Animal (veterinary) Bacterial Secretion Systems (Overview of the T3SS system)

Bacterial Secretion Systems

 Overview of the T3SS system:

Pathogenic bacterial strains are distinguished from non-pathogenic ones by the presence of specific set of genes that code for toxins, secretion systems, effectors that are meant to act extracellularly or effectors that should be delivered inside the host cell cytoplasm. These genes are usually tightly organized in operones that are located in chromosomal areas with a high distribution of mobile elements or can be found in virulence plasmids. Usually these chromosomal areas are called pathogenicity islands as they possess a different GC content from the rest of the genome, which implies  recent acquisition through horizontal gene transfer events. One of the most profound cases was a set of approximately 20-25 genes which together encode one of the best characterized pathogenic mechanisms termed “type III secretion”. By this mechanism extracellularly located bacteria that are in a close contact with a eukaryotic cell deliver proteins into the host cell cytosol. While the T3S apparatus is conserved in pathogens across the plant/animal phyllogenetic divide, the secreted proteins differ considerably. The genes coding for what are now recognized as structural T3SS components were first described as a contiguous cluster, esignated “hrp” in plant pathogens. Important insights into fundamental questions of bacterial pathobiology came with the recognition, in subsequent years, of the T3SS as a complex multiprotein channel dedicated to translocate the effectors from the pathogen to the host. Although originally linked to pathogenesis, T3SS are also found in members of the phylum proteobacteria that are symbiotic, commensal or otherwise associated with insects, nematodes, fishes, plants, as well as in obligatory bacterial parasites of the phylum Chlamydiae.

T3SS is a multicomponent apparatus with the following characteristics:

i)                  when fully developed it spans both bacterial membranes  and the periplasmic space;

ii)               it possesses a large extracellular appendage that reaches the eukaryotic host cell contributing to bacterial adherence;

iii)            it forms the translocation pore in the host cell membrane  to efficiently deliver proteins of bacterial origin inside the host cell;

iv)             a large number of T3SS cytosolic components form the export gate into the bacterial cytoplasm which sorts and prepares the substrates for secretion. 

The integral bacterial membrane part of the T3S apparatus consists of a series of rings. The protein that oligomerizes and forms the outer membrane and periplasmic rings belongs to the secretin family of proteins (which is also common to T2SS) and has a crucial role in T3S biogenesis.Secretins consist of various domains with the C-terminal one integrated in the outer membrane. The N-terminal domains are less conserved among secretion systems and are responsible for the formation of the periplasmic rings. An N-terminal signal targets secretins to the periplasmic space through the Sec pathway. From there  they are delivered to the outer membrane through a specific small lipidated protein, pilotin. Pilotins from various secretion systems possess different structures despite their common function, probably due to their interaction with the non-conserved C-terminal tail of various secretins. Thus, for example, the T3SS pilotin of Shigella flexneri possess an overall fold which differs from the fold of the T3SS pilotin of Pseudomonas aeruginosa or the T2SS pilotins of Neisseria meningitis and P. aeruginosa.

The T3SS inner membrane (IM) rings are formed by the proteins SctD and SctJ. SctD is a single-pass inner membrane protein that oligomerizes to form the most external inner membrane ring of the T3SS. Its N-terminal domain is facing the bacterial cytoplasm and its structure is homologous to forkhead-associated (FHA) domains. The inner membrane part of the  Salmonella typhimurium injecti- some.The inner membrane topology of six conserved components (HrcDSctD, HrcRSctR, HrcSSctS, HrcTSctT, HrcUSctU and HrcVSctV) of the T3SS from Xanthomonas campestris  by translational fusions to a dual alkaline phosphatase–ǃ-galactosidase reporter protein. Full IM rings have been modeled for PrgHSctD and PrgKSctJ based on docking of atomic structures of individual domains to cryo electron microscopy maps. The central density observed in the inner membrane rings (socket region) of a T3SS needle complex cryo electron microscopy reconstruction map from  Salmonella enterica sv. typhimurium is attributed to the SpaPSctR, SpaQSctT, SpaRSctS, SpaSSctU and InvASctV proteins.

 

In the socket region numerous cytosolic components are recruited to orchestrate the secretion of various T3SS substrates, like the ATPase SctN and its various subunits SctO, SctL. As biogenesis of the T3SS must take place before the secretion of the effectors, the first T3SS substrates to be secreted are the proteins that build the needle or pilus (SctF) and the inner rod (SctI), The proteins that form the translocator pore in the eukaryotic membrane along with the proteins found in the needle tip are the next substrates to be secreted prior to effector proteins secretion.

An additional cytoplasmic ring is believed to be formed around the T3SS export gate as in the case of the flagellum. Although never really observed by electron microscopy, recently Lara-Tejero and colleagues have reported the presence of a large platform in the T3SS of S. enterica sv. typhimurium that can sort substrates prior to secretion. This platform consists of SpaOSctQ, OrgASctK and OrgBSctL. Numerous crystal structure determinations of T3SS components have been reported: The structures of the C-terminal domain of HrcQBSctQ , the C-terminal domain of FliN  and the central part of FliM , all members of the SctQ/FliN,Y family and components of the cytoplasmic ring of the T3SS apparatus (C-ring) have been determined. Extended mutational and cross linking studies support a donut-shaped tetramer organization for the

FliN protein which is localized at the bottom of the C-ring. A model where the FliN tetramers alterates with the C-terminal domain of FliM (FliMC) seems to be in agreement with the major features observed in electron microscopic reconstructions. The side-wall of C-ring above the FliN4FliMC array is formed by the middle domain of FliM while the N-terminal domain interacts with the FliG which is localised in proximity with the inner membrane and is the connection unit  between the C-ring and the inner membrane, MS-ring. FliG has no homolog in non-flagellar T3SS and the homolog SctQ proteins are interacting to the T3SS injectisome through the SctD proteins.

 

The structures of EscUSctU and YscUSctU,  EPEC and  Yersinia homologs of HrcUSctU respectively provide insights into the properties of conserved  core components. The periplasmic domain of PrgHSctC from Salmonella  and the cytoplasmic domain of MxiDSctC from Structures of the periplasmic domains of the membrane components EscJSctJ from the enteropathogenic  Escherichia coli (EPEC) are also available.

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The T3SS secretion signal

Type III effector proteins (T3EPs) possess non-cleavable secretion signals in the N-terminal protein regions, but no discernible amino acid or peptide similarities can be found. Three different types of potential secretion signals have been discussed:

i)                  theN-terminus of the effector protein,

ii)               the ability of a chaperone to bind the effector before secretion,

iii)            the 5’-end region of the mRNA; this hypothesis is very controversial.

The prevailing view, supported by extensive biocomputing analyses, is that the amino acid composition of the N-terminal region of the effectors serves as secretion signal. The required N-terminal peptide length for secretion is usually 10–15 residues, whereas the minimum length needed for translocation is 50–60 residues. Additional targeting information is contained within the first 200 residues which provide binding sites for secretion chaperones. T3SS chaperones of mammal pathogens interact with their cognate effectors through a chaperone-binding domain (CBD) located within the first 100 amino acids of the effector, after the N-terminal export signal.

Analyses of effectors from pathogenic bacteria revealed that the 25 N-terminal residues are enriched in Ser and lack Leu. The N-terminal regions of T3EPs are probably unfolded, which is an important prerequisite for their transport through the narrow inner T3SS channel of presumably only 2.8 nm in diameter as was previously shown for the T3SS of several animal pathogenic

bacteria.

For some effectors however, the N-terminal secretion signal is not sufficient for maximal secretion and specific chaperone proteins are needed; these are usually located adjacent to the cognate effector genes, suggesting strong selection for their coexistence in the genome. T3S chaperones are proposed to play a role in targeting secretory cargo to the injectisome, either by providing targeting information, orfacilitating the exposure of the N-terminal export signal. Some chaperones are involved in the translocation of many substrate proteins, Class I chaperones (the chaperones of effectors) are soluble small, usually homodimeric proteins that bind effector proteins. Although diverse in their sequences, they belong to the structural class of  ǂ/ǃ proteins with a two-layer-sandwich architecture. For the chaperone-effector interaction a strand of the effector is added to extend the ǃ-sheet layer of the chaperone Class I chaperones have been further subclassified depending on whether they associate with one (class Ia) or several (class Ib) effectors. Class II chaperones are T3SS chaperones of the translocators. Experimental determinations of their structures  have confirmed earlier sequence analyses predicting an all-ǂ-helical domain structure, with the bulk of the protein consisting of three tandem tetratricopeptide repeats (TPRs) which are involved in protein-protein interactions.Their substrate is recognised and bound into a concave site of the chaperone. Class III chaperones prevent the premature polymerization of needle components in the bacterial cytoplasm. They are predicted to adopt extended ǂ-helical structures; this was confirmed by the crystal structure of the CesA which binds the EspA filament protein.

Many functions have been attributed to T3SS chaperones, but the exact role(s) of the entire family of chaperones remain to be determined. However, it has been proposed that one of the main roles of the T3SS chaperones is the stabilization of at least some effector proteins inside bacterial cell, as well as their maintainance in a secretion-competent state, i.e. a partially folded or unfolded conformation.