Structural determination ofRickettsialipid A without chemical extraction confirms shorter acyl chains in later-evolving Spotted Fever Group pathogens
Authors: Yang Hyojik , Verhoeve Victoria I. , Chandler Courtney E. , Nallar Shreeram , Snyder Greg A. , Ernst Robert K. , Gillespie Joseph J. 
Published: July 2023

ABSTRACTRickettsiae 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 thatR. akari(TRG),R. typhi(TG), andR. montanensis(SFG) produce lipid A with long 2’ secondary acyl chains (C16 or C18) compared to short 2’ secondary acyl chains (C12) inR. 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. rhipicephaliandR. parkeri) utilizing Fast Lipid Analysis Technique adopted for use with tandem mass spectrometry (FLATn). FLATnallowed 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 afterR. rhipicephalievolved shorter 2’ secondary acyl chains. Bioinformatics analysis ofRickettsiaLpxL 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 diverseRickettsiapathogens is unknown, our success using FLATnwill 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 inR. rickettsii(later-evolving SFG) relative toR. montanensis(basal SFG),R. typhi(TG), andR. akari(TRG). As lipid A structure influences recognition potential in vertebrate LPS sensors, further assessment ofRickettsialipid A structural heterogeneity is needed. Here, we sidestepped the difficulty ofex vivolipid 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 differentRickettsiapathogens and utilizing lipid A adjuvant or anti-inflammatory properties in vaccinology.