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The RTX toxin superfamily is a group of cytolysins and
cytotoxins 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 cell ...
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 sequence Protein primary structure is the linear sequence of amino acids in a peptide or protein. By convention, the primary structure of a protein is reported starting from the amino-terminal (N) end to the carboxyl-terminal (C) end. Protein biosynthesi ...
s, and extracellular secretion by the type I secretion systems (T1SS). The name RTX (repeats in toxin) refers to the
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 proteinogeni ...
and
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 ...
-rich repeats located at 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 is ...
of the toxin proteins, which facilitate export by a dedicated T1SS encoded within the ''rtx''
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 ...
.


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 acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha am ...
s. The repeats contain the common sequence structure , (where X represents any amino acid), but the number of repeats varies within RTX protein family members. These consensus regions function as sites for Ca2+ binding, which facilitate folding of the RTX protein following
export An export in international trade is a good produced in one country that is sold into another country or a service provided in one country for a national or resident of another country. The seller of such goods or the service provider is an ...
via an ATP-mediated type 1 secretion system (T1SS). Most of the T1SS proteins are encoded within the ''rtx''
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 ...
. 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 The ATP-binding cassette transporters (ABC transporters) are a transport system superfamily that is one of the largest and possibly one of the oldest gene families. It is represented in all extant phyla, from prokaryotes to humans. ABC transp ...
(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 ''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 ''Escher ...
'', ''
Pasteurella __NOTOC__ ''Pasteurella'' is a genus of Gram-negative, facultatively anaerobic bacteria. ''Pasteurella'' species are nonmotile and pleomorphic, and often exhibit bipolar staining ("safety pin" appearance). Most species are catalase- and oxidase-p ...
'' ''haemolytica'', and ''
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 oth ...
'', 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. The general ''rtx'' gene cluster encodes three protein types: the RTX toxin, an RTX activating
acyltransferase Acyltransferase is a type of transferase enzyme that acts upon acyl groups. Examples include: * Glyceronephosphate O-acyltransferase * Lecithin-cholesterol acyltransferase *Long-chain-alcohol O-fatty-acyltransferase In enzymology, a long-chain- ...
, and T1SS proteins. The toxin is inactive until
post-translational modification Post-translational modification (PTM) is the covalent and generally enzymatic modification of proteins following protein biosynthesis. This process occurs in the endoplasmic reticulum and the golgi apparatus. Proteins are synthesized by ribosome ...
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. 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. 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

Th
Transporter Classification Database
divides th
RTX-toxin superfamily
into 3 different families of homologues based on bioinformatic and phylogenetic analysis:
1.C.11
- The Pore-forming RTX Toxin (RTX-toxin) Family
1.C.56
- The Pseudomanas syringae HrpZ Cation Channel (HrpZ) Family
1.C.57
- The Clostridial Cytotoxin (CCT) Family RTX toxins were originally divided into
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 ...
s and leukotoxins. However, evidence has shown leukotoxic activity in the hemolysins, leading to reclassification of RTX toxin subgroups into two families: pore-forming leukotoxins
RTX-toxin family1.C.11.1.1
and the MARTX toxins
CCT family1.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 The ATP-binding cassette transporters (ABC transporters) are a transport system superfamily that is one of the largest and possibly one of the oldest gene families. It is represented in all extant phyla, from prokaryotes to humans. ABC transp ...
.


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). 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 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 ...
, production of inflammatory cytokines, degranulation, generation of oxygen-derived free radicals, and morphologic changes consistent with
apoptosis Apoptosis (from grc, ἀπόπτωσις, apóptōsis, 'falling off') is a form of programmed cell death that occurs in multicellular organisms. Biochemical events lead to characteristic cell changes (morphology) and death. These changes incl ...
. The C-terminal domain of the adenylate cyclase toxin (ACT or CyaA; TC
1.C.11.1.4
of ''
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 expres ...
'' 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. ACT uses a CD11b/CD18 (complement receptor 3) beta2 integrin receptor on myeloid phagocytic cells 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. CyaA may form two different types of pore-like structures, dependent on the orientation of the membrane potential and the pH.


Transport Reaction

The generalized transport reaction proposed for members of the RTX-toxin family is: :small molecules (in) → small molecules (out).


Examples

RTX toxins are produced by a variety of gram-negative bacteria. RTX toxin production and ''rtx'' ''genes'' have been discovered in many bacterial genera including ''Escherichia'', ''Proteus'', and ''Bordetella''. Members of the family ''
Pasteurellaceae The Pasteurellaceae comprise a large family of Gram-negative bacteria. Most members live as commensals on mucosal surfaces of birds and mammals, especially in the upper respiratory tract. Pasteurellaceae are typically rod-shaped, and are a notabl ...
'' also produce RTX toxins. The genus ''
Vibrio ''Vibrio'' is a genus of Gram-negative bacteria, possessing a curved-rod (comma) shape, several species of which can cause foodborne infection, usually associated with eating undercooked seafood. Being highly salt tolerant and unable to survive ...
'', which includes '' V. cholerae'' and '' V. vulnificus'', produces MARTX toxins, another class of RTX proteins.


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 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 following ...
in uropathogenic ''E. coli'' (UPEC), the leading cause of
urinary tract infection A urinary tract infection (UTI) is an infection that affects part of the urinary tract. When it affects the lower urinary tract it is known as a bladder infection (cystitis) and when it affects the upper urinary tract it is known as a kidney ...
s. The hly operon encodes the RTX toxin (HlyA), the HlyA activation protein HlyC (an
acyltransferase Acyltransferase is a type of transferase enzyme that acts upon acyl groups. Examples include: * Glyceronephosphate O-acyltransferase * Lecithin-cholesterol acyltransferase *Long-chain-alcohol O-fatty-acyltransferase In enzymology, a long-chain- ...

TC# 9.A.40.1.1
, and two proteins of the
T1SS 440px 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 ...
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.
Enterohaemorrhagic Shigatoxigenic ''Escherichia coli'' (STEC) and verotoxigenic ''E. coli'' (VTEC) are strains of the bacterium ''Escherichia coli'' that produce Shiga toxin (or verotoxin). Only a minority of the strains cause illness in humans. The ones that do ...
''Escherichia coli'' (EHEC) also produces an RTX toxin. EHEC haemolysin (EHEC-Hly) was discovered in the EHEC
serotype A serotype or serovar is a distinct variation within a species of bacteria or virus or among immune cells of different individuals. These microorganisms, viruses, or cells are classified together based on their surface antigens, allowing the epi ...
O157:H7. The EHEC-Hly operon contains four ''E. coli hly''
homologs A couple of homologous chromosomes, or homologs, are a set of one maternal and one paternal chromosome that pair up with each other inside a cell during fertilization. Homologs have the same genes in the same locus (genetics), loci where they pr ...
: EHEC-hlyA, EHEC-hlyC, EHEC-hlyB, and EHEC-hlyD.
Shiga toxins Shiga toxins are a family of related toxins with two major groups, Stx1 and Stx2, expressed by genes considered to be part of the genome of lambdoid prophages. The toxins are named after Kiyoshi Shiga, who first described the bacterial or ...
(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.


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 ''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 oth ...
'' 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. RtxA is a virulence factor involved in cholera which facilitates colonization of ''V. cholerae'' the small intestine. RtxA causes destruction of the actin
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 compos ...
in host cells through G-actin modification and destruction of
Rho Rho (uppercase Ρ, lowercase ρ or ; el, ρο or el, ρω, label=none) is the 17th letter of the Greek alphabet. In the system of Greek numerals it has a value of 100. It is derived from Phoenician letter res . Its uppercase form uses the sa ...
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.


In ''Bordetella pertussis''

Adenylate cyclase toxin (ACT or CyaA), is a primary virulence factor in ''
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 expres ...
''. 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.


References

{{reflist Bacterial toxins