Minimal Bacterial Cell JCVI-syn3B as a Chassis to Investigate Interactions between Bacteria and Mammalian Cells

• Published in: ACS synthetic biology 2024-04, Vol.13 (4), p.1128-1141
• Main Authors: Bittencourt, Daniela Matias de C., Brown, David M., Assad-Garcia, Nacyra, Romero, Michaela R., Sun, Lijie, Palhares de Melo, Luis Alberto M., Freire, Marcelo, Glass, John I.
• Format: Article
• Language: English
• ISSN: 2161-5063; 2161-5063
• DOI: 10.1021/acssynbio.3c00513

Mycoplasmas are atypical bacteria with small genomes that necessitate colonization of their respective animal or plant hosts as obligate parasites, whether as pathogens, or commensals. Some can grow axenically in specialized complex media yet show only host-cell-dependent growth in cell culture, where they can survive chronically and often through interactions involving surface colonization or internalization. To develop a mycoplasma-based system to identify genes mediating such interactions, we exploited genetically tractable strains of the goat pathogen Mycoplasma mycoides (Mmc) with synthetic designer genomes representing the complete natural organism (minus virulence factors; JCVI-syn1.0) or its reduced counterpart (JCVI-syn3B) containing only those genes supporting axenic growth. By measuring growth of surviving organisms, physical association with cultured human cells (HEK-293T, HeLa), and induction of phagocytosis by human myeloid cells (dHL-60), we determined that JCVI-syn1.0 contained a set of eight genes (MMSYN1-0179 to MMSYN1-0186, dispensable for axenic growth) conferring survival, attachment, and phagocytosis phenotypes. JCVI-syn3B lacked these phenotypes, but insertion of these genes restored cell attachment and phagocytosis, although not survival. These results indicate that JCVI-syn3B may be a powerful living platform to analyze the role of specific gene sets, from any organism, on the interaction with diverse mammalian cells in culture.