RTX toxin

Last updated

The RTX toxin superfamily is a group of cytolysins and cytotoxins produced by bacteria. [1] There are over 1000 known members with a variety of functions. [2] The RTX family is defined by two common features: characteristic repeats in the toxin protein sequences, and extracellular secretion by the type I secretion systems (T1SS). The name RTX (repeats in toxin) refers to the glycine and aspartate-rich repeats located at the C-terminus of the toxin proteins, which facilitate export by a dedicated T1SS encoded within the rtx operon. [3] [4]

Contents

Structure and function

RTX proteins range from 40 to over 600 kDa in size and all contain C-terminally located glycine and aspartate-rich repeat sequences of nine amino acids. The repeats contain the common sequence structure [GGXGXDX[L/I/V/W/Y/F]X], (where X represents any amino acid), but the number of repeats varies within RTX protein family members. [5] These consensus regions function as sites for Ca2+ binding, which facilitate folding of the RTX protein following export via an ATP-mediated type 1 secretion system (T1SS). Most of the T1SS proteins are encoded within the rtx operon. The T1SS proteins form a continuous channel spanning both the inner membrane (IM) and outer membrane (OM) of the bacterial cell, preventing RTX toxin exposure to the periplasmic space (between the IM and OM). Type 1 secretion system components include: an ABC transporter (TC# 3.A.1), a membrane fusion protein (MFP; TC# 8.A.1), and an outer membrane protein (OMF; TC# 1.B.17). The OMF is often encoded outside of the rtx operon as it may have multiple functions within the cell. In Escherichia coli , Pasteurella haemolytica, and Vibrio cholerae , TolC functions as the OMP in T1SS RTX toxin export. In each case, the tolC gene is located outside the rtx operon and encodes a conserved multifunctional protein. During transport, the T1SS recognizes the C-terminal repeats of the RTX toxin, and the C-terminus is transferred first through the channel. [3]

The general rtx gene cluster encodes three protein types: the RTX toxin, an RTX activating acyltransferase, and T1SS proteins. The toxin is inactive until post-translational modification by the cis-encoded RTX toxin activator, which typically occurs within the target cell. The RTX-activating acyltransferase catalyzes the attachment of acyl-linked fatty acids to internally located lysine residues within the RTX toxin. This modification is required in all RTX toxins; however, its exact function in RTX toxicity is not understood. Members of the RTX toxin family display a large range of functions, and typically multiple functional domains. [3] Pore-formation is the only known shared function in RTX cytotoxins, and pores are typically cation-selective allowing for an influx of Ca2+ in target cells. [6]

Members of the RTX superfamily (RTX (TC# 1.C.11); HrpZ (TC# 1.C.56) and CCT (TC# 1.C.57)) contain repeat sequences that are also found in autotransporters (e.g., 1.B.12.10.1 and 1.B.40.1.2) as well as TolA (2.C.1.2.1). These domains probably mediate protein-protein interactions.

Families

The Transporter Classification Database divides the RTX-toxin superfamily into 3 different families of homologues based on bioinformatic and phylogenetic analysis: [7] [8]

RTX toxins were originally divided into hemolysins and leukotoxins. [1] However, evidence has shown leukotoxic activity in the hemolysins, leading to reclassification of RTX toxin subgroups into two families: pore-forming leukotoxins (RTX-toxin family, 1.C.11.1.1) and the MARTX toxins (CCT family, 1.C.57.3.4) (multifunctional autoprocessing RTX toxins). MARTX toxins are much larger than RTX toxins and are exported by modified type 1 secretion systems containing an additional ABC-transporter. [3] [9]

The Pore-forming RTX Toxin (RTX-toxin) Family

The RTX-toxin family (TC# 1.C.11) (subfamily of RTX-toxin superfamily) is a large family of multidomain Gram-negative bacterial pore-forming exotoxins. They are secreted from the bacteria, and after processing, they insert into the membranes of animal cells. They exert both cell type- and species-specific effects (e.g., the leukotoxin of M. haemolytica interacts only with alveolar macrophages, neutrophils, and lymphocytes of ruminants and is believed to promote bacterial proliferation by killing or incapacitating these cells). [10] These toxins recognize protein receptors such as the β2-integrins, form pores at high concentrations, and cause cell rupture by mechanisms not well understood. Three transmembrane domains are believed to be involved in pore formation which in the E. coli HlyA protein (TC# 1.C.11.1.3) are at residues 299-319, 361-381 and 383-403. However, at low, sublytic concentrations, leukotoxin (TC# 1.C.11.1.1) causes activation of neutrophils, production of inflammatory cytokines, degranulation, generation of oxygen-derived free radicals, and morphologic changes consistent with apoptosis.

The C-terminal domain of the adenylate cyclase toxin (ACT or CyaA; TC# 1.C.11.1.4) of Bordetella pertussis forms a small cation-selective channel, disrupting the permeability barrier. This channel probably does not deliver the N-terminal adenylate cyclase to the host cell cytoplasm. However, mutations in residues in an amphipathic α-helix (Glu509 and Glu516) in the pore-forming domain block adenylate cyclase translocation and modulate as well the cation selectivity of the membrane channel, as translocation and pore-forming activities employ the same structural elements of the toxin molecule in an alternative and mutually independent way. [11] ACT uses a CD11b/CD18 (complement receptor 3) beta2 integrin receptor on myeloid phagocytic cells [12] but with low efficacy it can penetrate also cells lacking this receptor or insert into liposomes. Phosphatidylethanolamine and cholesterol stimulate ACT insertion. ACT also promotes lipid flip-flop suggesting that ACT forms trans-bilayer nonlamellar lipid structures when it inserts into the membrane. [13] CyaA may form two different types of pore-like structures, dependent on the orientation of the membrane potential and the pH. [14]

Transport Reaction

The generalized transport reaction proposed for members of the RTX-toxin family is: [8]

small molecules (in) → small molecules (out).

Examples

RTX toxins are produced by a variety of gram-negative bacteria. RTX toxin production and rtxgenes have been discovered in many bacterial genera including Escherichia, Proteus, and Bordetella. Members of the family Pasteurellaceae also produce RTX toxins. [15] The genus Vibrio , which includes V. cholerae and V. vulnificus , produces MARTX toxins, another class of RTX proteins. [3]

In Escherichia coli

RTX toxins have been found in numerous strains of pathogenic E. coli. The prototypical RTX toxin, α-haemolysin (HlyA; TC# 1.C.11.1.3), is a common virulence factor in uropathogenic E. coli (UPEC), the leading cause of urinary tract infections. The hly operon encodes the RTX toxin (HlyA), the HlyA activation protein HlyC (an acyltransferase; TC# 9.A.40.1.1), and two proteins of the T1SS machinery. The Hyl T1SS includes the ABC transporter HlyB (TC# 3.A.1.109.1), the membrane fusion protein HlyD (TC# 8.A.1.3.1), and the outer membrane protein TolC (TC# 1.B.17.1.1). While hlyB and hlyD genes are located within the hly operon, TolC is a multifunctional protein encodedd outside the hly operon. [3]

Enterohaemorrhagic Escherichia coli (EHEC) also produces an RTX toxin. EHEC haemolysin (EHEC-Hly) was discovered in the EHEC serotype O157:H7. The EHEC-Hly operon contains four E. coli hly homologs: EHEC-hlyA, EHEC-hlyC, EHEC-hlyB, and EHEC-hlyD. Shiga toxins (Stx) are the primary virulence factors in enterohaemorrhagic E. coli but EHEC produces several other virulence factors capable of damaging the vascular endothelium in EHEC infections. EHEC-Hly is expressed in numerous EHEC serogroups known to cause severe infections in humans. EHEC-Hly is transported within EHEC-secreted outer membrane vesicles (OMVs) in vitro. This mode of transport increases virulence by aiding in EHEC-Hly delivery to target cells. [16]

In Vibrio cholerae

RTX toxins in Vibrio bacteria represent an early discovery in RTX toxin research, but were only recently discovered to belong to a separate class of RTX toxins called MARTX toxins. In Vibrio cholerae the martx gene encodes six proteins: the MARTX toxin (RtxA), an acyltransferase (RtxC), a membrane fusion protein (RtxD), two ABC-transporters (RtxB and RtxE), and one protein with unknown function. [3] RtxA is a virulence factor involved in cholera which facilitates colonization of V. cholerae the small intestine. RtxA causes destruction of the actin cytoskeleton in host cells through G-actin modification and destruction of Rho GTPases. The toxin contains four functional domains: an actin cross-linking domain (ACD), a Rho-inactivating domain (RID), a cysteine protease domain (CPD), and an αβ-hydrolase. In V. cholerae infection, the CPD binds to inositol hexakisphosphate (InsP6, Phytic acid) inside eukaryotic host cells. This binding activates the autoproteolytic CPD which cleaves the MARTX protein into smaller independent proteins each containing only one of the effector domains ACD, RID, and αβ-hydrolase. This allows each effector to act independently within the host cell, this increases the effects of RtxA because the ACD and RID function in different locations within the cell. ACD cross-links monomeric G-actin in the host cell cytosol, preventing formation of actin microfilament, a major component of the cytoskeleton. RID inactivates membrane bound Rho-GTPases, which are regulators of cytoskeleton formation. [17]

In Bordetella pertussis

Adenylate cyclase toxin (ACT or CyaA), is a primary virulence factor in Bordetella pertussis . CyaA is a multifunctional RTX family toxin that targets myeloid phagocytes, impairing the innate immune response and promoting B. pertussis colonization. The cyaA operon encodes the five proteins CyaA (RTX toxin), CyaC (CyaA activation protein), and the three T1SS proteins: CyaB (an ABC transporter) CyaD (a membrane fusion protein), and CyaE (an outer membrane protein). The CyaA protein contains an adenylate cyclase domain (AC domain) and a hemolytic/cytolytic domain. The hemolytic function forms pores in target cells, while the cell-invasive AC domain translocates across cellular membrane into cell cytosol. CyaA binds the αMβ2 integrin, inserts itself into the cell membrane and opens a transient path for influx of Ca2+ ions that activate calpain. This enables repositioning of the CyaA toxin within the cell membrane into membrane microdomains enriched with cholesterol (lipid rafts) and translocation of the AC domain across the membrane into cell cytosol is completed. Once the AC domain is internalized into cytosol it is activated by binding of calmodulin and catalyzes unregulated conversion of cytosolic ATP to cAMP, thus raising it to cytotoxic levels. [6]

Related Research Articles

Adenylyl cyclase Enzyme with key regulatory roles in most cells

Adenylyl cyclase is an enzyme with key regulatory roles in essentially all cells. It is the most polyphyletic known enzyme: six distinct classes have been described, all catalyzing the same reaction but representing unrelated gene families with no known sequence or structural homology. The best known class of adenylyl cyclases is class III or AC-III. AC-III occurs widely in eukaryotes and has important roles in many human tissues.

Cyclic adenosine monophosphate Cellular second messenger

Cyclic adenosine monophosphate is a second messenger important in many biological processes. cAMP is a derivative of adenosine triphosphate (ATP) and used for intracellular signal transduction in many different organisms, conveying the cAMP-dependent pathway.

Exotoxin Toxin from bacteria that destroys or disrupts cells

An exotoxin is a toxin secreted by bacteria. An exotoxin can cause damage to the host by destroying cells or disrupting normal cellular metabolism. They are highly potent and can cause major damage to the host. Exotoxins may be secreted, or, similar to endotoxins, may be released during lysis of the cell. Gram negative pathogens may secrete outer membrane vesicles containing lipopolysaccharide endotoxin and some virulence proteins in the bounding membrane along with some other toxins as intra-vesicular contents, thus adding a previously unforeseen dimension to the well-known eukaryote process of membrane vesicle trafficking, which is quite active at the host-pathogen interface.

Secretion Controlled release of substances by cells or tissues

Secretion is the movement of material from one point to another, such as a secreted chemical substance from a cell or gland. In contrast, excretion is the removal of certain substances or waste products from a cell or organism. The classical mechanism of cell secretion is via secretory portals at the plasma membrane called porosomes. Porosomes are permanent cup-shaped lipoprotein structures embedded in the cell membrane, where secretory vesicles transiently dock and fuse to release intra-vesicular contents from the cell.

<i>Bordetella</i> Genus of bacteria

Bordetella is a genus of small, 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 to culture. All species can infect humans. The first three species to be described ; are sometimes referred to as the 'classical species'. Two of these are also motile.

Pertussis toxin

Pertussis toxin (PT) is a protein-based AB5-type exotoxin produced by the bacterium Bordetella pertussis, which causes whooping cough. PT is involved in the colonization of the respiratory tract and the establishment of infection. Research suggests PT may have a therapeutic role in treating a number of common human ailments, including hypertension, viral infection, and autoimmunity.

Cholera toxin Protein complex secreted by the bacterium Vibrio cholerae

Cholera toxin is AB5 multimeric protein complex secreted by the bacterium Vibrio cholerae. CTX is responsible for the massive, watery diarrhea characteristic of cholera infection. It is a member of the Heat-labile enterotoxin family.

Adenylate cyclase toxin is a virulence factor produced by some members of the genus Bordetella. Together with the pertussis toxin it is the most important virulence factor of the causative agent of whooping cough, Bordetella pertussis. Bordetella bronchiseptica and Bordetella parapertussis, also able to cause pertussis-like symptoms, also produce adenylate cyclase toxin. It is a toxin secreted by the bacteria to influence the host immune system.

Cytolysin refers to the substance secreted by microorganisms, plants or animals that is specifically toxic to individual cells, in many cases causing their dissolution through lysis. Cytolysins that have a specific action for certain cells are named accordingly. For instance, the cytolysins responsible for the destruction of red blood cells, thereby liberating hemoglobins, are named hemolysins, and so on. Cytolysins may be involved in immunity as well as in venoms.

Pore-forming toxin

Pore-forming proteins are usually produced by bacteria, and include a number of protein exotoxins but may also be produced by other organisms such as earthworms, who produce lysenin. They are frequently cytotoxic, as they create unregulated pores in the membrane of targeted cells.

The AB5 toxins are six-component protein complexes secreted by certain pathogenic bacteria known to cause human diseases such as cholera, dysentery, and hemolytic–uremic syndrome. One component is known as the A subunit, and the remaining five components are B subunits. All of these toxins share a similar structure and mechanism for entering targeted host cells. The B subunit is responsible for binding to receptors to open up a pathway for the A subunit to enter the cell. The A subunit is then able to use its catalytic machinery to take over the host cell's regular functions.

Hemolysin Molecule destroying the membrane of red blood cells

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 acquisition, many hemolysins produced by pathogens do not cause significant destruction of red blood cells during infection. However, hemolysins are often capable of lysing red blood cells in vitro.

Microbial toxins are toxins produced by micro-organisms, including bacteria, fungi, protozoa, dinoflagellates, and viruses. Many microbial toxins promote infection and disease by directly damaging host tissues and by disabling the immune system. Endotoxins most commonly refer to the lipopolysaccharide (LPS) or lipooligosaccharide (LOS) that are in the outer plasma membrane of Gram-negative bacteria. The botulinum toxin, which is primarily produced by Clostridium botulinum and less frequently by other Clostridium species, is the most toxic substance known in the world. However, microbial toxins also have important uses in medical science and research. Currently, new methods of detecting bacterial toxins are being developed to better isolate and understand these toxin. Potential applications of toxin research include combating microbial virulence, the development of novel anticancer drugs and other medicines, and the use of toxins as tools in neurobiology and cellular biology.

Adenylate cyclase toxin (CyaA) is released from bacterium Bordetella pertussis by the T1SS and released in the host’s respiratory tract in order to suppress its early innate and subsequent adaptive immune defense.

Heat-labile enterotoxin family Family of toxic protein complexes

In molecular biology, the heat-labile enterotoxin family includes Escherichia coli heat-labile enterotoxin and cholera toxin (Ctx) secreted by Vibrio cholerae.

The CTXφ bacteriophage is a filamentous bacteriophage. It is a positive-strand DNA virus with single-stranded DNA (ssDNA).

The type VI secretion system (T6SS) is molecular machine used by a wide range of Gram-negative bacterial species to transport effectors from the interior of a bacterial cell across the cellular envelope into an adjacent target cell. While often reported that the T6SS was discovered in 2006 by researchers studying the causative agent of cholera, Vibrio cholerae, the first study demonstrating that T6SS genes encode a protein export apparatus was actually published in 2004, in a study of protein secretion by the fish pathogen Edwardsiella tarda.

The Clostridial Cytotoxin (CCT) Family is a member of the RTX-toxin superfamily. There are currently 13 classified members belonging to the CCT family. A representative list of these proteins is available in the Transporter Classification Database. Homologues are found in a variety of Gram-positive and Gram-negative bacteria.

Contact-dependent growth inhibition (CDI) is a phenomenon where a bacterial cell may deliver a polymorphic toxin molecule into neighbouring bacterial cells upon direct cell-cell contact, causing growth arrest or cell death.

Bacterial secretion system Protein complexes present on the cell membranes of bacteria for secretion of substances

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 to invade the host cells. They can be classified into different types based on their specific structure, composition and activity. Generally, proteins can be secreted through two different processes. One process is a one-step mechanism in which proteins from the cytoplasm of bacteria are transported and delivered directly through the cell membrane into the host cell. Another involves a two-step activity in which the proteins are first transported out of the inner cell membrane, then deposited in the periplasm, and finally through the outer cell membrane into the host cell.

References

  1. 1 2 Lally ET, Hill RB, Kieba IR, Korostoff J (1999), "The interaction between RTX toxins and target cells", Trends in Microbiology, 7 (9): 356–361, doi:10.1016/S0966-842X(99)01530-9, PMID   10470043
  2. ↑ Linhartová, Irena; Bumba, Ladislav; Mašín, Jiří; Basler, Marek; Osička, Radim; Kamanová, Jana; Procházková, Kateřina; Adkins, Irena; Hejnová-Holubová, Jana (2010-11-01). "RTX proteins: a highly diverse family secreted by a common mechanism". FEMS Microbiology Reviews. 34 (6): 1076–1112. doi:10.1111/j.1574-6976.2010.00231.x. ISSN   1574-6976. PMC   3034196 . PMID   20528947.
  3. 1 2 3 4 5 6 7 Linhartová I, Bumba L, Mašín J, Basler M, Osička R, Kamanová J, Procházková K, Adkins I, Hejnová-Holubová J, Sadílková L, Morová J, Sebo P (November 2010). "RTX proteins: a highly diverse family secreted by a common mechanism". FEMS Microbiol. Rev. 34 (6): 1076–112. doi:10.1111/j.1574-6976.2010.00231.x. PMC   3034196 . PMID   20528947.
  4. ↑ Vigil, Patrick D.; Travis J. Wiles; Michael D. Engstrom; Lev Prasov; Matthew A. Mulvey; Harry L. T. Mobley (February 2012). "The Repeat-In-Toxin Family Member TosA Mediates Adherence of Uropathogenic Escherichia coli and Survival during Bacteremia". Infection and Immunity. 80 (2): 493–505. doi:10.1128/IAI.05713-11. PMC   3264304 . PMID   22083710.
  5. ↑ Satchell, Karla J. Fullner (November 2007). "MARTX, Multifunctional Autoprocessing Repeats-in-Toxin Toxins". Infection and Immunity. 75 (11): 5079–5084. doi:10.1128/IAI.00525-07. PMC   2168290 . PMID   17646359.
  6. 1 2 Bumba, Ladislav; Jiri Masin; Radovan Fiser; Peter Sebo (2010). "Bordetella Adenylate Cyclase Toxin Mobilizes Its b2 Integrin Receptor into Lipid Rafts to Accomplish Translocation across Target Cell Membrane in Two Steps". PLOS Pathogens. 6 (5): e1000901. doi:10.1371/journal.ppat.1000901. PMC   2869314 . PMID   20485565.
  7. ↑ Chen, Jonathan S.; Reddy, Vamsee; Chen, Joshua H.; Shlykov, Maksim A.; Zheng, Wei Hao; Cho, Jaehoon; Yen, Ming Ren; Saier, Milton H. (2011-01-01). "Phylogenetic characterization of transport protein superfamilies: superiority of SuperfamilyTree programs over those based on multiple alignments". Journal of Molecular Microbiology and Biotechnology. 21 (3–4): 83–96. doi:10.1159/000334611. ISSN   1660-2412. PMC   3290041 . PMID   22286036.
  8. 1 2 Saier, MH Jr. "RTX-toxin Superfamily". Transporter Classification Database. Saier Lab Bioinformatics Group / SDSC.
  9. ↑ Boardman, Bethany Kay & Fullner Satchell, Karla J. (December 2004). "Vibrio cholerae Strains with Mutations in an Atypical Type I Secretion System Accumulate RTX Toxin Intracellularly". Journal of Bacteriology. 186 (23): 8137–8143. doi:10.1128/JB.186.23.8137-8143.2004. PMC   529086 . PMID   15547287.
  10. ↑ Davies, R. L.; Whittam, T. S.; Selander, R. K. (2001-02-01). "Sequence diversity and molecular evolution of the leukotoxin (lktA) gene in bovine and ovine strains of Mannheimia (Pasteurella) haemolytica". Journal of Bacteriology. 183 (4): 1394–1404. doi:10.1128/JB.183.4.1394-1404.2001. ISSN   0021-9193. PMC   95014 . PMID   11157953.
  11. ↑ Osicková, A.; Osicka, R.; Maier, E.; Benz, R.; Sebo, P. (1999-12-31). "An amphipathic alpha-helix including glutamates 509 and 516 is crucial for membrane translocation of adenylate cyclase toxin and modulates formation and cation selectivity of its membrane channels". The Journal of Biological Chemistry. 274 (53): 37644–37650. doi: 10.1016/S0021-9258(19)52940-4 . ISSN   0021-9258. PMID   10608820.
  12. ↑ Guermonprez, P.; Khelef, N.; Blouin, E.; Rieu, P.; Ricciardi-Castagnoli, P.; Guiso, N.; Ladant, D.; Leclerc, C. (2001-05-07). "The Adenylate Cyclase Toxin of Bordetella pertussis Binds to Target Cells via the αMβ2 Integrin (Cd11b/Cd18)". Journal of Experimental Medicine. 193 (9): 1035–1044. doi:10.1084/jem.193.9.1035. ISSN   0022-1007. PMC   2193436 . PMID   11342588.
  13. ↑ Martín, César; Requero, M.-Asunción; Masin, Jiri; Konopasek, Ivo; Goñi, Félix M.; Sebo, Peter; Ostolaza, Helena (2004-06-01). "Membrane restructuring by Bordetella pertussis adenylate cyclase toxin, a member of the RTX toxin family". Journal of Bacteriology. 186 (12): 3760–3765. doi:10.1128/JB.186.12.3760-3765.2004. ISSN   0021-9193. PMC   419970 . PMID   15175289.
  14. ↑ Knapp, Oliver; Maier, Elke; Masín, Jirí; Sebo, Peter; Benz, Roland (2008-01-01). "Pore formation by the Bordetella adenylate cyclase toxin in lipid bilayer membranes: role of voltage and pH". Biochimica et Biophysica Acta (BBA) - Biomembranes. 1778 (1): 260–269. doi: 10.1016/j.bbamem.2007.09.026 . ISSN   0006-3002. PMID   17976530.
  15. ↑ Frey J (November 2011). "The role of RTX toxins in host specificity of animal pathogenic Pasteurellaceae". Veterinary Microbiology. 153 (1–2): 51–58. doi:10.1016/j.vetmic.2011.05.018. PMID   21645978.
  16. ↑ Aldick, Thomas; Bielaszewska, Martina; Uhlin, Bernt Eric; Humpf, Hans-Ulrich; Wai, Sun Nyunt; Karch, Helge (2009). "Vesicular stabilization and activity augmentation of enterohaemorrhagic Escherichia coli haemolysin". Molecular Microbiology. 71 (6): 1496–1508. doi: 10.1111/j.1365-2958.2009.06618.x . PMID   19210618.
  17. ↑ Prochazkova, Katerina; Ludmilla A. Shuvalova; George Minasov; Zdenek Voburka; Wayne F. Anderson; Karla J. F. Satchell (September 2009). "Structural and Molecular Mechanism for Autoprocessing of MARTX Toxin of Vibrio cholerae at Multiple Sites". Journal of Biological Chemistry. 284 (39): 26557–26568. doi: 10.1074/jbc.M109.025510 . PMC   2785344 . PMID   19620709.
This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.