Role of listeriolysin O and phospholipases C in <i>L. monocytogenes</i> intercellular protrusion dynamics, resolution, and autophagy avoidance
Bibliographic record
Abstract
ABSTRACT Cell-to-cell spread is a major mechanism used by the bacterial pathogen Listeria monocytogenes to disseminate within its host. In this mechanism, bacteria are directly transferred from the cytosol of an infected donor cell to a recipient cell via formation of an intercellular protrusion. The intercellular protrusion resolves into a vacuole that is disrupted by L. monocytogenes to reach the cytosol of the recipient cell, where it divides and starts new cell-to-cell spread cycles. Here, we studied the spatiotemporal dynamics of the donor and recipient host cell plasma membranes during intercellular protrusion formation, resolution, and disruption, as well as the role of the bacterial pore-forming toxin listeriolysin O (LLO) and two phospholipases C in these dynamics. Leveraging three-dimensional live-cell microscopy, we co-cultured and imaged infected epithelial cells expressing different fluorescent plasma membrane markers to distinguish donor from recipient cell membranes. We characterized, for the first time, the dynamics of the canonical cell-to-cell spread pathway in which protrusions resolve into double-membrane vacuoles, followed by the successive disruption of donor and recipient membranes. We found that LLO controls protrusion resolution into a double-membrane vacuole and disruption of donor and recipient membranes, whereas the phospholipases C (PLCs) control donor membrane disruption and prevent bacterial entrapment into autophagosomes. Furthermore, we identified a second, more efficient “non-canonical” cell-to-cell spread pathway strictly requiring LLO/PLCs cooperation, which resolves into a single-membrane vacuole that is rapidly disrupted. Altogether, this study provides the first detailed analysis of membrane dynamics within intercellular protrusions and vacuoles, establishing an advanced model for L. monocytogenes cell-to-cell spread. IMPORTANCE Intracellular bacterial pathogens, including L. monocytogenes , spread from cell to cell to effectively disseminate within host tissues and cause systemic infections. We developed a fluorescence microscopy-based method to visualize and distinguish in real time the dynamics of donor and recipient host cell plasma membranes during the different stages of the cell-to-cell spread process: (i) formation of the intercellular protrusion-containing L. monocytogenes , (ii) protrusion resolution into a vacuole, and (iii) disruption of the vacuolar membranes until the bacterium is released into the cytosol of the recipient cell. We measured the kinetics of the two identified—canonical and non-canonical—cell-to-cell spread pathways of L. monocytogenes in epithelial cells. This work also revealed that the virulence factors listeriolysin O and phospholipases C differentially control donor and recipient membrane remodeling and bacterial capture into autophagosomes, providing an advanced model for L. monocytogenes cell-to-cell spread.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".