Restriction-modification systems in bacteria Sites on bacterial chromosome

Restriction-modification systems in bacteria Sites on bacterial chromosome protected by proteins/methylation G A A T T C C T T A A G Eco. RI Sites on phage DNA/RNA unprotected and cut

Penicillin and other antibiotics • Discovered by Fleming in 1929 and turned into a useful treatment by Florey and Chain (1940 s) • Prevents the formation of the bacterial cell wall (by interfering with peptidoglycan cross-linking) – New cells subject to osmotic lysis – Only acts against Gram-positive bacteria • Other antibiotics – Streptomycin from bacterium Streptomyces acts on protein synthesis – Tetracycline, also from Streptomyces, also acts on protein synthesis on a wide range of bacteria

The immune system of Drosophila • Body fluid of insects contains haemolin – a member of the immunoglobulin superfamily, which binds to microbes • Also several short peptides that play an unknown role in antibacterial action – Defensins – like scorpion venom taxins – Cecropins and attacins (disrupt cell membrane) – Drosomycin (antifungal) – similar to plant substances – Rapid evolution of some these proteins indicates an ongoing evolutionary arms race • Cellular immunity – Haemocytes secrete antimicrobial peptides and are involved in coagulation, phagocytosis and encapsulation

Immune systems in vertebrates - overview • Mechanical barriers – Collagen/skin , mucous membranes, secretions • Non-specific defence (innate immunity) – Phagocytosis by neutrophils (70% WBC) – Macrophages (5% WBC) – The inflammatory response (histamine, chemokines, fever) – Antimicrobial proteins (complement, interferons) • Specific immune defence (adaptive immunity) – Humoral (antibodies, B cells) – Cell-mediated (Cytotoxic T cells, Helper T cells)

Antigen (1 st exposure) Engulfed by Free antigens directly activate Antigens displayed by infected cells activate Macrophage Stimulates B cell Gives rise to Stimulates Helper T cell Stimulates Memory helper T cell Stimulates Cytotoxic T cell Gives rise to Stimulates Antigen (2 nd exposure) Plasma cells Antibodies Memory B cells Stimulates Memory T cells Active cytotoxic T cells

B cells (made in bone marrow) • B cells secrete antibodies which recognise intact foreign particles outside cells and block their action/target them for destruction Infected cell B cell Antibody Each antigen can present many different antigenic determinants and be recognised by many different Antibodies Antigenic determinant Antigen

The structure of antibodies

The diversity of antibodies • Collectively referred to as immunoglobulins – Ig. M (pentamer) first to appear in response to infection – Ig. G (monomer) most abundant circulating form, confers maternal immunity – Ig. A (dimer) produced by mucous membranes – Ig. D (monomer) Differentiation of B cells – Ig. E (monomer) stimulate mast cells and histamine • Each has own heavy chain (a, d, e, g, m) • There are 2 types of light chain (k and l) • Both light and heavy chains are encoded by libraries of subunits which are shuffled to generate diversity

Light-chain diversity Variable (40 units) Joining (5 units) Constant Somatic recombination with imprecise joining TGGCTGGA x TGGCCAG TGGCAG TGGAG GCAGCC ‘Fine-tuning’ by somatic hypermutation (error-prone DNA repair process) Possible diversity – (prior to mutation) about 1012

Cell memory and clonal selection • Acquired immunity ensures that self-antigens are not attacked through – Clonal selection – Clonal deletion – Repeated editing – Absence of Helper T cells ensures that immune response is not mounted • Initially, B cells produce membrane bound antibodies • Cells stimulated by an antigenic response proliferate and differentiate into effector cells that secrete antibody • A fraction of the simulated cells differentiate instead into memory cells that are rapidly induced in subsequent antigenic challenges

T cells (made in thymus) • Only respond when foreign antigen displayed on surface of self-cells in periperal lymphoid organs • Recognise fragments of foreign particles displayed by MHC proteins • Cytotoxic T cells recognise and destroy infected cells • Helper T cells stimulate other cells – Macrophages – B cells – Cytotoxic T cells • T cell receptors are very like antibodies, but are membrane bound ONLY and are less variable

Cytotoxic killing • Cytotoxic cells release perforin (related to C 9) which makes the infected cell permeable to secreted serine proteases • Activate apoptosis (cell death) through capsase family

MHC proteins • Major histocompatibility complex (chromosome 6 in humans) • Class I MHC present to Cytotoxic T cells • Class II MHC present to Helper T cells • The most polymorphic loci in humans (some have >200 alleles) 6 p 21. 3 DP DQ DR 4 Mbp c. 127 genes C 4 C 2 TNFa, b HLA-B HLA-C HLA-A HLA-D 18 genes Class III Class I

Rapid evolution of MHC genes In both humans and house mice, the antigen-binding site (ABS) of class I and II MHC molecules (light blue) have a high rate of nonsynonymous versus synonymous nucleotide substitutions, which is the opposite pattern for genes under purifying selection, such as nonantigen-binding sites of MHC molecules (dark blue). Adapted from Potts WK and Wakeland EK (1990) Evolution of diversity at the major histocompatibility complex. Trends in Ecology and Evolution 5: 181– 187.

Helper T cell activation • Two signals are required – MHC bound peptide – Costimulatory signal • Different costimulatory receptors occur in different cell types – CD 3 complex on all T cells – CD 4 on helper T cells – CD 8 on cytotoxic T cells • Cytokines secreted by T cells (and B cells) provide communication between elements of the immune system inducing and suppressing reactions – Interleukins – Interferons – TNF-a

The immunoglobulin superfamily

How to evade the immune system • Once in, replicate really fast – Bacteria, viruses • Replicate within cells that are trying to destroy you – Mycobacterium tuberculosis replicates in macrophages • Trick normal cells into taking you in – Yersinia pseudotuberculosis expresses a protein that binds to E-cadherin and stimulates cells into forming a cell junction, through which the bacterium can enter • Present very diverse surface proteins which immune systems are unlikely to have experienced before – Trypanosomes somatic switching