NEET MDS Lessons
General Microbiology
PHAGOCYTOSIS AND INTRACELLULAR KILLING
A. Phagocytic cells
1. Neutrophiles/Polymorphonuclear cells
PMNs are motile phagocytic cells that have lobed nuclei. They can be identified by their characteristic nucleus or by an antigen present on the cell surface called CD66. They contain two kinds of granules the contents of which are involved in the antimicrobial properties of these cells.
The second type of granule found in more mature PMNs is the secondary or specific granule. These contain lysozyme, NADPH oxidase components, which are involved in the generation of toxic oxygen products, and characteristically lactoferrin, an iron chelating protein and B12-binding protein.
2. Monocytes/Macrophages
Macrophages are phagocytic cells . They can be identified morphologically or by the presence of the CD14 cell surface marker.
B. Response of phagocytes to infection
Circulating PMNs and monocytes respond to danger (SOS) signals generated at the site of an infection. SOS signals include N-formyl-methionine containing peptides released by bacteria, clotting system peptides, complement products and cytokines released from tissue macrophages that have encountered bacteria in tissue.
Some of the SOS signals stimulate endothelial cells near the site of the infection to express cell adhesion molecules such as ICAM-1 and selectins which bind to components on the surface of phagocytic cells and cause the phagocytes to adhere to the endothelium.
Vasodilators produced at the site of infection cause the junctions between endothelial cells to loosen and the phagocytes then cross the endothelial barrier by “squeezing” between the endothelial cells in a process called diapedesis.
Once in the tissue spaces some of the SOS signals attract phagocytes to the infection site by chemotaxis (movement toward an increasing chemical gradient). The SOS signals also activate the phagocytes, which results in increased phagocytosis and intracellular killing of the invading organisms.
C. Initiation of Phagocytosis
Phagocytic cells have a variety of receptors on their cell membranes through which infectious agents bind to the cells. These include:
1. Fc receptors – Bacteria with IgG antibody on their surface have the Fc region exposed and this part of the Ig molecule can bind to the receptor on phagocytes. Binding to the Fc receptor requires prior interaction of the antibody with an antigen. Binding of IgG-coated bacteria to Fc receptors results in enhanced phagocytosis and activation of the metabolic activity of phagocytes (respiratory burst).
2. Complement receptors – Phagocytic cells have a receptor for the 3rd component of complement, C3b. Binding of C3b-coated bacteria to this receptor also results in enhanced phagocytosis and stimulation of the respiratory burst.
3. Scavenger receptors – Scavenger receptors bind a wide variety of polyanions on bacterial surfaces resulting in phagocytosis of bacteria.
4. Toll-like receptors – Phagocytes have a variety of Toll-like receptors (Pattern Recognition Receptors or PRRs) which recognize broad molecular patterns called PAMPs (pathogen associated molecular patterns) on infectious agents. Binding of infectious agents via Toll-like receptors results in phagocytosis and the release of inflammatory cytokines (IL-1, TNF-alpha and IL-6) by the phagocytes.
D. Phagocytosis
The pseudopods eventually surround the bacterium and engulf it, and the bacterium is enclosed in a phagosome. During phagocytosis the granules or lysosomes of the phagocyte fuse with the phagosome and empty their contents. The result is a bacterium engulfed in a phagolysosome which contains the contents of the granules or lysosomes.
E. Respiratory burst and intracellular killing
During phagocytosis there is an increase in glucose and oxygen consumption which is referred to as the respiratory burst. The consequence of the respiratory burst is that a number of oxygen-containing compounds are produced which kill the bacteria being phagocytosed. This is referred to as oxygen-dependent intracellular killing. In addition, bacteria can be killed by pre-formed substances released from granules or lysosomes when they fuse with the phagosome. This is referred to as oxygen-independent intracellular killing.
1. Oxygen-dependent myeloperoxidase-independent intracellular killing
During phagocytosis glucose is metabolized via the pentose monophosphate shunt and NADPH is formed. Cytochrome B which was part of the specific granule combines with the plasma membrane NADPH oxidase and activates it. The activated NADPH oxidase uses oxygen to oxidize the NADPH. The result is the production of superoxide anion. Some of the superoxide anion is converted to H2O2 and singlet oxygen by superoxide dismutase. In addition, superoxide anion can react with H2O2 resulting in the formation of hydroxyl radical and more singlet oxygen. The result of all of these reactions is the production of the toxic oxygen compounds superoxide anion (O2-), H2O2, singlet oxygen (1O2) and hydroxyl radical (OH•).
2. Oxygen-dependent myeloperoxidase-dependent intracellular killing
As the azurophilic granules fuse with the phagosome, myeloperoxidase is released into the phagolysosome. Myeloperoxidase utilizes H2O2 and halide ions (usually Cl-) to produce hypochlorite, a highly toxic substance. Some of the hypochlorite can spontaneously break down to yield singlet oxygen. The result of these reactions is the production of toxic hypochlorite (OCl-) and singlet oxygen (1O2).
3. Detoxification reactions
PMNs and macrophages have means to protect themselves from the toxic oxygen intermediates. These reactions involve the dismutation of superoxide anion to hydrogen peroxide by superoxide dismutase and the conversion of hydrogen peroxide to water by catalase.
4. Oxygen-independent intracellular killing
In addition to the oxygen-dependent mechanisms of killing there are also oxygen–independent killing mechanisms in phagocytes: cationic proteins (cathepsin) released into the phagolysosome can damage bacterial membranes; lysozyme breaks down bacterial cell walls; lactoferrin chelates iron, which deprives bacteria of this required nutrient; hydrolytic enzymes break down bacterial proteins. Thus, even patients who have defects in the oxygen-dependent killing pathways are able to kill bacteria. However, since the oxygen-dependent mechanisms are much more efficient in killing, patients with defects in these pathways are more susceptible and get more serious infections.
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
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
Cell Functions:
-> Autolysis
- degradative reactions in cells caused by indigenous intracellular enzymes – usually occurs after cell death
- Irreversible (along with Coagulative necrosis or infarcts) – reversible: fatty degeneration, & hydropic degeneration
-> Autolysin:
• Ab causing cellular lysis in the presence of complement
• Autolytic enzymes produced by the organism degrade the cell’s own cell wall structures
-> In the presence of cephalosporins & penicillins, growing bacterial cells lyse
• W/o functional cell wall structures, the bacterial cell bursts
-> Heterolysis: cellular degradation by enzymes derived from sources extrinsic to the cell (e.g., bacteria)
-> Necrosis: sum of intracellular degradative reactions occurring after individual cell death w/in a living organism
Complement Fixation Test (CFT)
This test is based upon two properties of the complement viz:
a. Complent combines with all antigen-antibody complexes whether or not it is required for that reaction
b. Complement is needed in immunolytic reaction.
Test system
It contains an antigen and a serum suspected to be having antibody to that antigen. The serum is heat treated prior to the test to destroy its complement. Complement Is added in measured quantity to this system. This complement is the form of guinea pig serum which is considered a rich source of complement. The test system is incubated.
Indicator system
To test system, after incubation, is added the indicator system which consists of sheep
RBCs and antibody to sheep RBCs (haemolysin) and another incubation is allowed.
If there is specific antibody in the test system, it will bind to antigen and to this complex the complement will also get fixed. Hence, no complement will be available to combine with indicator system which though contains RBCs and their specific antibody, cannot undergo haemolysis unless complement gets attached. Absence of haemolysis shall indicated positive test or presence of specific antibody in the serum which has been added in the test system. Erythrocytes lysis is obtained in negative test.
ANTIGEN-ANTIBODY REACTIONS
Affinity of the antigen-antibody reaction refers to the intensity of the attraction between antigen and antibody molecule.
Antigen-antibody reactions
Reaction test Modified test
Precipitation -> Immunoelectrophoresis, Immunoprecipitation
Agglutination -> Latex agglutination, Indirect, Haemagglutination , Coagglutination ,Coombs test
Neutralization -> Measurement of LD, Plaque assays
Complement fixation -> Conglutination
Immunofluorescence -> Indirect immunofiuorescence, Immunoofluoremetric Assay
Enzyme immunoassay -> Enzyme linked, Immunosorbent assay
Radioimmunoassay -> Immunoradiometric assay
Avidity is the strength of the bond after the formation of antigen-antibody complex.
Sensitivity refers to the ability of the test to detect even very minute quantities of antigen or antibody. A test shall be called as highly sensitive if false negative results are absent or minimal.
Specificity refers to the ability of the test to detect reactions between homologous antigens and antibodies only, and with no other. In a highly specific test, false positive reactions will be minimal or absent.
CELLS ORGANELLES
Cell parts:
Mitochondrion – double MB structure responsible for cellular metabolism – powerhouse of the cell
Nucleus – controls synthetic activities and stores genetic information
Ribosome – site of mRNA attachment and amino acid assembly, protein synthesis
Endoplasmic reticulum – functions in intracellular transportation
Gogli apparatus/complex – composed of membranous sacs – involved in production of large CHO molecules & lysosomes
Lysosome – organelle contains hydrolytic enzymes necessary for intracellular digestion
Membrane bag containing digestive enzymes
Cellular food digestion – lysosome MB fuses w/ MB of food vacuole & squirts the enzymes inside. Digested food diffuses through the vacuole MB to enter the cell to be used for energy or growth. Lysosome MB keeps the cell iself from being digested
-Involved mostly in cells that like to phagocytose
-Involved in autolytic and digestive processes
-Formed when the Golgi complex packages up an especially large vesicle of digestive enzyme proteins
Phagosome
– vesicle that forms around a particle (bacterial or other) w/in the phagocyte that engulfed it
- Then separates from the cell membrane bag & fuses w/ lysozome to receive contents
- This coupling forms phagolysosomes in which digestion of the engulfed particle occurs
Microbodies:
- Contain catalase
- Bounded by a single membrane bag
- Compartments specialized for specific metabolic pathways
- Similar in function to lysosomes, but are smaller & isolate metabolic reactions involving H2O2
- Two general families:
· Peroxisomes: transfer H2 to O2, producing H2O2 – generally not found in plants
· Glyoxysomes: common in fat-storing tissues of the germinating seeds of plants
¨ Contain enzymes that convert fats to sugar to make the energy stored in the oils of the seed available
Inclusions
– transitory, non-living metabolic byproducts found in the cytoplasm of the cell
- May appear as fat droplets, CHO accumulations, or engulfed foreign matter.