Super-resolution in secretion systems:
Pathogenic bacteria have developed different types of systems to secrete
molecules into the extracellular space or to translocate them into host
cells (Filloux, 2022). The secreted or translocated molecules often
serve as virulence factors, for example, to compete with other bacterial
species, to invade mammalian host cells or to evade the host immune
system (Galan, 2009, Le et al., 2021). Bacterial secretion systems can
consist of a single protein or more than 20 different proteins that
combine to form complex macromolecular machines, as in the case of the
T3SS (Wagner et al., 2018, Jenkins et al., 2022). Most of our knowledge
about the structure and function of secretion systems comes from
genetic, biochemical and structural biological as well as electron
microscopic studies (Berger et al., 2021, Hu et al., 2018, Lunelli et
al., 2020, Marlovits et al., 2004, Worrall et al., 2016).
T3SSs, also called injectisomes, are found in numerous pathogens
including Yersinia, Pseudomonas, Shigella and Salmonella,and translocate effector proteins into eukaryotic host cells (Wagner et
al., 2018). Although the T3SSs of the various pathogens are highly
conserved, the effectors injected by them differ significantly in
structure and function and can manipulate a variety of cellular
processes. This ultimately determines the interaction of each pathogen
with the host and the outcome of the infection (Galan, 2009). T3SSs have
a width of ∼40 nm and a length of ∼150 nm and consist of both, stable
components (needle complex, export apparatus) and transiently associated
components (sorting platform, tip complex, pore complex) (Fig. 1A). The
needle complex is a multi-ringed cylindrical structure embedded in the
bacterial cell envelope and connected to a 30-70 nm long needle filament
that points into the extracellular space. Together with the export
apparatus, it forms a channel through which the structural and effector
proteins of the system are transported (Miletic et al., 2021). At the
distal end of the needle is the tip complex, which is involved in host
cell recognition, activation of secretion, and regulation of the
assembly of the pore complex upon host cell contact (Veenendaal et al.,
2007, Deane et al., 2006). Lastly, several cytoplasmic proteins form a
heteromultimeric complex known as the sorting platform, which is
involved in the selection and sorting of proteins destined for secretion
and translocation (Lara-Tejero et al., 2011).
Even though the complexity and presumed molecular dynamics of bacterial
secretion systems asks for an investigation with fluorescence
microscopic methods, the resolution of these methods has long been
insufficient for this purpose. However, novel super-resolution
microscopy techniques developed in the last two decades pushed the
resolution limit for fluorescently labeled molecules into the nanometer
range (Fig. 1A). Such techniques now also allow the study of bacterial
secretion systems at much higher resolution (Sahl et al., 2017). Some
relevant reports on this subject are presented below.
In an early systematic analysis, the suitability of the SLEs HaloTag and
SNAP-tag for super resolution microscopy of different Salmonella
enterica secretion system subunits was tested. The tags were
genetically linked to subunits of a type I secretion system (T1SS) and
T3SS (and to the flagellar rotor and a transcription factor), the tagged
proteins were labeled by cell-permeable dyes and analyzed by dSTORM and
SMT. This allowed determination of the number, subcellular localization
and dynamics of protein complexes in living bacteria (Barlag et al.,
2016). In a follow-up study, S. enterica T3SS effectors fused to
SLEs were found to be translocated into host cells where they remained
functional and were properly located (Goser et al., 2019). However, it
is important to consider that the SLEs may be secreted with greatly
varying efficiency depending on the type of tagged protein and T3SS
involved species (Singh and Kenney, 2021).
Using PALM, the intrabacterial distribution of the ATPase SecA, which is
the driving force for protein secretion by the SecYEG translocon, was
evaluated in E. coli . SecA was mostly localized as a homodimer
along the cytoplasmic membrane and diffused along it in three different
diffusion rate populations as found by SMT (Seinen et al., 2021).
The type I secretion system substrate hemolysin A (HlyA) was imaged on
the surface of E. coli using SIM. In contrast to other bacterial
secretion systems, HlyA showed no polarization on the cell surface and
its distribution was not influenced by cell growth and division cycle
(Beer et al., 2022).
Using SIM, the sfGFP-labeled inner membrane component VirB6 of theAgrobacterium tumefaciens type 4 secretion system was found to
preferentially localize to the cell poles (Mary et al., 2018). SIM was
also employed to subcellularly localize type 6 secretion system (T6SS)
assembly in response to cell-cell contact in Acinetobacter
baylyi , Acinetobacter baumanii and Burkholderia
thailandensis . Employing sfGFP-tagged sheath protein TssB, the
polymerization rate and time as well as the disassembly of the
contractile sheaths could be visualized. The individual T6SSs were
mainly assembled at the site of contact with neighboring bacterial
cells, whereby periplasmic proteins as well as the outer membrane
protein OmpA mediated this localization (Lin et al., 2022).
SIM was also used to show distributions of the type 9 secretion system
components GldL, GldM, GldK and GldN in Flavobacterium
johnsoniae . All of these proteins seem to be distributed in foci along
the bacterial circumference. GldK and GldN, which are part of the GldKN
complex, showed in average less foci per cell than GldL and GldM,
suggesting two subpopulations of GldLM complexes, one free and one
associated with GldKN rings (Vincent et al., 2022).
In a comprehensive super-resolution microscopy study of a T3SS, various
T3SS components in Salmonella Typhimurium were labeled with
fluorescent antibodies or the photoswitchable fluorophore mEos 3.2 and
visualized with 2D and 3D SMLM (Zhang et al., 2017). Thereby,
subcellular distributions and rough numbers of needle complexes, sorting
platform components, tip complex and an effector could be determined.
Needle complexes including export apparatus were almost exclusively
located at the bacterial plasma membrane, whereas a considerable
fraction of sorting platform components was also in the cytoplasm,
suggesting that sorting platforms are transiently and dynamically
associated with the needle complexes (Prindle et al., 2022) (Diepold et
al., 2017). The relative stochiometries of components of the sorting
platform and export apparatus could be determined, confirming previous
observations using other techniques (Diepold et al., 2015, Zilkenat et
al., 2016, Diepold et al., 2017). Further, due to the estimated
resolution of ∼ 35nm of the microscopic technique, the needle complex
protein PrgH (unified nomenclature: SctD) and the tip complex protein
SipD (unified nomenclature: SctA) could be visualized at a distance of ∼
100 nm in individual injectisomes (Fig. 1A). It was also found that
needle complexes are essential for the assembly of sorting platforms and
that the effector SopB is mainly found in clusters in the cytoplasm and
this does not depend on the parallel presence of needle complexes or
sorting platforms (Zhang et al., 2017).
STED microscopy and SIM were used to visualize the Yersinia
enterocolitica T3SS pore complex proteins YopB and YopD (unified
nomenclature: SctE and SctB, respectively) in infected host cells. Per
bacterium ∼ 30 what appeared to be single translocation pores at the tip
of injectisome needles formed upon host cell contact. The two pore
proteins YopB and YopD on one side and the needle complex/basal body
protein component YscD (unified nomenclature: SctD) on the other side of
single injectisomes could be resolved at a mean distance of ∼ 109 nm.
Further, 3D-STED microscopy allowed to localize YopB in translocation
pores which formed in a peculiar pre-vacuolar compartment in the
infected cells (Nauth et al., 2018). To minimize the label error for
MINFLUX nanoscopy, an ALFA-tag was introduced into YopD’s extracellular
domain (giving rise to YopD-ALFA). It was demonstrated that the ALFA-tag
did not compromise the central functions of YopD during protein
translocation by the Y. enterocolitica T3SS (Rudolph et al.,
2022). MINFLUX nanoscopy allowed to visualize single YopD-ALFA molecules
bound by fluorescent nanobodies in Yersinia translocation pores.
The localization precision was ~ 5 nm and thus the size
of the pore could be determined to be ~ 18 nm. Further,
clusters consisting of 12 molecules of sorting platform protein YscL
(unified nomenclature: SctL) fused with a HaloTag were recorded by 2D
and 3D MINFLUX microscopy. With an isotropic localization precision of
~ 5 nm, these experiments could reproduce the size of
the YscL structure determined by Cryo ET to be ~16 nm in
diameter (Carsten et al., 2022) (Berger et al., 2021). 3D MINFLUX
experiments performed in whole bacteria showed that the YscL complexes
localized almost exclusively at the plasma membrane and at very low
distances to each other (down to ~10 nm apart) (Carsten
et al., 2022).
SMT and SMLM in live Yersinia enterocolitica revealed distinct
diffusive states of the eYFP, eGFP and PAmCherry labelled sorting
platform components YscQ, YscL and YscN (unified nomenclature: SctQ,
SctL and SctN) and suggested that they form distinct cytosolic complexes
before binding to the needle complex (Rocha et al., 2018, Prindle et
al., 2022, Diepold et al., 2015). SMT of eGFP labelled YscD (unified
nomenclature: SctD) showed partial disassembly of the T3SS basal body
component at low external pH (Wimmi et al., 2021).
PALM was used to show that the Salmonella pathogenicity island-2 (SPI-2)
signaling proteins SsrA/B labeled with PAmCherry were induced under low
pH conditions. Furthermore, SMT identified pH-dependent DNA binding of
SsrB (Liew et al., 2019).
Recently Halo-tagged S. enterica effectors PipB2, SseF, SseJ and
SifA were visualized using SMT and SMLM. A bidirectional motility along
tubular membrane structures of SseF, SifA and PipB2 was revealed
providing novel and comprehensive information about the mobility ofSalmonella SPI-2 effectors. Co-motion tracking analysis showed
identical movement patterns of PipB2 together with the GFP labelled host
protein LAMP1 (Goser et al., 2023).