\)h the arms of the antibody molecule forms a Y. It is called the hinge region because there is some flexibility in the molecule at this point.
E. Domains
Three dimensional images of the immunoglobulin molecule show that it is not straight as depicted in figure 2A. Rather, it is folded into globular regions each of which contains an intra-chain disulfide bond (figure 2B-D). These regions are called domains.
1. Light Chain Domains - VL and CL
2. Heavy Chain Domains - VH, CH1 - CH3 (or CH4)
F. Oligosaccharides
Carbohydrates are attached to the CH2 domain in most immunoglobulins. However, in some cases carbohydrates may also be attached at other locations.
IMMUNOGLOBULIN FRAGMENTS: STRUCTURE/FUNCTION RELATIONSHIPS
Immunoglobulin fragments produced by proteolytic digestion –
A. Fab
Digestion with papain breaks the immunoglobulin molecule in the hinge region before the H-H inter-chain disulfide bond Figure 6. This results in the formation of two identical fragments that contain the light chain and the VH and CH1 domains of the heavy chain.
Antigen binding – These fragments are called the Fab fragments because they contained the antigen binding sites of the antibody. Each Fab fragment is monovalent whereas the original molecule was divalent. The combining site of the antibody is created by both VH and VL.
B. Fc
Digestion with papain also produces a fragment that contains the remainder of the two heavy chains each containing a CH2 and CH3 domain. This fragment was called Fc because it was easily crystallized.
Effector functions – The effector functions of immunoglobulins are mediated by this part of the molecule. Different functions are mediated by the different domains in this fragment .
Treatment of immunoglobulins with pepsin results in cleavage of the heavy chain after the H-H inter-chain disulfide bonds resulting in a fragment that contains both antigen binding sites . This fragment is called F(ab’)2because it is divalent. The Fc region of the molecule is digested into small peptides by pepsin. The F(ab’)2binds antigen but it does not mediate the effector functions of antibodies.
Method of Sterilization for common items
Autoclaving : Animal cages, Sugar tubes, Lab. Coats, Cotton , Filters, Instruments Culture media, Rubber, Gloves , Stopper, Tubing, Slides, Syringe and Wax needles , Test tubes, Enamel metal trays ,Wire baskets, Wood, Tongue depressor, Applicator, Endodontic instruments, Orthodontic pliers , Orthodontic kits, Saliva ejector, Handpieces Cavitron heads, Steel burs, Steel tumbler, Hand instruments
Hot air oven
Beakers, Flasks, Petri dish, Slides, Syringes, Test tubes, Glycerine, Needles ,Oil, Paper Saliva ejector, Matrix Band
Ethylene oxide
Fabric, Bedding, Blanket, Clothing, Matteresses, Pillows, Disposable instruments , Instruments, Blades, Knives, Scalpels, Scissors ,Talcum powder, Books, Cups, plates , Plastics., Flask, Petridish, Tubes, Tubing, Rubber , catheters, Drains, Gloves ,Special items - Bronchoscope, Cystoscope, Heart lung machine
Glutaraldehyde
Orthodontic kits, Orthodontic pliers , Steel burrs, 3 in 1 syringe tips ,Cystoscope ,Endoscope
Filtration
Antibiotics, Serum, Vaccines
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.
Classification:
Neutrophiles (pH = 7.0)
- P. aeruginosaqo
- Clostridium sporogenes
- Proteus species
Acidophiles (pH < 7.0)
- Thiobacillus thiooxidans
- Sulfollobus acidocaldaarius
- Bacillus acidocaldarius
Alkaliphiles (pH > 7.0)
- Nitrobacter species
- Streptococcus pneumoniae
INNATE (NON-SPECIFIC) IMMUNITY
The elements of the innate (non-specific) immune system include anatomical barriers, secretory molecules and cellular components.
Among the mechanical anatomical barriers are the skin and internal epithelial layers, the movement of the intestines and the oscillation of broncho-pulmonary cilia.
Associated with these protective surfaces are chemical and biological agents.
A. Anatomical barriers to infections
1. Mechanical factors
The epithelial surfaces form a physical barrier that is very impermeable to most infectious agents. Thus, the skin acts as our first line of defense against invading organisms. The desquamation of skin epithelium also helps remove bacteria and other infectious agents that have adhered to the epithelial surfaces.
2. Chemical factors
Fatty acids in sweat inhibit the growth of bacteria. Lysozyme and phospholipase found in tears, saliva and nasal secretions can breakdown the cell wall of bacteria and destabilize bacterial membranes. The low pH of sweat and gastric secretions prevents growth of bacteria. Defensins (low molecular weight proteins) found in the lung and gastrointestinal tract have antimicrobial activity. Surfactants in the lung act as opsonins (substances that promote phagocytosis of particles by phagocytic cells).
3. Biological factors
The normal flora of the skin and in the gastrointestinal tract can prevent the colonization of pathogenic bacteria by secreting toxic substances or by competing with pathogenic bacteria for nutrients or attachment to cell surfaces.
B. Humoral barriers to infection
Humoral factors play an important role in inflammation, which is characterized by edema and the recruitment of phagocytic cells. These humoral factors are found in serum or they are formed at the site of infection.
1. Complement system – The complement system is the major humoral non-specific defense mechanism (see complement chapter). Once activated complement can lead to increased vascular permeability, recruitment of phagocytic cells, and lysis and opsonization of bacteria.
2. Coagulation system – Depending on the severity of the tissue injury, the coagulation system may or may not be activated. Some products of the coagulation system can contribute to the non-specific defenses because of their ability to increase vascular permeability and act as chemotactic agents for phagocytic cells. In addition, some of the products of the coagulation system are directly antimicrobial. For example, beta-lysin, a protein produced by platelets during coagulation can lyse many Gram positive bacteria by acting as a cationic detergent.
3. Lactoferrin and transferrin – By binding iron, an essential nutrient for bacteria, these proteins limit bacterial growth.
4. Interferons – Interferons are proteins that can limit virus replication in cells.
5. Lysozyme – Lysozyme breaks down the cell wall of bacteria.
6. Interleukin -1 – Il-1 induces fever and the production of acute phase proteins, some of which are antimicrobial because they can opsonize bacteria.
C. Cellular barriers to infection
Part of the inflammatory response is the recruitment of polymorphonuclear eosinophiles and macrophages to sites of infection. These cells are the main line of defense in the non-specific immune system.
1. Neutrophils – Polymorphonuclear cells are recruited to the site of infection where they phagocytose invading organisms and kill them intracellularly. In addition, PMNs contribute to collateral tissue damage that occurs during inflammation.
2. Macrophages – Tissue macrophages and newly recruited monocytes , which differentiate into macrophages, also function in phagocytosis and intracellular killing of microorganisms. In addition, macrophages are capable of extracellular killing of infected or altered self target cells. Furthermore, macrophages contribute to tissue repair and act as antigen-presenting cells, which are required for the induction of specific immune responses.
3. Natural killer (NK) and lymphokine activated killer (LAK) cells – NK and LAK cells can nonspecifically kill virus infected and tumor cells. These cells are not part of the inflammatory response but they are important in nonspecific immunity to viral infections and tumor surveillance.
4. Eosinophils – Eosinophils have proteins in granules that are effective in killing certain parasites.
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.
The cell cycle
1) Labile cells (GI tract, blood cells)
- Described as parenchymal cells that are normally found in the G0 phase that can be stimulated to enter the G1
- Undergo continuous replication, and the interval between two consecutive mitoses is designated as the cell cycle
- After division, the cells enter a gap phase (G1), in which they pursue their own specialized activities
• If they continue in the cycle, after passing the restriction point (R), they are committed to a new round of division
• The G1 phase is followed by a period of nuclear DNA synthesis (S) in which all chromosomes are replicated
• The S phase is followed by a short gap phase (G2) and then by mitosis
• After each cycle, one daughter cell will become committed to differentiation, and the other will continue cycling
2) Stable cells (Hepatocytes, Kidney)
- After mitosis, the cells take up their specialized functions (G0).
- They do not re-enter the cycle unless stimulated by the loss of other cells
3) Permanent cells (neurons)
- Become terminally differentiated after mitosis and cannot re-enter the cell cycle
- Which cells do not have the ability to differentiate -> Cardiac myocytes