A non-pathogenic Mycoplasma pneumoniae-based chassis is leading the development of live biotherapeutic products (LBPs) for respiratory diseases. However, reports connecting Guillain-Barré syndrome (GBS) cases to prior M. pneumoniae infections represent a concern for exploiting such a chassis. Galactolipids, especially galactocerebroside (GalCer), are considered the most likely M. pneumoniae antigens triggering autoimmune responses associated with GBS development. In this work, we generated different strains lacking genes involved in galactolipids biosynthesis. Glycolipid profiling of the strains demonstrated that some mutants show a complete lack of galactolipids. Cross-reactivity assays with sera from GBS patients with prior M. pneumoniae infection showed that certain engineered strains exhibit reduced antibody recognition. However, correlation analyses of these results with the glycolipid profile of the engineered strains suggest that other factors different from GalCer contribute to sera recognition, including total ceramide levels, dihexosylceramide (DHCer), and diglycosyldiacylglycerol (DGDAG). Finally, we discuss the best candidate strains as potential GBS-free Mycoplasma chassis.

This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme ERC LUNG-BIOREPAIR (101020135). We also acknowledge the support of the Spanish Ministry of Science and Innovation through the Plan Nacional PID2021-122341NB-I00 and the Centro de Excelencia Severo Ochoa (CEX2020-001049-S, MCIN/AEI /10.13039/501100011033), the Generalitat de Catalunya through the CERCA programme, the Center for Industrial Technology Development (CDTI) through the Neotec programme (SNEO 20211019) and to the EMBL partnership. C.P.-L. acknowledges the support of ‘Programa Torres Quevedo’ grant [PTQ2020-011048] funded by MCIN/AEI/10.13039/501100011033; European Union ‘NextGenerationEU/PRTR’. The proteomics analyses were performed in the CRG/UPF Proteomics Unit which is part of the Spanish National Infrastructure for Omics Technologies (ICTS OmicsTech). We thank T. Hoogenboezem and C. Gago da Graça (Department of Pediatrics, Erasmus MC–Sophia Children's Hospital, University Medical Centre, Rotterdam, The Netherlands) for excellent technical assistance.

Peer reviewed