Biology subjects

Gahlmann, A.

Publications and source records attributed to Gahlmann, A..

9 recordsLinked to original sources

bioRxiv2025

Cooperative Binding of Cytosolic Type III Secretion System Proteins to the Injectisome Revealed by Live-Cell Single-Molecule Localization Microscopy

Type III secretion systems (T3SSs) are employed by many Gram-negative bacteria to translocate virulent effector proteins into host cells. Secretion occurs through the T3SS injectisome, a multi-membrane-spanning biomolecular machine. While the overall structure of the injectisome is becoming increasingly well defined, the structural dynamics that regulate secretion remain unresolved. Particularly important for the functional regulation of type 3 secretion are the cytosolic injectisome proteins which transiently associate with injectisomes and whose structural dynamics and function remain incompletely understood. Here, we use long-exposure single-molecule localization microscopy to quantify the bound times of the cytosolic components SctQ, SctL, and SctN, at the injectisome of Yersinia enterocolitica both in controllably induced secretion ON and OFF states and in the presence and absence of effector proteins. In the absence of effector proteins, each component exhibits distinct short-lived binding behavior on a timescale of seconds. However, the presence of the effector protein YopE increases bound times and synchronizes binding behavior across components, specifically without active secretion. Upon activation of secretion, a subpopulation of long-lived binding events emerges for SctQ, SctL, and SctN even within the same injectisomes, indicating that binding behavior between these proteins is secretion state-dependent and cooperative. These findings establish that the interaction kinetics between cytosolic injectisome proteins are modulated by the presence of secretion substrates and secretion activating signals, and reveal a new layer of heterogeneity in the functional regulation of type 3 secretion.

microbiology↗

bioRxiv2024

Targeting Iron - Respiratory Reciprocity Promotes Bacterial Death

Discovering new bacterial signaling pathways offers unique antibiotic strategies. Here, through an unbiased resistance screen of 3,884 gene knockout strains, we uncovered a previously unknown non-lytic bactericidal mechanism that sequentially couples three transporters and downstream transcription to lethally suppress respiration of the highly virulent P. aeruginosa strain PA14 - one of three species on the WHOs Priority 1: Critical list. By targeting outer membrane YaiW, cationic lacritin peptide N-104 translocates into the periplasm where it ligates outer loops 4 and 2 of the inner membrane transporters FeoB and PotH, respectively, to suppress both ferrous iron and polyamine uptake. This broadly shuts down transcription of many biofilm-associated genes, including ferrous iron-dependent TauD and ExbB1. The mechanism is innate to the surface of the eye and is enhanced by synergistic coupling with thrombin peptide GKY20. This is the first example of an inhibitor of multiple bacterial transporters.

microbiology↗

bioRxiv2022

Dimerization of iLID Optogenetic Proteins Observed Using 3D Single-Molecule Tracking in Live Bacterial Cells

3D single molecule tracking microscopy has enabled measurements of protein diffusion in living cells, offering information about protein dynamics and cellular environments. For example, different diffusive states can be resolved and assigned to protein complexes of different size and composition. However, substantial statistical power and biological validation, often through genetic deletion of binding partners, are required to support diffusive state assignments. When investigating cellular processes, transient perturbation to protein spatial distributions is preferable to permanent genetic deletion of an essential protein. In this context, optogenetic dimerization systems can be used to manipulate protein spatial distributions which could offer a means to deplete specific diffusive states observed in single molecule tracking experiments. Here, we evaluate the performance of the iLID optogenetic system in living E. coli cells using diffraction-limited microscopy and 3D single-molecule tracking. We observed a robust optogenetic response in protein spatial distributions after 488 nm laser activation. Surprisingly, 3D single-molecule tracking results indicate activation of the optogenetic response when illuminating with high intensity light with wavelengths at which there is minimal photon absorbance by the LOV2 domain. The pre-activation can be minimized through the use of iLID system mutants, and titration of protein expression levels. SIGNIFICANCE STATEMENTWe describe the combination of 3D single-molecule tracking microscopy and optogenetic manipulation of protein spatial distributions in living cells. Such a combination is impactful, because optogenetic systems enable sample perturbation in real time using light, which provides more flexibility than gene deletion and gene editing approaches that result in permanent changes to the specimen. We specifically investigate the performance of the iLID optogenetic system in a knocksideways experiment, in which cytosolic prey proteins (SspB) are sequestered to the membrane by interacting with membrane-anchored bait proteins (iLID). We quantified the magnitude of the optogenetic effect using both diffraction-limited imaging and 3D single-molecule tracking microscopy. We found, surprisingly, that the iLID optogenetic response is activated substantially by high intensity light at wavelengths for which there is negligible photon absorption by the iLID protein. Quantification of this alternative activation mechanism is a necessary component before optogenetic tools, such as iLIDs, are employed in single-molecule knocksideways experiment that are designed to provide new biological insights.

biophysics↗

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↗

bioRxiv2021

BCM3D 2.0: Accurate segmentation of single bacterial cells in dense biofilms using computationally generated intermediate image representations

Accurate detection and segmentation of single cells in three-dimensional (3D) fluorescence timelapse images is essential for observing individual cell behaviors in large bacterial communities called biofilms. Recent progress in machine-learning-based image analysis is providing this capability with every increasing accuracy. Leveraging the capabilities of deep convolutional neural networks (CNNs), we recently developed bacterial cell morphometry in 3D (BCM3D), an integrated image analysis pipeline that combines deep learning with conventional image analysis to detect and segment single biofilm-dwelling cells in 3D fluorescence images. While the first release of BCM3D (BCM3D 1.0) achieved state-of-the-art 3D bacterial cell segmentation accuracies, low signal-to-background ratios (SBRs) and images of very dense biofilms remained challenging. Here, we present BCM3D 2.0 to address this challenge. BCM3D 2.0 is entirely complementary to the approach utilized in BCM3D 1.0. Instead of training CNNs to perform voxel classification, we trained CNNs to translate 3D fluorescence images into intermediate 3D image representations that are, when combined appropriately, more amenable to conventional mathematical image processing than a single experimental image. Using this approach, improved segmentation results are obtained even for very low SBRs and/or high cell density biofilm images. The improved cell segmentation accuracies in turn enable improved accuracies of tracking individual cells through 3D space and time. This capability opens the door to investigating timedependent phenomena in bacterial biofilms at the cellular level.

microbiology↗

bioRxiv2020

Non-Invasive Single-Cell Morphometry in Living Bacterial Biofilms

Fluorescence microscopy enables spatial and temporal measurements of live cells and cellular communities. However, this potential has not yet been fully realized for investigations of individual cell behaviors and phenotypic changes in dense, three-dimensional (3D) bacterial biofilms. Accurate cell detection and cellular shape measurement in densely packed biofilms are challenging because of the limited resolution and low signal to background ratios (SBRs) in fluorescence microscopy images. In this work, we present Bacterial Cell Morphometry 3D (BCM3D), an image analysis workflow that combines deep learning with mathematical image analysis to accurately segment and classify single bacterial cells in 3D fluorescence images. In BCM3D, deep convolutional neural networks (CNNs) are trained using simulated biofilm images with experimentally realistic SBRs, cell densities, labeling methods, and cell shapes. We systematically evaluate the segmentation accuracy of BCM3D using both simulated and experimental images. Compared to state-of-the-art bacterial cell segmentation approaches, BCM3D consistently achieves higher segmentation accuracy and further enables automated morphometric cell classifications in multi-population biofilms.

microbiology↗

bioRxiv2018

Computational Correction of Spatially-Variant Optical Aberrations in 3D Single Molecule Localization Microscopy

3D single-molecule localization microscopy relies on fitting the shape of point-spread-functions (PSFs) recorded on a wide-field detector. However, optical aberrations distort those shapes, which compromise the accuracy and precision of single-molecule localization microscopy. Here we employ a computational phase retrieval based on a vectorial PSF model to quantify the spatially-variance of optical aberrations in a two-channel ultrawide-field single-molecule localization microscope. The use of a spatially-variant PSF model enables accurate and precise emitter localization in x, y- and z-directions throughout the entire field-of-view.\n\nOCIS Codes(100.6640) Superresolution; (180.2520) Fluorescence microscopy; (180.6900) Three-dimensional microscopy; (100.5070) Phase retrieval; (150.1135) Algorithms; (220.1010) Aberrations (global).

biophysics↗

bioRxiv2018

Resolving Cytosolic Diffusive States in Bacteria by Single-Molecule Tracking

The trajectory of a single protein in the cytosol of a living cell contains information about its molecular interactions in its native environment. However, it has remained challenging to accurately resolve and characterize the diffusive states that can manifest in the cytosol using analytical approaches based on simplifying assumptions. Here, we show that multiple intracellular diffusive states can be successfully resolved if sufficient single-molecule trajectory information is available to generate well-sampled distributions of experimental measurements and if experimental biases are taken into account during data analysis. To address the inherent experimental biases in camera-based and MINFLUX-based single-molecule tracking, we use an empirical data analysis framework based on Monte Carlo simulations of confined Brownian motion. This framework is general and adaptable to arbitrary cell geometries and data acquisition parameters employed in 2D or 3D single-molecule tracking. We show that, in addition to determining the diffusion coefficients and populations of prevalent diffusive states, the timescales of diffusive state switching can be determined by stepwise increasing the time window of averaging over subsequent single-molecule displacements. Time-averaged diffusion (TAD) analysis of single-molecule tracking data may thus provide quantitative insights into binding and unbinding reactions among rapidly diffusing molecules that are integral for cellular functions.

biophysics↗

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↗