Type three secretion system
Encyclopedia
Type three secretion system (often written Type III secretion system and abbreviated TTSS or T3SS) is a protein
appendage found in several Gram-negative
bacteria
.
In pathogenic bacteria, the needle-like structure is used as a sensory probe to detect the presence of eukaryotic organism
s and secrete
proteins that help the bacteria infect
them. The proteins are secreted directly from the bacterial cell
into the eukaryotic cell, also known as "the host" cell.
found in Gram-negative bacteria. Many bacteria possess a T3SS and can transfer the T3SS gene cassette horizontally
to other species. The most researched T3SSs are from species of Shigella
(causes bacillary dysentery
), Salmonella
(typhoid fever
), Escherichia coli
(food poisoning), Burkholderia
(glanders
), Yersinia
(plague), Chlamydia
(sexually transmitted disease
), Pseudomonas
(infects human
s, animal
s and plant
s) and the plant pathogens Erwinia
, Ralstonia
, Rhizobium
, Vibrio
(gastroenteritis
and diarrhea
), and Xanthomonas
.
The T3SS is composed of approximately 30 different proteins, making it one of the most complex secretion systems. Its structure shows many similarities with bacterial flagella (long, rigid, extracellular structures used for motility
). Some of the proteins participating in T3SS share amino-acid sequence homology to flagellar proteins. Some of the bacteria possessing a T3SS have flagella as well and are motile (Salmonella, for instance), and some do not (Shigella, for instance). Technically speaking, type III secretion is used both for secreting infection-related proteins and flagellar components. However, the term "type III secretion" is used mainly in relation to the infection apparatus. The bacterial flagellum shares a common ancestor with the type III secretion system,
T3SSs are essential for the pathogenicity (the ability to infect) of many pathogenic bacteria. Defects in the T3SS may render a bacterium non-pathogenic. It has been suggested that some non-invasive strains of gram-negative bacteria have lost the T3SS because the energetically costly system is no longer of use. Although traditional antibiotics were effective against these bacteria in the past, antibiotic-resistant strains constantly emerge. Understanding the way the T3SS works and developing drugs targeting it specifically have become since the late 1990s an important goal of many research groups around the world.
is excluded; see below). Bacterial proteins that need to be secreted pass from the bacterial cytoplasm
through the needle directly into the host cytoplasm. Three membrane
s separate the two cytoplasms: the double membrane (inner and outer membranes) of the Gram-negative bacterium and the eukaryotic membrane. The needle provides a smooth passage through those highly selective and almost impermeable membranes. A single bacterium can have several hundred needle complexes spread across its membrane. It has been proposed that the needle complex is a universal feature of all T3SSs of pathogenic bacteria.
The needle complex starts at the cytoplasm of the bacterium, crosses the two membranes and protrudes from the cell. The part anchored in the membrane is the base (or basal body) of the T3SS. The extracellular part is the needle. A so-called inner rod connects the needle to the base. The needle itself, although the biggest and most prominent part of the T3SS, is made out of many units of one protein. The majority of the different T3SS proteins are therefore those that build the base and those that are secreted into the host. As mentioned above, the needle complex shares similarities with bacterial flagella. More specifically, the base of the needle complex is structurally very similar to the flagellar base; the needle itself is analogous to the flagellar hook, a structure connecting the base to the flagellar filament.
The base is composed of several circular rings and is the first structure that is built in a new needle complex. Once the base is completed, it serves as a secretion machine for the outer proteins (the needle). Once the whole complex is completed the system switches to secreting proteins that are intended to be delivered into host cells. The needle is presumed to be built from bottom to top; units of needle monomer
protein pile upon each other, so that the unit at the tip of the needle is the last one added. The needle subunit is one of the smallest T3SS proteins, measuring at around 9 kDa
. 100−150 subunits comprise each needle.
The T3SS needle measures around 60−80 nm in length and 8 nm in external width. It needs to have a minimal length so that other extracellular bacterial structures (adhesin
s and the lipopolysaccharide
layer, for instance) do not interfere with secretion. The hole of the needle has a 3 nm diameter. Most folded effector proteins are too large to pass through the needle opening, so most secreted proteins must pass through the needle unfolded
, a task carried out by the ATPase
at the base of the structure
Most T3SS genes are laid out in operon
s. These operons are located on the bacterial chromosome in some species and on a dedicated plasmid
in other species. Salmonella, for instance, has a chromosomal region in which most T3SS genes are gathered, the so-called Salmonella pathogenicity island (SPI). Shigella, on the other hand, has a large virulence plasmid on which all T3SS genes reside. It is important to note that many pathogenicity islands and plasmids contain elements that allow for frequent horizontal gene transfer of the island/plasmid to a new species.
Effector proteins that are to be secreted through the needle need to be recognized by the system, since they float in the cytoplasm together with thousands of other proteins. Recognition is done through a secretion signal—a short sequence of amino acids located at the beginning (the N-terminus) of the protein (usually within the first 20 amino acids), that the needle complex is able to recognize. Unlike other secretion systems, the secretion signal of T3SS proteins is never cleaved off the protein.
ion
s in the growth medium
(for Yersinia and Pseudomonas; done by adding a chelator such as EDTA
or EGTA
) and by adding the aromatic dye
Congo red
to the growth medium (for Shigella), for instance. These methods and other are used in laboratories to artificially induce type III secretion.
Induction of secretion by external cues other than contact with host cells also takes place in vivo
, in infected organisms. The bacteria sense such cues as temperature
, pH
, osmolarity and oxygen
levels, and use them to "decide" whether to activate their T3SS. For instance, Salmonella can replicate and invade better in the ileum
rather than in the cecum
of animal intestine
. The bacteria are able to know where they are thanks to the different ions present in these regions; the ileum contains formate
and acetate
, while the cecum does not. The bacteria sense these molecules, determine that they are at the ileum and activate their secretion machinery. Molecules present in the cecum, such as propionate
and butyrate
, provide a negative cue to the bacteria and inhibit secretion. Cholesterol
, a lipid
found in most eukaroytic cell membranes, is able to induce secretion in Shigella.
The external cues listed above either regulate secretion directly or through a genetic mechanism. Several transcription factor
s that regulate the expression of T3SS genes are known. Some of the chaperones that bind T3SS effectors also act as transcription factors. A feedback mechanism has been suggested: when the bacterium does not secrete, its effector proteins are bound to chaperones and float in the cytoplasm. When secretion starts, the chaperones detach from the effectors and the latter are secreted and leave the cell. The lone chaperones then act as transcription factors, binding to the genes encoding their effectors and inducing their transcription and thereby the production of more effectors.
bacteria that lack translocators are able to secrete proteins but are not able to deliver them into host cells. In general each T3SS includes three translocators. Some translocators serve a double role; after they participate in pore formation they enter the cell and act as bona fide effectors.
T3SS effectors manipulate host cells in several ways. The most striking effect is the promoting of uptake of the bacterium by the host cell. Many bacteria possessing T3SSs must enter host cells in order to replicate and propagate infection. The effectors they inject into the host cell induce the host to engulf the bacterium and to practically "eat" it. In order for this to happen the bacterial effectors manipulate the actin
polymerization
machinery of the host cell. Actin is a component of the cytoskeleton
and it also participates in motility and in changes in cell shape. Through its T3SS effectors the bacterium is able to utilize the host cell's own machinery for its own benefit. Once the bacterium has entered the cell it is able to secrete other effectors more easily and it can penetrate neighboring cells and quickly infect the whole tissue
.
T3SS effectors have also been shown to tamper with the host's cell cycle
and some of them are able to induce apoptosis
. One of the most researched T3SS effector is IpaB from Shigella flexneri
. It serves a double role, both as a translocator, creating a pore in the host cell membrane, and as an effector, exerting multiple detrimental effects on the host cell. It was shown in 1994 that IpaB induces apoptosis in macrophage
s—cells of the animal immune system
—after being engulfed by them. It was later shown that IpaB does that by interacting with caspase 1
, a major regulatory protein in eukaryotic cells.
Another well characterized class of T3SS effectors are Transcription Activator-like effectors (TAL effector
s) from Xanthomonas
. When injected into plants, these proteins can enter the nucleus of the plant cell, bind plant promoter sequences, and activate transcription of plant genes that aid in bacterial infection. TAL effector-DNA recognition has recently been demonstrated to comprise a simple code and this has greatly improved the understanding of how these proteins can alter the transcription of genes in the host plant cells.
Since the beginning of the 1990s new T3SS proteins are being found in different bacterial species at a steady rate. Abbreviations have been given independently for each series of proteins in each organism, and the names usually do not reveal much about the protein's function. Some proteins discovered independently in different bacteria have later been shown to be homologous
; the historical names, however, have mostly been kept, a fact that might cause confusion. For example, the proteins SicA, IpgC and SycD are homologs from Salmonella, Shigella and Yersinia, respectively, but the last letter (the "serial number") in their name does not show that.
Below is a summary of the most common protein-series names in several T3SS-containing species:
Following those abbreviations is a letter or a number. Letters usually denote a "serial number", either the chronological order of discovery or the physical order of appearance of the gene in an operon
. Numbers, the rarer case, denote the molecular weight of the protein in kDa
. Examples: IpaA, IpaB, IpaC; MxiH, MxiG, MxiM; Spa9, Spa47.
Several key elements appear in all T3SSs: the needle monomer, the inner rod of the needle, the ring proteins, the two translocators, the needle-tip protein, the ruler protein (which is thought to determine the needle's length; see above) and the ATPase
, which supplies energy for secretion. The following table shows some of these key proteins in four T3SS-containing bacteria:
For the isolation, bacteria are grown in a large volume of liquid growth medium
until they reach log phase
. They are then centrifuge
d; the supernatant (the medium) is discarded and the pellet
(the bacteria) is resuspended in a lysis buffer
typically containing lysozyme
and sometimes a detergent
such as LDAO or Triton X-100. This buffer disintegrates the cell wall
. After several rounds of lysis and washing, the opened bacteria are subjected to a series of ultracentrifugations. This treatment enriches large macromolecular structures and discards smaller cell components. Optionally, the final lysate is subjected to further purification by CsCl
density gradient
.
An additional approach for further purification uses affinity chromatography
. Recombinant
T3SS proteins that carry a protein tag
(a histidine tag, for instance) are produced by molecular cloning
and then introduced (transformed) into the researched bacteria. After initial NC isolation, as described above, the lysate is passed through a column coated with particles with high affinity to the tag (in the case of histidine tags: nickel
ion
s). The tagged protein is retained in the column, and with it the entire needle complex. High degrees of purity can be achieved using such methods. This purity is essential for many delicate assays that were later used for NC characterization.
The isolation of NCs was a major step in T3SS research. Type III effectors were known since the beginning of the 1990s, but the way in which they are delivered into host cells was a complete mystery. The homology between many flagellar
and T3SS proteins led researchers to suspects the existence of an outer T3SS structure similar to flagella. The identification and subsequent isolation of the needle structure enabled researchers to:
were digitally analyzed and averaged to obtain a first semi-3D structure of the NC. The helical structure of NCs from Shigella flexneri was resolved at a resolution of 16 Å
using X-ray
fiber diffraction
in 2003, and a year later a 17-Å
3D structure of NCs from Salmonella typhimurium was published. Recent advances and approaches have allowed high-resolution 3D images of the NC, further clarifying the complex structure of the NC.
Numerous T3SS proteins have been crystallized over the years. These include structural proteins of the NC, effectors and chaperones. The first structure of a needle-complex monomer was of MxiH from Shigella flexneri, which was resolved in 2006. Crystal structures often give detailed insight into the function of the protein at the molecular and atom
level. Many issues regarding the assembly and structure of the NC, the way effectors damage host cells, and the function of chaperones, have been clarified using crystallography.
. The bands that appear after staining can be individually excised from the gel and analyzed using protein sequencing
and mass spectrometry
. The structural components of the NC can be separated from each other (the needle part from the base part, for instance), and by analyzing those fractions the proteins participating in each one can be deduced. Alternatively, isolated NCs can be directly analyzed by mass spectrometry, without prior electrophoresis
, in order to obtain a complete picture of the NC proteome
.
Manipulation of T3SS components can have influence on several aspects of bacterial function and pathogenicity. Examples of possible influences:
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...
appendage found in several Gram-negative
Gram-negative
Gram-negative bacteria are bacteria that do not retain crystal violet dye in the Gram staining protocol. In a Gram stain test, a counterstain is added after the crystal violet, coloring all Gram-negative bacteria with a red or pink color...
bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
.
In pathogenic bacteria, the needle-like structure is used as a sensory probe to detect the presence of eukaryotic organism
Organism
In biology, an organism is any contiguous living system . In at least some form, all organisms are capable of response to stimuli, reproduction, growth and development, and maintenance of homoeostasis as a stable whole.An organism may either be unicellular or, as in the case of humans, comprise...
s and secrete
Secretion
Secretion is the process of elaborating, releasing, and oozing chemicals, or a secreted chemical substance from a cell or gland. In contrast to excretion, the substance may have a certain function, rather than being a waste product...
proteins that help the bacteria infect
Infection
An infection is the colonization of a host organism by parasite species. Infecting parasites seek to use the host's resources to reproduce, often resulting in disease...
them. The proteins are secreted directly from the bacterial cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
into the eukaryotic cell, also known as "the host" cell.
Overview
The term Type III secretion system was coined in 1993. This secretion system is distinguished from at least five other secretion systemsSecretion
Secretion is the process of elaborating, releasing, and oozing chemicals, or a secreted chemical substance from a cell or gland. In contrast to excretion, the substance may have a certain function, rather than being a waste product...
found in Gram-negative bacteria. Many bacteria possess a T3SS and can transfer the T3SS gene cassette horizontally
Horizontal gene transfer
Horizontal gene transfer , also lateral gene transfer , is any process in which an organism incorporates genetic material from another organism without being the offspring of that organism...
to other species. The most researched T3SSs are from species of Shigella
Shigella
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...
(causes bacillary dysentery
Bacillary dysentery
Bacillary dysentery is a type of dysentery, and is a severe form of shigellosis.Bacillary dysentery is associated with species of bacteria from the Enterobacteriaceae family. The term is usually restricted to Shigella infections....
), Salmonella
Salmonella
Salmonella is a genus of rod-shaped, Gram-negative, non-spore-forming, predominantly motile enterobacteria with diameters around 0.7 to 1.5 µm, lengths from 2 to 5 µm, and flagella which grade in all directions . They are chemoorganotrophs, obtaining their energy from oxidation and reduction...
(typhoid fever
Typhoid fever
Typhoid fever, also known as Typhoid, is a common worldwide bacterial disease, transmitted by the ingestion of food or water contaminated with the feces of an infected person, which contain the bacterium Salmonella enterica, serovar Typhi...
), Escherichia coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
(food poisoning), 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;...
(glanders
Glanders
Glanders is an infectious disease that occurs primarily in horses, mules, and donkeys. It can be contracted by other animals such as dogs, cats and goats...
), Yersinia
Yersinia
Yersinia is a genus of bacteria in the family Enterobacteriaceae. Yersinia are Gram-negative rod shaped bacteria, a few micrometers long and fractions of a micrometer in diameter, and are facultative anaerobes. Some members of Yersinia are pathogenic in humans; in particular, Y. pestis is the...
(plague), Chlamydia
Chlamydia (bacterium)
Chlamydia is a genus of bacteria that are obligate intracellular parasites. Chlamydia infections are the most common bacterial sexually transmitted infections in humans and are the leading cause of infectious blindness worldwide....
(sexually transmitted disease
Sexually transmitted disease
Sexually transmitted disease , also known as a sexually transmitted infection or venereal disease , is an illness that has a significant probability of transmission between humans by means of human sexual behavior, including vaginal intercourse, oral sex, and anal sex...
), Pseudomonas
Pseudomonas
Pseudomonas is a genus of gammaproteobacteria, belonging to the family Pseudomonadaceae containing 191 validly described species.Recently, 16S rRNA sequence analysis has redefined the taxonomy of many bacterial species. As a result, the genus Pseudomonas includes strains formerly classified in the...
(infects human
Human
Humans are the only living species in the Homo genus...
s, animal
Animal
Animals are a major group of multicellular, eukaryotic organisms of the kingdom Animalia or Metazoa. Their body plan eventually becomes fixed as they develop, although some undergo a process of metamorphosis later on in their life. Most animals are motile, meaning they can move spontaneously and...
s and plant
Plant
Plants are living organisms belonging to the kingdom Plantae. Precise definitions of the kingdom vary, but as the term is used here, plants include familiar organisms such as trees, flowers, herbs, bushes, grasses, vines, ferns, mosses, and green algae. The group is also called green plants or...
s) and the plant pathogens Erwinia
Erwinia
Erwinia is a genus of Enterobacteriaceae bacteria containing mostly plant pathogenic species which was named for the first phytobacteriologist, Erwin Smith. It is a gram negative bacterium related to E. coli, Shigella, Salmonella and Yersinia. It is primarily a rod-shaped bacteria. A well-known...
, Ralstonia
Ralstonia
Ralstonia is a genus of proteobacteria, previously included in the genus Pseudomonas. It is named after the American bacteriologist E. Ralston.-Genomics:* *...
, Rhizobium
Rhizobium
Rhizobium is a genus of Gram-negative soil bacteria that fix nitrogen. Rhizobium forms an endosymbiotic nitrogen fixing association with roots of legumes and Parasponia....
, Vibrio
Vibrio
Vibrio is a genus of Gram-negative bacteria possessing a curved rod shape, several species of which can cause foodborne infection, usually associated with eating undercooked seafood. Typically found in saltwater, Vibrio are facultative anaerobes that test positive for oxidase and do not form...
(gastroenteritis
Gastroenteritis
Gastroenteritis is marked by severe inflammation of the gastrointestinal tract involving both the stomach and small intestine resulting in acute diarrhea and vomiting. It can be transferred by contact with contaminated food and water...
and diarrhea
Diarrhea
Diarrhea , also spelled diarrhoea, is the condition of having three or more loose or liquid bowel movements per day. It is a common cause of death in developing countries and the second most common cause of infant deaths worldwide. The loss of fluids through diarrhea can cause dehydration and...
), and Xanthomonas
Xanthomonas
Xanthomonas is a genus of Proteobacteria, many of which cause plant diseases. Most varieties of Xanthomonas are available from the National Collection of Plant Pathogenic Bacteria in the United Kingdom and other international culture collections such as ICMP in New Zealand, CFBP in France, and...
.
The T3SS is composed of approximately 30 different proteins, making it one of the most complex secretion systems. Its structure shows many similarities with bacterial flagella (long, rigid, extracellular structures used for motility
Motility
Motility is a biological term which refers to the ability to move spontaneously and actively, consuming energy in the process. Most animals are motile but the term applies to single-celled and simple multicellular organisms, as well as to some mechanisms of fluid flow in multicellular organs, in...
). Some of the proteins participating in T3SS share amino-acid sequence homology to flagellar proteins. Some of the bacteria possessing a T3SS have flagella as well and are motile (Salmonella, for instance), and some do not (Shigella, for instance). Technically speaking, type III secretion is used both for secreting infection-related proteins and flagellar components. However, the term "type III secretion" is used mainly in relation to the infection apparatus. The bacterial flagellum shares a common ancestor with the type III secretion system,
T3SSs are essential for the pathogenicity (the ability to infect) of many pathogenic bacteria. Defects in the T3SS may render a bacterium non-pathogenic. It has been suggested that some non-invasive strains of gram-negative bacteria have lost the T3SS because the energetically costly system is no longer of use. Although traditional antibiotics were effective against these bacteria in the past, antibiotic-resistant strains constantly emerge. Understanding the way the T3SS works and developing drugs targeting it specifically have become since the late 1990s an important goal of many research groups around the world.
Structure
The hallmark of T3SS is the needle (more generally, the needle complex (NC) or the T3SS apparatus (T3SA); also called injectisome when the ATPaseATPase
ATPases are a class of enzymes that catalyze the decomposition of adenosine triphosphate into adenosine diphosphate and a free phosphate ion. This dephosphorylation reaction releases energy, which the enzyme harnesses to drive other chemical reactions that would not otherwise occur...
is excluded; see below). Bacterial proteins that need to be secreted pass from the bacterial cytoplasm
Cytoplasm
The cytoplasm is a small gel-like substance residing between the cell membrane holding all the cell's internal sub-structures , except for the nucleus. All the contents of the cells of prokaryote organisms are contained within the cytoplasm...
through the needle directly into the host cytoplasm. Three membrane
Cell membrane
The cell membrane or plasma membrane is a biological membrane that separates the interior of all cells from the outside environment. The cell membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells. It basically protects the cell...
s separate the two cytoplasms: the double membrane (inner and outer membranes) of the Gram-negative bacterium and the eukaryotic membrane. The needle provides a smooth passage through those highly selective and almost impermeable membranes. A single bacterium can have several hundred needle complexes spread across its membrane. It has been proposed that the needle complex is a universal feature of all T3SSs of pathogenic bacteria.
The needle complex starts at the cytoplasm of the bacterium, crosses the two membranes and protrudes from the cell. The part anchored in the membrane is the base (or basal body) of the T3SS. The extracellular part is the needle. A so-called inner rod connects the needle to the base. The needle itself, although the biggest and most prominent part of the T3SS, is made out of many units of one protein. The majority of the different T3SS proteins are therefore those that build the base and those that are secreted into the host. As mentioned above, the needle complex shares similarities with bacterial flagella. More specifically, the base of the needle complex is structurally very similar to the flagellar base; the needle itself is analogous to the flagellar hook, a structure connecting the base to the flagellar filament.
The base is composed of several circular rings and is the first structure that is built in a new needle complex. Once the base is completed, it serves as a secretion machine for the outer proteins (the needle). Once the whole complex is completed the system switches to secreting proteins that are intended to be delivered into host cells. The needle is presumed to be built from bottom to top; units of needle monomer
Monomer
A monomer is an atom or a small molecule that may bind chemically to other monomers to form a polymer; the term "monomeric protein" may also be used to describe one of the proteins making up a multiprotein complex...
protein pile upon each other, so that the unit at the tip of the needle is the last one added. The needle subunit is one of the smallest T3SS proteins, measuring at around 9 kDa
Atomic mass unit
The unified atomic mass unit or dalton is a unit that is used for indicating mass on an atomic or molecular scale. It is defined as one twelfth of the rest mass of an unbound neutral atom of carbon-12 in its nuclear and electronic ground state, and has a value of...
. 100−150 subunits comprise each needle.
The T3SS needle measures around 60−80 nm in length and 8 nm in external width. It needs to have a minimal length so that other extracellular bacterial structures (adhesin
Adhesin
Adhesins are cell-surface components or appendages of bacteria that facilitate bacterial adhesion or adherence to other cells or to inanimate surfaces. Adhesins are a type of virulence factor....
s and the lipopolysaccharide
Lipopolysaccharide
Lipopolysaccharides , also known as lipoglycans, are large molecules consisting of a lipid and a polysaccharide joined by a covalent bond; they are found in the outer membrane of Gram-negative bacteria, act as endotoxins and elicit strong immune responses in animals.-Functions:LPS is the major...
layer, for instance) do not interfere with secretion. The hole of the needle has a 3 nm diameter. Most folded effector proteins are too large to pass through the needle opening, so most secreted proteins must pass through the needle unfolded
Protein folding
Protein folding is the process by which a protein structure assumes its functional shape or conformation. It is the physical process by which a polypeptide folds into its characteristic and functional three-dimensional structure from random coil....
, a task carried out by the ATPase
ATPase
ATPases are a class of enzymes that catalyze the decomposition of adenosine triphosphate into adenosine diphosphate and a free phosphate ion. This dephosphorylation reaction releases energy, which the enzyme harnesses to drive other chemical reactions that would not otherwise occur...
at the base of the structure
T3SS proteins
The T3SS proteins can be grouped into three categories:- Structural proteins: build the base, the inner rod and the needle.
- Effector proteins: get secreted into the host cell and promote infection.
- Chaperones: bind effectors in the bacterial cytoplasm, protect them from aggregation and degradationProteolysisProteolysis is the directed degradation of proteins by cellular enzymes called proteases or by intramolecular digestion.-Purposes:Proteolysis is used by the cell for several purposes...
and direct them towards the needle complex.
Most T3SS genes are laid out in operon
Operon
In genetics, an operon is a functioning unit of genomic DNA containing a cluster of genes under the control of a single regulatory signal or promoter. The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo trans-splicing to create...
s. These operons are located on the bacterial chromosome in some species and on a dedicated plasmid
Plasmid
In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
in other species. Salmonella, for instance, has a chromosomal region in which most T3SS genes are gathered, the so-called Salmonella pathogenicity island (SPI). Shigella, on the other hand, has a large virulence plasmid on which all T3SS genes reside. It is important to note that many pathogenicity islands and plasmids contain elements that allow for frequent horizontal gene transfer of the island/plasmid to a new species.
Effector proteins that are to be secreted through the needle need to be recognized by the system, since they float in the cytoplasm together with thousands of other proteins. Recognition is done through a secretion signal—a short sequence of amino acids located at the beginning (the N-terminus) of the protein (usually within the first 20 amino acids), that the needle complex is able to recognize. Unlike other secretion systems, the secretion signal of T3SS proteins is never cleaved off the protein.
Induction of secretion
Contact of the needle with a host cell triggers the T3SS to start secreting; not much is known about this trigger mechanism (see below). Secretion can also be induced by lowering the concentration of calciumCalcium
Calcium is the chemical element with the symbol Ca and atomic number 20. It has an atomic mass of 40.078 amu. Calcium is a soft gray alkaline earth metal, and is the fifth-most-abundant element by mass in the Earth's crust...
ion
Ion
An ion is an atom or molecule in which the total number of electrons is not equal to the total number of protons, giving it a net positive or negative electrical charge. The name was given by physicist Michael Faraday for the substances that allow a current to pass between electrodes in a...
s in the growth medium
Growth medium
A growth medium or culture medium is a liquid or gel designed to support the growth of microorganisms or cells, or small plants like the moss Physcomitrella patens.There are different types of media for growing different types of cells....
(for Yersinia and Pseudomonas; done by adding a chelator such as EDTA
EDTA
Ethylenediaminetetraacetic acid, widely abbreviated as EDTA , is a polyamino carboxylic acid and a colourless, water-soluble solid. Its conjugate base is named ethylenediaminetetraacetate. It is widely used to dissolve limescale. Its usefulness arises because of its role as a hexadentate ligand...
or EGTA
EGTA (chemical)
EGTA is a polyamino carboxylic acid, a chelating agent that is related to the better known EDTA, but with a much higher affinity for calcium than for magnesium ions...
) and by adding the aromatic dye
Dye
A dye is a colored substance that has an affinity to the substrate to which it is being applied. The dye is generally applied in an aqueous solution, and requires a mordant to improve the fastness of the dye on the fiber....
Congo red
Congo red
Congo red is the sodium salt of 3,3'-bis. It is a secondary diazo dye...
to the growth medium (for Shigella), for instance. These methods and other are used in laboratories to artificially induce type III secretion.
Induction of secretion by external cues other than contact with host cells also takes place in vivo
In vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
, in infected organisms. The bacteria sense such cues as temperature
Temperature
Temperature is a physical property of matter that quantitatively expresses the common notions of hot and cold. Objects of low temperature are cold, while various degrees of higher temperatures are referred to as warm or hot...
, pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
, osmolarity and oxygen
Oxygen
Oxygen is the element with atomic number 8 and represented by the symbol O. Its name derives from the Greek roots ὀξύς and -γενής , because at the time of naming, it was mistakenly thought that all acids required oxygen in their composition...
levels, and use them to "decide" whether to activate their T3SS. For instance, Salmonella can replicate and invade better in the ileum
Ileum
The ileum is the final section of the small intestine in most higher vertebrates, including mammals, reptiles, and birds. In fish, the divisions of the small intestine are not as clear and the terms posterior intestine or distal intestine may be used instead of ileum.The ileum follows the duodenum...
rather than in the cecum
Cecum
The cecum or caecum is a pouch, connecting the ileum with the ascending colon of the large intestine. It is separated from the ileum by the ileocecal valve or Bauhin's valve, and is considered to be the beginning of the large intestine. It is also separated from the colon by the cecocolic...
of animal intestine
Intestine
In human anatomy, the intestine is the segment of the alimentary canal extending from the pyloric sphincter of the stomach to the anus and, in humans and other mammals, consists of two segments, the small intestine and the large intestine...
. The bacteria are able to know where they are thanks to the different ions present in these regions; the ileum contains formate
Formate
Formate or methanoate is the ion CHOO− or HCOO− . It is the simplest carboxylate anion. It is produced in large amounts in the hepatic mitochondria of embryonic cells and in cancer cells by the folate cycle Formate or methanoate is the ion CHOO− or HCOO− (formic acid minus one hydrogen ion). It...
and acetate
Acetate
An acetate is a derivative of acetic acid. This term includes salts and esters, as well as the anion found in solution. Most of the approximately 5 billion kilograms of acetic acid produced annually in industry are used in the production of acetates, which usually take the form of polymers. In...
, while the cecum does not. The bacteria sense these molecules, determine that they are at the ileum and activate their secretion machinery. Molecules present in the cecum, such as propionate
Propionate
The propanoate or propionate ion is C2H5COO− .A propanoic or propionic compound is a salt or ester of propanoic acid....
and butyrate
Butyrate
Butyrate is the traditional name for the conjugate base of butanoic acid . The formula of the butanoate ion is C4H7O2-. The archaic name is used as part of the name of butyrates or butanoates, or esters and salts of butyric acid, a short chain fatty acid...
, provide a negative cue to the bacteria and inhibit secretion. Cholesterol
Cholesterol
Cholesterol is a complex isoprenoid. Specifically, it is a waxy steroid of fat that is produced in the liver or intestines. It is used to produce hormones and cell membranes and is transported in the blood plasma of all mammals. It is an essential structural component of mammalian cell membranes...
, a lipid
Lipid
Lipids constitute a broad group of naturally occurring molecules that include fats, waxes, sterols, fat-soluble vitamins , monoglycerides, diglycerides, triglycerides, phospholipids, and others...
found in most eukaroytic cell membranes, is able to induce secretion in Shigella.
The external cues listed above either regulate secretion directly or through a genetic mechanism. Several transcription factor
Transcription factor
In molecular biology and genetics, a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the flow of genetic information from DNA to mRNA...
s that regulate the expression of T3SS genes are known. Some of the chaperones that bind T3SS effectors also act as transcription factors. A feedback mechanism has been suggested: when the bacterium does not secrete, its effector proteins are bound to chaperones and float in the cytoplasm. When secretion starts, the chaperones detach from the effectors and the latter are secreted and leave the cell. The lone chaperones then act as transcription factors, binding to the genes encoding their effectors and inducing their transcription and thereby the production of more effectors.
T3SS-mediated Infection
T3SS effectors enter the needle complex at the base and make their way inside the needle towards the host cell. The exact way in which effectors enter the host is mostly unknown. It has been previously suggested that the needle itself is capable of puncturing a hole in the host cell membrane; this theory has been refuted. It is now clear that some effectors, collectively named translocators, are secreted first and produce a pore or a channel (a translocon) in the host cell membrane, through which other effectors may enter. MutatedMutation
In molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...
bacteria that lack translocators are able to secrete proteins but are not able to deliver them into host cells. In general each T3SS includes three translocators. Some translocators serve a double role; after they participate in pore formation they enter the cell and act as bona fide effectors.
T3SS effectors manipulate host cells in several ways. The most striking effect is the promoting of uptake of the bacterium by the host cell. Many bacteria possessing T3SSs must enter host cells in order to replicate and propagate infection. The effectors they inject into the host cell induce the host to engulf the bacterium and to practically "eat" it. In order for this to happen the bacterial effectors manipulate the 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...
polymerization
Polymerization
In polymer chemistry, polymerization is a process of reacting monomer molecules together in a chemical reaction to form three-dimensional networks or polymer chains...
machinery of the host cell. Actin is a component of the cytoskeleton
Cytoskeleton
The cytoskeleton is a cellular "scaffolding" or "skeleton" contained within a cell's cytoplasm and is made out of protein. The cytoskeleton is present in all cells; it was once thought to be unique to eukaryotes, but recent research has identified the prokaryotic cytoskeleton...
and it also participates in motility and in changes in cell shape. Through its T3SS effectors the bacterium is able to utilize the host cell's own machinery for its own benefit. Once the bacterium has entered the cell it is able to secrete other effectors more easily and it can penetrate neighboring cells and quickly infect the whole tissue
Tissue (biology)
Tissue is a cellular organizational level intermediate between cells and a complete organism. A tissue is an ensemble of cells, not necessarily identical, but from the same origin, that together carry out a specific function. These are called tissues because of their identical functioning...
.
T3SS effectors have also been shown to tamper with the host's cell cycle
Cell cycle
The cell cycle, or cell-division cycle, is the series of events that takes place in a cell leading to its division and duplication . In cells without a nucleus , the cell cycle occurs via a process termed binary fission...
and some of them are able to induce apoptosis
Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
. One of the most researched T3SS effector is IpaB from Shigella flexneri
Shigella flexneri
Shigella flexneri is a species of Gram-negative bacteria in the genus Shigella that can cause diarrhea in humans. There are several different serogroups of Shigella; S. flexneri belongs to group B. S. flexneri infections can usually be treated with antibiotics although some strains have become...
. It serves a double role, both as a translocator, creating a pore in the host cell membrane, and as an effector, exerting multiple detrimental effects on the host cell. It was shown in 1994 that IpaB induces apoptosis in macrophage
Macrophage
Macrophages are cells produced by the differentiation of monocytes in tissues. Human macrophages are about in diameter. Monocytes and macrophages are phagocytes. Macrophages function in both non-specific defense as well as help initiate specific defense mechanisms of vertebrate animals...
s—cells of the animal immune system
Immune system
An immune system is a system of biological structures and processes within an organism that protects against disease by identifying and killing pathogens and tumor cells. It detects a wide variety of agents, from viruses to parasitic worms, and needs to distinguish them from the organism's own...
—after being engulfed by them. It was later shown that IpaB does that by interacting with caspase 1
Caspase 1
Caspase 1 is an enzyme that proteolytically cleaves other proteins, such as the precursor forms of the inflammatory cytokines interleukin 1-β and interleukin 18, into active mature peptides...
, a major regulatory protein in eukaryotic cells.
Another well characterized class of T3SS effectors are Transcription Activator-like effectors (TAL effector
TAL effector
TAL effectors are proteins secreted by Xanthomonas bacteria via their type III secretion system when they infect various plant species...
s) from Xanthomonas
Xanthomonas
Xanthomonas is a genus of Proteobacteria, many of which cause plant diseases. Most varieties of Xanthomonas are available from the National Collection of Plant Pathogenic Bacteria in the United Kingdom and other international culture collections such as ICMP in New Zealand, CFBP in France, and...
. When injected into plants, these proteins can enter the nucleus of the plant cell, bind plant promoter sequences, and activate transcription of plant genes that aid in bacterial infection. TAL effector-DNA recognition has recently been demonstrated to comprise a simple code and this has greatly improved the understanding of how these proteins can alter the transcription of genes in the host plant cells.
Unresolved issues
Hundreds of articles on T3SS have been published since the mid-nineties. However, numerous issues regarding the system remain unresolved:- T3SS proteins. Of the approximately 30 T3SS proteins less than 10 in each organism have been directly detected using biochemicalBiochemistryBiochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...
methods. The rest, being perhaps rare, have proven difficult to detect and they remain theoretical (although genetic rather than biochemical studies have been performed on many T3SS genes/proteins). The localization of each protein is also not entirely known. - The length of the needle. It is not known how the bacterium "knows" when a new needle has reached its proper length. Several theories exist, among them the existence of a "ruler protein" that somehow connects the tip and the base of the needle. Addition of new monomers to the tip of the needle should stretch the ruler protein and thereby signal the needle length to the base.
- Energetics. The force that drives the passage of proteins inside the needle is not completely known. An ATPaseATPaseATPases are a class of enzymes that catalyze the decomposition of adenosine triphosphate into adenosine diphosphate and a free phosphate ion. This dephosphorylation reaction releases energy, which the enzyme harnesses to drive other chemical reactions that would not otherwise occur...
is associated with the base of the T3SS and participates in directing proteins into the needle; but whether it supplies the energy for transport is not clear. - Secretion signal. As mentioned above, the existence of a secretion signal in effector proteins is known. The signal allows the system to distinguish T3SS-tranported proteins from any other protein. Its nature, requirements and the mechanism of recognition are poorly understood, but methods for predicting which bacterial proteins can be transported by the Type III secretion system have recently been developed.
- Activation of secretion. The bacterium must know when the time is right to secrete effectors. Unnecessary secretion, when no host cell is in vicinity, is wasteful for the bacterium in terms of energy and resources. The bacterium is somehow able to recognize contact of the needle with the host cell. How this is done is still being researched, and the method may well be dependant on the pathogen. Some theories postulate a delicate conformational change in the structure of the needle upon contact with the host cell; this change perhaps serves as a signal for the base to commence secretion. One method of recognition has been discovered in Salmonella, which relies on sensing host cell cytosolic pHPHIn chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
through the pathogenicity island 2-encoded T3SS in order to switch on secretion of effectors. - Binding of chaperones. It is not known when chaperones bind their effectors (whether during or after translation) and how they dissociate from their effectors before secretion.
- Effector mechanisms. Although much was revealed since the beginning of the 21st century about the ways in which T3SS effectors manipulate the host, the majority of effects and pathways remains unknown.
- Evolution. As mentioned, the T3SS is closely related to the bacterial flagellum. There are three competing hypotheses: first, that the flagellum evolved first and the T3SS is derived from that structure, second, that the T3SS evolved first and the flagellum is derived from it, and third, that the two structures are derived from a common ancestor. Those who support the hypothesis that the T3SS evolved from flagella cite evidence that Eukaryotes evolved after Prokaryotes. Thus, the need for motility would have caused selection for the development of flagella before an injectisome. However this suggestion can be seen as ‘reductive evolution,’ and receives no topological support from the phylogenetic trees. Thus, the hypothesis that the two structures derived from a common ancestor accounts for the protein homology between the two structures, as well as their functional diversity.
Nomenclature of T3SS proteins
Protein homology
Protein homology is biological homology between proteins, meaning that the proteins are derived from a common "ancestor". The proteins may be in different species, with the ancestral protein being the form of the protein that existed in the ancestral species...
; the historical names, however, have mostly been kept, a fact that might cause confusion. For example, the proteins SicA, IpgC and SycD are homologs from Salmonella, Shigella and Yersinia, respectively, but the last letter (the "serial number") in their name does not show that.
Below is a summary of the most common protein-series names in several T3SS-containing species:
- Yersinia
- Yop: Yersinia outer protein
- Ysc: Yersinia secretion (component)
- Ypk: Yersinia protein kinase
- Salmonella
- Spa: Surface presentation of antigen
- Sic: Salmonella invasion chaperone
- Sip: Salmonella invasion protein
- Prg: PhoP-repressed gene
- Inv: Invasion
- Org: Oxygen-regulated gene
- Ssp: Salmonella-secreted protein
- Iag: Invasion-associated gene
- Shigella
- Ipg: Invasion plasmid gene
- Ipa: Invasion plasmid antigen
- Mxi: Membrane expression of Ipa
- Spa: Surface presentation of antigen
- Osp: Outer Shigella protein
- Escherichia
- Tir: Translocated intimin receptor
- Sep: Secretion of E. coli proteins
- Esc: Escherichia secretion (component)
- Esp: Escherichia secretion protein
- Ces: Chaperone of E. coli secretion
- Pseudomonas
- Hrp: Hypersensitive response and pathogenicity
- Hrc: Hypersensitive response conserved (or Hrp conserved)
- In several species:
- Vir: Virulence
Following those abbreviations is a letter or a number. Letters usually denote a "serial number", either the chronological order of discovery or the physical order of appearance of the gene in an operon
Operon
In genetics, an operon is a functioning unit of genomic DNA containing a cluster of genes under the control of a single regulatory signal or promoter. The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo trans-splicing to create...
. Numbers, the rarer case, denote the molecular weight of the protein in kDa
KDA
KDA may refer to:* Karachi Development Authority* Kongsberg Defence & Aerospace* Kotelawala Defence Academy* Kramer Design Associates* Lithium diisopropylamide, KDA is the potassium analogue of lithium diisopropylamideOr kDa may refer to:...
. Examples: IpaA, IpaB, IpaC; MxiH, MxiG, MxiM; Spa9, Spa47.
Several key elements appear in all T3SSs: the needle monomer, the inner rod of the needle, the ring proteins, the two translocators, the needle-tip protein, the ruler protein (which is thought to determine the needle's length; see above) and the ATPase
ATPase
ATPases are a class of enzymes that catalyze the decomposition of adenosine triphosphate into adenosine diphosphate and a free phosphate ion. This dephosphorylation reaction releases energy, which the enzyme harnesses to drive other chemical reactions that would not otherwise occur...
, which supplies energy for secretion. The following table shows some of these key proteins in four T3SS-containing bacteria:
| ↓ Function / Genus → | Shigella | Salmonella | Yersinia | Escherichia |
|---|---|---|---|---|
| Needle monomer | MxiH | PrgI | YscF | EscF |
| Inner rod | MxiI | PrgJ | YscI | EscI |
| Needle-tip protein | IpaD | SipD | LcrV | EspA |
| Translocator | IpaB | SipB | YopB | EspD |
| Translocator | IpaC | SipC | YopD | EspB |
| Chaperone for the two translocators | IpgC | SicA | SycD | CesD |
| ATPase | Spa47 | InvC | YscN | SepB (EscN) |
| Ruler protein | Spa32 | InvJ | YscP | Orf16 |
| Switch | Spa40 | SpaS | YscU | EscU |
| Gatekeeper | MxiC | InvE | YopN (TyeA) | SepL |
Isolation of T3SS needle complexes
The isolation of large, fragile, hydrophobic membrane structures from cells has constituted a challenge for many years. By the end of the 1990s, however, several approaches have been developed for the isolation of T3SS NCs. In 1998 the first NCs were isolated from Salmonella typhimurium.For the isolation, bacteria are grown in a large volume of liquid growth medium
Growth medium
A growth medium or culture medium is a liquid or gel designed to support the growth of microorganisms or cells, or small plants like the moss Physcomitrella patens.There are different types of media for growing different types of cells....
until they reach log phase
Bacterial growth
250px|right|thumb|Growth is shown as L = log where numbers is the number of colony forming units per ml, versus T Bacterial growth is the division of one bacterium into two daughter cells in a process called binary fission. Providing no mutational event occurs the resulting daughter cells are...
. They are then centrifuge
Centrifuge
A centrifuge is a piece of equipment, generally driven by an electric motor , that puts an object in rotation around a fixed axis, applying a force perpendicular to the axis...
d; the supernatant (the medium) is discarded and the pellet
Precipitation (chemistry)
Precipitation is the formation of a solid in a solution or inside anothersolid during a chemical reaction or by diffusion in a solid. When the reaction occurs in a liquid, the solid formed is called the precipitate, or when compacted by a centrifuge, a pellet. The liquid remaining above the solid...
(the bacteria) is resuspended in a lysis buffer
Lysis buffer
A lysis buffer is a buffer solution used for the purpose of lysing cells for use in molecular biology experiments that analyze the compounds of the cells . There are many different kind of lysis buffer that one can apply, depending on what analysis the cell lysate will be used for e.g. red blood...
typically containing lysozyme
Lysozyme
Lysozyme, also known as muramidase or N-acetylmuramide glycanhydrolase, are glycoside hydrolases, enzymes that damage bacterial cell walls by catalyzing hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan and between...
and sometimes a detergent
Detergent
A detergent is a surfactant or a mixture of surfactants with "cleaning properties in dilute solutions." In common usage, "detergent" refers to alkylbenzenesulfonates, a family of compounds that are similar to soap but are less affected by hard water...
such as LDAO or Triton X-100. This buffer disintegrates the cell wall
Cell wall
The cell wall is the tough, usually flexible but sometimes fairly rigid layer that surrounds some types of cells. It is located outside the cell membrane and provides these cells with structural support and protection, and also acts as a filtering mechanism. A major function of the cell wall is to...
. After several rounds of lysis and washing, the opened bacteria are subjected to a series of ultracentrifugations. This treatment enriches large macromolecular structures and discards smaller cell components. Optionally, the final lysate is subjected to further purification by CsCl
CSCL
CSCL can refer to:* Caesium chloride , a chemical compound.* Computer Supported Collaborative Learning, a research topic on supporting collaborative learning with the assistance of computer artifacts....
density gradient
Density Gradient
Density gradient is a spatial variation in density over an area. The term is used in the natural sciences to describe varying density of matter, but can apply to any quantity whose density can be measured...
.
An additional approach for further purification uses affinity chromatography
Affinity chromatography
Affinity chromatography is a method of separating biochemical mixtures and based on a highly specific interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.-Uses:Affinity chromatography can be used to:...
. Recombinant
Recombinant DNA
Recombinant DNA molecules are DNA sequences that result from the use of laboratory methods to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms...
T3SS proteins that carry a protein tag
Protein tag
Protein tags are peptide sequences genetically grafted onto a recombinant protein. Often these tags are removable by chemical agents or by enzymatic means, such as proteolysis or intein splicing. Tags are attached to proteins for various purposes....
(a histidine tag, for instance) are produced by molecular cloning
Molecular cloning
Molecular cloning refers to a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms...
and then introduced (transformed) into the researched bacteria. After initial NC isolation, as described above, the lysate is passed through a column coated with particles with high affinity to the tag (in the case of histidine tags: nickel
Nickel
Nickel is a chemical element with the chemical symbol Ni and atomic number 28. It is a silvery-white lustrous metal with a slight golden tinge. Nickel belongs to the transition metals and is hard and ductile...
ion
Ion
An ion is an atom or molecule in which the total number of electrons is not equal to the total number of protons, giving it a net positive or negative electrical charge. The name was given by physicist Michael Faraday for the substances that allow a current to pass between electrodes in a...
s). The tagged protein is retained in the column, and with it the entire needle complex. High degrees of purity can be achieved using such methods. This purity is essential for many delicate assays that were later used for NC characterization.
The isolation of NCs was a major step in T3SS research. Type III effectors were known since the beginning of the 1990s, but the way in which they are delivered into host cells was a complete mystery. The homology between many flagellar
Flagellum
A flagellum is a tail-like projection that protrudes from the cell body of certain prokaryotic and eukaryotic cells, and plays the dual role of locomotion and sense organ, being sensitive to chemicals and temperatures outside the cell. There are some notable differences between prokaryotic and...
and T3SS proteins led researchers to suspects the existence of an outer T3SS structure similar to flagella. The identification and subsequent isolation of the needle structure enabled researchers to:
- characterize the three-dimensional structure of the NC in detail, and through this to draw conclusions regarding the mechanism of secretion (for example, that the narrow width of the needle requires unfolding of effectors prior to secretion),
- analyze the protein components of the NC, this by subjecting isolated needles to proteomic analysis (see below),
- assign roles to various NC components, this by knocking outGene knockoutA gene knockout is a genetic technique in which one of an organism's genes is made inoperative . Also known as knockout organisms or simply knockouts, they are used in learning about a gene that has been sequenced, but which has an unknown or incompletely known function...
T3SS genes, isolating NCs from the mutated bacteria and examining the changes that the mutations caused.
Microscopy and crystallography
The visualization of T3SS NCs is only possible with electron microscopy. The first images of NCs (1998) showed needle structures protruding from the cell wall of live bacteria and flat, two-dimensional isolated NCs. In 2001 images of NCs from Shigella flexneriShigella flexneri
Shigella flexneri is a species of Gram-negative bacteria in the genus Shigella that can cause diarrhea in humans. There are several different serogroups of Shigella; S. flexneri belongs to group B. S. flexneri infections can usually be treated with antibiotics although some strains have become...
were digitally analyzed and averaged to obtain a first semi-3D structure of the NC. The helical structure of NCs from Shigella flexneri was resolved at a resolution of 16 Å
Ångström
The angstrom or ångström, is a unit of length equal to 1/10,000,000,000 of a meter . Its symbol is the Swedish letter Å....
using X-ray
X-ray
X-radiation is a form of electromagnetic radiation. X-rays have a wavelength in the range of 0.01 to 10 nanometers, corresponding to frequencies in the range 30 petahertz to 30 exahertz and energies in the range 120 eV to 120 keV. They are shorter in wavelength than UV rays and longer than gamma...
fiber diffraction
Fiber diffraction
Fiber diffraction is a subarea of scattering, an area in which molecular structure is determined from scattering data . In fiber diffraction the scattering pattern does not change, as the sample is rotated about a unique axis...
in 2003, and a year later a 17-Å
Ångström
The angstrom or ångström, is a unit of length equal to 1/10,000,000,000 of a meter . Its symbol is the Swedish letter Å....
3D structure of NCs from Salmonella typhimurium was published. Recent advances and approaches have allowed high-resolution 3D images of the NC, further clarifying the complex structure of the NC.
Numerous T3SS proteins have been crystallized over the years. These include structural proteins of the NC, effectors and chaperones. The first structure of a needle-complex monomer was of MxiH from Shigella flexneri, which was resolved in 2006. Crystal structures often give detailed insight into the function of the protein at the molecular and atom
Atom
The atom is a basic unit of matter that consists of a dense central nucleus surrounded by a cloud of negatively charged electrons. The atomic nucleus contains a mix of positively charged protons and electrically neutral neutrons...
level. Many issues regarding the assembly and structure of the NC, the way effectors damage host cells, and the function of chaperones, have been clarified using crystallography.
Proteomics
Several methods have been employed in order to identify the array of proteins that comprise the T3SS. Isolated needle complexes can be separated with SDS-PAGESDS-PAGE
SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, describes a collection of related techniques widely used in biochemistry, forensics, genetics and molecular biology to separate proteins according to their electrophoretic mobility...
. The bands that appear after staining can be individually excised from the gel and analyzed using protein sequencing
Protein sequencing
Protein sequencing is a technique to determine the amino acid sequence of a protein, as well as which conformation the protein adopts and the extent to which it is complexed with any non-peptide molecules...
and mass spectrometry
Mass spectrometry
Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles.It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and...
. The structural components of the NC can be separated from each other (the needle part from the base part, for instance), and by analyzing those fractions the proteins participating in each one can be deduced. Alternatively, isolated NCs can be directly analyzed by mass spectrometry, without prior electrophoresis
Electrophoresis
Electrophoresis, also called cataphoresis, is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. This electrokinetic phenomenon was observed for the first time in 1807 by Reuss , who noticed that the application of a constant electric...
, in order to obtain a complete picture of the NC proteome
Proteome
The proteome is the entire set of proteins expressed by a genome, cell, tissue or organism. More specifically, it is the set of expressed proteins in a given type of cells or an organism at a given time under defined conditions. The term is a portmanteau of proteins and genome.The term has been...
.
Genetic and functional studies
The T3SS in many bacteria has been manipulated by researchers. Observing the influence of individual manipulations can be used to draw insights into the role of each component of the system. Examples of manipulations are:- Deletion of one or more T3SS genes (gene knockoutGene knockoutA gene knockout is a genetic technique in which one of an organism's genes is made inoperative . Also known as knockout organisms or simply knockouts, they are used in learning about a gene that has been sequenced, but which has an unknown or incompletely known function...
). - OverexpressionGene expressionGene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
of one or more T3SS genes (in other words: production in vivoIn vivoIn vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
of a T3SS protein in quantities larger than usual). - Point or regional changes in T3SS genes or proteins. This is done in order to define the function of specific amino acids or regions in a protein.
- The introduction of a gene or a protein from one species of bacteria into another (cross-complementation assay). This is done in order to check for differences and similarities between two T3SSs.
Manipulation of T3SS components can have influence on several aspects of bacterial function and pathogenicity. Examples of possible influences:
- The ability of the bacteria to invade host cells, in the case of intracellular pathogens. This can be measured using an invasion assay (gentamicin protection assayGentamicin protection assayThe gentamicin protection assay or survival assay or invasion assay is a method used in microbiology. It is used to quantify the ability of pathogenic bacteria to invade eukaryotic cells....
). - The ability of intracellular bacteria to migrate between host cells.
- The ability of the bacteria to kill host cells. This can be measured by several methods, for instance by the LDHLactate dehydrogenaseLactate dehydrogenase is an enzyme present in a wide variety of organisms, including plants and animals.Lactate dehydrogenases exist in four distinct enzyme classes. Two of them are cytochrome c-dependent enzymes, each acting on either D-lactate or L-lactate...
-release assay, in which the enzymeEnzymeEnzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
LDH, which leaks from dead cells, is identified by measuring its enzymatic activity. - The ability of a T3SS to secrete a specific protein or to secrete at all. In order to assay this, secretion is induced in bacteria growing in liquid medium. The bacteria and medium are then separated by centrifugation, and the medium fraction (the supernatant) is then assayed for the presence of secreted proteins. In order to prevent a normally secreted protein from being secreted, a large molecule can be artificially attached to it. If the then non-secreted protein stays "stuck" at the bottom of the needle complex, the secretion is effectively blocked.
- The ability of the bacteria to assemble an intact needle complex. NCs can be isolated from manipulated bacteria and examined microscopically. Minor changes, however cannot always be detected by microscopy.
- The ability of bacteria to infect live animals or plants. Even if manipulated bacteria are shown in vitroIn vitroIn vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
to be able to infect host cells, their ability to sustain an infection in a live organism cannot be taken for granted. - The expression levels of other genes. This can be assayed in several ways, notably northern blotNorthern blotThe northern blot is a technique used in molecular biology research to study gene expression by detection of RNA in a sample. With northern blotting it is possible to observe cellular control over structure and function by determining the particular gene expression levels during differentiation,...
and RT-PCR. The expression levels of the entire genomeGenomeIn modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
can be assayed by microarrayMicroarrayA microarray is a multiplex lab-on-a-chip. It is a 2D array on a solid substrate that assays large amounts of biological material using high-throughput screening methods.Types of microarrays include:...
. Many type III transcription factors and regulatory networks were discovered using these methods. - The growth and fitness of bacteria.
Type III signal peptide prediction tools
Further reading
- Instant insight outlining the chemistry of the injectisome from the Royal Society of ChemistryRoyal Society of ChemistryThe Royal Society of Chemistry is a learned society in the United Kingdom with the goal of "advancing the chemical sciences." It was formed in 1980 from the merger of the Chemical Society, the Royal Institute of Chemistry, the Faraday Society and the Society for Analytical Chemistry with a new...
- Host-Pathogen Interaction in Pseudomonas syringae pv. tomato and tomato plant leading to bacterial speck disease.