Structural determination of Rickettsia lipid A without chemical extraction confirms shorter acyl chains in later-evolving Spotted Fever Group pathogens

Rickettsiae are Gram-negative obligate intracellular parasites of numerous eukaryotes. Human pathogens of the Transitional Group (TRG), Typhus Group (TG), and Spotted Fever Group (SFG) rickettsiae infect blood-feeding arthropods, have dissimilar clinical manifestations, and possess unique genomic and morphological attributes. Lacking glycolysis, rickettsiae pilfer numerous metabolites from host cytosol to synthesize peptidoglycan and lipopolysaccharide (LPS). For LPS, O-antigen immunogenicity varies between SFG and TG pathogens; however, lipid A proinflammatory potential is unknown. We previously demonstrated that R. akari (TRG), R. typhi (TG), and R. montanensis (SFG) produce lipid A with long 2 secondary acyl chains (C16 or C18) compared to short 2 secondary acyl chains (C12) in R. rickettsii (SFG) lipid A. To further probe this structural heterogeneity and estimate a time point when shorter 2 secondary acyl chains originated, we generated lipid A structures for two additional SFG rickettsiae (R. rhipicephali and R. parkeri) utilizing Fast Lipid Analysis Technique adopted for use with tandem mass spectrometry (FLATn). FLATn allowed analysis of lipid A structure directly from host cell-purified bacteria, providing substantial improvement over lipid A chemical extraction. FLATn-derived structures indicate SFG rickettsiae diverging after R. rhipicephali evolved shorter 2 secondary acyl chains. Bioinformatics analysis of Rickettsia LpxL late acyltransferases revealed discrete active sites and hydrocarbon rulers for long versus short 2 secondary acyl chain addition. While the significance of different lipid A structures for diverse Rickettsia pathogens is unknown, our success using FLATn will facilitate determining how structural heterogeneity impacts interactions with host lipid A receptors and overall inflammatory potential. IMPORTANCEDeforestation, urbanization, and homelessness lead to spikes in Rickettsioses. Vector-borne human pathogens of Transitional Group (TRG), Typhus Group (TG), and Spotted Fever Group (SFG) rickettsiae differ by clinical manifestations, immunopathology, genome composition, and morphology. We previously showed that lipid A (or endotoxin), the membrane anchor of Gram-negative bacterial lipopolysaccharide (LPS), structurally differs in R. rickettsii (later-evolving SFG) relative to R. montanensis (basal SFG), R. typhi (TG), and R. akari (TRG). As lipid A structure influences recognition potential in vertebrate LPS sensors, further assessment of Rickettsia lipid A structural heterogeneity is needed. Here, we sidestepped the difficulty of ex vivo lipid A chemical extraction by utilizing FLATn, a new procedure for generating lipid A structures directly from host cell-purified bacteria. These data confirm later-evolving SFG pathogens synthesize structurally distinct lipid A. Our findings impact interpreting immune responses to different Rickettsia pathogens and utilizing lipid A adjuvant or anti-inflammatory properties in vaccinology.