Characterization of novel actin‐associated proteins at enteropathogenic <i>Escherichia coli</i> and <i>Listeria monocytogenes</i> actin‐rich structures
Bibliographic record
Abstract
Enteropathogenic Escherichia coli (EPEC) and Listeria monocytogenes generate actin‐rich structures that are used for their colonization of host cells. EPEC remains extracellular, attaches to the host plasma membrane and secretes a variety of effector proteins that are used to control the host cells. The most dramatic morphological phenotype generated during these infections is the formation of actin‐rich protrusions called pedestals that are needed for E. coli ‐based disease. L. monocytogenes enters its host cell and once in the cytosol it generates a bacterial protein (called ActA) that recruits actin polymerizing proteins to 1 pole of the microbe forming a comet tail that is used for movement within and amongst the host cells Previously, our lab conducted a mass spectrometry analysis of concentrated EPEC pedestals and over 90 novel proteins were identified. From this list, we selected a subset for confirmatory analysis and to determine whether their presence required the pre‐formation of the actin‐rich structures. These proteins included a calponin protein (CNN), dihydropyrimidinase‐like protein (CRMP4), nucleoside kinase (NK), mitogen‐activated protein pathway kinase (MK), and a ubiquitin‐conjugating enzyme (Ube2N). Because these novel proteins were observed in EPEC pedestals, we hypothesized that these five protein candidates are important for EPEC pedestals and L. monocytogenes comet tails. To study this, we first confirmed that these proteins were enriched in EPEC pedestals by immunolocalizing the proteins in EPEC‐infected cells. CNN, and NK were found throughout the full length of the pedestal while CRMP4, MK, and Ube2N were concentrated at the apical tip of EPEC pedestals. In L. monocytogenes ‐infected cells, CNN and NK were found in actin clouds, comet tails, and listeriopods. CRMP4 immunolocalized only at invasion sites and listeriopods while MK was only present at listeriopods. To ensure that recruitment of these proteins were independent of bacterial attachment on the host cell and required formation of the actin‐rich structures, we infected cells with either a tir ‐deficient EPEC mutant or an actA ‐deficient L. monocytogenes mutant as both mutants cannot generate actin‐structures. None of the five proteins were recruited to the mutants suggesting that their presences are dependent on the formation of the actin‐rich structures. From these results, we have identified novel proteins that are important for the various stages of EPEC and L. monocytogenes infections. By identifying these proteins, we have begun to elucidate the complex web of proteins involved in the formation and maintenance of actin structures during these infections. Support or Funding Information This study was funded through NSERC. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| 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 source (direct Gemma or distilled Codex), 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".