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.

Animal(veterinary) Bacterial Secretion Systems (introduction)

Bacterial Secretion Systems

 Introduction 

 Secretion in unicellular species is the transport or translocation of molecules, for example proteins, from the interior of the cell to its exterior. In bacteria secretion is a very important mechanism, either modulating their interactions with their environment for adaptation and survival or establishing interactions with  their eukaryotic hosts for pathogenesis or symbiosis. To overcome the physical barriers of membranes, Gram-negative bacteria use a variety of molecular machines which have been elaborated to secrete a wide range of proteins and other molecules; their functions include biogenesis of organelles (e.g. pili and flagella), virulence, efflux of toxins etc. As  in some cases the secreted proteins are destined to enter host cells (effectors, toxins), some of the secretion systems include extracellular appendices to translocate proteins across the plasma membrane of the host.

With the rapid accumulation of bacterial  genome sequences, our knowledge of the complexity of bacterial protein secretion systems has expanded and several secretion systems have been identified. Gene Ontology has been very useful for describing the components and functions of these systems,  and for capturing the similarities among the diverse systems (Tseng et al., 2009). These analyses along with numerous biochemical studies have revealed the existence of at least six major mechanisms of protein secretion.These pathways are highly conserved throughout the Gram-negative bacterial species and are functionally independent with respect to outer membrane translocation; commonalities exist in the inner membrane transport steps of some systems, with most of them being terminal branches of the general secretion pathway (Sec). The pathways have been numbered Type I, II, III, IV, V and VI. 

In Gram-negative bacteria, some secreted proteins are exported across the inner and outer membranes in a single step via the Type I, III, IV or VI pathways. Other proteins are first exported into the periplasmic space using the universal Sec or two-arginine (Tat) pathways and then translocated across the outer membrane via the Type II, V or less commonly, the Type I or IV machinery. In Gram-positive bacteria, secreted proteins are commonly translocated across the single membrane by the Sec pathway, the two-arginine (Tat) pathway, or the recently identified type VII secretion system. In the following we will briefly survey the six Gram-negative bacterial secretion systems known to modulate interactions with host organisms:

Type I secretion system: 

This system (T1SS) forms a contiguous channel traversing the inner and outer membranes of Gram-negative bacteria. It is a simple system, which consists of only three major components: ATP-binding cassette transporters, Outer Membrane Factors, and Membrane Fusion Proteins. T1SS transports ions and various molecules including proteins of various sizes (20 900 kDa) and non-proteinaceous substrates like cyclic ǃ-glucans and polysaccharides. 

Type II secretion system:

This system (T2SS) is encoded by at least 12 genes and supports the transport of a group of seemingly unrelated proteins across the outer membrane. In  order for these proteins to enter the type II secretion pathway, they have to first translocate across the cytoplasmic membrane via the Sec-system and then fold into a translocation competent conformation in the periplasm. Proteins secreted by T2SS include proteases, cellulases, pectinases, phospholipases, lipases, and toxins which contribute to cell damage and disease. Although Sec-dependent translocation is universal, the T2SS is found only in Gram-negative proteobacteria phylum. A bacterial species may have more than one T2SS.

Type III secretion system: 

These systems (T3SS) are essential mediators of the interaction of many Gram-negative pathogenic proteobacteria (ǂ,  ǃ,  DŽ and  Dž subdivisions) with their human, animal, or plant hosts  and are evolutionarily related to bacterial flagella. The machinery of the T3SS, termed the injectisome, appears to have a common evolutionary origin with the flagellum and translocates a diverse repertoire  of effector proteins either to extracellular locations or directly into eukaryotic cells, in a Sec-independent manner. The T3SS effectors(T3EPs) modulate the function of crucial host regulatory molecules and trigger a range of highly dynamic cellular responses which determine pathogen-host recognition, pathogen/symbiont accommodation and elicitation or suppression of defense responses by the eukaryotic hosts. In some cases however, effector proteins are simply secreted out of the cell. T3SS have evolved into seven families. Some bacteria may harbor more than one T3SS, usually from different families. T3SS genes are encoded in pathogenicity islands and/or are located on plasmids, and are commonly subject to horizontal gene transfer. 

Type IV secretion system: 

In comparison to other secretion systems, T4SS is unique in its ability to transport nucleic acids in addition to proteins into plant and animal cells, as well as into yeast and other bacteria. Usually T4SS comprises 12 proteins that can be identified as homologs of the VirB1–11 and VirD4 proteins of the Agrobacterium tumefaciens Ti plasmid transfer system. T4SS spans both membranes of Gram-negative bacteria, using a specific transglycosylase, VirB1, to digest the intervening murein . While many organisms have homologous type IV secretion systems, not all systems contain the same sets of genes. The only common protein is VirB10 (TrbI) among all T4SS systems. 

Type V secretion system:

T5SS is  the simplest protein secretion mechanism. Proteins are

secreted via the autotransporter system (type Va or AT-1), the two-partner secretion pathway (type Vb), and the oligomeric autotransporters (type Vc or AT-2 system). Proteins secreted via these pathways have similarities in their primary structures as well as striking similarities in their modes of biogenesis.There are three sub-classes of T5SS. The archetypal bacterial proteins secreted via the T5SS (T5aSS subclass) consist of a N-terminal passenger domain of 40-400 kD in size and a conserved C-terminal domain. The proteins are synthesized with a N-terminal signal peptide that directs their export into the periplasm via the Sec machinery. 

Type VI secretion system:

In T6SS 13 genes are thought to constitute the minimal number needed to produce a functional apparatus. TheT6SS gene clusters (T6SS loci) often occur in multiple, non-orthologous  copies per genome and have probably been acquired via horizontal gene transfer. Each T6SS probably assumes a different role in the interactions of the harbouring organism with others. Although the T6SS has been studied primarily  in the context of pathogenic bacteria-host interactions, it has been suggested that it may also function to promote commensal or mutualistic relationships between bacteria and eukaryotes, as well as to mediate cooperative or competitive interactions between bacterial species.  The T6SS machinery constitutes a phage-tail-spike-like injectisome that has the potential to introduce effector proteins directly into the cytoplasm of host cells, analogous to the T3SS and T4SS machineries.

Genetic, structural and biochemical studies of the above bacterial secretion systems along with massive  in silico analyses of microbial genomes have been used to distinguish pathogens from their non-pathogenic relatives. These studies have established the presence of characteristic conserved features within  individual types of secretion systems, along with considerable sequence and structural diversities within each system at the level of specific components and effector proteins.

Despite the complexity of these systems however, the problem of identifying conserved features and properties within each secretion system type, or across several types of systems is of particular importance, going beyond  a fundamental understanding of how bacterial secretion works. Even for well studied pathogens, not all virulence factors have been identified, making it possible that e.g. effector proteins that are associated with different diseases are still unknown. In less well characterized bacterial species there is certainly a wide spectrum of unknown effectors. This  situation may be now changing through new approaches that use advanced machine learning algorithms to identify within individual types of secretion systems common themes for effectors and other system components thatgo beyond simple amino acid motifs), or through the identification of important structural and physicochemical properties as universal signatures of virulence factors.

This review will focus on the well-characterized T3SS proteins where the prevalence of coiled-coil domains along with pronounced structural flexibility/disorder have been proposed to be characteristic properties associated with a protein-protein interaction mode within T3SS and as essential requirements for secretion. Common themes with other secretion systems (T4SS, T6SS) will be also discussed.