SBM 2044 Lecture 8 Bacillus and Corynebacterium

1. SBM 2044 Lecture 8 Bacillus and Corynebacterium

2. AIMS: Bacillus • To provide an overview of Bacillus species • To introduce the anthrax disease • To explain the pathogenesis of B. anthracis, including the mechanisms of the secreted toxins

3. Bacillus • Aerobic, G+ rods in chains, spores are located in center of the non-motile bacilli • Found in soil, water, air and vegetation • Spores are viable for decades. • B. cereus – produce enterotoxin and cause food poisoning. • B. anthracis – infection in human through injured skin (cutaneous anthrax), mucous membranes (GI anthrax), or inhalation of spores into lung.

4. Spores • Why do bacteria produce spores? • Survival • Classification • Definition = a resting cell, highly resistant to dessication, heat, and chemical agents; when returned to favourable conditions bacteria re-activated, the spores germinate to produce single vegetative cells.

5. Bacillus anthracis

7. Anthrax • Primarily a disease of herbivores • In animals, portal of entry is through ingestion of spores: • Injured skin (cutaneous anthrax) • Mucous membranes (gastrointestinal anthrax) • Inhalation of spores into the lung (inhalation anthrax) • Spores germinate, and growth of vegetative organisms result in formation of a gelatinous oedema and congestion. • Spread via lymphatics to bloodstream and multiply freely in blood and tissues.

8. Typical anthrax lesion on arm at 10th day.

9. Bacillus anthracis • Capsulated, poly-D-glutamic acid capsule is antiphagocytic • Anthrax toxin is made up of three proteins: • protective antigen (PA) • oedema factor (EF) • lethal factor (LF). • PA binds to specific host cell receptors and forms membrane channel that mediate entry of EF and LF into the cytosol. • EF is an adenylate cyclase • EF sustains the activation of host cAMP-dependent signalling pathways. • LF is a metalloproteinase that site-specifically cleaves MKKs (mitogen-activated protein kinase kinases). • cleavage of MKKs by LF prevents them from activating their downstream MAPK (mitogen-activated protein kinase) substrates and subsequently the cell is unable to respond to any stimuli. • http://www.sumanasinc.com/webcontent/anisamples/microbiology/ani_anthrax.swf

10. Bacillus • Anthrax can be successfully treated with antibiotics if they are administered prophylactically after spore exposure. • Treatment: ciprofloxacin, penicillin G along with gentamicin and streptomycin. Also fluoroquinolones and tetracyclines. • Vaccine with live spores and a toxoid used to protect livestock in endemic areas.

11. SBM 2044: Lecture 8 Diphtheria AIMS: • To discuss diphtheria in detail - paradigm of: • - an early success in microbiology - first toxoid vaccine • - approaches used to study a bacterial protein toxin

12. Diphtheria • Acute, transmissible, infection of upper respiratory tract • - mostly in children aged 2 - 9 • Caused by Corynebacterium diphtheriae • - Gram-positive, aerobic, non-motile, ‘club-shaped’ rod Black colonies on tellurite medium ‘Halos’ on Tinsdale’s medium

14. Diphtheria Inflammatory exudate ‘pseudomembranous’ appearance Bullneck diphtheria

15. suggested bacteria might release a “poison” Milestones in Microbiology Loeffler (1884)– isolatedC. diphtheriae • pure culture virulent in animals • C. diphtheriae localised, but lesions at other sites Roux & Yersin (1888) Confirmed lesions due to a TOXIN

16. Diphtheria – key early success in Microbiology von Behring & colleagues (1890s) C. diphtheriae culture filtrates treated with iodine tri-chloride induced specific immunity in animals + immunity could be passively transferred in blood to other animals First TOXOID vaccine By 1920s Diphtheria toxoid vaccine widely available in USA (Later included as part of the triple DPT vaccine)

17. Current incidence of diphtheria Endemic in many economically-deprived countries that cannot afford widespread vaccination Very rare in developed countries (e.g. 0-2 cases/year in US) (e.g. contrast with 1921: >200,000 cases in USA) Nevertheless - no room for complacency. Potential dangers illustrated by resurgence of diphtheria in Russia during early 1990s.

18. Reported Russian cases of diphtheria between 1965 - 1994 • Resurgence occurred despite high vaccine • uptake (> 90% children) • High proportion of cases in young adults 50,000 40,000 30,000 20,000 10,000 81 90 94 65 72 Year

19. Reasons for resurgence? • Inadequate vaccination of children in ‘70’s ?? • - strongly denied - no increase in children in 1970s • More subtle explanation ? Insufficient data to provide clear answer, but good example of how even long-controlled diseases can re-emerge to cause serious problems In Russia, aggressive efforts to boost anti-toxin immunity by vaccination of adults since 1994 worked - cases began to decrease in 1995

20. Diphtheria Toxin Another key early finding 1959: First observed that addition of DTx to cultured cells inhibits protein synthesis But, further advance had to await more effective techniques for e.g. protein purification & analysis.

21. Exotoxins • Exotoxins usually secretedy by the bacterium by the Type I or Type II secretion system • Exotoxins are synthesised as protoxins and must be activated on binding to host cell membrane. • Activation involves a proteolytic cleavage and reduction of a disulphide bond (that holds A + B domains together). • Please read Chapter 9 Schaechter’s

22. A (21K) -C N- s s B (37K) -N s s s s -C Late 1960s + 1970s: Biochemical studies on DTx DTx purified: Toxicity greatly increased by ‘nicking’ intact toxin with trypsin (a protease) SDS-PAGE Intact - low activity Mr 58K single polypeptide ‘nicked’ - high activity linked A + B fragments N- s s -C

23. A (21K) -C N- disulphide bonds s s X-S-S-X X-SH + HS-X -N oxidation reduction s s -C -C B (37K) -C N- B (37K) Reduction Purified individual A and B fragments A (21K) N- Compared each with intact ‘nicked’ toxin

24. Comparing linked A + B with purified A or B alone Inhibition of protein synthesis Toxicity intact cells cell extracts A + B A B Conclusions: • DTx kills cells by inhibiting protein synthesis • Inhibitory activity resides in fragment A, but first A must • separate from B • Possible that Fragment B ‘delivers’ A to cell cytoplasm

25. DTx Entry • Early observations (1960s): • Lag of 20 – 30 min between binding and killing • suggests involvement of entry ‘pathway’ • NH4Cl protected cells against DTx • suggests involved of an acidic compartment Late 1970s • Discovery of RME (receptor-mediated endocytosis) via • clatherin-coated pits and acidification of endocytic vesicles Involved in DTx entry ??

26. RME

27. Clathrin-coated vesicles

28. Inside the host cell • Once inside the endosomal vesicle, reduction of disulphide bond takes place and separates A from B • Acidic conditions in vesicle promotes translocation of A domain into cytosol • A domain ADP ribosylates elongation factor-2 (EF-2), hence blocks protein synthesis • EF-2 the only known substrate for DTx – due to its specific modified histidine residues (called ‘diphthamide’) • http://www.sumanasinc.com/webcontent/animations/content/diphtheria.html

29. Early 1980s Possible involvement of RME supported by additional circumstantial evidence suggesting ‘productive’ DTx entry facilitated by a low pH. Example: Lag between Cells in buffer DTx binding and killing pH 7.0 20-30 min pH 5.5 < 5 min DTx-A could cross cell membrane directly at low pH

30. Other ‘ADP-ribosyl transferase’ A-B subunit toxins Pathogen Toxin Target Effect inhibit protein synthesis C. diphtheriae Diphtheria EF-2 Pseudomonas Exotoxin A aeruginosa (ETA) Vibrio cholerae Cholera increase cAMP production Gia E. coli (ETEC) Heat-labile (LT) Bordetella pertussis Pertussis Gsa Clostridium perfringens iota-toxin Destroy actin filaments Actin Several sp. of iota-like Clostridium