Actin assembly-inducing protein
Encyclopedia
The Actin assembly-inducing protein (ActA) is a protein
encoded and used by Listeria monocytogenes to propel itself through a mammalian host cell. ActA is a bacterial surface protein comprising a membrane-spanning region. In a mammalian cell the bacterial ActA interacts with the Arp2/3 complex
and actin
monomers to induce actin polymerization on the bacterial surface generating an actin comet tail. The gene encoding ActA is named actA or prtB.
cells and rapidly try to escape their internalization vacuole. In the cytosol they start to polymerize actin on their surface by the help of the ActA protein. It has been shown that ActA is not only necessary but also sufficient to induce motility of bacteria in the absence of other bacterial factors.
-negative Tn917-lac Listeria mutants because of the phenotype that they were unable to spread from cell to cell. These mutant bacteria still escaped from the phagosomes as efficiently as wild-type bacteria and multiplied within the infected cells but they were not surrounded by actin like wild-type bacteria. Further analysis showed, that Tn917-lac had inserted into actA, the second gene of an operon. The third gene of this operon, plcB, encodes the L. monocytogenes lecithinase. To determine whether actA itself, plcB or other co-transcribed downstream regions are involved in actin assembly, mutations in the appropriate genes were generated. All mutants except the actA mutants were similar to wild-type concerning association with F-actin and cell-cell spreading. Complementation with actA restored wild-type phenotype in the actA mutants.
(WASP), a nucleation promoting factor (NPF) present in host cells. NPFs in general recruit and bind to the already in the mammalian cell existing actin-related-protein 2 and 3 complex
(Arp2/3 complex) and induce an activating conformational change of the Arp2/3 complex. Due to this conformational change, NPFs initiate polymerization of a new actin filament at a 70° angle, which leads to the characteristic Y-branched actin structures in the leading edge of motile cells. ActA localizes to the old pole of the bacterium and spans both the bacterial cell membrane and the cell wall, lateral diffusion is inhibited; thus ActA localizes in a polarized and anchored manner on the bacterial surface. Consequently actin polymerization only starts in this region on the surface of the bacterium. Expression of ActA is induced only after entering a mammalian host cell.
Actin filament assembly generates the force that pushes the bacterium in the mammalian host cytoplasm forward. Continuous actin polymerization is sufficient for motility in the cytoplasm and even for infection of adjacent cells.
.
The ActA chain can be divided into three functional domains (Fig. 2):
The N-terminal portion of ActA plays an important role in actin polymerization. The domain displays consensus elements present in eukaryotic WASP family NPFs which include an actin monomer-binding region as well as an Arp2/3 binding C (central or cofilin homology) and A (acidic) region. The actin monomer-binding region of ActA has functional properties like the WASP-Homology-2 (WH2) or V domain, but differs in the sequence. Thus in WASP-family NPFs the order of the domains is WH2 followed by C,and then by A, which is not the case in ActA.
, vinculin
and palladin
, known to associate with focal adhesion
s or stress fibers. The vasodilator-stimulated phosphoprotein (VASP
) can bind through its Ena/VASP homology 1 domain (EVH1 domain
) to the central proline-rich region and recruits profilin
, an actin monomer binding protein, which itself promotes polymerization at barbed ends of actin filaments. Furthermore, VASP seems to interact with F-actin through its carboxy-terminal EVH2 domain, which provides a linkage of the bacterium to the tail. This statement is supported by the fact that ActA can bind multiple Ena/VASP proteins simultaneously and has a high affinity between ActA and Ena/VASP. VASP has been shown to reduce the frequency actin-Y-branches in vitro and thus increases the proportion of filaments which are organized in a parallel alignment in comet tails.
In summary, besides
, a Rho-family small GTPase, WASP/N-WASP activates the Arp2/3 complex, which leads to rapid actin polymerization.
the protein IcsA activates N-WASP
, which in non-infected mammalian cells is activated by the GTPase Cdc42. Active N-WASP/WASP leads to actin polymerization by activating the Arp2/3 complex. In contrast, the Listeria ActA protein interacts with and activates directly the Arp2/3 complex.
The Rickettsia
RickA protein is also able to activate the Arp2/3 complex in a WASP-like manner. In contrast to Listeria, the actin filaments are organized in long, unbranched parallel bundles. The Arp2/3 complex is only localized near the bacterial surface and thus it is assumed that a more frequent Arp2/3 complex-independent elongation occurs.
In Burkholderia
pseudomallei BimA initiates actin polymerization in vitro. It is assumed that intracellular migration of this bacterium functions independently of the Arp2/3 complex.
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
encoded and used by Listeria monocytogenes to propel itself through a mammalian host cell. ActA is a bacterial surface protein comprising a membrane-spanning region. In a mammalian cell the bacterial ActA interacts with the Arp2/3 complex
Arp2/3 complex
Arp2/3 complex is a seven-subunit protein that plays a major role in the regulation of the actin cytoskeleton. It is a major component of the actin cytoskeleton and is found in most in actin cytoskeleton-containing eukaryotic cells....
and actin
Actin
Actin is a globular, roughly 42-kDa moonlighting protein found in all eukaryotic cells where it may be present at concentrations of over 100 μM. It is also one of the most highly-conserved proteins, differing by no more than 20% in species as diverse as algae and humans...
monomers to induce actin polymerization on the bacterial surface generating an actin comet tail. The gene encoding ActA is named actA or prtB.
Introduction
As soon as L. monocytogenes bacteria are ingested by humans, they get internalized into intestinal epitheliumIntestinal epithelium
The intestinal epithelium is the epithelium that covers the small and large intestine. It is simple columnar and nonciliated.They primarily take part in the digestive system. However, they also express TLR 4 receptors, and are thus a part of the immune system, both as a barrier and as a first-line...
cells and rapidly try to escape their internalization vacuole. In the cytosol they start to polymerize actin on their surface by the help of the ActA protein. It has been shown that ActA is not only necessary but also sufficient to induce motility of bacteria in the absence of other bacterial factors.
Discovery of ActA
ActA was discovered by analysing lecithinaseLecithinase
Lecithinase is a type of phospholipase that acts upon lecithin.It can be produced by Clostridium perfringens or Listeria Monocytogenes . C. perfringens alpha toxin causes myonecrosis and hemolysis....
-negative Tn917-lac Listeria mutants because of the phenotype that they were unable to spread from cell to cell. These mutant bacteria still escaped from the phagosomes as efficiently as wild-type bacteria and multiplied within the infected cells but they were not surrounded by actin like wild-type bacteria. Further analysis showed, that Tn917-lac had inserted into actA, the second gene of an operon. The third gene of this operon, plcB, encodes the L. monocytogenes lecithinase. To determine whether actA itself, plcB or other co-transcribed downstream regions are involved in actin assembly, mutations in the appropriate genes were generated. All mutants except the actA mutants were similar to wild-type concerning association with F-actin and cell-cell spreading. Complementation with actA restored wild-type phenotype in the actA mutants.
Function
ActA is a protein which acts as a mimic of Wiskott-Aldrich syndrome proteinWiskott-Aldrich syndrome protein
The Wiskott–Aldrich Syndrome Protein is a 502-amino acid protein that is expressed in cells of the hematopoietic system. In the inactive state, WASp exists in an auto-inhibited conformation with sequences near its C-terminus binding to a region near its N-terminus...
(WASP), a nucleation promoting factor (NPF) present in host cells. NPFs in general recruit and bind to the already in the mammalian cell existing actin-related-protein 2 and 3 complex
Arp2/3 complex
Arp2/3 complex is a seven-subunit protein that plays a major role in the regulation of the actin cytoskeleton. It is a major component of the actin cytoskeleton and is found in most in actin cytoskeleton-containing eukaryotic cells....
(Arp2/3 complex) and induce an activating conformational change of the Arp2/3 complex. Due to this conformational change, NPFs initiate polymerization of a new actin filament at a 70° angle, which leads to the characteristic Y-branched actin structures in the leading edge of motile cells. ActA localizes to the old pole of the bacterium and spans both the bacterial cell membrane and the cell wall, lateral diffusion is inhibited; thus ActA localizes in a polarized and anchored manner on the bacterial surface. Consequently actin polymerization only starts in this region on the surface of the bacterium. Expression of ActA is induced only after entering a mammalian host cell.
Actin filament assembly generates the force that pushes the bacterium in the mammalian host cytoplasm forward. Continuous actin polymerization is sufficient for motility in the cytoplasm and even for infection of adjacent cells.
Research
New data indicates that ActA plays a role also in vacuolar disruption. A deletion mutant of ActA was defective in permeabilizing the vacuole. An 11 amino acid stretch of the N-terminus of the acidic region (32-42) was shown to be important for disruption of the phagosomePhagosome
In cell biology, a phagosome is a vacuole formed around a particle absorbed by phagocytosis. The vacuole is formed by the fusion of the cell membrane around the particle. A phagosome is a cellular compartment in which pathogenic microorganisms can be killed and digested...
.
Structure
The primary proteinous product of the actA gene consists of 639 amino acids and includes the signal peptide (first N-terminal 29 amino acids) and the ActA chain (C-terminal 610 amino acids). Therefore the sequence of the mature ActA protein consist of 610 amino acids. ActA has a molecular weight of 70,349 Da and is a surface protein.The ActA chain can be divided into three functional domains (Fig. 2):
- N-terminal domain that is highly charged: amino acid residues 1-234
- central domain with proline-rich repeats: amino acid residues 235-394
- C-terminal domain with a transmembrane domain: amino acid residues 395-610
N-terminal Domain
The first 156 amino acids of the N-terminal domain consist of three regions (Fig. 2):- A-region with a stretch of acidic residues: 32-45
- AB-region, an actin monomer-binding region: 59-102
- C-region, a cofilin homology sequence: 145-156
The N-terminal portion of ActA plays an important role in actin polymerization. The domain displays consensus elements present in eukaryotic WASP family NPFs which include an actin monomer-binding region as well as an Arp2/3 binding C (central or cofilin homology) and A (acidic) region. The actin monomer-binding region of ActA has functional properties like the WASP-Homology-2 (WH2) or V domain, but differs in the sequence. Thus in WASP-family NPFs the order of the domains is WH2 followed by C,and then by A, which is not the case in ActA.
Central Domain
The central proline-rich region of ActA is crucial for ensuring efficient bacterial motility. There are four proline-rich repeats containing either FPPPP or FPPIP motifs. These regions mimic those of the host cell cytoskeletal protein zyxinZyxin
Zyxin is a protein that in humans is encoded by the ZYX gene.-Interactions:Zyxin has been shown to interact with ENAH, LASP1, LATS1, Actinin, alpha 1 and Vasodilator-stimulated phosphoprotein.-External links:* Info with links in the...
, vinculin
Vinculin
In mammalian cells, vinculin is a membrane-cytoskeletal protein in focal adhesion plaques that is involved in linkage of integrin adhesion molecules to the actin cytoskeleton...
and palladin
Palladin
Palladin is a protein that in humans is encoded by the PALLD gene. Palladin is a component of actin-containing microfilaments that control cell shape, adhesion, and contraction.- Discovery :...
, known to associate with focal adhesion
Focal adhesion
In cell biology, focal adhesions are specific types of large macromolecular assemblies through which both mechanical force and regulatory signals are transmitted. More precisely, they can be considered as sub-cellular macromolecules that mediate the regulatory effects In cell biology, focal...
s or stress fibers. The vasodilator-stimulated phosphoprotein (VASP
Vasodilator-stimulated phosphoprotein
Vasodilator-stimulated phosphoprotein is a protein that in humans is encoded by the VASP gene.- Function :Vasodilator-stimulated phosphoprotein is a member of the Ena-VASP protein family. Ena-VASP family members contain an N-terminal EVH1 domain that binds proteins containing E/DFPPPPXD/E motifs...
) can bind through its Ena/VASP homology 1 domain (EVH1 domain
EVH1 domain
The EVH1 domain is an evolutionary conserved protein domain.The EVH1 domain is found in multi-domain proteins implicated in a diverse range of signalling, nuclear transport and cytoskeletal events. This domain of around 115 amino acids is present in species ranging from yeast to mammals...
) to the central proline-rich region and recruits profilin
Profilin
Profilin is an actin-binding protein involved in the dynamic turnover and restructuring of the actin cytoskeleton. It is found in all eukaryotic organisms in most cells. Profilin is important for spatially and temporally controlled growth of actin microfilaments, which is an essential process in...
, an actin monomer binding protein, which itself promotes polymerization at barbed ends of actin filaments. Furthermore, VASP seems to interact with F-actin through its carboxy-terminal EVH2 domain, which provides a linkage of the bacterium to the tail. This statement is supported by the fact that ActA can bind multiple Ena/VASP proteins simultaneously and has a high affinity between ActA and Ena/VASP. VASP has been shown to reduce the frequency actin-Y-branches in vitro and thus increases the proportion of filaments which are organized in a parallel alignment in comet tails.
C-terminal Domain
The C-terminal domain of ActA has a hydrophobic region which anchors the protein in the bacterial membrane.In summary, besides
- the absence of sequence homology in the actin-binding-region and
- an alteration in the sequence of ARP2/3 activating domains typical for WASP-family NPFs (V(WH2)-C-A),
- a major difference between ActA and host NPFs is that ActA does not have elements that bind to regulatory proteins such as Rho family GTPases. This structural difference between ActA and host NPFs can be advantageous for L. monocytogenes and its pathogenesis because the actin nucleation activity of L. monocytogenes is independent of host regulation.
Analogues
WASP/N-WASP, which is functionally mimicked by ActA, is highly conserved in eukaryotes. It is an important actin-cytoskeleton organizer and is critical for processes such as endocytosis and cell motility. Activated by Cdc42CDC42
Cell division control protein 42 homolog also known as CDC42 is a protein involved in regulation of the cell cycle. In humans, CDC42 is encoded by the CDC42 gene.- Function :...
, a Rho-family small GTPase, WASP/N-WASP activates the Arp2/3 complex, which leads to rapid actin polymerization.
Actin-based Motility of other Pathogens
In ShigellaShigella
Shigella is a genus of Gram-negative, nonspore forming, non-motile, rod-shaped bacteria closely related to Escherichia coli and Salmonella. The causative agent of human shigellosis, Shigella causes disease in primates, but not in other mammals. It is only naturally found in humans and apes. During...
the protein IcsA activates N-WASP
WASL (gene)
Neural Wiskott-Aldrich syndrome protein is a protein that in humans is encoded by the WASL gene.The Wiskott-Aldrich syndrome family of proteins share similar domain structure, and are involved in transduction of signals from receptors on the cell surface to the actin cytoskeleton...
, which in non-infected mammalian cells is activated by the GTPase Cdc42. Active N-WASP/WASP leads to actin polymerization by activating the Arp2/3 complex. In contrast, the Listeria ActA protein interacts with and activates directly the Arp2/3 complex.
The Rickettsia
Rickettsia
Rickettsia is a genus of non-motile, Gram-negative, non-sporeforming, highly pleomorphic bacteria that can present as cocci , rods or thread-like . Being obligate intracellular parasites, the Rickettsia survival depends on entry, growth, and replication within the cytoplasm of eukaryotic host cells...
RickA protein is also able to activate the Arp2/3 complex in a WASP-like manner. In contrast to Listeria, the actin filaments are organized in long, unbranched parallel bundles. The Arp2/3 complex is only localized near the bacterial surface and thus it is assumed that a more frequent Arp2/3 complex-independent elongation occurs.
In Burkholderia
Burkholderia
Burkholderia is a genus of proteobacteria probably best known for its pathogenic members:Burkholderia mallei, responsible for glanders, a disease that occurs mostly in horses and related animals;...
pseudomallei BimA initiates actin polymerization in vitro. It is assumed that intracellular migration of this bacterium functions independently of the Arp2/3 complex.
External links
- YoutubeVideo from Nature, Listeria monocytogenes [2:00-4:12]