Resolving Injectisome Binding Dynamics in <em>Yersinia enterocolitica</em> and Bactofilin Organization in <em>Helicobacter pylori</em> by Single-Molecule Localization and Tracking Microscopy 17 views

The organization and dynamics of proteins within living bacterial cells operate at spatial and temporal scales that have historically been inaccessible to light microscopy. Single-molecule localization and tracking microscopy (SMLM/SMT) overcomes the diffraction limit to resolve individual protein positions and motions in living cells, providing readouts of diffusion, spatial organization, and binding kinetics at the nanometer scale. This dissertation applies these methods to two bacterial pathogens, Yersinia enterocolitica and Helicobacter pylori, and develops new computational tools for analyzing the resulting data. We first discuss methods in single molecule tracking and present DiffusionNet, a machine learning framework for classifying diffusion states directly from single-molecule images. DiffusionNet uses a double attention architecture with latent variable entropy minimization to classify diffusive states. Our approach can be used to pinpoint protein binding and unbinding events from SMLM experiments in living cells in an automated pipeline. However, this requires a priori knowledge of the true number of diffusive states for a protein of interest. We then apply SMLM/SMT to characterize the cytosolic complex of the Y. enterocolitica Type III Secretion System (T3SS). Using AlphaFold-Multimer predictions to identify conserved interface residues of the ATPase SctN, we designed and validated a panel of point mutants that are oligomerization and/or secretion incompetent. Single-molecule tracking of these mutants reveals that disrupting SctN oligomerization reduces its membrane-associated fraction, linking ATPase self-assembly to injectisome incorporation. A charge-retaining substitution produces a particularly informative phenotype: it decouples middle and late secretion, and full-length YopE selectively rescues the middle secretion defect, establishing an allosteric role for the effector at the cytosolic complex. In a complementary study, we use long-exposure SMLM to measure the bound times of SctQ, SctL, and SctN at the injectisome. Under effector-free conditions, each protein exhibits distinct short-lived binding kinetics. In the presence of YopE, bound times increase and under secreting conditions, a population of long-lived binding events emerge. Both short- and long-lived binding modes can occur at the same injectisome, suggesting asymmetry rather than distinct classes of injectisomes. These results support a model of effector-dependent cooperative binding of the cytosolic complex to the injectisome. Finally, we apply live-cell SMLM/SMT to the bactofilin CcmA in H. pylori, which contributes to the helical cell shape. CcmA partitions between a semi-mobile population consistent with small oligomeric species and an immobile population consistent with more stable polymer. Loss of the outer membrane protein Csd5 increases the immobile, clustered fraction rather than dispersing it. This supports a model in which Csd5 spatially organizes CcmA polymerization at the inner membrane and regulates polymerization itself. Together, these studies demonstrate the power of single-molecule microscopy to resolve protein assembly, dynamics, and spatial organization in living bacterial pathogens, contributing both methodological and biological advances to the study of bacterial virulence and cell biology.

PHD (Doctor of Philosophy)

Yersinia enterocolitica; Helicobacter pylori; Type III Secretion System; Bactofilin; Single-Molecule Localization Microscopy ; Single Molecule Tracking

English

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2026-04-23