SBM 2044 Lecture 8
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SBM 2044 Lecture 8 Bacillus and Corynebacterium. 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. Bacillus.

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SBM 2044 Lecture 8 Bacillus and Corynebacterium

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Sbm 2044 lecture 8 bacillus and corynebacterium

SBM 2044 Lecture 8

Bacillus and Corynebacterium


Sbm 2044 lecture 8 bacillus and corynebacterium

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


Bacillus

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.


Spores

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.


Bacillus anthracis

Bacillus anthracis


Anthrax

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.


Sbm 2044 lecture 8 bacillus and corynebacterium

Typical anthrax lesion on arm at 10th day.


Sbm 2044 lecture 8 bacillus and corynebacterium

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


Sbm 2044 lecture 8 bacillus and corynebacterium

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.


Sbm 2044 lecture 8 bacillus and corynebacterium

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


Sbm 2044 lecture 8 bacillus and corynebacterium

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


Sbm 2044 lecture 8 bacillus and corynebacterium

Diphtheria

Inflammatory exudate

‘pseudomembranous’ appearance

Bullneck

diphtheria


Sbm 2044 lecture 8 bacillus and corynebacterium

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


Sbm 2044 lecture 8 bacillus and corynebacterium

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)


Sbm 2044 lecture 8 bacillus and corynebacterium

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.


Sbm 2044 lecture 8 bacillus and corynebacterium

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


Sbm 2044 lecture 8 bacillus and corynebacterium

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


Sbm 2044 lecture 8 bacillus and corynebacterium

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.


Exotoxins

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


Sbm 2044 lecture 8 bacillus and corynebacterium

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


Sbm 2044 lecture 8 bacillus and corynebacterium

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


Sbm 2044 lecture 8 bacillus and corynebacterium

Comparing linked A + B with purified A or B alone

Inhibition of protein synthesis

Toxicity

intact cellscell 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


Sbm 2044 lecture 8 bacillus and corynebacterium

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 ??


Sbm 2044 lecture 8 bacillus and corynebacterium

RME


Clathrin coated vesicles

Clathrin-coated vesicles


Inside the host cell

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


Sbm 2044 lecture 8 bacillus and corynebacterium

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


Sbm 2044 lecture 8 bacillus and corynebacterium

Other ‘ADP-ribosyl transferase’ A-B subunit toxins

PathogenToxin TargetEffect

inhibit

protein

synthesis

C. diphtheriaeDiphtheria

EF-2

Pseudomonas Exotoxin A

aeruginosa (ETA)

Vibrio choleraeCholera

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


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