Biology 216 - Lecture 25 Corynebacterium I. Corynebacterium (general) - Taxonomic mess (coryneform gp) - related to acid fast (C32) Found in soil, water, skin, mucus membranes A. Propionibacterium acnes (Used to Coryne) - anaerobe; lipolytic enz; acne; tetracycline B. Corynebacterium diphtheriae - exotoxin -> cell death C. Diphtheroid - common skin, usually not speciated since contaminant. D. JK group - Prosthetic valve, neutropenics, resist antibiotics II. C. diphtheriae - man only First disease completely described by action of an exotoxin. A. Bacteriology - Gm+ pleomorphic rod; Koryne (club) especially in inadequate media Loeffler - coagulated serum; metachromatic granules (LMB) - polyphosphates Snapping division - chromosome shapes Colony types (biotypes) - 3 types on tellurite (mitis, gravis, intermedius) Originally reported difference in virulence - not true B. Diphtheria toxin - 1888, cell-free simulates disease (heart, CNS necrosis) Local infection (nasopharyngeal) - systemic toxin effects; LD50 for guinea pig, 25 ng The major virulence factor and protective antigen; Single serotype 1. Toxin production - phage encoded and iron regulated transcription. Corynephage - integrates into bacterial chromosome -> lysogeny Iron regulation: Phage + coryne extract -> toxin Phage + coryne extract + Fe -> no toxin Phage + e. coli extract -> toxin Phage + e. coli + Fe -> toxin Model - C. diphth codes aporepressor (dtxR = diphtheria toxin regulation) which binds Fe to block transcription. 2. Toxin structure - 2 subunits (A and B linked by disulfide) A/B endocytosed, pH of endocytic vessicle drops, toxin shape changes, cross. 3. MOA - ADP-ribosylates EFII (at modified histidine - diphthamide): Blocks peptide elongation. All eucaryotes have this. Only cell protein with this modified diphthamide. 4. DT cell receptor - L. Eidels (Friday Seminar) a. Characterize receptor by DT + 125I membrane lysate, ppt w/ anti DT, gel b. Mouse cells (resistant) + cDNA from Monkey -> sensitive cell (replica) c. Sequence cDNA = EGF receptor, ie DT recruited this receptor. 5. CRM - cross reactive material - genetic toxoid C. Pathogenesis - few cases/yr in US (1900s, 200K/yr in US; prior to vaccination) 1. Epidemiology - Used to be disease of children, now 40 yr olds (no herd immunity) When endemic, children develope immunity, so only young get infected. (still is childhood disease in underdeveloped) In naive population, all populations get it (ex. American Indians) dT every 10 years, often not given. 2. Transmission - man only host; droplets from asymptomatic, infected, convalescent. Survives in pseudomembranous exudate for months. Predisposition: crowding; slum; lack of immunity. 3. Colonization - nasopharynx, pharynx, tonsil, trachea -> lymphadenitis (similar to mono, strept) 4. Local infection - degeneration of epithelia (toxin) Inflammation - fibrinous exudate, pseudomembrane, blood, fibrin, PMN, C.diph Coalesce to form large membrane -> suffocation Sore throat, fever, malaise - clinically similar to strept throat. 5. Systemic toxin - heart, kidney, CNS - w eeks into disease. a. Myocarditis - fatty tissue, inflammation, edema, enlargement, weakness, circ collapse (damage reversible) b. CNS - myelin degeneration late in infection (week 4) Schwann cells (produce myelin) - insulation for nerve impulse Cranial first - soft palate (difficulty swallow; nasal regurgitation) Oculomotor paralysis (eyes); Diaphragm paralysis (resp failure); reversible D. Lab diagnosis - Isolation, ID, proof of toxicity Therapy begun based upon clinical and immunity history. Attached pseudomembrane. 1. Specimen - nasopharynx, throat, pseudomembrane - direct exam (vincent's disease) 2. Culture - BAP (Strept); Tellurite (selective diff); Loeffler (granules and club) 3. ID - biochemicals 4. Toxin production - CDC or reference lab - experts since iron regulates tox production a. Rabbit back test (shaved) - toxin neutralization test Grow in low Fe To - subq inject supernatant T5 - inject antitoxin iv T5.5 - inject supernatant Observe necrosis and neutralization at 48 hr b. Chinese hampster ovary cells (CHO) - cell line sensitive to dipth tox (die) Normally, cells grow, produce acid -> yellow indicator Cells + tox -> death (pink) Cells + tox + anti tox -> neutralization (yellow) c. Elek - immunodiffusion - less sensitive Antitoxin soaked filter strip on agar Streak isolate perpendicular (low Fe media) 24 hour check for pptn line at 45 degree angle E. Treatment - immediate neutralization of toxin 1. Passive immunization - 1901 Emil von Behring (Nobel) Use antitoxin raised in horses (high titer) Strategy - neutralize unbound toxin (bound toxin unaffected) Test patient hypersensitivity (10 % of patients) 1:10 serum into eye or subq (immediate arthus rxn in 30 min) Give antitoxin iv or im; small doses if hypersensitive (epinephrine ready) 2. Antibiotics - little effect on course; shut down source of toxin. Eliminate carrier state; Treat contacts (antibiotics and booster vaccine) 3. Respiratory support and anti-inflammatory for heart inflammation. F. Prevention - formalin toxoid (single serotype) - DPT (toxin + formalin 37 C -> binds SH of active site of A subunit) 1. Given 2,4,6,18, 5 yrs as DPT Boost with dT every 10 yrs (alum ppt since no adjuvant) - hypersensitivity to D 2. Adult immunity is weak since low incidence and no natural booster. 3. Antibody is to B-subunit - blocks attachment of holotoxin 4. Vaccine has little effect on colonization. G. Applications of diphtheria toxin (none have worked) NOTE: Patient anti-A does not seem to affect this strategy 1. ConjugateA-subunit to cancer antibodies. 2. ConjugateA-subunit to anti-HIV surface Ag