Structural determination of
Rickettsia
lipid 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: January 2024

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 the 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
Rickettsia akari
(TRG),
Rickettsia typhi
(TG), and
Rickettsia montanensis
(SFG) produce lipid A with long 2′ secondary acyl chains (C16 or C18) compared to short 2′ secondary acyl chains (C12) in
Rickettsia 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 (
Rickettsia rhipicephali
and
Rickettsia parkeri
) utilizing fast lipid analysis technique adopted for use with tandem mass spectrometry (FLAT
n
). FLAT
n
allowed analysis of lipid A structure directly from host cell-purified bacteria, providing a substantial improvement over lipid A chemical extraction. FLAT
n
-derived structures indicate SFG rickettsiae diverging after
R. rhipicephali
evolved shorter 2′ secondary acyl chains. While 2′ secondary acyl chain lengths do not distinguish
Rickettsia
pathogens from non-pathogens,
in silico
analyses of
Rickettsia
LpxL late acyltransferases revealed discrete active sites and hydrocarbon rulers for long versus short 2′ secondary acyl chain addition. Our collective data warrant determining
Rickettsia
lipid A inflammatory potential and how structural heterogeneity impacts lipid A-host receptor interactions.

IMPORTANCE

Deforestation, 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
Rickettsia rickettsii
(later-evolving SFG) relative to
Rickettsia montanensis
(basal SFG),
Rickettsia typhi
(TG), and
Rickettsia 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 fast lipid analysis technique adopted for use with tandem mass spectrometry, a new procedure for generating lipid A structures directly from host cell-purified bacteria. These data confirm that 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.