Biology subjects

Diepold, A.

Publications and source records attributed to Diepold, A..

13 recordsLinked to original sources

bioRxiv2026

A simple platform for in situ DNA-PAINT optimization enables improved multiplexed super-resolution imaging in Gram-negative bacteria

DNA-PAINT enables super-resolution imaging with high multiplexing capacity, yet it is rarely used in nanoscale bacteriology. A likely reason is the lack of straightforward protocols and of systematic characterization of imager-docking strand pairs inside intact bacterial cells, whose envelope and dense cytoplasm differ markedly from both the DNA origami in vitro systems used to develop them, and eukaryotic cells. In this work, we establish a screening platform based on RNA-FISH, in which we extend the 16S rRNA-directed EUB338 probe with docking sequences of choice. This allows imager-docking pairs to be benchmarked directly inside Escherichia coli cells and characterized for parameters such as specificity, binding kinetics, and localization precision. We screened 12 different DNA-PAINT sequences and used an optimized set to perform four-target 3D Exchange-PAINT in single E. coli cells. We further applied our strategy to image three effector mRNAs in the Gram-negative pathogen Yersinia enterocolitica and quantified RNA clusters and their 3D distribution. Our work provides a framework and practical guidelines for high-quality 3D DNA-PAINT imaging in Gram-negative bacteria and should facilitate its broader adoption in nanoscale bacteriology.

microbiology↗

bioRxiv2025

Modulating gene expression and protein secretion in the bacterial predator Bdellovibrio bacteriovorus

The predatory bacterium Bdellovibrio bacteriovorus kills and consumes other bacteria, thrives in diverse environments and holds great potential to address major challenges in medicine, agriculture, and biotechnology. As a bacterial predator it represents an alternative to traditional antimicrobial strategies to combat multidrug-resistant bacterial pathogens and prevent food waste, while the multitude of predatory enzymes it produces hold potential for biotechnological applications. However, the limited availability of versatile genetic tools and secretion assays constrain both fundamental studies and bioengineering of B. bacteriovorus. Here, we developed a molecular toolbox for B. bacteriovorus by systematically tuning gene expression and secretion of a reporter protein. We investigated functional native and synthetic promoters from the Anderson library with varying expression levels and demonstrated their efficacy in driving expression of the fluorescent reporter protein mScarletI3 at both the population and single-cell level. Additionally, we evaluated different ribosomal binding sites (RBS) to fine-tune gene expression. To examine secretion, we established a novel protocol to quantify extracellular release of a Nanoluc luciferase reporter protein in B. bacteriovorus using different native Sec-dependent signal sequences. We anticipate that the newly developed genetic toolkit and techniques will advance research on this fundamental predator-prey system, laying the foundation for its broader application and future bioengineering efforts. This work will pave the way for tailored applications of B. bacteriovorus in microbial ecology, agriculture, biotechnology, and medicine.

microbiology↗

bioRxiv2025

Activation of the Yersinia type III secretion system induces large-scale chromosomal and virulence plasmid DNA rearrangements

The type III secretion system (T3SS) is used by Gram-negative bacteria, including important pathogens, to manipulate eukaryotic target cells by injecting effector proteins. Type III secretion and bacterial physiology are known to be tightly interconnected and influence each other. Most notably, secreting cells undergo growth arrest in the T3SS model organisms Yersinia, Salmonella and Shigella. The molecular basis of this phenotype, referred to as secretion-associated growth inhibition, is debated. In Yersinia, T3SS genes are encoded extra-chromosomally in the plasmid of Yersinia virulence (pYV), whose copy number increases upon induction of T3SS secretion. In this study, we characterize the link between T3SS activity and subcellular organization by localizing and quantifying the pYV and chromosomal DNA in Yersinia enterocolitica. We find that activation of secretion not only increases the number of pYV plasmids per bacterium, but that the plasmids also move towards the membrane and poles. This relocalization is not caused by transertion (coupled transcription, translation and translocation) of effectors, but part of a broader DNA rearrangement, leading to a distinct relocalization of chromosomal DNA to mid-cell. We hypothesize that these striking DNA rearrangements occurring during secretion are a main factor in the secretion-associated growth inhibition of pathogenic bacteria.

microbiology↗

bioRxiv2024

Yersinia actively downregulates type III secretion and adhesion at higher cell densities

The T3SS injectisome is used by Gram-negative bacteria, including important pathogens, to manipulate eukaryotic target cells by injecting effector proteins. While in some bacterial species, T3SS-negative bacteria benefit from the activity of their T3SS-positive siblings, the T3SS model organism Yersinia enterocolitica was thought to uniformly express and assemble injectisomes. In this study, we found that Yersinia actively suppress T3SS expression, assembly and activity at higher cell densities, such as inside microcolonies. This effect is highly specific to the T3SS, reversible, and distinct from stationary phase adaptation. It is conferred by the main T3SS transcription factor VirF, which is downregulated at higher densities and whose in trans expression restores T3SS activity. Transcript analysis showed that this effect is mediated by increased levels of the regulatory RNAs CsrBC, which sequester the regulatory protein CsrA and destabilize the virF transcript. The concomitant downregulation of the VirF-dependent adhesin YadA led to a drastic reduction in bacterial cell adhesion. We propose that this active suppression of T3SS secretion and cell attachment at higher local bacterial densities promotes a switch during Yersinia infection from a T3SS-active colonization stage to a bacterial replication and dissemination phase.

microbiology↗

bioRxiv2023

Repertoire and abundance of secreted virulence factors shape the pathogenic capacity of Pseudomonas syringae pv. aptata

Pseudomonas syringae pv. aptata is a member of the sugar beet pathobiome and the causative agent of leaf spot disease. Like many pathogenic bacteria, P. syringae relies on the secretion of toxins, which manipulate host-pathogen interactions, to establish and maintain an infection. This study analyzes the secretome of six pathogenic P. syringae pv. aptata strains with different defined virulence capacities in order to identify common and strain-specific features, and correlate the secretome with disease outcome. All strains show a high type III secretion system (T3SS) and type VI secretion system (T6SS) activity under apoplast-like conditions mimicking the infection. Surprisingly, we found that low pathogenic strains show a higher secretion of most T3SS substrates, whereas a distinct subgroup of four effectors was exclusively secreted in medium and high pathogenic strains. Similarly, we detected two T6SS secretion patterns: while one set of proteins was highly secreted in all strains, another subset consisting of known T6SS substrates and previously uncharacterized proteins was exclusively secreted in medium and high virulence strains. Taken together, our data show that P. syringae pathogenicity is correlated with the repertoire and fine-tuning of effector secretion and indicate distinct strategies for establishing virulence of P. syringae pv. aptata in plants.

microbiology↗

bioRxiv2022

Distinct complexes containing the cytosolic type III secretion system ATPase resolved by 3D single-molecule tracking in live Yersinia enterocolitica

The membrane-embedded injectisome, the structural component of the virulence-associated type III secretion system (T3SS), is used by gram-negative bacterial pathogens to inject species-specific effector proteins into eukaryotic host cells. The cytosolic injectisome proteins are required for export of effectors and display both stationary, injectisome-bound populations as well as freely-diffusing cytosolic populations. How the cytosolic injectisome proteins interact with each other in the cytosol and associate with membrane-embedded injectisomes remains unclear. Here, we utilize 3D single-molecule tracking to resolve distinct cytosolic complexes of injectisome proteins in living Yersinia enterocolitica cells. Tracking of the eYFP-labeled ATPase, YeSctN, and its regulator, YeSctL, reveals that these proteins form a cytosolic complex with each other and then further with YeSctQ. YeSctNL and YeSctNLQ complexes can be observed both in wild type cells and in {Delta}sctD mutants, which cannot assemble injectisomes. In {Delta}sctQ mutants, the relative abundance of the YeSctNL complex is considerably increased. These data indicate that distinct cytosolic complexes of injectisome proteins can form prior to injectisome binding, which has important implications for how injectisomes are functionally regulated. ImportanceInjectisomes are membrane-embedded, multiprotein assemblies used by bacterial pathogens to inject virulent effector proteins into eukaryotic host cells. Protein secretion is regulated by cytosolic proteins that dynamically bind and unbind at injectisomes. However, how these regulatory proteins interact with each other remains unknown. By measuring the diffusion rates of single molecules in living cells, we show that cytosolic injectisome proteins form distinct oligomeric complexes with each other prior to binding to injectisomes. We additionally identify the molecular compositions of these complexes and quantify their relative abundances. Quantifying to what extent cytosolic proteins exist as part of larger complexes in living cells has important implications for deciphering the complexity of biomolecular mechanisms. The results and methods reported here are thus relevant for advancing our understanding of how injectisomes, and related multiprotein assemblies, such as bacterial flagellar motors, are functionally regulated.

microbiology↗

bioRxiv2022

Pilotins are mobile T3SS components involved in assembly and substrate specificity of the bacterial type III secretion system

In animal pathogens, assembly of the type III secretion system injectisome requires the presence of so-called pilotins, small lipoproteins that assist the formation of the secretin ring in the outer membrane. Using a combination of functional assays, interaction studies, proteomics, and live-cell microscopy, we determined the contribution of the pilotin to the assembly, function, and substrate selectivity of the T3SS and identified potential new downstream roles of pilotin proteins. In absence of its pilotin SctG, Yersinia enterocolitica forms few, largely polar injectisome sorting platforms and needles. In line, most export apparatus subcomplexes are mobile in these strains, suggesting the absence of fully assembled injectisomes. Remarkably, while absence of the pilotin all but prevents export of early T3SS substrates, such as the needle subunits, it has little effect on secretion of late T3SS substrates, including the virulence effectors. We found that pilotins transiently interact with other injectisome components such as the secretin in the outer membrane, but mostly form transient mobile clusters in the bacterial membrane, which do not colocalize with assembled injectisomes. Together, these findings provide a new view on the role of pilotins during and after assembly of type III secretion injectisomes.

microbiology↗

bioRxiv2021

Visualization of bacterial type 3 secretion system components down the molecular level by MINFLUX nanoscopy

Type 3 secretion systems (T3SS) are essential virulence factors of numerous bacterial pathogens and inject effector proteins into host cells. The needle-like T3SS machinery consists of more than 20 components, has a length of around 100 nm and a diameter of up to 30 nm according to EM studies. Its intrabacterial components are highly dynamic and in permanent exchange with other bacterial structures. Therefore, a temporally and spatially resolved visualization of the T3SS using fluorescence microscopy techniques has been challenging. In the present study, novel labeling strategies were combined with super-resolution microscopy such as STED, STORM and MINFLUX. MINFLUX nanoscopy allowed to visualize components of the T3SS machinery such as the dynamic sorting platform component YscL and the extrabacterial pore protein YopD at unprecedented resolutions. The presented results represent the basis for an in depth investigation of T3SS structure and function and therefore gain new insights into the infection process of human pathogens in order to develop novel treatment and prevention strategies.

microbiology↗

bioRxiv2019

Dynamic relocalization of the cytosolic type III secretion system components ensures specific protein secretion

Many bacterial pathogens use a type III secretion system (T3SS) to manipulate host cells. Protein secretion by the T3SS injectisome is activated upon contact to any host cell, and it has been unclear how premature secretion is prevented during infection. We found that in gastrointestinal pathogens, cytosolic injectisome components are temporarily released from the proximal interface of the injectisome at low external pH, preventing protein secretion in acidic environments, such as the stomach. In Yersinia enterocolitica, low external pH is detected in the periplasm and leads to a partial dissociation of the inner membrane injectisome component SctD, which in turn causes the dissociation of the cytosolic T3SS components. This effect is reversed upon restoration of neutral pH, allowing a fast activation of the T3SS at the native target regions within the host. These findings indicate that the cytosolic components form an adaptive regulatory interface, which regulates T3SS activity in response to environmental conditions.

microbiology↗

bioRxiv2019

LITESEC-T3SS - Light-controlled protein delivery into eukaryotic cells with high spatial and temporal resolution

Many bacteria employ a type III secretion system (T3SS), also called injectisome, to translocate proteins into eukaryotic host cells through a hollow extracellular needle. The system can efficiently transport heterologous cargo, which makes it a uniquely suited tool for the translocation of proteins into eukaryotic cells. However, the injectisome indiscriminately injects proteins into any adjoining eukaryotic cell, and this lack of target specificity currently limits its application in biotechnology and healthcare. In this study, we exploit the dynamic nature of the T3SS to control protein secretion and translocation into eukaryotic cells by light. By combining optogenetic interaction switches with the dynamic cytosolic T3SS component SctQ, the cytosolic availability of SctQ and in consequence T3SS-dependent effector secretion can be regulated by external light. The resulting system, which we call LITESEC-T3SS (Light-induced translocation of effectors through sequestration of endogenous components of the T3SS), allows rapid, specific, and reversible activation or deactivation of the T3SS upon illumination. We demonstrate the application of the system for light-regulated translocation of a heterologous reporter protein into cultured eukaryotic cells. LITESEC-T3SS represents a new method to achieve unparalleled spatial and temporal resolution for the controlled protein translocation into eukaryotic host cells.

microbiology↗

bioRxiv2018

Flagellar stators stimulate c-di-GMP production by Pseudomonas aeruginosa

Flagellar motility is critical for surface attachment and biofilm formation in many bacteria. A key regulator of flagellar motility in Pseudomonas aeruginosa and other microbes is cyclic diguanylate (c-di-GMP). High levels of this second messenger repress motility and stimulate biofilm formation. C-di-GMP levels regulate motility in P. aeruginosa in part by influencing the localization of its two flagellar stator sets, MotAB and MotCD. Here we show that just as c-di-GMP can influence the stators, stators can impact c-di-GMP levels. We demonstrate that the swarming motility-driving stator MotC physically interacts with the transmembrane region of the diguanylate cyclase SadC. Furthermore, we demonstrate that this interaction is capable of stimulating SadC activity. We propose a model by which the MotCD stator set interacts with SadC to stimulate c-di-GMP production in conditions not permissive to motility. This regulation implies a positive feedback loop in which c-di-GMP signaling events cause MotCD stators to disengage from the motor; then disengaged stators stimulate c-di-GMP production to reinforce a biofilm mode of growth. Our studies help define the bidirectional interactions between c-di-GMP and the motility machinery.\n\nImportance.The ability of bacterial cells to control motility during early steps in biofilm formation is critical for the transition to a non-motile, biofilm lifestyle. Recent studies have clearly demonstrated the ability of c-di-GMP to control motility via a number of mechanisms, including through controlling transcription of motility-related genes and modulating motor function. Here we provide evidence that motor components can in turn impact c-di-GMP levels. We propose that communication between motor components and c-di-GMP synthesis machinery allows the cell to have a robust and sensitive switching mechanism to control motility during early events in biofilm formation.

microbiology↗

bioRxiv2018

Visualization and regulation of translocons in Yersinia type III protein secretion machines during host cell infection

Type III secretion systems (T3SSs) are essential virulence factors of numerous bacterial pathogens. Upon host cell contact the T3SS machinery - also named injectisome - assembles a pore complex/translocon within host cell membranes that serves as an entry gate for the bacterial effectors. Whether and how translocons are physically connected to injectisome needles, whether their phenotype is related to the level of effector translocation and which target cell factors trigger their assembly have remained unclear. We employed the superresolution fluorescence microscopy techniques Stimulated Emission Depletion (STED) and Structured Illumination Microscopy (SIM) as well as immunogold electron microscopy to visualize Y. enterocolitica translocons during infection of different target cell types. Thereby we were able to resolve translocon and needle complex proteins within the same injectisomes and demonstrate that these fully assembled injectisomes are generated in a prevacuole, a PI(4,5)P2 enriched host cell compartment inaccessible to large extracellular proteins like antibodies. Furthermore, the putatively operable translocons were produced by the yersiniae to a much larger degree in macrophages (up to 25% of bacteria) than in HeLa cells (2% of bacteria). However, when the Rho GTPase Rac1 was activated in the HeLa cells, uptake of the yersiniae into the prevacuole, translocon formation and effector translocation were strongly enhanced reaching the same levels as in macrophages. Our findings indicate that operable T3SS translocons can be visualized as part of fully assembled injectisomes with superresolution fluorescence microscopy techniques. By using this technology we provide novel information about the spatiotemporal organisation of T3SS translocons and their regulation by host cell factors.\n\nAuthor SummaryMany human, animal and plant pathogenic bacteria employ a molecular machine termed injectisome to inject their toxins into host cells. Because injectisomes are crucial for these bacterias infectious potential they have been considered as targets for antiinfective drugs. Injectisomes are highly similar between the different bacterial pathogens and most of their overall structure is well established at the molecular level. However, only little information is available for a central part of the injectisome named the translocon. This pore-like assembly integrates into host cell membranes and thereby serves as an entry gate for the bacterial toxins. We used state of the art fluorescence microscopy to watch translocons of the diarrheagenic pathogen Yersinia enterocolitica during infection of human host cells. Thereby we could for the first time - with fluorescence microscopy - visualize translocons connected to other parts of the injectisome. Furthermore, because translocons mark functional injectisomes we could obtain evidence that injectisomes only become active when the bacteria are almost completely enclosed by host cells. These findings provide a novel view on the organisation and regulation of bacterial translocons and may thus open up new strategies to block the function of infectious bacteria.

microbiology↗

bioRxiv2017

Single-molecule tracking in live Yersinia enterocolitica reveals distinct cytosolic complexes of injectisome subunits

In bacterial type 3 secretion, substrate proteins are actively transported from the bacterial cytoplasm into the host cell cytoplasm by a large membrane-embedded machinery called the injectisome. Injectisomes transport secretion substrates in response to specific environmental signals, but the molecular details by which the cytosolic secretion substrates are selected and transported through the type 3 secretion pathway remain unclear. Secretion activity and substrate selectivity are thought to be controlled by a sorting platform consisting of the proteins SctK, SctQ, SctL, and SctN, which together localize to the cytoplasmic side of membrane-embedded injectisomes. However, recent work revealed that sorting platform proteins additionally exhibit substantial cytosolic populations and that SctQ reversibly binds to and dissociates from the cytoplasmic side of membrane-embedded injectisomes. Based on these observations, we hypothesized that dynamic molecular turnover at the injectisome and cytosolic assembly among sorting platform proteins is a critical regulatory component of type 3 secretion. To determine whether sorting platform complexes exist in the cytosol, we measured the diffusive properties of the two central sorting platform proteins, SctQ and SctL, using live cell high-throughput 3D single-molecule tracking microscopy. Single-molecule trajectories, measured in wild-type and mutant Yersinia enterocolitica cells, reveal that both SctQ and SctL exist in several distinct diffusive states in the cytosol, indicating that these proteins form stable homo- and hetero-oligomeric complexes in their native environment. Our findings provide the first diffusive state-resolved insights into the dynamic regulatory network that interfaces stationary membrane-embedded injectisomes with the soluble cytosolic components of the type 3 secretion system.

biophysics↗