Bacterial Genetics

1. Bacterial Genetics Medical Microbiology

2. You have known… • Science of genetics describes and analyze heredity of physiologic functions that form the properties of organism. • These properties are determined by the total of all the genetic information named genome. • Basic unit of genetics isgene, a segment of DNA that carries in its nucleotide sequence information for a specific biochemical or physiologic property. • A gene is relatively stable but occasionally may undergo a nucleotide change, such a change is called as mutation. • Mutations may occur spontaneously or can beinduced by a number of physical or chemical agents.

3. Bacteria may have changes including: • Morphological and/or structural changes • Variations of cultural characteristics and biochemical reactions • Changes in virulence • Variation in antigenicity • Changes in drug resistance These variations can bedivided into two types: • ▲Phenotypic variation:non-heritable • ▲Genotypic variation:heritable (mutation)

4. Bacterial Genome DNA/Genome: Genetic materials relative to bacterial mutation. A. chromosome B. out of chromosome:a) plasmid b) phage c) transposable genetic element

5. Bacterial chromosome consists of a single, circle of double-stranded DNA. ▲2 mm long in average ▲Usually < 5 Mb (1 Mb = 1024 Kb) 1. Chromosomal DNA

6. 2. Plasmid • Transfer property ▲Conjugative plasmid：mediate conjugation through sex pilus ▲Non-conjugative plasmid:can not mediate conjugation because of no gene for encoding sex pilus • Phenotypic effect ▲Fertility plasmid (F factor):has a sex pilus-encoding gene ▲Resistance plasmid (R factors):contains genes that encoding enzymes to destroy antibiotics

7. RTF Tn 10 Tn 8 Tn 9 Tn 21 R determinant Structure of R Factor • RTF (Resistance Transfer Factor) ▲ Conjugative plasmid ▲ Transfer genes • R determinant ▲ Resistance genes ▲ Transposons (Tn, 转座子)

8. 3. Phage • Phages are obligate intracellular parasites that specially propagate in bacteria (bacterial virus). • ▲Nucleic acid: DNA (dsDNA or ssDNA) or RNA (dsRNA or ss RNA). • ▲Protein:function in infection and protect nucleic acid.

9. Head Capsid DNA Tail Contractile Sheath Base Plate Tail Fibers • Structure of T4 phage ▲Head: DNA or RNA+protein coat (capsid) ▲ Tail: is composed of a hollow core surrounded by a sheath withbase plate andat the end.

10. Interaction between phages and bacteria • Phages are wide spread in nature but usually has a specificity of host bacteria. • Infection with avirulent phageresults in phage replication with the production of new phage particles and their subsequent release causes death of host bacteria. adsorption penetration biosynthesis maturation / release • Infection with atemperate phagedoes not necessarily lead to bacterial death, but phage’s nucleic acidintegrates into bacterial chromosome without new phase production. adsorption penetration integration

11. Virulent phage I. Adsorption Recognition of host bacterial surface receptors by tail fibers II. Penetration Phage’s nucleic acid is injected through hollow core into bacterial cytoplasm.

12. Virulent phage IV. Maturation and Release III. Biosynthesis

13. Temperate Phage (Prophage formation) II.peneration I.adsorption III. integrate of phage DNA into host genome IV. prophage replicates along with chromosome of lysogenic bacterium

14. 4. Transposable Genetic Elements • Definition: segments of DNA that have the capacity to move fromonebacterial DNA molecule (bacterial chromosome or plasmid)to anotheror fromonelocationtoanotherin the same one DNA molecule (jumping gene / movable gene) • Types of Transposable Genetic Elements ▲ Insertion sequences (IS) ▲ Transposons (Tn)

15. GFEDCBA ABCDEFG Transposase Insertion sequence (IS) ▲Definition:a type oftransposable Genetic Elementsonly has a transposase-encoding gene alone. ▲ Structure:a small DNA has reverse repeated sequences at the two ends that are involved in transposition. In the middle there is a transposase coding gene. ▲Function:introduction of an insertion sequence into a bacterial gene will result ininactivation of the gene.

16. IS Resistance Gene(s) IS IS Resistance Gene(s) IS Transposon (Tn) ▲Definition:a type oftransposable Genetic Elementshas both insertion sequences (IS) and other genes. ▲Structure:the extra genes are located between the terminal repeated sequences. ▲Function:Since transposons can jump from one DNA molecule to another and frequently carryantibiotic-resistant genes, the transposons mediate drug resistance in bacteria.

17. Types of Mutation • Self Mutations:Spontaneously occur with low frequency. • Gene transfer and recombination: high frequency. One bacterium (recipient)uptakeexogenous DNA segment from another bacterium (donor) or phage(Gene transfer)and then the DNA segment isincorporatedinto DNA of itself (recombination). • Major mechanisms of bacterial gene transfer ▲ Transformation ▲ Transduction ▲ Conjugation ▲ Lysogenic conversion

18. Transformation • Definition:a bacterial recipient uptake naked DNA segment offered by bacterial donor in environment and then the DNA segment recombinate with the recipient’schromosomal DNA.

19. Significance for transformation • Transformation occurs in nature and it can lead to increased virulence ( e.g. S. pneumoniae) and drug resistance.

20. Transduction • Definition:a bacterial donor’s chromosomal DNA segment transferred to abacterial recipientby way of aphage, and then the DNA segmentrecombinate with the recipient’s chromosomal DNA . ▲Generalized Transduction:incorrect assemblage (any genes). ▲Specialized transduction: Sometimes, during activation of prophage, the excisedphage’s DNA contains host DNA segments at its ends.

21. Lysogenic conversion • Definition:a bacterial recipient is infected with phage from a bacterial donor, andthe phage’s genes recombinate with recipient’s chromosomal DNA. • As an example, Corynebacterium diphtheriae will produces diphtheria toxin after it is infected by the β- phage, because the gene encoding the toxin is carried by the phage.

22. Donor Recipient Conjugation • Definition:Gene transfer from a donor to a recipient by direct contact between two bacterial cells throughsex pili. ▲Donor:a bacterium withF factorcan producesex pili. ▲Recipient:a F factor-absent bacterium.

23. F+ F- F+ F- F+ F- F+ F+ F+ F+ F+ factor-depecdent Conjugation • The F+ bacterium transfers extra chain of F+ factor and then the completed F+ factors in the two bacteria is synthesized by rolling circle replication.

24. Hfr F- Hfr Hfr F- F- Hfr F- High-frequency recombination (Hfr) • F factor is integrated into bacterial chromosome. • Bacterial DNAis transferred with a high frequency. • F factor is difficulty to be transferred.

25. F’ F’ F’ F’ F- Hfr F’ factor-depecdent Conjugation • F factor in bacterial DNA is excised but it carries bacterial chromosomal DNAat its two sides (F’).

26. Summary • The most important contents in this lecture are displayed as the followings: • The Agents (plasmid, phage, bacterial chromosomal DNA) associated with bacterial mutation. • Concepts of Transformation, Transduction, Conjugation and Lysogenic conversion. • The Significanceof bacterial mutation (e.g. bacterial virulence, drug resistance and antigenicity).

27. Medical Microbiology Bacterial Infection

28. Bacterial Pathogenicity Terms : • Pathogen:disease-causing microbe. • Opportunistic pathogen:A microbe does not cause diseases in normal conditions, but can cause diseases in some certain conditions. • Pathogenicity:ability of a microbe to cause diseases. • Virulence:quantitative ability of a microbe to cause disease (invasiveness and toxigenicity). • LD50:the number of pathogen required to cause death in half of the exposed hosts.

29. Source of infection • Exogenous infection • Infectious agents come from environment or hosts (patient, diseased animal or carrier). • ▲carrier:individuals (human and animal) infected with microbes but no clinical signs or symptoms. • Endogenous infection • Normal flora act as infections agents under some certain conditions (opportunistic infection).

30. Normal flora Microbial populations inhabit on skin and mucosa of healthy normal persons. ▲ Gastrointestinal tract ▲Urogenital tract ▲Skin and Conjunctiva (结膜)

31. Physiological role of normal flora ▲Antagonism:a) biolfilm; b) antimicrobial agents. ▲Trophism:a)digestion of foods; b) production of vitamins (K and B). ▲Immunoenhancement:promotes development of mucosal immune system. ▲Anti-tumorigenesis and anti-apolexis(衰老): e.g. eliminate nitrite and anti-oxidation (SOD).

32. Opportunistic infections The conditions required by normal flora to cause diseases. ▲ Translocation of normal flora. ▲ Suppression of normal flora. ▲ Low immunity of human body.

33. Hospital acquired infections (Nosocomial infections) New infections after 48 hours of hospital admission.

34. Pathogenic process of bacteria Generally, infection process caused by a bacterial pathogen involves the four steps as the following: ▲Adhesion (chemotaxis) ▲Survival / propagation ▲ Penetration and spread ▲ Tissue injury Virulence: adhesins, invasive enzymes and toxins.

35. BACTERIUM adhesin receptor EPITHELIUM 1. Adhesion A process of recognition and combination of microbial adhesins with receptors on the surface of host cells ▲lipoteichoic acid(G+ bacteria) ▲ordinary pilus and outer membrane proteins (G- bacteria)

36. 2. Survival / propagation • Anti-phagocytosis: e.g. capsules. • propagation: probably due to stress.

37. 3. Penetration and Spread ▲Collagenase: hydrolyze collagens. ▲Hyaluronidas: hydrolyze hyaluronic acid. ▲Coagulase: agglutinate fibrinogen. ▲Streptokinase/fibrinolysin: the former activates fibrinogenase to thrombin, and the latter directly hydrolyze fibrin. ▲Cytolysins: 1) hemolysin (to lyse erythrocyte or tissue cells) 2) Leukocisin (to kill leukocyte or tissue cells )

38. 4. Tissue injury ▲Exotoxin ▲Endotoxin ▲Immunopathological reaction

39. 4.1. Exotoxin ▲Cytotoxin: e.g. diphtheria toxin ▲Nuerotoxin: e.g. tetanospasmin ▲Enterotoxin: e.g. cholera enterotoxin diphtheria toxin

40. 4.1. Exotoxin tetanospasmin cholera enterotoxin

41. Cell surface Toxic Binding A B A-B type exotoxins ▲ Subunit A is toxic, while subunit B is the ligand of cell’s receptor to mediate A subunit into host cells.

42. 4.2. Endotoxin (LPS) ▲Fever: a typical pyrogen. ▲Leukocytoreaction: leukopenia/leukocytosis. ▲DIC: disseminated intravascular coagulation. ▲Shock: dilatation of small blood vessels ▲Inflammation: IL-1β, TNF-αand IL-6. LPS

43. Property Endotoxin Exotoxin Chemical nature Lipopolysaccharide ( MW = 800-1, 000 kDa ) Protein or peptide ( MW = 50-1, 000 kDa ) Relationship to bacteria Part of outer membrane Extracellular Denatured by boiling No Usually Antigenicity Yes Yes Form toxoid No Yes Toxicity Relatively low ( > 100 µg ) Relatively high ( approximate 1 µg ) Pyrogenicity Yes Occasionally Specificity to host cells Low degree High degree Enzymatic activity No Usually Major difference between endotoxin and exotoxin

44. 4.3. Immunopathological reaction • Human immune responses to bacteria may cause tissue injury by: • Numerous cytokines and complement activation. • Continuously generated bacterial antigens will subsequently causet humoral antibodies and cell mediated immunity, which resulting in chronic immunopathological injury. • Some of bacterial antigens (e.g. M protein of Streptococcus) react with host tissue antigens to cause autoimmunity.

45. Number and route ▲Invaded mumber of bacteria: Different pathogens need different bacterial number to cause diseases. ▲Suitable invading route of bacteria: Most of bacteria require suitablec invading routes to cause diseases (e.g. Clostridum tetani causes disease through wounds and Mycobacterium tuberculosis has multiple invading routes).

46. According to infectious state: ▲Inapparent or subclinical infection:The infection with no manifesting clinical signs and symptoms. ▲Latent infection:The infection is inactive but maintain potential to cause diseases. ▲ Apparent infection:clinical signs and symptoms. Types of infection According to infectious site: ▲Local infection; ▲ Systemic infection

47. Systemic infection ▲Bacteremia:Bacteria enter bloodstream without propagation in bloodstream. ▲Toxemia:Exotoxin or endotoxin rather than bacteriaenters bloodstream. ▲Septicemia:Bacteria enter bloodstream with propagation and release virulent factors. ▲Pyemia:Pyogenic bacteria enter bloodstream with propagation and release virulent factors, and then spread through bloodstream into the target organs to form pyogenic foci.

48. Summary • The most important contents in this lecture are displayed as the followings: • Concepts of virulence, normal flora, hospital acquired infection, latent infection, toxemia, septicemia,endotoxemiaandpyemia. • The physiologic role of normal flora. • Theconditions for generation of opportunistic infection. • The difference between exotoxinand endotoxin. • The pathogenic effects of endotoxin.

49. Anti-infection Immunity Medical Microbiology

50. Innate Immunity • Phagocytosis: mononuclear-macrophages, neutrophils. • Complements: lyse bacteria • Others: lysozyme, antibacterial peptide.