Andreas Diepold

Deciphering the assembly of the Yersinia type III secretion injectisome

The assembly of the Yersinia enterocolitica type III secretion injectisome was investigated by grafting fluorescent proteins onto several components, YscC (outer‐membrane (OM) ring), YscD (forms the inner‐membrane (IM) ring together with YscJ), YscN (ATPase), and YscQ (putative C ring). The recombinant injectisomes were functional and appeared as fluorescent spots at the cell periphery. Epistasis experiments with the hybrid alleles in an array of injectisome mutants revealed a novel outside‐in assembly order: whereas YscC formed spots in the absence of any other structural protein, formation of YscD foci required YscC, but not YscJ. We therefore propose that the assembly starts with YscC and proceeds through the connector YscD to YscJ, which was further corroborated by co‐immunoprecipitation experiments. Completion of the membrane rings allowed the subsequent assembly of cytosolic components. YscN and YscQ attached synchronously, requiring each other, the interacting proteins YscK and YscL, but no further injectisome component for their assembly. These results show that assembly is initiated by the formation of the OM ring and progresses inwards to the IM ring and, finally, to a large cytosolic complex.

In situ structural analysis of the Yersinia enterocolitica injectisome

Injectisomes are multi-protein transmembrane machines allowing pathogenic bacteria to inject effector proteins into eukaryotic host cells, a process called type III secretion. Here we present the first three-dimensional structure of Yersinia enterocolitica and Shigella flexneri injectisomes in situ and the first structural analysis of the Yersinia injectisome. Unexpectedly, basal bodies of injectisomes inside the bacterial cells showed length variations of 20%. The in situ structures of the Y. enterocolitica and S. flexneri injectisomes had similar dimensions and were significantly longer than the isolated structures of related injectisomes. The crystal structure of the inner membrane injectisome component YscD appeared elongated compared to a homologous protein, and molecular dynamics simulations documented its elongation elasticity. The ring-shaped secretin YscC at the outer membrane was stretched by 30–40% in situ, compared to its isolated liposome-embedded conformation. We suggest that elasticity is critical for some two-membrane spanning protein complexes to cope with variations in the intermembrane distance. DOI: http://dx.doi.org/10.7554/eLife.00792.001

Assembly of the bacterial type III secretion machinery

Many bacteria that live in contact with eukaryotic hosts, whether as symbionts or as pathogens, have evolved mechanisms that manipulate host cell behaviour to their benefit. One such mechanism, the type III secretion system, is employed by Gram-negative bacterial species to inject effector proteins into host cells. This function is reflected by the overall shape of the machinery, which resembles a molecular syringe. Despite the simplicity of the concept, the type III secretion system is one of the most complex known bacterial nanomachines, incorporating one to more than hundred copies of up to twenty different proteins into a multi-MDa transmembrane complex. The structural core of the system is the so-called needle complex that spans the bacterial cell envelope as a tripartite ring system and culminates in a needle protruding from the bacterial cell surface. Substrate targeting and translocation are accomplished by an export machinery consisting of various inner membrane embedded and cytoplasmic components. The formation of such a multimembrane-spanning machinery is an intricate task that requires precise orchestration. This review gives an overview of recent findings on the assembly of type III secretion machines, discusses quality control and recycling of the system and proposes an integrated assembly model.

The assembly of the export apparatus (YscR,S,T,U,V) of the Yersinia type III secretion apparatus occurs independently of other structural components and involves the formation of an YscV oligomer

YscV (FlhA in the flagellum) is an essential component of the inner membrane (IM) export apparatus of the type III secretion injectisome. It contains eight transmembrane helices and a large C‐terminal cytosolic domain. YscV was expressed at a significantly higher level than the other export apparatus components YscR, YscS, YscT, and YscU, and YscV‐EGFP formed bright fluorescent spots at the bacterial periphery, colocalizing in most cases with YscC‐mCherry. This suggested that YscV is the only protein of the export apparatus that oligomerizes. Oligomerization required the cytosolic domain of YscV, as well as YscR, ‐S, ‐T, but no other Ysc protein, indicating that an IM platform can assemble independently from the membrane‐ring forming proteins YscC, ‐D, ‐J. However, in the absence of YscC, ‐D, ‐J, this IM platform moved laterally at the bacterial surface. YscJ, but not YscD could be recruited to the IM platform in the absence of the secretin YscC. As YscJ was shown earlier to be also recruited by the outer membrane (OM) platform made of YscC and YscD, we infer that assembly of the injectisome proceeds through the independent assembly of an IM and an OM platform that merge through YscJ.

Type III secretion systems: the bacterial flagellum and the injectisome.

The flagellum and the injectisome are two of the most complex and fascinating bacterial nanomachines. At their core, they share a type III secretion system (T3SS), a transmembrane export complex that forms the extracellular appendages, the flagellar filament and the injectisome needle. Recent advances, combining structural biology, cryo-electron tomography, molecular genetics, in vivo imaging, bioinformatics and biophysics, have greatly increased our understanding of the T3SS, especially the structure of its transmembrane and cytosolic components, the transcriptional, post-transcriptional and functional regulation and the remarkable adaptivity of the system. This review aims to integrate these new findings into our current knowledge of the evolution, function, regulation and dynamics of the T3SS, and to highlight commonalities and differences between the two systems, as well as their potential applications.

Composition, Formation, and Regulation of the Cytosolic C-ring, a Dynamic Component of the Type III Secretion Injectisome

The injectisome is a membrane complex through which some bacteria can inject effector proteins into host cells. This study reveals that the cytosolic C-ring structure has a dynamic relationship to the rest of the injectisome, with implications for the regulation of secretion.

Assembly of the Yersinia injectisome: the missing pieces

The assembly of the type III secretion injectisome culminates in the formation of the needle. In Yersinia, this step requires not only the needle subunit (YscF), but also the small components YscI, YscO, YscX and YscY. We found that these elements act after the completion of the transmembrane export apparatus. YscX and YscY co‐purified with the export apparatus protein YscV, even in the absence of any other protein. YscY‐EGFP formed fluorescent spots, suggesting its presence in multiple copies. YscO and YscX were required for export of the early substrates YscF, YscI and YscP, but were only exported themselves after the substrate specificity switch had occurred. Unlike its flagellar homologue FliJ, YscO was not required for the assembly of the ATPase YscN. Finally, we investigated the role of the small proteins in export across the inner membrane. No export of the reporter substrate YscP1–137‐PhoA into the periplasm was observed in absence of YscI, YscO or YscX, confirming that these proteins are required for export of the first substrates. In contrast, YscP1–137‐PhoA accumulated in the periplasm in the absence of YscF, suggesting that YscF is not required for the function of the export apparatus, but that its polymerization opens the secretin YscC.

Visualization of the Serratia Type VI Secretion System Reveals Unprovoked Attacks and Dynamic Assembly

Summary The Type VI secretion system (T6SS) is a bacterial nanomachine that fires toxic proteins into target cells. Deployment of the T6SS represents an efficient and widespread means by which bacteria attack competitors or interact with host organisms and may be triggered by contact from an attacking neighbor cell as a defensive strategy. Here, we use the opportunist pathogen Serratia marcescens and functional fluorescent fusions of key components of the T6SS to observe different subassemblies of the machinery simultaneously and on multiple timescales in vivo. We report that the localization and dynamic behavior of each of the components examined is distinct, revealing a multi-stage and dynamic assembly process for the T6SS machinery. We also show that the T6SS can assemble and fire without needing a cell contact trigger, defining an aggressive strategy that broadens target range and suggesting that activation of the T6SS is tailored to survival in specific niches.

PilZ Domain Protein FlgZ Mediates Cyclic Di-GMP-Dependent Swarming Motility Control in Pseudomonas aeruginosa

UNLABELLED The second messenger cyclic diguanylate (c-di-GMP) is an important regulator of motility in many bacterial species. In Pseudomonas aeruginosa, elevated levels of c-di-GMP promote biofilm formation and repress flagellum-driven swarming motility. The rotation of P. aeruginosas polar flagellum is controlled by two distinct stator complexes, MotAB, which cannot support swarming motility, and MotCD, which promotes swarming motility. Here we show that when c-di-GMP levels are elevated, swarming motility is repressed by the PilZ domain-containing protein FlgZ and by Pel polysaccharide production. We demonstrate that FlgZ interacts specifically with the motility-promoting stator protein MotC in a c-di-GMP-dependent manner and that a functional green fluorescent protein (GFP)-FlgZ fusion protein shows significantly reduced polar localization in a strain lacking the MotCD stator. Our results establish FlgZ as a c-di-GMP receptor affecting swarming motility by P. aeruginosa and support a model wherein c-di-GMP-bound FlgZ impedes motility via its interaction with the MotCD stator. IMPORTANCE The regulation of surface-associated motility plays an important role in bacterial surface colonization and biofilm formation. c-di-GMP signaling is a widespread means of controlling bacterial motility, and yet the mechanism whereby this signal controls surface-associated motility in P. aeruginosa remains poorly understood. Here we identify a PilZ domain-containing c-di-GMP effector protein that contributes to c-di-GMP-mediated repression of swarming motility by P. aeruginosa We provide evidence that this effector, FlgZ, impacts swarming motility via its interactions with flagellar stator protein MotC. Thus, we propose a new mechanism for c-di-GMP-mediated regulation of motility for a bacterium with two flagellar stator sets, increasing our understanding of surface-associated behaviors, a key prerequisite to identifying ways to control the formation of biofilm communities.

Yersinia enterocolitica Type III secretion injectisomes form regularly spaced clusters which incorporate new machines upon activation

Bacterial type III secretion systems or injectisomes are multiprotein complexes directly transporting bacterial effector proteins into eukaryotic host cells. To investigate the distribution of injectisomes in the bacterium and the influence of activation of the system on that distribution, we combined in vivo fluorescent imaging and high‐resolution in situ visualization of Yersinia enterocolitica injectisomes by cryo‐electron tomography. Fluorescence microscopy showed the injectisomes as regularly distributed spots around the bacterial cell. Under secreting conditions (absence of Ca2+), the intensity of single spots significantly increased compared with non‐secreting conditions (presence of Ca2+), in line with an overall up‐regulation of expression levels of all components. Single injectisomes observed by cryo‐electron tomography tended to cluster at distances less than 100 nm, suggesting that the observed fluorescent spots correspond to evenly distributed clusters of injectisomes, rather than single injectisomes. The up‐regulation of injectisome components led to an increase in the number of injectisomes per cluster rather than the formation of new clusters. We suggest that injectisome clustering may allow more effective secretion into the host cells.