Protein Toxins

 Information from CDC

Biological Toxins. Biological toxins (also referred to as biotoxins) are nonliving toxic proteins that are naturally produced by many different types of living organisms. Biotoxins are:

• Thousands of times more toxic by mass than chemical warfare agents.
  
  
• Considered to pose the same level of risk as the microorganisms that produce them.
  
  
• Not themselves infectious or contagious after exposure. However, a biotoxin-producing organism may be infectious or contagious after exposure.

Bacterial Toxins: Friends or Foes?

Clare K. Schmitt, Karen C. Meysick, and Alison D. O'Brien
Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA

Many emerging and reemerging bacterial pathogens synthesize toxins that serve as primary virulence factors. We highlight seven bacterial toxins produced by well-established or newly emergent pathogenic microbes. These toxins, which affect eukaryotic cells by a variety of means, include Staphylococcus aureus -toxin, Shiga toxin, cytotoxic necrotizing factor type 1, Escherichia coli heat-stable toxin, botulinum and tetanus neurotoxins, and S. aureus toxic-shock syndrome toxin. For each, we discuss the information available on its synthesis and structure, mode of action, and contribution to virulence. We also review the role certain toxins have played in unraveling signal pathways in eukaryotic cells and summarize the beneficial uses of toxins and toxoids. Our intent is to illustrate the importance of the analysis of bacterial toxins to both basic and applied sciences.

Since diphtheria toxin was isolated by Roux and Yersin in 1888, microbial toxins have been recognized as the primary virulence factor(s) for a variety of pathogenic bacteria. Bacterial toxins have been defined as "soluble substances that alter the normal metabolism of host cells with deleterious effects on the host". Indeed, the major symptoms associated with disease caused by Corynebacterium diphtheriae (diphtheria), Bordetella pertussis (whooping cough), Vibrio cholerae (cholera), Bacillus anthracis (anthrax), Clostridium botulinum (botulism), Clostridium tetani (tetanus), and enterohemorrhagic Escherichia coli (bloody diarrhea and hemolytic uremic syndrome) are all related to the activities of the toxins produced by these organisms. With the recognition of the central role of toxin in these and other diseases has come the application of inactive toxins (toxoids) as vaccines. Such toxoid vaccines have had an important positive impact on public health.

In this review, we provide a summary overview (Table) of a variety of bacterial toxins categorized according to mode of action: damaging cell membranes, inhibiting protein synthesis, activating second messenger pathways, inhibiting the release of neurotransmitters, or activating the host immune response. We also describe in detail seven toxins: Staphylococcus aureus -toxin, Shiga toxin (Stx), cytotoxic necrotizing factor type 1 (CNF1), E. coli heat-stable toxin (ST), botulinum and tetanus neurotoxins, and toxic-shock syndrome toxin (TSST) produced by S. aureus. We emphasize these toxins because they are produced by emerging (Stx of enterohemorrhagic E. coli) or reemerging (-toxin of multidrug-resistant S. aureus) pathogens or illustrate different structures or modes of action (ST, CNF1, neurotoxins, and TSST).

Table. Characteristics of bacterial toxinsa
Organism/toxin	Mode of action	Target	Disease	Toxin implicated in diseaseb
Damage membranes
Aeromonas    hydrophila/aerolysin	Pore-former	Glycophorin	Diarrhea	(yes)
Clostridium    perfringens/	Pore-former	Cholesterol	Gas gangrenec	?
perfringolysin O
Escherichia coli/    hemolysind	Pore-former	Plasma membrane	UTIs	(yes)
Listeria monocytogenes/	Pore-former	Cholesterol	Foodborne systemic	(yes)
listeriolysin O			illness meningitis
Staphyloccocus aureus/    -toxin	Pore-former	Plasma membrane	Abcessesc	(yes)
Streptococcus      pneumoniae/	Pore-former	Cholesterol	Pneumoniac	(yes)
pneumolysin
Streptococcus pyogenes/	Pore-former	Cholesterol	Strep throat Sfc	?
streptolysin O
Inhibit protein synthesis
Corynebacterium    diphtheriae/	ADP- ribosyltransferase	Elongation factor 2	Diphtheria	yes
diphtheria toxin
E. coli/Shigella    dysenteriae/	N-glycosidase	28S rRNA	HC and HUS	yes
Shiga toxins
Pseudomonas    aeruginosa/	ADP- ribosyltransferase	Elongation factor 2	Pneumoniac	(yes)
exotoxin A
Activate second messenger pathways
E.coli
CNF	Deamidase	Rho G-proteins	UTIs	?
LT	ADP- ribosyltransferase	G-proteins	Diarrhea	yes
STd	Stimulates guanylate cyclase	guanylate cyclase receptor	Diarrhea	yes
CLDTd	G2 block	Unknown	Diarrhea	(yes)
EAST	ST-like?	Unknown	Diarrhea	?
Bacillus anthracis/    edema factor	Adenylate cyclase	ATP	Anthrax	yes
Bordetella pertussis/
dermonecrotic toxin	Deamidase	Rho G-proteins	Rhinitis	(yes)
pertussis toxin	ADP- ribosyltransferase	G-protein(s)	Pertussis	yes
Clostridium botulinum/    C2 toxin	ADP- ribosyltransferase	Monomeric G-actin	Botulism	?
C. botulinum/C3 toxin	ADP- ribosyltransferase	Rho G-protein	Botulism	?
Clostridium difficile/
toxin A	Glucosyltransferase	Rho G-protein(s)	Diarrhea/PC	(yes)
toxin B	Glucosyltransferase	Rho G-protein(s)	Diarrhea/PC	?
Vibrio cholerae/cholera    toxin	ADP- ribosyltransferase	G-protein(s)	Cholera	yes
Activate immune response
S. aureus/
enterotoxins	Superantigen	TCR and MHC II	Food poisoningc	yes
exfoliative toxins	Superantigen (and serine protease?)	TCR and MHC II	SSSc	yes
toxic-shock toxin	Superantigen	TCR and MHC II	TSSc	yes
S. pyogenes/pyrogenic    exotoxins	Superantigens	TCR and MHC II	SF/TSSc	yes
Protease
B. anthracis/lethal factor	Metalloprotease	MAPKK1/MAPKK2	Anthrax	yes
C. botulinum/neurotoxins      A-G	Zinc-metalloprotease	VAMP/synaptobrevin   SNAP-25 syntaxin	Botulism	yes
Clostridium tetani/      tetanus toxin	Zinc-metalloprotease	VAMP/synaptobrevin	Tetanus	yes
aAbbreviations: CNF, cytotoxic necrotizing factor; LT, heat-labile toxin; ST, heat-stable toxin; CLDT, cytolethal distending toxin; EAST, enteroaggregative E. coli heat-stable toxin; TCR, T-cell receptor; MHC II, major histocompatibility complex class II; MAPKK, mitogen-activated protein kinase kinase; VAMP, vesicle-associated membrane protein; SNAP-25, synaptosomal-associated protein; UTI, urinary tract infection; HC, hemorrhagic colitis; HUS, hemolytic uremic syndrome; PC, antibiotic-associated pseudomembranous colitis; SSS, scalded skin syndrome; SF, scarlet fever; TSS, toxic-shock syndrome. bYes, strong causal relationship between toxin and disease; (yes), role in pathogenesis has been shown in animal model or appropriate cell culture; ?, unknown. cOther diseases are also associated with the organism. dToxin is also produced by other genera of bacteria

When It Rains, It Pores

Many bacterial exotoxins have the capacity to damage the extracellular matrix or the plasma membrane of eukaryotic cells. The damage not only may result in the direct lysis of cells but also can facilitate bacterial spread through tissues. Toxins that mediate this cellular damage do so by either enzymatic hydrolysis or pore formation. Bacterial hyaluronidases, collagenases, and phospholipases have the capacity to degrade cellular membranes or matrices. Specific examples of these types of toxins include the -toxin of Clostridium perfringens, which has phospholipase C activity; Streptococcus pyogenes streptokinase, which can hydrolyze plasminogen to plasmin and dissolve clots; and the clostridial collagenases. Pore-forming toxins, as the name suggests, disrupt the selective influx and efflux of ions across the plasma membrane by inserting a transmembrane pore. This group of toxins includes the RTX (repeats in toxin) toxins from gram-negative bacteria, streptolysin O produced by S. pyogenes, and the S. aureus -toxin (described below).

S. aureus -toxin can be considered the prototype of oligomerizing pore-forming cytotoxins. The -toxin gene resides as a single copy on the chromosome of most pathogenic S. aureus strains, and its expression is environmentally regulated at the transcriptional level by the staphylococcal accessory gene regulator (agr) locus. The -toxin is synthesized as a 319 amino acid precursor molecule that contains an N-terminal signal sequence of 26 amino acids. The secreted mature toxin, or protomer, is a hydrophilic molecule that lacks cysteine residues and has a molecular mass of approximately 33 kDa. Recently, the crystallographic structure of the fully assembled -toxin pore was solved. On the plasma membrane, seven toxin protomers assemble to form a 232 kDa mushroom-shaped heptamer comprising three distinct domains (Figure 1A). The cap and rim domains of the -toxin heptamer are situated at the surface of the plasma membrane, while the stem domain serves as the transmembrane channel.

Figure 1. Diagrammatic representation of the mode of action of several bacterial toxins. A. Damage to cellular membranes by Staphylococcus aureus -toxin. After binding and oligomerization, the stem of the mushroom-shaped -toxin heptamer inserts into the target cell and disrupts membrane permeability as depicted by the influx and efflux of ions represented by red and green circles. B. Inhibition of protein synthesis by Shiga toxins (Stx). Holotoxin, which consists of an enzymatically active (A) subunit and five binding (B) subunits, enters cells through the globotriasylceramide (Gb3) receptor. The N-glycosidase activity of the A subunit then cleaves an adenosine residue from 28S ribosomal RNA, which halts protein synthesis. C. Examples of bacterial toxins that activate secondary messenger pathways. Binding of the heat-stable enterotoxins (ST) to a guanylate cyclase receptor results in an increase in cyclic GMP (cGMP) that adversely effects electrolyte flux. By ADP-ribosylation or glucosylation respectively, the C3 exoenzyme (C3) of Clostridium botulinum and the Clostridium difficile toxins A and B (CdA & CdB) inactivate the small Rho GTP-binding proteins. Cytotoxic necrotizing factor (CNF) of E. coli and the dermonecrotic toxin (DNT) of Bordetella species activate Rho by deamidation.

Alpha-toxin is cytolytic to a variety of cell types, including human monocytes, lymphocytes, erythrocytes, platelets, and endothelial cells. For -toxin to damage cellular membranes, three sequential events are required. Toxin protomers must first bind to target membranes by either unidentified high-affinity receptors or through nonspecific absorption to substances such as phosphotidylcholine or cholesterol on the lipid bilayer. Second, membrane-bound protomers must oligomerize into a nonlytic prepore heptamer complex. Third, the heptamer must undergo a series of conformational changes that create the stem domain of the toxin, which is then inserted into the membrane. The -toxin pore allows the influx and efflux of small molecules and ions that eventually lead to the swellin