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).