Medical Microbiology

Microbial Toxins & Cholera

Kelly Doran BIOL 584 Spring 12

Schaechters ch 9,20,16

Lecture Outline/Learning Objectives

! Exotoxins
! Membrane disruption ! Superantigens ! AB Toxins

! Endotoxins ! Vibrio cholera and cholera toxin

! Inhibition of Protein Synthesis ! Inactivation via ADP-ribosylation

Bacterial Toxins
LPS

Staphylococcus aureus Group B streptococcus

Corynebacterium diphtheriae

Vibrio cholera

AB toxins

Membrane disrupting toxins

2 TYPES
! Channel- or pore-forming
! Inserts itself into the host cell membrane ! Creates a channel or pore ! Multiple pores cause leakage of cell interior and inrush of water, lysis, death

! Phospholipases or hemolysins
! Destroy integrity of host cell membrane lipids

Pore-forming toxin
Cytolytic toxins damage cell membranes ! lysis ! death

! Staph. aureus !-toxin protein inserts into membrane ! pores or channels

Phospholipases or hemolysins
Cytolytic toxins damage cell membranes ! lysis ! death e.g. Phospholipases hydrolyse phospholipids in cell membranes

Streptococcus " hemolysin

cell necrosis toxemia shock

phospholipase

"-hemolysin Group B Streptococcus

Toxins Subvert Host Function

! protein exotoxins
! Block protein synthesis ! Block 2nd messenger pathways

Toxins Subvert Host Function

! AB Toxins
! B subunit binds to host cell
! Delivers A subunit to cell ! Often 5 B subunits form a pore for A entry ! ADP-Ribosyltransferase
! Diphtheria toxin ! Cholera toxin

! A subunit has toxic activity

! Cholera toxin

ADP-Ribosylating Toxins

! Ribosylates to overactivate adenylate cyclase ! cAMP activates ion transport, water follows ! Uncontrollable diarrhea

! Diphtheria toxin
! Ribosylates elongation factor 2 ! Blocks ribosome function, cell dies
Corynebacterium diphtheriae

! Forms pseudomembrane over trachea

Neurotoxins ! Clostridium botulinum ! Clostridium tetani

Toxins with an intracellular site of action:

Botulinum toxin Tetanus toxin

AB Toxins

Inhibitors of protein synthesis ! Corynebacterium diphtheriae ! Pseudomonas aeruginosa ! Shigella spp. Adenylate cyclase enzymes ! Bacillus anthracis ! Bordetella pertussis ! Vibrio cholerae ! Bordetella pertussis

Diphtheria toxin Exotoxin A Shiga toxin Oedema toxin adenylate cyclase CT (cholera toxin) Pertussis toxin

Diphtheria toxin: DT
Corynebacterium diphtheriae: ! Colonises nose, throat, skin ! DT acts locally, heart, nervous system ! death ! Toxin ! necrosis of mucosal cells ! pseudomembrane in throat ! Genes for DT - temperate phage ! DT toxoid protective component of DTP vaccine ! ADP-ribosylation of EF-2 ! inhibits translation ! cell death

Centres for Disease Control & Prevention!

Diphtheria Toxin

T Domain

Botulism and Tetanus

! Not and infection but an intoxication! ! Symptoms caused by ingestion of the toxins, not by colonization of bacteria ! Clostridium botulinum ! Clostridium tetani

! Clostridium toxins

Central Nervous System Infections

! C. botulinumbotulin toxin (Botox)

! Anaerobe, grows in canned food ! Spores survive unless autoclaved ! Toxin blocks release of acetylcholine
! Prevents muscle movement

! C. tetanitetanus toxin

! Anaerobe, grows in puncture wounds

! Toxin blocks release of GABA, inhibitory transmitter

! Blood flow interrupted, tissue becomes anaerobic

! Muscles contract uncontrollably, lethal spasms

Clostridium Toxins: Paralytics

Tetanus: spastic paralysis (rigid), lockjaw Botulism: flaccid paralysis (sagging)

Bacterial Toxins

The structure of the Gram-negative cell wall:

Endotoxin

www.cat.cc.md.us!

LPS

ENDOTOXIN
Gram negative LPS from outer membrane
! Mainly cell associated but some shed (lysis, blebbing) ! cell free form ! Less toxic, but more heat stable than exotoxins ! Lipid A toxic moiety ! Effects on host ! inflammation, tissue damage (localised infections) ! fever, widespread tissue damage, septic shock (systemic infections) ! death (20-50%) ! Mechanism of action over stimulation of immune system during systemic infection. ! Peptidoglycan fragments ! Lipoteichoic acid

Gram positives septic shock

Antigen Presentation
Major Histocompatability Complex MHC class II molecules "1 present extracellular peptides processed in lysozomes (eg. bacterial proteins) to T "2 + helper cells (CD4 ), triggering inflammation and specific immunity against the pathogen.
!1

!2

Superantigens

S. aureus: Toxic Shock Syndrome Toxin-1 (TSST-1)

Vibrio cholerae
! ! ! ! ! Gram negative Curved rod Motile Polar flagellum Facultative anaerobe ! Serotypes based on O Antigen

Epidemiology
! Epidemiology is the study of factors affecting the health and illness of individuals and populations ! Endemic infection refers to infection or disease that occurs regularly at low or moderate frequency. ! Epidemic is a sudden increases in frequency above endemic levels. ! Pandemics are global epidemics. The size of outbreaks is dependant upon factors such as the ratio of susceptible to immune subjects, period of infectivity, population density, etc.

John Snow, the first epidemiologist

Cholera outbreak London 1854

Cholera - Disease
! ! ! ! ! Acute intestinal illness Abdominal pain Watery diarrhea Vomiting dehydration

vibrios in both end-on and horizontal modes close to tips of microvilli.

Vibrio cholerae - Encounter

! Environment
! Water ! Algae ! Marine invertebrates

! Infected humans
! Asymptomatic carriers

Cholera - Entry

V. Cholerae - Spread & Multiplication

! ! ! ! ! ! Adhesins - common pili Adhesins - TCP pili (toxin coregulated) Neuraminidase Protease - mucinase Motility/chemotaxis Toxin productin - CT toxin

Cholera Toxin: CT
Cholera toxin responsible for most of pathology: ! B - pentameric ring, binds to GM1 receptor on www.itb.uni-stuttgart.de! intestinal cells ! A = A1 + A2 disulphide bonded ! A1 enters cell; ADP-ribosylates Gs ! permanently stimulate adenylate cyclase ! " cAMP ! affect activity of ion channels ! " secretion of Cl- by intestinal crypt cells ! # absorption of Na+ ! net flow of ions & H20 into intestinal lumen ! profuse watery rice water diarrhea up to 20L/ day

Effect of Cholera Toxin on Intestinal Epithelium

Cholera Toxin
Another molecular Switch

Normal Situation

Adenylate cyclase (AC) is activated normally by a regulatory protein (GS) and GTP; however activation is normally brief because another regulatory protein (Gi), hydrolyzes GTP

Cholera Toxin

The A1 fragment catalyzes the attachment of ADPRibose (ADPR) to the regulatory protein forming GsADPR from which GTP cannot be hydrolyzed. Since GTP hydrolysis is the event that inactivates the adenylate cyclase, the enzyme remains continually activated.

V. Cholerae - Treatment
! Oral rehydration ! Interrupt fecal-oral transmission ! Vaccines in other countries ! Oral, live attenuated strains

! film based on novel by W. Somerset Maugham. ! Set in the 1920s ! Cholera epidemic in China