A step toward synthetic endosymbiosis: introducing Escherichia coli and the trypanosomatid endosymbiont Candidatus Kinetoplastibacterium crithidii into mammalian cells using a polyethylene glycol-based fusion protocol
- 1University of Duesseldorf
Abstract
Synthetic endosymbiosis, the deliberate introduction of bacteria into eukaryotic host cells, provides a tractable model for how endosymbiotic relationships become established, and a route to cells with new metabolic capabilities that never evolved naturally in that lineage. Current methods for introducing bacteria into mammalian cells rely on microinjection, engineered invasion, listeriolysin-mediated phagosomal escape, or naturally permissive hosts. These methods are invasive, low-throughput, or costly, and most require the prospective endosymbiont to be genetically tractable, which excludes endosymbiotic bacteria which are of great interest as potential organelle precursors. Here we describe a protocol based on polyethylene glycol (PEG), long used to fuse mammalian cells, that delivers Escherichia coli into HeLa cells without genetic modification of either partner or the use of specialised equipment. We optimised the governing parameters in two stages: first, bacterial density, gentamicin concentration, and PEG molecular weight and concentration were optimised; then, medium composition and the timing of fusion and recovery. Across four parameters screened, 10% PEG-3350 applied for 4 min to HeLa cells 12 h after seeding, followed by 4 h in gentamicin-containing medium, gave the most consistent delivery. Under these conditions, confocal microscopy and z-stack reconstruction detected mCherry-tagged E. coli inside HeLa cells. Applying the same protocol to Candidatus Kinetoplastibacterium crithidii, a naturally occurring {beta}-proteobacterial endosymbiont of the trypanosomatid Angomonas deanei, we observed the endosymbiont inside the HeLa cells. This protocol offers an accessible entry point for constructing artificial endosymbioses.
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BibTeX
@misc{pednekar2026step,
title = {{A step toward synthetic endosymbiosis: introducing Escherichia coli and the trypanosomatid endosymbiont Candidatus Kinetoplastibacterium crithidii into mammalian cells using a polyethylene glycol-based fusion protocol}},
author = {R. Pednekar and N. van Geelen-Kuenzel and C. M. Diehl and L.-A. Koch and B. Llorente and E. C. M. Nowack and M. D. Zurbriggen},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.20.753003},
url = {https://www.biorxiv.org/content/10.64898/2026.09.20.753003v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - A step toward synthetic endosymbiosis: introducing Escherichia coli and the trypanosomatid endosymbiont Candidatus Kinetoplastibacterium crithidii into mammalian cells using a polyethylene glycol-based fusion protocol
AU - R. Pednekar
AU - N. van Geelen-Kuenzel
AU - C. M. Diehl
AU - L.-A. Koch
AU - B. Llorente
AU - E. C. M. Nowack
AU - M. D. Zurbriggen
PY - 2026
DA - 2026/09/20
PB - bioRxiv
DO - 10.64898/2026.09.20.753003
UR - https://www.biorxiv.org/content/10.64898/2026.09.20.753003v1
AB - Synthetic endosymbiosis, the deliberate introduction of bacteria into eukaryotic host cells, provides a tractable model for how endosymbiotic relationships become established, and a route to cells with new metabolic capabilities that never evolved naturally in that lineage. Current methods for introducing bacteria into mammalian cells rely on microinjection, engineered invasion, listeriolysin-mediated phagosomal escape, or naturally permissive hosts. These methods are invasive, low-throughput, or costly, and most require the prospective endosymbiont to be genetically tractable, which excludes endosymbiotic bacteria which are of great interest as potential organelle precursors. Here we describe a protocol based on polyethylene glycol (PEG), long used to fuse mammalian cells, that delivers Escherichia coli into HeLa cells without genetic modification of either partner or the use of specialised equipment. We optimised the governing parameters in two stages: first, bacterial density, gentamicin concentration, and PEG molecular weight and concentration were optimised; then, medium composition and the timing of fusion and recovery. Across four parameters screened, 10% PEG-3350 applied for 4 min to HeLa cells 12 h after seeding, followed by 4 h in gentamicin-containing medium, gave the most consistent delivery. Under these conditions, confocal microscopy and z-stack reconstruction detected mCherry-tagged E. coli inside HeLa cells. Applying the same protocol to Candidatus Kinetoplastibacterium crithidii, a naturally occurring {beta}-proteobacterial endosymbiont of the trypanosomatid Angomonas deanei, we observed the endosymbiont inside the HeLa cells. This protocol offers an accessible entry point for constructing artificial endosymbioses.
ER -