BIOLOGY UNIT 4 – THE NATURAL ENVIRONMENT AND SPECIES SURVIVALTopic 6 – Infection, immunity and forensics

Investigating time of death

• Temperature of a body can give some indication of how long a person has been dead.

• The presence of absence of rigor mortis also can be used to estimate time of death.

• The stage of succession of organisms on a corpse combined with information on the species life cycles in similar conditions and temperatures can help to estimate time of death (known as forensic entomology).

Transcription

• The process which forms mRNA.• Double helix is unzipped as hydrogen bonds between bases

are broken by RNA polymerase. • The prime DNA strand (template strand) is used to order the

sequence of nucleotides in an RNA transcript. RNA polymerase joins many small nucleotide units together to form mRNA.

• Every triplet code of DNA gives rise to a complementary codon on the mRNA. Every thymine is replaced with uracil.

• mRNA passes through the pores in the nuclear membrane where they then move to the surface of the ribosomes.

Translation

• tRNA is found in the cytoplasm, and consists of a unit of three bases known as the anticodon and an amino acid.

• Ribosomal RNA holds together the mRNA, tRNA and the enzymes controlling protein synthesis.

• Each mRNA strand has a start codon (AUG) and a stop codon (UAA, UAC or UGA). The mRNA becomes attached to a ribosome and the ribosome starts reading it and coding for the complementary amino acid.

• Then, tRNA lines up its anticodon alongside the complementary codon in the mRNA. Hydrogen bonds form between bases, peptide bonds join between the amino acids. When the end is reached, a completed polypeptide chain is formed. (Then primary, secondary, tertiary structure…)

Post-transcriptional changes• In recent years, it appears to have been shown that the ‘one

gene, one polypeptide’ theory is over simplistic. • The RNA transcribed from DNA is now called ‘pre-mRNA’ and

contains some nonsense sections that do not code for any protein, known as introns.

• The areas of the RNA that do code for the polypeptide chains are known as exons.

• Before the mRNA lines up on ribosomes after transcription, the ends are capped so that it is not attacked by enzymes, the introns are removed, and the exons are joined together in a process called RNA splicing. This is carried out by enzymes known as spliceosomes.

• These changes lead to more variety in phenotype, as the areas that are removed can be varied.

DNA profiling • Individuals and species can be identified through patterns in

their DNA. Introns are the regions used in DNA profiling. • There are short sequences of DNA within introns that are

repeated many times. Mini-satellites have 20-50 bases repeated from 50 to several hundred times. Micro-satellites have 2-4 bases repeated 5-15 times.

• DNA strands are chopped into fragments using restriction enzymes (e.g. restriction endonucleases), which cut the DNA molecule at specific base sequences known as recognition sites.

• The fragments are separated using gel electrophoresis. DNA fragments are placed in wells in agarose gel medium in a buffering solution to maintain a constant pH, along with known DNA fragments.

DNA profiling • Gel contains a dye that binds to the DNA fragments. The dye

will fluoresce when placed under UV light, so the known DNA can be compared with the unknown DNA to identify it following completion.

• An electric current is passed through the apparatus and the DNA fragments move at different rates towards the positive anode (due to negatively charged phosphate groups).

• The next stage is Southern blotting. An alkaline buffer solution is added to the gel and a nylon filter placed over it. Dry absorbent paper is used to draw the solution containing the DNA to the filter, which then ‘blots’ onto it. The alkaline solution denatures the DNA fragments so the base sequences are exposed.

Polymerase chain reaction (PCR)

• PCR is used to amplify the DNA to allow for DNA profiling to take place by adapting the natural process in which DNA is replicated in the cell.

• The DNA sample to be amplified, along with DNA polymerase, primers and a good supply of the four nucleotide bases are mixed together in a PCR vial and placed in a PCR machine where they are heated to 90-95°C. This causes the DNA strands to separate as the hydrogen bonds break, and when the mixture is then cooled to 55-60°C the primers can bind to the single DNA strands.

• It is then reheated to 75°C where polymerase works to build the complementary strands of DNA. This is repeated around 30 times to give around 1 billion copies of the original DNA.

Viruses• Smallest of all the microorganisms• They are arrangements of genetic material and protein that

invade other living cells and take over their biochemistry to reproduce

• They contain DNA or RNA with either double or single stranded nucleic acid

• Viral DNA acts directly as a template for both new viral DNA and for the mRNAs needed to synthesise viral protein

• Viral RNA uses the enzyme reverse transcriptase to produce DNA molecules corresponding to the viral genome, which acts as a template for new viral proteins

• They have protein coats made up of capsomeres (protein units)• They may or may not have an envelope

Viruses

There is considerable variation in the structure of viruses, but they all contain a few key features.

Virus life cycles1. Bacteriophage attacks bacterium and attaches to it2. Viral (phage) DNA is injected into host cell, bringing about synthesis of

viral enzymes3a. Viral DNA incorporated into host cell DNA. Viral DNA replicatedeach time the bacterium divides. (lysogenic pathway)OR 3b. Phage DNA inactivates the host DNA and takes over cell chemistry. (lytic pathway).4. Phage DNA is replicated. New phage particles are assembled and protein coats form around the viral DNA. Lysozyme is synthesised or released. 5. Lysis – bacterial cell burst due to action of lysozyme, releasing up to 1000 new phages to infect more bacteria.[Some types of virus have both the lysogenic and lytic pathway in their life cycle, but others move straight to the lytic stage after infecting a cell. During the period of lysogeny, the virus is said to be dormant. During the lytic pathway, the virus is virulent (disease-causing)].

Virus life cycles

Retrovirus life cycle• Retroviruses (e.g. HIV) contain viral RNA, and so cannot be

used as mRNA but must be translated into DNA by the enzyme reverse transcriptase in the cell cytoplasm.

• This viral DNA then passes into the nucleus of the host cell where it is inserted into the host DNA. This is then transcribed by transcriptase enzymes to make viral mRNA and new viral genome RNA.

• New viral material is synthesized, and leaves the cell via exocytosis, taking some of the host cell membrane and eventually killing the cell through repeatedly puncturing the cell as more viruses are replicated.

Bacteria structure*= not present in all bacteria

Bacteria structure

• Capsule – protects the bacterium from phagocytosis by white blood cells. Also covers the cell markers on the cell membrane which identify the cell, making it easier for bacterium to be pathogenic.

• Flagella – helps to move the bacterium by rapid rotations.• Some plasmids also code for particular aspects such as the

production of a toxin or resistance to an antibiotic.• Bacterial cells walls have a peptidoglycan layer, made up of many

parallel polysaccharide chains with peptide cross-linkages.• Gram staining can be used to identify Gram-positive bacteria (thick

peptidoglycan layer, turns teichoic acid purple/blue) and Gram-negative bacteria (thinner peptidoglycan layer, has no teichoic acid so turns red).

How bacteria reproduce

• Most commonly, bacteria reproduce through asexual reproduction by splitting in two (binary fission). Once a bacterium reaches a certain size, the genetic material replicates into two identical sections and the cell splits.

• However, there are rare forms of other reproduction that sometimes take place:

• Transformation – a short piece of DNA is released by a doner and actively taken up by a recipient cell.

• Transduction – a small amount of DNA is transferred from one bacterium to another by a bacteriophage.

• Conjugation – genetic information is transferred by direct contact.

How are pathogens transmitted?

• Vectors – A living organisms transmits infection from one host to another. Many insects are vectors. (e.g. malaria)

• Fomites – inanimate objects that carry pathogens, such as hospital towels and bedding. (e.g. Staphylococcus)

• Direct contact – direct contact spreads many skin diseases. (e.g. gonorrhoea)

• Inhalation – coughing and sneezing release droplets which can spread infections if inhaled. (e.g. influenza)

• Ingestion – passed through contaminated food or drink. (e.g. salmonella)

• Inoculation – directly through break in the skin. (e.g. HIV)

Barriers to entry

• Skin – toughened by keratin to prevent penetration. Produces sebum that inhibits the growth of microorganisms.

• Mucus (e.g. in respiratory system) – traps microorganisms. Contains lysozymes, enzymes which rupture bacteria by disrupting their cell walls.

• Lysozymes are also present tears, milk and saliva. • Blood clotting – prevents entry of further pathogens when

skin breaks.• Saliva and earwax – bactericidal properties, kills bacteria.• Hydrochloric acid in stomach – destroys ingested

microorganisms.

Non-specific immune response

• The non-specific response simply recognises the difference between self and non-self and reacts against anything foreign.

• Inflammation – mast cells and damaged white blood cells release chemicals called histamines, triggering vasodilation causing local heat and redness. The raised temperature reduces pathogen effectiveness. The histamines also make the capillary walls leaky, so plasma, white blood cells and antibodies are forced out of the capillaries causing swelling (oedema), and these can then destroy the pathogen.

• Fever – the hypothalamus resets to a higher body temperature, which reduces the pathogen’s ability to reproduce and allows the specific immune response to work more effectively.

Non-specific: Phagocytosis

• Phagocyte describes white blood cells which engulf and digest pathogens. There are two main types: neutrophils and macrophages. They accumulate at site of infection to attack.

• A phagocyte recognises the antigens on a pathogen.• The cytoplasm of the phagocyte moves round the pathogen,

engulfing it.• The pathogen is now contained in a phagocytic vacuole in the

cytoplasm of the phagocyte.• A lysosome fuses with the phagocytic vacuole. The lysosomal

enzymes break down the pathogen• The phagocyte then presents the pathogens antigens. It sticks

the antigens on its surface to activate other immune system cells

Specific immune response

• There are two main types of lymphocytes involved in the immune system; B cells, made in the bone marrow and mature in the lymph glands, and T cells, made in the bone marrow but mature in the thymus gland.

• T cells are mainly T killer and T helper cells. T killer cells produce chemicals that destroy pathogens. T helper cells are involved in producing antibodies.

• The process of these cells working is reliant on special proteins known as MHC proteins, which display antigens on the cell surface membrane.

Specific: The humoral response

• Consists of the T helper activation stage and the effector stage.

• In the T helper activation stage, following phagocytosis, the macrophage presents the pathogenic antigen as an MHC protein complex on the cell surface and becomes known as an antigen-presenting cell (APC).

• CD4 receptors on the membrane of T helper cells enable it to bind to the specific antigen on the MHC complex. This triggers T helper cells to produce clones; most become active T helper cells, but some become T memory cells which remain in the body and rapidly become active if the same antigen is encountered a second time.

Specific: The humoral response

• During the effector stage, B cells and T cells are active. • B cells engulf pathogens and can also present the antigens on

MHS complexes, becoming another type of APC.• A T helper cell from the active clone recognizes the specific

antigen on the B cell and binds to it, triggering the release of cytokines from the T helper cell which stimulate the B cell to form clones. These can be B effector cells (which then differentiate into plasma cell clones to produce antibodies) or B memory cells.

• Antibodies work in a variety of ways. They can reduce the ability of pathogens to invade host cells, help prevent them spreading, and the antigen-antibody complexes may stimulate other reactions to destroy the antigen.

Specific: The cell-mediated response

• When the pathogen is inside a host cell (e.g. virus), the humoral response is not very effective, so the cell-mediated response is used.

• The pathogen is digested and antigens presented on MHC complexes on an APC.

• T killer cells bind to the antigen/MHC complex. If exposed to cytokines from an active T helper cell, this then triggers the production of clone T killer cells and T killer memory cells.

• T killer cells release enzymes which perforate the membrane of infected cells, making them swell with water and burst (induced apoptosis). Any pathogens released are labelled with antibodies and destroyed.

Antibiotics

• Work by the principle of selective toxicity – they interfere with the metabolism or function of the pathogen with little damage to the human host.

Antimicrobial action Example antibioticAntimetabolites – interrupt metabolic pathways, e.g. blocking nucleic acid synthesis

Sulphonamides

Prevent formation of cross-liking in cell walls so bacteria burst

Beta-lactams, e.g. penicillin

Damage the cell membrane so metabolites leak out/water leaks in

Some penicillins

Protein synthesis inhibitors prevent successful transcription/translation so protein is affected

Tetracyclines

DNA gyrase inhibitors stop bacterial DNA coiling so it doesn’t fit in the bacterium

Quinolone

Antibiotics

• Bacteriostatic – inhibits the growth of the microorganism.• Bacteriocidal – will destroy almost all of the pathogen

present. • Broad spectrum antibiotics target a wide range of harmful

bacteria, pathogens, and neutral and good bacteria.• Narrow spectrum antibiotics target one or two specific

pathogens.• Effectiveness depends on – concentration of drug in affected

area of body, local pH, susceptibility of pathogen, dosage etc.

Infection prevention and control

There are several ways to prevent infection, particularly in hospital settings where healthcare-acquired infections are problematic:• Controlling the use of antibiotics (prevents resistant bacteria)• Hygiene measures (prevents spreading through contact)• Isolation of patients (when infected, to prevent spread of

infection)• Prevention of infection coming into the hospital• Monitoring levels of healthcare-acquired infections

Different types of immunity

• Natural active immunity – e.g. coming into contact with foreign antigen. Your own body produces the antibodies.

• Natural passive immunity – e.g. breastfeeding. Antibodies received through mother’s milk.

• Artificial passive immunity – e.g. injection containing antibodies to prevent development of a disease, but does not give prolonged immunity.

• Artificial active immunity – e.g. immunisation/vaccination. Own body produces antibodies, but source is artificial.

Tuberculosis

• Spread by droplet infection, drinking infected milk or working in close contact with cattle

• Active TB symptoms – fever, night sweats, loss of appetite, loss of weight, tiredness, coughing (blood).

• If immune system is healthy upon infection, a mass of tissue called a tubercule will form containing the bacteria. After about 8 weeks the immune system controls the bacteria and inflammation dies down.

• Some tuberculosis can avoid the immune system, so may survive. The bacteria produce waxy layers which protect from enzymes, and can lie dormant for years until the person is weakened, and may then produce active tuberculosis.

HIV/AIDS

• HIV attaches to the CD4 receptors on T helper cells and infects them. It is a retrovirus, so takes over the host DNA and replicates (see previous slide on retrovirus replication). When it leaves the host T helper cell, it is destroyed.

• At the same time, the host T killer cells recognise and destroy some of the heavily infected T helper cells. The result is a great reduction in the number of T helper cells, which means that the activation of many macrophages and T killer cells does not take place, so immunity is lowered and the individual is vulnerable to secondary infections. (progresses to AIDS)

HIV/AIDS