Adenylate cyclase toxin is a

virulence factor Virulence factors (preferably known as pathogenicity factors or effectors in plant science) are cellular structures, molecules and regulatory systems that enable microbial pathogens (bacteria, viruses, fungi, and protozoa) to achieve the followin ...

produced by some members of the genus ''

Bordetella ''Bordetella'' () is a genus of small (0.2 – 0.7 µm), gram-negative coccobacilli of the phylum Pseudomonadota. ''Bordetella'' species, with the exception of '' B. petrii'', are obligate aerobes, as well as highly fastidious, or difficult ...

''. Together with the pertussis toxin it is the most important virulence factor of the causative agent of

whooping cough Whooping cough, also known as pertussis or the 100-day cough, is a highly contagious bacterial disease. Initial symptoms are usually similar to those of the common cold with a runny nose, fever, and mild cough, but these are followed by two o ...

, ''

Bordetella pertussis ''Bordetella pertussis'' is a Gram-negative, aerobic, pathogenic, encapsulated coccobacillus of the genus '' Bordetella'', and the causative agent of pertussis or whooping cough. Like '' B. bronchiseptica'', ''B. pertussis'' is motile and expr ...

''. ''

Bordetella bronchiseptica ''Bordetella bronchiseptica'' is a small, gram-negative, rod-shaped bacterium of the genus '' Bordetella''. It can cause infectious bronchitis in dogs and other animals, but rarely infects humans. Closely related to '' B. pertussis''—the ob ...

'' and ''

Bordetella parapertussis ''Bordetella parapertussis'' is a small Gram-negative bacterium of the genus ''Bordetella'' that is adapted to colonise the mammalian respiratory tract. Pertussis caused by ''B. parapertussis'' manifests with similar symptoms to '' B. pertussis' ...

,'' also able to cause pertussis-like symptoms, also produce adenylate cyclase toxin. It is a

toxin A toxin is a naturally occurring organic poison produced by metabolic activities of living cells or organisms. Toxins occur especially as a protein or conjugated protein. The term toxin was first used by organic chemist Ludwig Brieger (1849 ...

secreted by the

bacteria Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were am ...

to influence the host

immune system The immune system is a network of biological processes that protects an organism from diseases. It detects and responds to a wide variety of pathogens, from viruses to parasitic worms, as well as Tumor immunology, cancer cells and objects such ...

Structure

Adenylate cyclase toxin from ''Bordetella pertussis'' is a 1706

amino acid residue Protein structure is the three-dimensional arrangement of atoms in an amino acid-chain molecule. Proteins are polymers specifically polypeptides formed from sequences of amino acids, the monomers of the polymer. A single amino acid monomer may ...

long

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respon ...

. The protein consists of three domains: from the

N-terminus The N-terminus (also known as the amino-terminus, NH2-terminus, N-terminal end or amine-terminus) is the start of a protein or polypeptide, referring to the free amine group (-NH2) located at the end of a polypeptide. Within a peptide, the ami ...

up to roughly residue 400, there is an adenylate-cyclase domain; between residues 500 and 700, there is a

hydrophobic In chemistry, hydrophobicity is the physical property of a molecule that is seemingly repelled from a mass of water (known as a hydrophobe). In contrast, hydrophiles are attracted to water. Hydrophobic molecules tend to be nonpolar and, ...

domain; and from residue 1000 to the

C-terminus The C-terminus (also known as the carboxyl-terminus, carboxy-terminus, C-terminal tail, C-terminal end, or COOH-terminus) is the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH). When the protein i ...

, there are

calcium Calcium is a chemical element with the symbol Ca and atomic number 20. As an alkaline earth metal, calcium is a reactive metal that forms a dark oxide-nitride layer when exposed to air. Its physical and chemical properties are most similar t ...

binding repeats. Two

acylation In chemistry, acylation (or alkanoylation) is the chemical reaction in which an acyl group () is added to a compound. The compound providing the acyl group is called the acylating agent. Because they form a strong electrophile when treated wit ...

sites are located at

lysine Lysine (symbol Lys or K) is an α-amino acid that is a precursor to many proteins. It contains an α-amino group (which is in the protonated form under biological conditions), an α-carboxylic acid group (which is in the deprotonated &minu ...

residues K860 and K983. The part of the toxin from residue 400 to the C-terminus, called

hemolysin Hemolysins or haemolysins are lipids and proteins that cause lysis of red blood cells by disrupting the cell membrane. Although the lytic activity of some microbe-derived hemolysins on red blood cells may be of great importance for nutrient acqu ...

, is structurally related to a large family of bacterial toxins -

RTX toxin The RTX toxin superfamily is a group of cytolysins and cytotoxins produced by bacteria. There are over 1000 known members with a variety of functions. The RTX family is defined by two common features: characteristic repeats in the toxin protein se ...

s. Differences between the toxins of different ''Bordetella'' species are mainly in the calcium-binding domain.

Folding and secretion

The toxin is secreted by the

Type I secretion system Bacterial secretion systems are protein complexes present on the cell membranes of bacteria for secretion of substances. Specifically, they are the cellular devices used by pathogenic bacteria to secrete their virulence factors (mainly of protein ...

, which spans both

membranes A membrane is a selective barrier; it allows some things to pass through but stops others. Such things may be molecules, ions, or other small particles. Membranes can be generally classified into synthetic membranes and biological membranes. Bi ...

and

periplasm The periplasm is a concentrated gel-like matrix in the space between the inner cytoplasmic membrane and the bacterial outer membrane called the ''periplasmic space'' in gram-negative bacteria. Using cryo-electron microscopy it has been found tha ...

space, allowing the toxin to be secreted from the

cytoplasm In cell biology, the cytoplasm is all of the material within a eukaryotic cell, enclosed by the cell membrane, except for the cell nucleus. The material inside the nucleus and contained within the nuclear membrane is termed the nucleoplasm. ...

straight outside the cell. A large proportion of the toxin remains associated with the bacterium exterior proteins, mainly filamentous haemagglutinin, but these toxin molecules are not active. Besides attachment to bacterial proteins,

aggregation Aggregation may refer to: Business and economics * Aggregation problem (economics) * Purchasing aggregation, the joining of multiple purchasers in a group purchasing organization to increase their buying power * Community Choice Aggregation, the ...

also inactivates the toxin. This quick inactivation highlights the necessity of close contact between secreting bacterium and target cell.

RTX toxins

RTX stands for 'repeats in toxins,' but not all members of the family are toxins. Repeating

aspartate Aspartic acid (symbol Asp or D; the ionic form is known as aspartate), is an α-amino acid that is used in the biosynthesis of proteins. Like all other amino acids, it contains an amino group and a carboxylic acid. Its α-amino group is in the pro ...

and

glycine Glycine (symbol Gly or G; ) is an amino acid that has a single hydrogen atom as its side chain. It is the simplest stable amino acid ( carbamic acid is unstable), with the chemical formula NH2‐ CH2‐ COOH. Glycine is one of the proteinog ...

rich nonapeptides (repeats 9 amino acids long) are a characteristic feature of this family of proteins, and are able to bind calcium

ion An ion () is an atom or molecule with a net electrical charge. The charge of an electron is considered to be negative by convention and this charge is equal and opposite to the charge of a proton, which is considered to be positive by conven ...

s. A feature of the RTX proteins is their ability to form pores in cell membranes, allowing ions to leak. This may manifest as a

hemolytic Hemolysis or haemolysis (), also known by several other names, is the rupturing (lysis) of red blood cells (erythrocytes) and the release of their contents (cytoplasm) into surrounding fluid (e.g. blood plasma). Hemolysis may occur in vivo o ...

activity on

erythrocytes Red blood cells (RBCs), also referred to as red cells, red blood corpuscles (in humans or other animals not having nucleus in red blood cells), haematids, erythroid cells or erythrocytes (from Greek ''erythros'' for "red" and ''kytos'' for "holl ...

, leading to this group of toxins being called 'hemolysins'. The cell types which are vulnerable to this pore-forming activity varies among the toxins. The acylation of lysines is required for the pore-forming

cytotoxic Cytotoxicity is the quality of being toxic to cells. Examples of toxic agents are an immune cell or some types of venom, e.g. from the puff adder (''Bitis arietans'') or brown recluse spider (''Loxosceles reclusa''). Cell physiology Treating ...

effects of all the RTX proteins. Toxins from many known

gram-negative Gram-negative bacteria are bacteria that do not retain the crystal violet stain used in the Gram staining method of bacterial differentiation. They are characterized by their cell envelopes, which are composed of a thin peptidoglycan cell wa ...

pathogenic bacteria are in the RTX family. An example is α-hemolysin from ''

Escherichia coli ''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus '' Esc ...

'' or RtxA from ''

Vibrio cholerae ''Vibrio cholerae'' is a species of Gram-negative, facultative anaerobe and comma-shaped bacteria. The bacteria naturally live in brackish or saltwater where they attach themselves easily to the chitin-containing shells of crabs, shrimps, and ot ...

.''

Function

Molecular mechanisms

Adenylate cyclase toxin binds to target cells by the

complement receptor 3 Macrophage-1 antigen (or integrin αMβ2 or macrophage integrin or Mac-1) is a complement receptor ("CR3") consisting of CD11b (integrin αM) and CD18 (integrin β2). The integrin α chain is noncovalently bound to the integrin β chain. It binds t ...

(CD11b/CD18, or Mac-1). Target cell are therefore myeloid lineage cells, mainly

phagocyte Phagocytes are cells that protect the body by ingesting harmful foreign particles, bacteria, and dead or dying cells. Their name comes from the Greek ', "to eat" or "devour", and "-cyte", the suffix in biology denoting "cell", from the Greek ...

s, such as

neutrophil Neutrophils (also known as neutrocytes or heterophils) are the most abundant type of granulocytes and make up 40% to 70% of all white blood cells in humans. They form an essential part of the innate immune system, with their functions varying in ...

s. Binding to cells without the CR3 also happens, but at a much lower rate. The portion responsible for binding to the receptor is inside the calcium binding repeats, from residues 1166 to 1287. The hemolysin portion of the protein then binds to the target membrane and inserts itself into the

bilayer A bilayer is a double layer of closely packed atoms or molecules. The properties of bilayers are often studied in condensed matter physics, particularly in the context of semiconductor devices, where two distinct materials are united to form junc ...

. The adenylate cyclase (AC) domain is then translocated across the cytoplasmic membrane into the cytoplasm. Translocation of the AC domain is independent of cytotoxic pore-forming activity, as these two activities require to toxin to adopt different conformations. The transiently opened pores do, however, contribute to AC domain function by potassium leakage and calcium influx into the target cell, which slows

endocytosis Endocytosis is a cellular process in which substances are brought into the cell. The material to be internalized is surrounded by an area of cell membrane, which then buds off inside the cell to form a vesicle containing the ingested material. E ...

of CR3/adenylate cyclase toxin clusters, also, the CR3/toxin complex is mobilized by detachment from the

cytoskeleton The cytoskeleton is a complex, dynamic network of interlinking protein filaments present in the cytoplasm of all cells, including those of bacteria and archaea. In eukaryotes, it extends from the cell nucleus to the cell membrane and is comp ...

. The complex is then recruited into

cholesterol Cholesterol is any of a class of certain organic molecules called lipids. It is a sterol (or modified steroid), a type of lipid. Cholesterol is biosynthesized by all animal cells and is an essential structural component of animal cell membr ...

-rich

lipid raft The cell membrane, plasma membranes of cells contain combinations of glycosphingolipids, cholesterol and protein Receptor (biochemistry), receptors organised in glycolipoprotein lipid microdomains termed lipid rafts. Their existence in cellular mem ...

s. Calcium influx by itself has many negative effects on target cells, such as deregulation of

cellular signalling In biology, cell signaling (cell signalling in British English) or cell communication is the ability of a cell to receive, process, and transmit signals with its environment and with itself. Cell signaling is a fundamental property of all cellular ...

. The adenylate cyclase domain has intrinsic enzymatic activity. Translocation of the AC domain into the cell starts the main process by which this toxin influences target cells: the AC domain binds

calmodulin Calmodulin (CaM) (an abbreviation for calcium-modulated protein) is a multifunctional intermediate calcium-binding messenger protein expressed in all eukaryotic cells. It is an intracellular target of the secondary messenger Ca2+, and the bi ...

, and

catalyzes Catalysis () is the process of increasing the rate of a chemical reaction by adding a substance known as a catalyst (). Catalysts are not consumed in the reaction and remain unchanged after it. If the reaction is rapid and the catalyst recycl ...

unregulated production of cAMP from

ATP ATP may refer to: Companies and organizations * Association of Tennis Professionals, men's professional tennis governing body * American Technical Publishers, employee-owned publishing company * ', a Danish pension * Armenia Tree Project, non ...

. cAMP is an important

second messenger Second messengers are intracellular signaling molecules released by the cell in response to exposure to extracellular signaling molecules—the first messengers. (Intercellular signals, a non-local form or cell signaling, encompassing both first m ...

molecule and its massive overproduction affects many cellular processes. In phagocytes, most of the

bactericidal A bactericide or bacteriocide, sometimes abbreviated Bcidal, is a substance which kills bacteria. Bactericides are disinfectants, antiseptics, or antibiotics. However, material surfaces can also have bactericidal properties based solely on their ...

functions are stopped by cAMP-mediated activation of

PKA PKA may refer to: * Professionally known as: ** Pen name ** Stage persona * p''K''a, the symbol for the acid dissociation constant at logarithmic scale * Protein kinase A, a class of cAMP-dependent enzymes * Pi Kappa Alpha, the North-American s ...

and Epac.

Effect on target cells

The above described effects of the adenylate cyclase toxin, mainly the cAMP overproduction, have a profound effect on target cells. Although phagocytic immune cells migrate to the site of

infection An infection is the invasion of tissues by pathogens, their multiplication, and the reaction of host tissues to the infectious agent and the toxins they produce. An infectious disease, also known as a transmissible disease or communicable d ...

in the lungs, they are not able to mount an effective response. Not only the phagocytic uptake of bacteria is blocked, but subsequent production of ROS by neutrophils and

monocyte Monocytes are a type of leukocyte or white blood cell. They are the largest type of leukocyte in blood and can differentiate into macrophages and conventional dendritic cells. As a part of the vertebrate innate immune system monocytes also i ...

s, NETs by neutrophils, and NO by macrophages, is also inhibited. The effect on neutrophils is most important in early infection with ''Bordetella'', impairing most of their

antimicrobial An antimicrobial is an agent that kills microorganisms or stops their growth. Antimicrobial medicines can be grouped according to the microorganisms they act primarily against. For example, antibiotics are used against bacteria, and antifungals ...

functions. Intoxication with the adenylate cyclase toxin leads to shift in

polarization Polarization or polarisation may refer to: Mathematics *Polarization of an Abelian variety, in the mathematics of complex manifolds *Polarization of an algebraic form, a technique for expressing a homogeneous polynomial in a simpler fashion by ...

of macrophages from M1 (

proinflammatory Inflammation (from la, inflammatio) is part of the complex biological response of body tissues to harmful stimuli, such as pathogens, damaged cells, or irritants, and is a protective response involving immune cells, blood vessels, and molec ...

) phenotype to M2 (immunoregulatory)

phenotype In genetics, the phenotype () is the set of observable characteristics or traits of an organism. The term covers the organism's morphology or physical form and structure, its developmental processes, its biochemical and physiological prop ...

and may lead to macrophage apoptosis. cAMP accumulation after adenylate cyclase intoxication also interferes with IRF signalling in

dendritic cell Dendritic cells (DCs) are antigen-presenting cells (also known as ''accessory cells'') of the mammalian immune system. Their main function is to process antigen material and present it on the cell surface to the T cells of the immune system. ...

s, which leads to lower IL-12 production. IL-12 is important for

T-cell A T cell is a type of lymphocyte. T cells are one of the important white blood cells of the immune system and play a central role in the adaptive immune response. T cells can be distinguished from other lymphocytes by the presence of a T-cell ...

response polarization. Other effects of cAMP on dendritic cell interaction with T-cells are also detrimental to the immune response. Although cAMP induces dendritic cell migration into

lymph node A lymph node, or lymph gland, is a kidney-shaped organ of the lymphatic system and the adaptive immune system. A large number of lymph nodes are linked throughout the body by the lymphatic vessels. They are major sites of lymphocytes that inc ...

s, it lowers their capacity to interact with T-cells and present antigen. This has a tolerogenic effect on the T-cell population.

Pertussis vaccination

Vaccination Vaccination is the administration of a vaccine to help the immune system develop immunity from a disease. Vaccines contain a microorganism or virus in a weakened, live or killed state, or proteins or toxins from the organism. In stimulating ...

against ''Bordetella pertussis'' is used in infancy to prevent whooping cough. The recent switch from whole-cell

pertussis vaccine Pertussis vaccine is a vaccine that protects against whooping cough (pertussis). There are two main types: whole-cell vaccines and acellular vaccines. The whole-cell vaccine is about 78% effective while the acellular vaccine is 71–85% effectiv ...

to acellular component vaccine in many countries has led to the fact that adenylate cyclase toxin is not present in most vaccines. Although not included in current vaccines, research shows that

immunization Immunization, or immunisation, is the process by which an individual's immune system becomes fortified against an infectious agent (known as the immunogen). When this system is exposed to molecules that are foreign to the body, called ''non-s ...

with adenylate cyclase toxin elicits neutralizing antibodies. Neutralizing antibodies can block binding of the toxin to CR3. Antibodies against adenylate cyclase toxin are also present in the serum of humans infected with ''B. pertussis.'' Adenylate cyclase toxin based constructs have been proven to elicit the production of neutralizing antibodies, but lack the cytotoxicity associated with the complete toxin. Genetically detoxified adenylate cyclase toxin also serves in promoting the Th1/Th17 response, acting as an

adjuvant In pharmacology, an adjuvant is a drug or other substance, or a combination of substances, that is used to increase the efficacy or potency of certain drugs. Specifically, the term can refer to: * Adjuvant therapy Adjuvant therapy, also known ...

Other roles for adenylate cyclase toxin

Adenylate cyclase toxin, or its parts, also serve as a tool for cell biologists. The AC domain finds use as a reporter protein. This reporter activity is based on activating cAMP production if translocated into a cell, conjugated to a studied protein. The AC domain consists of two subdomains, both are required for cAMP production. Conjugating each subdomain to a different protein allows protein-protein interactions to be studied, because cAMP production indicates close interaction of the proteins. Similarly, the two subdomains can be linked by a studied protein, which is then digested by

protease A protease (also called a peptidase, proteinase, or proteolytic enzyme) is an enzyme that catalyzes (increases reaction rate or "speeds up") proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the form ...

s. Loss of cAMP production indicates cleavage by protease.{{Cite journal, last1=Dautin, first1=N, last2=Karimova, first2=G, last3=Ladant, first3=D, date=October 2002, title=Bordetella pertussis adenylate cyclase toxin: a versatile screening tool, journal=Toxicon, language=en, volume=40, issue=10, pages=1383–1387, doi=10.1016/S0041-0101(02)00158-7, pmid=12368108

References

Bacterial toxins Virulence factors