Bacterial membranes are complex and dynamic, arising from an array of evolutionary pressures. One enzyme that alters membrane compositions through covalent lipid modification is MprF. We recently identified that Streptococcus agalactiae MprF synthesizes lysyl-phosphatidylglycerol (Lys-PG) from anionic PG, and a novel cationic lipid, lysyl-glucosyl-diacylglycerol (Lys-Glc-DAG), from neutral glycolipid Glc-DAG. This unexpected result prompted us to investigate whether Lys-Glc-DAG occurs in other MprF-containing bacteria, and whether other novel MprF products exist. Here, we studied protein sequence features determining MprF substrate specificity. First, pairwise analyses identified several streptococcal MprFs synthesizing Lys-Glc-DAG. Second, a restricted Boltzmann machine-guided approach led us to discover an entirely new substrate for MprF in Enterococcus, diglucosyl-diacylglycerol (Glc₂-DAG), and an expanded set of organisms that modify glycolipid substrates using MprF. Overall, we combined the wealth of available sequence data with machine learning to model evolutionary constraints on MprF sequences across the bacterial domain, thereby identifying a novel cationic lipid.

Toxoplasma gondii is an intracellular parasite that subverts host cell functions via secreted virulence factors. Up to 70% of parasite-controlled changes in the host transcriptome rely on the MYR1 protein, which is required for the translocation of secreted proteins into the host cell. Mice infected with MYR1 knock-out (KO) strains survive infection, supporting a paramount function of MYR1-dependent secreted proteins in Toxoplasma virulence and proliferation. However, we have previously shown that MYR1 mutants have no growth defect in pooled in vivo CRISPR-Cas9 screens in mice, suggesting that the presence of parasites that are wild-type at the myr1 locus in pooled screens can rescue the phenotype. Here, we demonstrate that MYR1 is not required for the survival in IFN-γ-activated murine macrophages, and that parasites lacking MYR1 are able to expand during the onset of infection. While ΔMYR1 parasites have restricted growth in single-strain murine infections, we show that the phenotype is rescued by co-infection with wild-type (WT) parasites in vivo, independent of host functional adaptive immunity or key pro-inflammatory cytokines. These data show that the major function of MYR1-dependent secreted proteins is not to protect the parasite from clearance within infected cells. Instead, MYR-dependent proteins generate a permissive niche in a paracrine manner, which rescues ΔMYR1 parasites within a pool of CRISPR mutants in mice. Our results highlight an important limitation of otherwise powerful in vivo CRISPR screens and point towards key functions for MYR1-dependent Toxoplasma-host interactions beyond the infected cell.