Haemophilus influenzae as a chassis for directed endosymbiosis

The transition from prokaryotes to eukaryotic life represents one of the most consequential events in the history of life on Earth. Through the understanding of endosymbiotic theory, namely the origins of organelles via partnerships between hosts and intracellular symbionts, this evolutionary transition has become clearer over recent decades. Inspired by this evolutionary history, synthetic biologists have begun to experimentally recreate endosymbiosis through directed endosymbiosis: the engineering of stable intracellular partnerships between distinct species. This thesis explores Haemophilus influenzae as a candidate chassis for directed endosymbiosis with Saccharomyces cerevisiae. H. influenzae is small, genetically tractable, naturally competent for DNA uptake, facultatively intracellular, and dependent on host-relevant cofactors including NAD and hemin. These features make it attractive for engineering bacterial endosymbionts. First, H. influenzae strains lacking the restriction endonuclease genes HindIIR and HindIIIR were shown to transform more efficiently by electroporation than wild-type. A pSU20-derived plasmid series was then developed to provide selectable and fluorescent markers for H. influenzae. Second, co-culture experiments showed that YPAD supplemented with NAD and hemin supports the growth of both species, whereas synthetic complete yeast medium requires yeast extract or peptone to support H. influenzae. H. influenzae also rescued the adenine-linked red phenotype of S. cerevisiae VL6-48 and promoted yeast growth under nutrient-limiting conditions, suggesting metabolic compatibility between the two species. Finally, cell engulfment was quantified using fluorescence microscopy and automated Z-stack image analysis. Productive genome transfer was rare, while microscopy detected four candidate engulfment events among 6,636 segmented yeast cells. Together, these results establish H. influenzae as a tractable candidate endosymbiont chassis and define the main barriers that must be overcome to engineer synthetic organelles.

Name thesis:degree_name

Ph D

Discipline thesis:degree_discipline

Biochemistry

Grantor dc:publisher

The University of Western Ontario

Year dc:date.issued

2026

Author dc:creator

• Hamadache, Samir

Advisor dc:contributor.advisor

• Karas, Bogumil J

• synthetic organelles
• endosymbiosis
• Haemophilus influenzae
• Saccharomyces cerevisiae
• syntrophy
• cell engulfment
• genome transfer
• genetic tools
• coculture
• evolution

Language dc:language.iso

en

Handle dc:identifier.uri

https://hdl.handle.net/20.500.14721/39996

OAI identifier oai:identifier

oai:uwo.scholaris.ca:20.500.14721/39996