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.
