cytolethal distending toxin
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Cytolethal distending toxins (abbreviated CDTs) are a class of hetero trimeric toxins produced by certain
gram-negative bacteria 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 wall ...
that display
DNase Deoxyribonuclease (DNase, for short) refers to a group of glycoprotein endonucleases which are enzymes that catalyze the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA. The role of the DNase enzyme in cells ...
activity. These toxins trigger G2/M
cell cycle The cell cycle, or cell-division cycle, is the series of events that take place in a cell that cause it to divide into two daughter cells. These events include the duplication of its DNA (DNA replication) and some of its organelles, and sub ...
arrest in specific mammalian cell lines, leading to the enlarged or distended cells for which these toxins are named. Affected cells die by apoptosis. Each toxin consists of three distinct subunits named alphabetically in the order that their coding genes appear in the ''cdt
operon In genetics, an operon is a functioning unit of DNA containing a cluster of genes under the control of a single promoter. The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo splic ...
''. Cytolethal distending toxins are classified as
AB toxins The AB toxins are two-component protein complexes secreted by a number of pathogenic bacteria, though there is a pore-forming AB toxin found the eggs of a snail. They can be classified as Type III toxins because they interfere with internal cell ...
, with an active ("A") subunit that directly damages DNA and a binding ("B") subunit that helps the toxin attach to the target cells. CdtB is the active subunit and a
homolog In biology, homology is similarity due to shared ancestry between a pair of structures or genes in different taxa. A common example of homologous structures is the forelimbs of vertebrates, where the wings of bats and birds, the arms of prima ...
to mammalian
DNase I Deoxyribonuclease I (usually called DNase I), is an endonuclease of the DNase family coded by the human gene DNASE1. DNase I is a nuclease that cleaves DNA preferentially at phosphodiester linkages adjacent to a pyrimidine nucleotide, yielding ...
, whereas CdtA and CdtC make up the binding subunit. Cytolethal distending toxins are produced by
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 from the phylum '' Pseudomonadota''. Many of these bacteria, including ''
Shigella dysenteriae ''Shigella dysenteriae'' is a species of the rod-shaped bacterial genus ''Shigella''. ''Shigella'' species can cause shigellosis ( bacillary dysentery). Shigellae are Gram-negative, non-spore-forming, facultatively anaerobic, nonmotile bacteri ...
'', ''
Haemophilus ducreyi ''Haemophilus ducreyi'' is a fastidious gram-negative coccobacillus bacteria. It causes the sexually transmitted disease chancroid, a major cause of genital ulceration in developing countries characterized by painful sores on the genitalia. Ch ...
'', and ''
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 ...
'', infect humans. Bacteria that produce CDTs often persistently colonize their host.


History

The first recorded observation of a cytolethal-distending toxin was in 1987 in a pathogenic strain in '' E. coli'' isolated from a young patient. Later that year, scientists W.M. Johnson and H. Lior published the journal article "Production of Shiga toxin and a cytolethal distending toxin (CLDT) by serogroups of ''
Shigella ''Shigella'' is a genus of bacteria that is Gram-negative, facultative anaerobic, non-spore-forming, nonmotile, rod-shaped, and genetically closely related to '' E. coli''. The genus is named after Kiyoshi Shiga, who first discovered it in 1 ...
spp.''" in ''Microbiology Letters''. The discovery of other bacteria producing CDT toxins continues to this day. In 1994 Scott and Kaper cloned and sequenced a ''cdt operon'' from another ''E. coli'' strain, publishing in ''Infection and Immunity''. The three genes discovered were denoted ''cdtA'', ''cdtB'', and ''cdtC''. In 1997, the first paper of many to show G2/M cell cycle arrest caused by a cytolethal distending toxin was published in '' Molecular Microbiology''. The study focused on another ''E. coli'' strain. This paper was followed by a 1999 publication in ''Infectious Immunity'', which demonstrated that '' H. ducreyi'' CDT causes cell death via apoptosis. This finding was also confirmed for other cytolethal distending toxins in subsequent studies. The discovery of the
homology Homology may refer to: Sciences Biology *Homology (biology), any characteristic of biological organisms that is derived from a common ancestor * Sequence homology, biological homology between DNA, RNA, or protein sequences *Homologous chrom ...
of ''cdtB'' to mammalian
DNase I Deoxyribonuclease I (usually called DNase I), is an endonuclease of the DNase family coded by the human gene DNASE1. DNase I is a nuclease that cleaves DNA preferentially at phosphodiester linkages adjacent to a pyrimidine nucleotide, yielding ...
and the current AB model for the toxin were published in early 2000. Further research and the publication of crystal structures for the CDT toxins from two different species continues to support this model.


Sources

All known cytolethal distending toxins are produced by gram-negative bacteria in the '' Gammaproteobacteria'' and ''
Campylobacterota Campylobacterota are a phylum of bacteria. All species of this phylum are Gram-negative. The Campylobacterota consist of few known genera, mainly the curved to spirilloid ''Wolinella'' spp., ''Helicobacter'' spp., and ''Campylobacter'' spp. Most ...
''. In several cases, the bacteria producing CDT are human
pathogens In biology, a pathogen ( el, πάθος, "suffering", "passion" and , "producer of") in the oldest and broadest sense, is any organism or agent that can produce disease. A pathogen may also be referred to as an infectious agent, or simply a ger ...
. Medically important CDT producers include: *''
Haemophilus ducreyi ''Haemophilus ducreyi'' is a fastidious gram-negative coccobacillus bacteria. It causes the sexually transmitted disease chancroid, a major cause of genital ulceration in developing countries characterized by painful sores on the genitalia. Ch ...
'' ( chancroids) *''
Aggregatibacter actinomycetemcomitans ''Aggregatibacter actinomycetemcomitans'' is a Gram-negative, facultative anaerobe, nonmotile bacterium that is often found in association with localized aggressive periodontitis, a severe infection of the periodontium. It is also suspected to b ...
'' (
periodontitis Periodontal disease, also known as gum disease, is a set of inflammatory conditions affecting the tissues surrounding the teeth. In its early stage, called gingivitis, the gums become swollen and red and may bleed. It is considered the main cau ...
) *''
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 ...
'' (various diseases) *''
Shigella dysenteriae ''Shigella dysenteriae'' is a species of the rod-shaped bacterial genus ''Shigella''. ''Shigella'' species can cause shigellosis ( bacillary dysentery). Shigellae are Gram-negative, non-spore-forming, facultatively anaerobic, nonmotile bacteri ...
'' (
dysentery Dysentery (UK pronunciation: , US: ), historically known as the bloody flux, is a type of gastroenteritis that results in bloody diarrhea. Other symptoms may include fever, abdominal pain, and a feeling of incomplete defecation. Complications ...
) *''
Salmonella enterica ''Salmonella enterica'' (formerly ''Salmonella choleraesuis'') is a rod-headed, flagellate, facultative anaerobic, Gram-negative bacterium and a species of the genus ''Salmonella''. A number of its serovars are serious human pathogens. Epidemi ...
'' serotype Typhi (
typhoid fever Typhoid fever, also known as typhoid, is a disease caused by '' Salmonella'' serotype Typhi bacteria. Symptoms vary from mild to severe, and usually begin six to 30 days after exposure. Often there is a gradual onset of a high fever over several ...
) *'' Campylobacter upsaliensis'' (
enterocolitis Enterocolitis is an inflammation of the digestive tract, involving enteritis of the small intestine and colitis of the colon. It may be caused by various infections, with bacteria, viruses, fungi, parasites, or other causes. Common clinical manif ...
) *''
Campylobacter jejuni ''Campylobacter jejuni'' () is a species of pathogenic bacteria, one of the most common causes of food poisoning in Europe and in the US. The vast majority of cases occur as isolated events, not as part of recognized outbreaks. Active surveillan ...
'' (enterocolitis) CDT-producing bacteria are often associated with mucosal linings, such as those in the stomach and intestines, and with persistent infections. The toxins are either secreted freely or associated with the membrane of the producing bacteria.


Nomenclature

Individual cytolethal distending toxins are named for the bacterial species that they are isolated from. As of 2011, most scientists have adopted the practice of placing the first letter of both the genus and species in front of the toxin name to reflect its source (i.e., the CDT from ''Haemaphilus ducreyi'' is referred to as HdCDT). If several subspecies produce different toxins, as in the case of ''E. coli'', Roman numerals may be added after the second letter. Both complete toxins and individual subunits are labeled using this convention. In response to the continued discovery of additional cytolethal distending toxins, a 2011 review has proposed that the toxin names be expanded to include the first three letters of the species (i.e., HducCDT for ''Haemaphilus ducreyi'' CDT).


Cellular effects

CDT toxins are
genotoxins Genotoxicity is the property of chemical agents that damage the genetic information within a cell causing mutations, which may lead to cancer. While genotoxicity is often confused with mutagenicity, all mutagens are genotoxic, but some genotoxic su ...
capable of directly damaging DNA in target cells. They are the only AB-type toxins discovered that display
DNase Deoxyribonuclease (DNase, for short) refers to a group of glycoprotein endonucleases which are enzymes that catalyze the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA. The role of the DNase enzyme in cells ...
activity, allowing them to introduce breaks into the target cell's DNA. In many
cell lines An immortalised cell line is a population of cells from a multicellular organism which would normally not proliferate indefinitely but, due to mutation, have evaded normal cellular senescence and instead can keep undergoing division. The cells ...
including human
fibroblasts A fibroblast is a type of biological cell that synthesizes the extracellular matrix and collagen, produces the structural framework ( stroma) for animal tissues, and plays a critical role in wound healing. Fibroblasts are the most common cells ...
,
epithelial cells Epithelium or epithelial tissue is one of the four basic types of animal tissue, along with connective tissue, muscle tissue and nervous tissue. It is a thin, continuous, protective layer of compactly packed cells with a little intercell ...
,
endothelial cells The endothelium is a single layer of squamous endothelial cells that line the interior surface of blood vessels and lymphatic vessels. The endothelium forms an interface between circulating blood or lymph in the lumen and the rest of the vessel ...
, and
keratinocytes Keratinocytes are the primary type of cell found in the epidermis, the outermost layer of the skin. In humans, they constitute 90% of epidermal skin cells. Basal cells in the basal layer (''stratum basale'') of the skin are sometimes referre ...
, CDTs cause G2/M
cell cycle The cell cycle, or cell-division cycle, is the series of events that take place in a cell that cause it to divide into two daughter cells. These events include the duplication of its DNA (DNA replication) and some of its organelles, and sub ...
arrest, cytoplasmic distension, and eventual cell death via apoptosis. Most publications attribute the G2/M cycle arrest to the buildup of irreversible DNA damage from the toxin's DNase activity as the trigger for the G2/M cell cycle arrest, but other research suggests that this model is incomplete. The cytoplasmic distension is a direct result of the G2/M cell cycle arrest. The cell enlarges in preparation for mitosis, but cannot divide to restore its normal size. Aside from classical apoptosis, signs of cellular senescence has also been observed in normal and cancer cell lines (fibroblasts, HeLa and U2-OS) after CDT intoxication In lymphocytes, cell death occurs quickly and is not preceded by significant cytoplasmic distension. The ability of these toxins to effect lymphocytes differently may be advantageous to the bacteria that utilize these toxins, but the mechanism behind this phenomenon is not yet well understood.


Toxin structure

The active, assembled toxin is a
tripartite Tripartite means composed of or split into three parts, or refers to three parties. Specifically, it may also refer to any of the following: * 3 (number) * Tripartite language * Tripartite motto * Tripartite System in British education * Triparti ...
structure with three distinct subunits- CdtA, CdtB, and CdtC. In terms of function, it is an
AB toxin The AB toxins are two-component protein complexes secreted by a number of pathogenic bacteria, though there is a pore-forming AB toxin found the eggs of a snail. They can be classified as Type III toxins because they interfere with internal cell ...
. In this context, the CdtB subunit is actually the catalytically active "A" subunit, and the CdtA and CdtC together form the binding "B" subunit, which helps the toxin bind and enter target cells. Some literature refers to the toxin structure as AB2 to reflect the presence of both CdtA and CdtC. Different from all other CDTs, ''Salmonella enterica'' serovar Typhi CDT (SeCDT) has no CdtA and CdtC homologues. However, encoded closely to the active subunit ''cdtb'', the Pertussis-like toxin A and B (pltA/pltB) have been shown to be essential for cellular intoxication. PltA and PltB have a different structure from CdtA and CdtC, thus promoting CdtB activity in a different way. Both PltA and PltB have been found to bind directly to CdtB ''in vitro''. In addition, different from all other CDTs, ''Salmonella'' genotoxin is produced only upon bacterial internalization in infected cells, thus the SeCDT traffic may differ remarkably from the canonical ones.


CdtB

CdtB is considered the active subunit of the CDT holotoxin. Microinjection of CdtB into susceptible cells without CdtA or CdtC results in the G2/M cell cycle arrest and cytoplasmic distension characteristic of CDT toxins. The structure of CdtB is well-conserved between different bacteria. The CdtB subunit is the most sequentially conserved between species. The molecular weight of CdtB ranges from 28 kDa to 29 kDa, depending on the species. As the active subunit, CdtB is termed the "A" subunit according to the
AB toxin The AB toxins are two-component protein complexes secreted by a number of pathogenic bacteria, though there is a pore-forming AB toxin found the eggs of a snail. They can be classified as Type III toxins because they interfere with internal cell ...
model. This confusing nomenclature is due to the naming of the toxin's subunits before their individual functions were understood.


Activity

CdtB exhibits at least two enzymatic activities-
DNase Deoxyribonuclease (DNase, for short) refers to a group of glycoprotein endonucleases which are enzymes that catalyze the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA. The role of the DNase enzyme in cells ...
activity capable of introducing double-strand breaks in DNA, and a phosphatase activity that resembles phosphatidylinositol 3,4,5-triphosphatase. Both activities can be demonstrated ''
in vitro ''In vitro'' (meaning in glass, or ''in the glass'') studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called " test-tube experiments", these studies in biology ...
'' in the absence of the other two subunits. The relative importance of each activity ''
in vivo Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and ...
'' is unclear. Mutations that reduce either activity also reduce the toxin's ability to induce G2/M phase arrest in at least some of the susceptible
cell lines An immortalised cell line is a population of cells from a multicellular organism which would normally not proliferate indefinitely but, due to mutation, have evaded normal cellular senescence and instead can keep undergoing division. The cells ...
.


Similarities to mammalian DNase I

CdtB is functionally homologous to mammalian
DNase I Deoxyribonuclease I (usually called DNase I), is an endonuclease of the DNase family coded by the human gene DNASE1. DNase I is a nuclease that cleaves DNA preferentially at phosphodiester linkages adjacent to a pyrimidine nucleotide, yielding ...
and contains a conserved penta
peptide Peptides (, ) are short chains of amino acids linked by peptide bonds. Long chains of amino acids are called proteins. Chains of fewer than twenty amino acids are called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides. ...
sequence found in all DNase I enzymes to date. In addition, several residues critical to DNase I's ability to break the
phosphodiester bonds In chemistry, a phosphodiester bond occurs when exactly two of the hydroxyl groups () in phosphoric acid react with hydroxyl groups on other molecules to form two ester bonds. The "bond" involves this linkage . Discussion of phosphodiesters is ...
in the DNA backbone are found in the CdtB structure. A 2002 paper studying the effect of
point mutations A point mutation is a genetic mutation where a single nucleotide base is changed, inserted or deleted from a DNA or RNA sequence of an organism's genome. Point mutations have a variety of effects on the downstream protein product—consequence ...
on five of these residues found that four of the five mutations tested abolished both CdtB's ability to degrade DNA in cell-free extracts and to cause G2/M arrest upon microinjection. The fifth mutation moderately reduced CdtB's activity.


CdtA and CdtC

CdtA and CdtC make up the B subunit of the CDT holotoxin responsible for targeting the CdtB against susceptible cells. Neither subunit appears highly conserved, with sequence identities between different species often lower than 30%. The molecular weight of CdtA ranges from 23 kDa to 30 kDa, whereas CdtC ranges from 19 kDa to 21 kDa depending on the species.


Activity

CdtA and CdtC are both believed to bind to the surface of target cells. The exact mechanism of this binding is unclear, and may not be conserved between CDT toxins from different species. Proposed targets of CdtA and CdtC binding have included cholesterol, N-linked glycans, and glycosphingolipids. Current research has produced conflicting results on the actual importance of these proposed targets. Both CdtA and CdtC contain lectin domains, suggesting that the toxin may bind via carbohydrates on the target cell's surface, whereas other research has suggested that the targets are surface proteins.


Notes

{{Reflist Toxins