type three secretion system | Assignments Plant pathology

Type III secretion systems (T3SSs) are protein transport nanomachines that are

used by numerous important Gram-negative bacterial pathogens and symbionts

to establish trans-kingdom interactions with different hosts. They are essential

virulence factors for many notorious bacterial pathogens, including the agents

of plague and typhoid fever.

T3SSs evolved from the flagellum, which is a key organelle for bacterial

motility, and the core components that are involved in the assembly of these

complex nanomachines are highly conserved. Although the flagellar systems

are largely inherited vertically, the non-flagellar T3SSs can be transmitted

through horizontal gene transfer.

T3SSs are syringe-shaped, multi-megadalton complexes that are composed of

more than 20 proteins and a series of ring structures embedded in the bacterial

inner and outer membranes, as well as translocation pores in the host cell

membrane. Encircled by these rings is a hollowed conduit that enables the

delivery of partially unfolded virulence effector proteins into the host cell.

Integrative imaging technologies that combine cryo-electron microscopy (cryo-

EM), X-ray crystallography, NMR and computer modelling have enabled the

high-resolution visualization of key substructures of the T3SSs and the

nanomachine in action. Assembly of the T3SS apparatus and substrate

secretion occur in a defined temporal order and hierarchy, and genetic analyses

and molecular biology have enabled the identification and functional

characterization of the key regulators that control these processes.

Effector proteins that are secreted through T3SSs carry out various functions

within the host cell, including the manipulation of host immune responses and

actin cytoskeletal dynamics, subverting gene expression and post-translational

modifications, hijacking signal transduction pathways, and interrupting vesicle

transport and endocytic trafficking, all of which can promote bacterial

colonization, survival and replication.

T3SSs are attractive targets for vaccines and therapeutics owing to their

essential roles in bacterial virulence and pathogenicity. By targeting bacterial

virulence mechanisms instead of growth, inhibitors of T3SSs may exert less

selective pressure on pathogens to develop drug resistance. Structural and

functional characterization of T3SSs should facilitate mechanism-based drug

design

The type III secretion system (T3SS) is a membrane-embedded nanomachine found in several

Gram-negative bacteria. Upon contact between bacteria and host cells, the syringelike T3SS

transfers proteins termed effectors from the bacterial cytosol to the cytoplasm or the plasma

membrane of a single target cell. This is a major difference from secretion systems that merely

release molecules into the extracellular milieu, where they act on potentially distant target cells

expressing the relevant surface receptors. The syringe architecture is conserved at the structural

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 Type III secretion systems (T3SSs) are protein transport nanomachines that are

used by numerous important Gram-negative bacterial pathogens and symbionts

to establish trans -kingdom interactions with different hosts. They are essential

virulence factors for many notorious bacterial pathogens, including the agents

of plague and typhoid fever.

 T3SSs evolved from the flagellum, which is a key organelle for bacterial

motility, and the core components that are involved in the assembly of these

complex nanomachines are highly conserved. Although the flagellar systems

are largely inherited vertically, the non-flagellar T3SSs can be transmitted

through horizontal gene transfer.

 T3SSs are syringe-shaped, multi-megadalton complexes that are composed of

more than 20 proteins and a series of ring structures embedded in the bacterial

inner and outer membranes, as well as translocation pores in the host cell

membrane. Encircled by these rings is a hollowed conduit that enables the

delivery of partially unfolded virulence effector proteins into the host cell.

 Integrative imaging technologies that combine cryo-electron microscopy (cryo-

EM), X-ray crystallography, NMR and computer modelling have enabled the

high-resolution visualization of key substructures of the T3SSs and the

nanomachine in action. Assembly of the T3SS apparatus and substrate

secretion occur in a defined temporal order and hierarchy, and genetic analyses

and molecular biology have enabled the identification and functional

characterization of the key regulators that control these processes.

 Effector proteins that are secreted through T3SSs carry out various functions

within the host cell, including the manipulation of host immune responses and

actin cytoskeletal dynamics, subverting gene expression and post-translational

modifications, hijacking signal transduction pathways, and interrupting vesicle

transport and endocytic trafficking, all of which can promote bacterial

colonization, survival and replication.

 T3SSs are attractive targets for vaccines and therapeutics owing to their

essential roles in bacterial virulence and pathogenicity. By targeting bacterial

virulence mechanisms instead of growth, inhibitors of T3SSs may exert less

selective pressure on pathogens to develop drug resistance. Structural and

functional characterization of T3SSs should facilitate mechanism-based drug

design

The type III secretion system (T3SS) is a membrane-embedded nanomachine found in several Gram-negative bacteria. Upon contact between bacteria and host cells, the syringelike T3SS transfers proteins termed effectors from the bacterial cytosol to the cytoplasm or the plasma membrane of a single target cell. This is a major difference from secretion systems that merely release molecules into the extracellular milieu, where they act on potentially distant target cells expressing the relevant surface receptors. The syringe architecture is conserved at the structural

and functional level and supports injection into a great variety of hosts and tissues. However, the pool of effectors is species specific and determines the outcome of the interaction, via modulation of target-cell function. The T3SS-mediated phenomenon of contact-dependent translocation of bacterial proteins into host cells was first reported for Yersinia pestis 20 years ago. Since then, studies have shown that bacteria use this system to communicate with organisms belonging to other kingdoms, including protists, fungi, plants and animals. Both commensal and pathogenic bacteria express T3SSs. Nevertheless, the T3SS has received far more attention in microorganisms that cause disease in humans, livestock or crops because of the social and economic consequences. In fact, the T3SS is a crucial virulence factor in many bacteria, as highlighted by the reduced, if not abolished, virulence of bacterial mutants with impaired secretion. Table 1 gives an overview of model bacteria endowed with T3SSs. Two classes of T3SSs have been described: the flagellar and the nonflagellar T3SS, which is also termed the injectisome. This Primer will only discuss non-flagellar T3SSs. Structure and assembly of the injectisome The T3SS is an approximately 3.5 MDa complex that spans the Magazine R785 Gram-negative double membrane and protrudes into the extracellular: around 25 structural and ancillary proteins are required for its assembly. It encompasses an ATPase-containing cytoplasmic domain, membrane-integrated inner rings, a periplasm- traversing domain, membrane-integrated outer rings, a hollow filament establishing the connection between the bacterial cytosol and the target cell, and translocators forming a pore in the plasma membrane of target cells Additionally, the T3SS can possess a tip protein, which blocks secretion until host contact is established. The filament connecting the bacterial cytosol to the target cell is a pilus in microbes interacting with plants, but a needle in microbes that interact with other organisms. The outer membrane ring is related to secretins of type II secretion systems and type IV pili. After Sec- or Tatdependent transport of secretion substrates from the bacterial cytosol to the periplasm, the type II secretion system transfers proteins across a multimeric pore complex (the secretin) to the extracellular milieu. Such a pore complex is also present in type IV pili, which can deliver proteins or DNA to target cells (similarly to T3SSs), but can also take up DNA from the extracellular milieu. For its assembly, the T3SS exports its own periplasmic and extracellular components: secretion is hierarchical, starting with subunits that are closer to the base. Peptidoglycandegrading enzymes assist the T3SS with its insertion into the periplasmic mesh. Needle length is determined by molecular rulers and lies within the nanometre range, whilst pili are several micrometres long to allow for penetration of the thick wall of plant cells. Furthermore, to facilitate pilus insertion the T3SS translocates enzymes termed harpins, which modify the plant cell wall. Regulation of secretion Secretion signal in effectors After injectisome assembly, a substrate switch allows for the secretion of effectors, which can be delayed with respect to assembly. At the molecular level, the substrate switch is poorly understood. In most species, the injectisome is assembled long before encounter with a target cell, but in some species assembly takes place just before injection of effectors. To what extent a hierarchy of injection exists for co-expressed effectors and how it is regulated is poorly understood. T3SS effectors do not have a specific secretion signal. Rather, the amino-acid composition of a large amino-terminal region of the effector seems crucial to the recognition of secretion substrates. Secreted hybrid proteins can be constructed by fusing the portion of the effector gene encoding the amino terminus to a gene of interest. The aminoterminal secretion

Genetic and functional studies [edit]

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 knockout).  Overexpression of one or more T3SS genes (in other words: production in vivo 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 assay).  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 LDH-release assay, in which the enzyme 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 vitro 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 blot and RT-PCR. The expression levels of the entire genome can be assayed by microarray. Many type III transcription factors and regulatory networks were discovered using these methods.  The growth and fitness of bacteria.

Function

Activation of Secretion.

Yet, a signal is required to allow either the hook/filament transition or induction of secretion by TTSSs. Induction of secretion is the instantaneous, minimum 50% posttranslational release of proteins with translocator or effector functions. Cotranslational secretion probably occurs at a slower rate Assembling TTSSs secrete proteins that are not effectors, but external parts of the machine or regulators of assembly. Beyond this stage, bacteria continue to secrete these proteins and also early effectors at low levels much as flagella do before the hook/filament transition The natural signal for secretion may be the lipid membrane of animal cells or the cell wall of plant cells Presumably, the signal is transmitted from the distal needle tip to the cytoplasmic ATPase powering secretion. The needle of the NC reaches to the periplasmic leaflet of the inner membrane Flagellar filament switching from right- to left-handed supercoiled helical conformations is thought to occur by a 0.8-Å increase in the distance between flagellin molecules within some protofilament(s), leading to discreet alterations of helical parameters If needle subunits also pack helically, similar switch(es) in their straight superstructure might transmit tactile signal(s). How the flagellar hook endogenously signals its completion, leading to FlgK, FlgL, and FliD secretion, is unclear. The hook cap FlgD is displaced by FlgK, allowing filament growth The NC cap may sense host cell proximity, an external signal, via a related mechanism and become lost at TTSS activation

The ATPase: Energy Requirements and Transport Intermediates.

Are effector motifs recognized by the secreton?

Recently determined structures of effectors alone or in complex with their chaperone suggest that ( i ) the binding of the chaperone partially unfolds the underlying effector region, preventing the N-terminal amphipathic helix from folding against the rest of the protein, and ( ii ) the part of the effector beyond the chaperonebinding domain is folded during storage (see Supporting Text ). If and how part(s) of the secreton recognize the opened helix is unknown. How a motif as common as a loose amphipathic helix serves as a selective

type three secretion system, Assignments of Plant pathology

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Download type three secretion system and more Assignments Plant pathology in PDF only on Docsity!

 Type III secretion systems (T3SSs) are protein transport nanomachines that are

used by numerous important Gram-negative bacterial pathogens and symbionts

to establish trans -kingdom interactions with different hosts. They are essential

virulence factors for many notorious bacterial pathogens, including the agents

of plague and typhoid fever.

 T3SSs evolved from the flagellum, which is a key organelle for bacterial

motility, and the core components that are involved in the assembly of these

complex nanomachines are highly conserved. Although the flagellar systems

are largely inherited vertically, the non-flagellar T3SSs can be transmitted

through horizontal gene transfer.

 T3SSs are syringe-shaped, multi-megadalton complexes that are composed of

more than 20 proteins and a series of ring structures embedded in the bacterial

inner and outer membranes, as well as translocation pores in the host cell

membrane. Encircled by these rings is a hollowed conduit that enables the

delivery of partially unfolded virulence effector proteins into the host cell.

 Integrative imaging technologies that combine cryo-electron microscopy (cryo-

EM), X-ray crystallography, NMR and computer modelling have enabled the

high-resolution visualization of key substructures of the T3SSs and the

nanomachine in action. Assembly of the T3SS apparatus and substrate

secretion occur in a defined temporal order and hierarchy, and genetic analyses

and molecular biology have enabled the identification and functional

characterization of the key regulators that control these processes.

 Effector proteins that are secreted through T3SSs carry out various functions

within the host cell, including the manipulation of host immune responses and

actin cytoskeletal dynamics, subverting gene expression and post-translational

modifications, hijacking signal transduction pathways, and interrupting vesicle

transport and endocytic trafficking, all of which can promote bacterial

colonization, survival and replication.

 T3SSs are attractive targets for vaccines and therapeutics owing to their

essential roles in bacterial virulence and pathogenicity. By targeting bacterial

virulence mechanisms instead of growth, inhibitors of T3SSs may exert less

selective pressure on pathogens to develop drug resistance. Structural and

functional characterization of T3SSs should facilitate mechanism-based drug

design

Genetic and functional studies [edit]

Function

Activation of Secretion.

The ATPase: Energy Requirements and Transport Intermediates.

Are effector motifs recognized by the secreton?