NEET MDS Lessons
General Microbiology
COMPLEMENT
The complement system primarily serves to fight bacterial infections.
The complement system can be activated by at least three separate pathways.
1) alternative pathway -
- The alternative pathway of complement activation starts with the spontaneous hydroysis of an internal thioester bond in the plasma complement component C3 to result in C3(H2O).
- The smaller cleavage products C3a, C4a, C5a, sometimes called "anaphylatoxins", act as phagocytes, they cause mast cell degranulation and enhance vessel permeability, thereby facilitating access of plasma proteins and leukocytes to the site of infection
- alternative pathway provides a means of non-specific resistance against infection without the participation of antibodies and hence provides a first line of defense against a number of infectious agents.
2) Lecithin Pathway
The lectin pathway of complement activation exploits the fact that many bacterial surfaces contain mannose sugar molecules in a characteristic spacing. The oligomeric plasma protein mannan-binding lectin (MBL; lectins are proteins binding sugars) binds to such a pattern of mannose moieties, activating proteases MASP-1 and MASP-2 (MASP=MBL activated serine protease, similar in structure to C1r and C1s). These, by cleaving C4 and C2, generate a second type of C3 convertase consisting of C4b and C2b, with ensuing events identical to those of the alternative pathway.
3) classical pathway
The classical pathway usually starts with antigen-bound antibodies recruiting the C1q component, followed by binding and sequential activation of C1r and C1s serine proteases. C1s cleaves C4 and C2, with C4b and C2b forming the C3 convertase of the classical pathway. Yet, this pathway can also be activated in the absence of antibodies by the plasma protein CRP (C-reactive protein), which binds to bacterial surfaces and is able to activate C1q.
Pharmacology cross reference: humanized monoclonal antibody Eculizumab binds to complement component C5, inhibiting its cleavage and preventing activation of the lytic pathway. This is desirable when unwanted complement activation causes hemolysis, as in paroxysmal nocturnal hemoglobinuria or in some forms of hemolytic uremic syndrome. For the lytic pathway's importance in fighting meningococcal infections, Eculizumab treatment increases the risk of these infections, which may be prevented by previous vaccination.
BIOLOGICALLY ACTIVE PRODUCTS OF COMPLEMENT ACTIVATION
Activation of complement results in the production of several biologically active molecules which contribute to resistance, anaphylaxis and inflammation.
Kinin production
C2b generated during the classical pathway of C activation is a prokinin which becomes biologically active following enzymatic alteration by plasmin. Excess C2b production is prevented by limiting C2 activation by C1 inhibitor (C1-INH) also known as serpin which displaces C1rs from the C1qrs complex (Figure 10). A genetic deficiency of C1-INH results in an overproduction of C2b and is the cause of hereditary angioneurotic edema. This condition can be treated with Danazol which promotes C1-INH production or with ε-amino caproic acid which decreases plasmin activity.
Anaphylotoxins
C4a, C3a and C5a (in increasing order of activity) are all anaphylotoxins which cause basophil/mast cell degranulation and smooth muscle contraction. Undesirable effects of these peptides are controlled by carboxypeptidase B (C3a-INA).
Chemotactic Factors
C5a and MAC (C5b67) are both chemotactic. C5a is also a potent activator of neutrophils, basophils and macrophages and causes induction of adhesion molecules on vascular endothelial cells.
Opsonins
C3b and C4b in the surface of microorganisms attach to C-receptor (CR1) on phagocytic cells and promote phagocytosis.
Other Biologically active products of C activation
Degradation products of C3 (iC3b, C3d and C3e) also bind to different cells by distinct receptors and modulate their functions.
Application of agglutination reactions
Agglutination reaction Example
Tube agglutination -> Widal test, Weil Felix reaction, Standard tube test for brucellosis
Slide agglutination -> Typing of pneumococci,Diagnosis of Salmonella,Diagnosis of Shigella
Agglutination Absorption test -> Salmonella diagnosis
Coagglutination -> Grouping of streptococci, Identification of gonococci, Detection of Haemophilus, Antigen in CSF
Passive agglutination
Latex agglutination Detection of HBs Ag, ASO, CRP
Precipitation Reaction
This reaction takes place only when antigen is in soluble form. Such an antigen when
comes in contact with specific antibody in a suitable medium results into formation of an insoluble complex which precipitates. This precipitate usually settles down at the bottom of the tube. If it fails to sediment and remains suspended as floccules the reaction is known as flocculation. Precipitation also requires optimal concentration of NaCl, suitable temperature and appropriate pH.
Zone Phenomenon
Precipitation occurs most rapidly and abundantly when antigen and antibody are in optimal proportions or equivalent ratio. This is also known as zone of equivalence. When antibody is in great excess, lot of antibody remains uncombined. This is called zone of antibody excess or prozone. Similarly a zone of antigen excess occurs in which all antibody has combined with antigen and additional uncombined antigen is present.
Applications of Precipitation Reactions
Both qualitative determination as well as quantitative estimation of antigen and antibody can be performed with precipitation tests. Detection of antigens has been found to be more sensitive.
Agglutination
In agglutination reaction the antigen is a part of the surface of some particulate material such as erythrocyte, bacterium or an inorganic particle e.g. polystyrene latex which has been coated with antigen. Antibody added to a suspension of such particles combines with the surface antigen and links them together to form clearly visible aggregate which is called as agglutination.
Application of precipitation reactions
Precipitation reaction Example
Ring test Typing of streptococci, Typing of pneumococci
Slide test (flocculation) VDRL test
Tube test (flocculation) Kahn test
Immunodiffusion Eleks test
Immunoelectrophoresis Detection Of HBsAg, Cryptococcal antigen in CSF
Types of microscopy used in bacteriology
Light microscopy
Phase contrast microscopy
Fluorescence microscopy
Darkfield microscopy
Transmission electron microscopy
Scanning electron microscopy
Fluorescent microscopy in which ultraviolet rays are used to examine cells after treatment with fluorescent days.
Phase contrast microscope enhances the refractive index differences of the cell components. This microscopy can be used to reveal details of the internal structures as well as capsules, endospores and motility
Electron microscope The resolving power is more than 200 times that of light microscope.
Measurement of Bacterial of Growth
A convenient method is to determine turbidity by photoelectric colorimeter or spectrophotometer.
The cell number can be counted as total cell number as well as viable count. Viable Count Viable number of bacteria can be counted by inoculating the suspension onto solid growth medium and counting the number of colonies. Since each colony is the end product of one viable bacterium, their count gives the number of viable bacteria in the suspension.
Total number of bacteria can be ascertained in specially designed chambers such as Coulter counter.
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.
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.