Microbiology and Immunology — Bacteria, Operons, Pathogens, Innate and Adaptive Immunity

• Leeuwenhoek first described microbes with single-lens microscopes in the 1670s. Pasteur disproved spontaneous generation with swan-neck flasks and showed fermentation is microbial; Koch tied specific microbes to specific diseases (anthrax, tuberculosis) and set out his postulates; Fleming found penicillin in 1928. Woese, comparing 16S rRNA sequences, split prokaryotes into Bacteria and Archaea, giving the three-domain tree with Eukarya.
• Gram-positive walls are a thick peptidoglycan layer threaded with teichoic acids, which retains the crystal violet–iodine complex through alcohol decolourisation and stains purple. Gram-negative walls have a thin peptidoglycan layer in a periplasm beneath an outer membrane whose outer leaflet is lipopolysaccharide (lipid A is the endotoxin); alcohol extracts the dye and the safranin counterstain makes them pink. Mycobacteria are acid-fast because of mycolic acids.
• Archaea have ether-linked isoprenoid membrane lipids (bacteria and eukaryotes use ester-linked fatty acids), lack peptidoglycan (some have pseudomurein, so lysozyme does not lyse them), and have transcription and translation machinery closer to eukaryotes.
• Methods in microbiology: sterilisation (autoclaving at 121 °C), aseptic technique, pure culture by streak, spread and pour plates, staining and microscopy, enumeration by serial dilution and colony counting (CFU) or by the most-probable-number method, and identification by biochemical tests and 16S rRNA sequencing.

Microbes are classed by energy and carbon source — photo- or chemotrophs, auto- or heterotrophs — so a chemolithoautotroph oxidises inorganic compounds for energy and fixes CO₂. A batch culture passes through lag, exponential (log), stationary and death phases. In the exponential phase N = N₀·2ⁿ, so the number of generations is n = log₂(N/N₀) = 3.32 log₁₀(N/N₀) and the generation time is g = t/n; the specific growth rate is μ = ln 2/g. Worked: 2 × 10³ cells become 1.28 × 10⁵ in 3 h; N/N₀ = 64 = 2⁶, so n = 6 and g = 180/6 = 30 min.

Nitrogen fixation is catalysed by nitrogenase — the Fe protein (dinitrogenase reductase) passing electrons to the MoFe protein (dinitrogenase): N₂ + 8H⁺ + 8e⁻ + 16 ATP → 2NH₃ + H₂ + 16 ADP + 16 Pᵢ. Nitrogenase is irreversibly damaged by O₂, so fixers protect it: leghaemoglobin buffers O₂ in legume root nodules colonised by Rhizobium, cyanobacteria such as Anabaena fix in thick-walled heterocysts that lack PSII, and free-living Azotobacter respires fast enough to keep its interior anaerobic.

A pathogen must adhere (pili, adhesins), invade or colonise, evade immunity (capsules, antigenic variation, IgA proteases) and damage the host. Exotoxins are secreted proteins, often A–B toxins in which B binds the cell and A is the enzyme — cholera toxin ADP-ribosylates Gαs, diphtheria toxin ADP-ribosylates elongation factor 2. Endotoxin is the lipid A of Gram-negative LPS, released as bacteria lyse, and it causes fever and septic shock through TLR4. Many Gram-negative pathogens inject effectors through a type III secretion system.

Resistance spreads fastest horizontally, on plasmids, transposons and integrons carried by conjugation, transformation and transduction. A two-component system is the bacterial sensing circuit: a membrane sensor histidine kinase autophosphorylates on a histidine when it detects its signal and transfers the phosphoryl group to an aspartate of a response regulator, usually a transcription factor (EnvZ–OmpR senses osmolarity, PhoR–PhoB phosphate). Quorum sensing couples gene expression to population density: Gram-negative bacteria secrete acyl-homoserine lactones (LuxI makes, LuxR senses in Vibrio fischeri bioluminescence), Gram-positive bacteria use oligopeptides read by two-component systems, and AI-2 serves between species. Viruses are nucleic acid in a protein capsid (helical, icosahedral or complex), sometimes enveloped; the Baltimore classification groups them by genome and mRNA route into seven classes — dsDNA, ssDNA, dsRNA, (+)ssRNA, (−)ssRNA, ssRNA with reverse transcription (retroviruses) and dsDNA with reverse transcription (hepadnaviruses). Phages such as λ choose between lytic growth and lysogeny.

Primary lymphoid organs are where lymphocytes are made and selected — bone marrow (B cells, and all haematopoiesis) and thymus (T cells). Secondary lymphoid organs are where they meet antigen — lymph nodes, spleen and mucosa-associated lymphoid tissue such as Peyer’s patches and tonsils. Innate immunity is immediate and germline-encoded: barriers, phagocytes (neutrophils, macrophages), NK cells, and pattern-recognition receptors such as the Toll-like receptors that read pathogen-associated molecular patterns (TLR4 LPS, TLR5 flagellin, TLR3 double-stranded RNA, TLR9 unmethylated CpG DNA). Inflammation — redness, heat, swelling and pain — is vasodilation and leakage that bring cells and plasma proteins to the site. Cytokines carry the signals: IL-1, TNF-α and IL-6 drive inflammation and fever, IL-2 drives T-cell proliferation, IFN-γ activates macrophages (the Th1 signature), IL-4 drives Th2 responses and the switch to IgE, IL-10 and TGF-β damp responses, and type I interferons are antiviral. Chemokines are chemotactic cytokines — CXCL8 (IL-8) recruits neutrophils.

An antibody is two identical heavy and two identical light chains; the Fab arms carry the antigen-binding site made of the hypervariable CDRs, and the Fc stem sets the effector function. IgG is the most abundant serum class and the only one to cross the placenta; IgM is a pentamer and the first class made in a primary response; IgA is a dimer in secretions; IgE arms mast cells and basophils in allergy and anti-helminth defence; IgD sits with IgM on naive B cells. Humoral immunity is antibody neutralising, opsonising and fixing complement; cellular immunity is cytotoxic CD8⁺ T cells killing infected cells and CD4⁺ helper T cells directing the response.

• Antibody diversity comes from V(D)J recombination — RAG1/RAG2 cut at recombination signal sequences (the 12/23 rule) and join one V, one D and one J segment for a heavy chain, one V and one J for a light chain — multiplied by the pairing of any heavy with any light chain, by junctional diversity (imprecise joining and N-nucleotides added by TdT), and after antigen by somatic hypermutation in germinal centres. AID drives both hypermutation and class switching, which changes the constant region and so the isotype while keeping the specificity.
• T cells develop in the thymus: double-negative thymocytes become CD4⁺CD8⁺ double positives, which are positively selected in the cortex for recognising self-MHC and negatively selected, mainly in the medulla, if they bind self-peptide–MHC too strongly (AIRE lets medullary cells display tissue-specific self-antigens). Survivors become single-positive CD4⁺ or CD8⁺. B cells develop in the bone marrow, and self-reactive ones are deleted, edited or made anergic.
• MHC class I is on all nucleated cells and presents endogenous peptides of 8–10 residues — made by the proteasome and pumped into the ER by TAP — to CD8⁺ T cells. MHC class II is on professional antigen-presenting cells (dendritic cells, macrophages, B cells) and presents exogenous peptides of roughly 13–25 residues from endosomes — the invariant chain holds the groove until its CLIP fragment is exchanged by HLA-DM — to CD4⁺ T cells. Dendritic cells can also cross-present exogenous antigen on class I.
• Memory: a secondary response is faster, larger, dominated by IgG and of higher affinity, because memory B and T cells persist after the primary response. Polyclonal antibodies (from an immunised animal) recognise many epitopes; monoclonal antibodies come from one B-cell clone, immortalised as a hybridoma, and recognise one epitope.

Tolerance is central (deletion of strongly self-reactive lymphocytes in thymus and marrow) and peripheral: a T cell that receives antigen (signal 1) without co-stimulation from B7–CD28 (signal 2) becomes anergic, and CD4⁺CD25⁺FoxP3⁺ regulatory T cells suppress responses through IL-10, TGF-β and CTLA-4. Autoimmunity is tolerance failing — myasthenia gravis (antibodies against the acetylcholine receptor), Graves’ disease (stimulating antibodies against the TSH receptor), systemic lupus erythematosus (immune complexes), type 1 diabetes and rheumatoid arthritis (T-cell driven). Immunodeficiency is primary — severe combined immunodeficiency from adenosine deaminase deficiency or the X-linked common γ-chain defect, X-linked agammaglobulinaemia from BTK loss, DiGeorge syndrome from thymic aplasia — or secondary, above all HIV destroying CD4⁺ T cells. Graft-versus-host disease follows bone-marrow or haematopoietic stem-cell transplantation when mature donor T cells attack the recipient’s tissues; it is the reverse of graft rejection.

Immunisation is active (the host makes its own response and memory) or passive (pre-formed antibody, immediate but short-lived, as with antivenom or maternal IgG). Vaccines are live attenuated (MMR, BCG, oral polio), inactivated (injected polio vaccine), subunit or recombinant (hepatitis B surface antigen made in yeast), toxoid (tetanus, diphtheria), conjugate (a polysaccharide coupled to a carrier protein so that T-cell help and memory are recruited, as for Haemophilus influenzae type b) and nucleic acid (mRNA). Adjuvants such as alum boost the innate signal that non-living vaccines lack.