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

2. Bacillus AIMS: 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.
B. cereus food poisoning is a problem in immunocompromised humans (endocarditis, conjuctivitis)

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
Morphology: square ends and in long chains; spores are located in the centre of nonmotile bacilli.

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.
Anthrax is not known to spread from one person to another.

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.

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. Diphtheria SBM 2044: Lecture 8 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 ‘Halos’ on Tinsdale’s medium
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
Can be lethal - killed 5% of population of the American colony New Hampshire in 1735
First bacterial species identified clearly as a specific etiologically agent of a disease
Loeffler isolated C. diphtheriae from throats of patients + grew in pure culture + demonstrated virulence for animals
Roux & Yersin (1888) showed sterile filtrates from C. diphtheriae cultures kill guinea pigs
+ produce lesions identical to those seen when animals infected with the live organism. Thus confirmed lesions
due to a ‘Toxin’
‘Halos’ on
Tinsdale’s
medium

14. ‘pseudomembranous’ appearance
Diphtheria
Inflammatory exudate
‘pseudomembranous’ appearance
Bullneck
diphtheria

15. Milestones in Microbiology
Loeffler (1884) – isolated C. diphtheriae
pure culture virulent in animals
C. diphtheriae localised, but lesions at other sites
suggested bacteria might release a “poison”
Can be lethal - killed 5% of population of the American colony New Hampshire in 1735
First bacterial species identified clearly as a specific etiologically agent of a disease
Loeffler isolated C. diphtheriae from throats of patients + grew in pure culture + demonstrated virulence for animals
Roux & Yersin (1888) showed sterile filtrates from C. diphtheriae cultures kill guinea pigs
+ produce lesions identical to those seen when animals infected with the live organism. Thus confirmed lesions
due to a ‘Toxin’
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.
Due primarily to effective vaccination programmes

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
Protective immunity to Diphtheria is associated with anti-toxin antibodies, but titre drops with age.
One suggested explanation was that inadequate vaccination of children in 1970s led to susceptibility some 20 years later, but
10,000 65 72 81 90 94
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
insufficient data to provide clear answer
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. 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
A (21K) s -C N-
B (37K) -N N- s s -C

23. A (21K) B (37K) A (21K) B (37K) -C N- X-S-S-X X-SH + HS-X
oxidation reduction
disulphide bonds
B (37K) s -C -N
Reduction
Purified individual
A and B fragments
A (21K) N- -C -C N-
B (37K)
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
DTx can bind to cells at 4 C, but does not kill cell until temp raised to 37 C, so can easily distinguish between binding and entry.
NH4CL neutralises low pH
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’)

29. Possible involvement of RME supported by additional
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 min
pH < 5 min
If low pH necessary for DTx-A to get cross membrane, under normal physiological conditions, where likely to cross from the inside
of an acidic cellular compartment, e.g. acified endosome
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
Why do so many act as ADP-ribosyl transferases?
Mammalian cells have an endogenous ADP-ribosyl transferase that modifies the diphthamide residue
on EF-2. Similarly, others modify Gia and Gia at exactly the same sites as cholera and pertussis toxins respectively.
These endogenous ADP-ribosyl transferases regulate the activities of their various ‘target’ proteins, but do so in a
very carefully controlleed manner
Bacterial toxins have simply ‘hijacked’ an activity that already exists in their target cells, but unlike endogenous
enzymes, the target cell has no control over the toxin
Clostridium
perfringens
iota-toxin
Destroy
actin
filaments
Actin
Several sp. of iota-like
Clostridium