Structural determination of Rickettsia lipid A without chemical extraction confirms shorter acyl chains in later-evolving spotted fever group pathogens

ABSTRACT 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 (FLATn). FLATn allowed analysis of lipid A structure directly from host cell-purified bacteria, providing a substantial improvement over lipid A chemical extraction. FLATn-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.

G enus Rickettsia (Alphaproteobacteria: Rickettsiales) comprises species of obligate intracellular parasites of numerous eukaryotes (1).Across the Rickettsia phylogeny, all known agents of human disease are vector-borne pathogens of the transitional group (TRG), typhus group (TG), or spotted fever group (SFG) rickettsiae, which are later-evolv ing clades relative to basal lineages of numerous invertebrate and protist endosym bionts (2).Compared to other Rickettsiales species with human health relevance (e.g., Orientia tsutsugamushi, Wolbachia, species of Neorickettsia, Anaplasma, Neoehrlichia, and Ehrlichia), rickettsiae alone synthesize lipopolysaccharide (LPS) (3), which comprises extracellular polysaccharide chains (O-antigen) linked to a membrane phosphoglycolipid (lipid A) by a core oligosaccharide.As all Rickettsia species lack glycolytic enzymes, they are the only known bacteria to synthesize a canonical Gram-negative cell envelope rich in LPS, as well as peptidoglycan (4,5), from metabolites derived from host cytosol (6).
All described rickettsioses are caused by arthropod-borne pathogens that differ in their protein secretome (7,8) and presumably LPS composition.For LPS, Rickettsia O-antigen contains the sugar quinovosamine (9)(10)(11) that is required for S-layer formation and vertebrate pathogenicity (12).While the proinflammatory nature of Rickettsia lipid A remains unknown, it is a candidate for the well-characterized triggering of mamma lian MD-2/TLR4 receptor and non-canonical inflammasome activation during infection (13)(14)(15)(16)(17)(18).We previously demonstrated that human pathogens Rickettsia akari (TRG) and Rickettsia typhi (TG), as well as the non-pathogen Rickettsia montanensis (SFG), produce lipid A with longer 2′' secondary acyl chains (C16 or C18) relative to shorter chains (C12) in the pathogen Rickettsia rickettsii (SFG) lipid A (19).As R. rickettsii is later evolving in the SFG rickettsiae relative to R. montanensis, we surmised that a switch from longer to shorter 2′ secondary acyl chains occurred later in SFG rickettsial evolution.This laterevolving clade is dominated by other notable human pathogens, including the agents of Japanese spotted fever (Rickettsia japonica), Flinders Island spotted fever (Rickettsia honei), Pacific Coast tick fever (Rickettsia philipii), Mediterranean spotted fever (Rickettsia conorii), Siberian tick typhus (Rickettsia sibirica), African tick-bite fever (Rickettsia africae), and Dermacentor-borne necrosis erythema and lymphadenopathy (Rickettsia raoultii and Rickettsia slovaca).However, the occurrence of other non-pathogens in this clade (e.g., Rickettsia peacockii and the seal fur louse endosymbiont) and uncertain vertebrate infectivity dynamics for many Rickettsia species raise questions as to the nature of Rickettsia lipid A and its impact on host cell immune systems.
To further evaluate Rickettsia lipid A structural heterogeneity, we selected two additional species, Rickettsia rhipicephali and Rickettsia parkeri, for lipid A structural analysis.In recent phylogeny estimations, R. rhipicephali groups in a clade that diverges after R. montanensis (long 2′ secondary acyl chains) but before R. rickettsii (short 2′ secondary acyl chain), while R. parkeri belongs to a clade that is sister to the R. rickettsiicontaining clade (1,7).Thus, our strategy zeros in on the evolutionary timepoint for the transition to short 2′ secondary acyl chains in SFG rickettsiae.Furthermore, we utilized a different analytical approach to generate new structures called fast lipid analysis technique adopted for use with tandem mass spectrometry (FLAT n ), allowing assessment of prior structure determinations that were based on lipid A chemical extraction.Briefly, FLAT n is a method for the on-surface and/or on-tissue release of lipid A from LPS that allows its detection by matrix-assisted laser desorption ionization mass spectrometry in the negative ion mode (20,21) and has been shown to facilitate direct analysis of lipid A structure from a single bacteria colony (22).Therefore, FLAT n circumvents time-, labor-, and sample-intensive techniques previously required to chemically extract lipid A prior to MS analysis.Thus, we reasoned that FLAT n would yield Rickettsia lipid A structures from a minimal sample of bacteria partially purified from far fewer host cells than our prior approach.FLAT n allowed for the direct analysis of R. rhipicephali and R. parkeri lipid A structures from host cells, providing substantial improvement and efficiency over lipid A chemical extraction (Fig. 1).Bacterial samples that were purified from host cells using either beador sucrose gradient-based strategies sufficed to generate MS spectra at either 1,936.37m/z (C16 2′ secondary acyl chains) or 1,880.31m/z (C12 2′ secondary acyl chains), consistent with prior analyses of chemically extracted Rickettsia lipid A (19) (Fig. 1A  and B).Subsequent derivatization of these single ions for R. rhipicephali (Fig. 1C) and R. parkeri (Fig. 1D) yielded fragmentation products that supported structural elucidation.The FLAT n -derived structure for R. rhipicephali lipid A corroborates those of R. akari, R. typhi, and R. montanensis with long 2′ secondary acyl chains (Fig. 1E).In contrast, the R. parkeri FLAT n -derived lipid A structure matches that determined for R. rickettsii strains with short 2′ secondary acyl chains (Fig. 1F).Thus, as with prior results (19), we have discovered distinct lipid A in later-evolving SFG rickettsiae that is more structurally similar to inflammatory lipid A of certain Enterobacteriaceae species than other Rickettsia lipid A with longer 2′ secondary acyl chains.
While bolstering prior results that indicated Rickettsia lipid A structural heterogeneity, FLAT n -derived structures also helped refine the evolutionary time point when short 2′ secondary acyl chains originated within SFG rickettsiae (Fig. 2A).In light of phylog eny estimation, our collective data indicate SFG rickettsiae diverging after R. rhipice phali evolved shorter 2′ secondary acyl chains (Fig. 2A, red shading).A parsimonious interpretation entails all members of the R. rhipicephali-containing clade synthesize lipid A with long 2′ secondary acyl chains, whereas all later-evolving SFG rickettsiae synthesize lipid A with short 2′ secondary acyl chains.Aside from many human pathogens in the later-evolving lineages, R. peacockii and the endosymbiont of the seal fur louse (Proechinophthirus fluctus) are non-pathogenic species that do not infect vertebrates (23,24).Assuming that these endosymbionts synthesize lipid A [both of their genomes encode the full suite of Raetz pathway enzymes (3)], short 2′ secondary acyl chains could be considered a trait that emerged without selective pressure from the vertebrate host environment.Alternatively, the non-pathogenic endosymbionts of this clade may have lost the ability to invade vertebrate hosts due to the pseudogenization of numerous genes implicated in vertebrate cell invasion (23,24), including uncharacterized LPSmodification enzymes that may only be necessary for survival in vertebrate hosts (25).Generating lipid A structures for other species in this clade will be necessary to further evaluate our observations, as will determining if rickettsiae alter their lipid A acyl chain lengths in vertebrate versus invertebrate hosts.
Finally, we inspected Rickettsia LpxL proteins for possible active site traits that correlate with lipid A structural heterogeneity (Fig. 2A and B).LpxL is the late acyl transferase in the Raetz pathway that acylates 2′ primary acyl chains (see Fig. S1).Of only five variable residues across Rickettsia LpxL proteins, two (positions 100 and 130) have distinct properties defining later-evolving SFG rickettsiae (Fig. 2A and B).While nonetheless interesting with short chain (Ala/Thr) replacing charged (Lys/Glu) residues, position 100 is outside of the LpxL active site.However, position 130 (highlighted yellow in Fig. 2A and B) has a conserved Iso in place of Val/Leu and is proximal to active site post-infection, host cells were recovered and lysed with 3 mm beads, host debris was removed via low-speed centrifugation (5,000 rpm), and Rickettsia was collected by high-speed centrifugation (8,000 rpm).Purification via sucrose gradient followed our prior protocol (19).Bacterial pellets were then analyzed via FLAT n .Asterisks indicate the expected size for Rickettsia lipid A with C16 (~1,936.37 m/z) or C12 (1,880.31m/z) 2′ secondary acyl chains based on our prior report (19).(C and D) Derivatization of a single ion for the (C) R. rhipicephali sample (1,936.37m/z) and the (D) R. parkeri sample illustrating five and six, respectively, major fragmentation products.These products are named in the tables with theoretical and experimental sizes shown, with error calculation illustrating robust interpretation.They are also color-coded to facilitate the interpretation of the spectra above and predicted structures.(E and F) FLAT n -derived structure predictions for lipid A of (E) R. rhipicephali, which is similar to previously determined R. akari, R. typhi, and R. montanensis structures, and (F) R. parkeri, which is similar to previously determined structures for R. rickettsii strains Shelia Smith and Iowa.Sites yielding fragmentation products are yellow, with corresponding nomenclature described in the tables in panels C and D. The inset describes the conserved and variable lipid A acylation of Rickettsia lipid A, with colored symbols mapped on structures (see Fig. S1 for more details).Observation mSphere residue Glu-127, which has been implicated in substrate binding for the analogous late acyltransferase LpxM (30) (Fig. 2C).This compelling correlation of acyl chain length and active site composition across Rickettsia phylogeny warrants characterizing substrate specificities for divergent Rickettsia LpxL enzymes.In summary, our work (i) bolsters Rickettsia lipid A structural heterogeneity, (ii) shows FLAT n is effective for analyzing obligate intracellular bacterial lipid A, and (iii) identifies LpxL properties that may explain variable acyl chain length addition.We anticipate our findings to impact studies comparing host immune responses to divergent pathogens and inform on the efficacy of lipid A in Rickettsia vaccinology.

FIG 1
FIG 1 Rickettsia lipid A structures determined by FLAT n confirm variable acyl chain lengths for different rickettsiae.(A and B) FLAT-MS spectra from (A) R. rhipicephali partially purified from host cells using either a bead (B) or sucrose gradient (S) purification strategy, and from (B) R. parkeri partially purified from host cells using sucrose gradient purification strategy.Briefly, Vero76 cells were grown to confluence in T-25 flasks with cells infected at an MOI of 10.At 3 days (Continued on next page)

4 FIG 2
FIG 2The evolution of variable acyl chain lengths in Rickettsia lipid A. (A) Superimposition of determined lipid A structures and late acyltransferase LpxL characteristics over an estimated Rickettsia phylogeny.Tree is redrawn from a recent study(7).BG, Bellii group and TIG, Tamurae/Ixodes group.Taxon names in gray boxes were determined to synthesize lipid A with palmitate or stearate (C16 or C18) added to the primary 2′ acyl chain; taxon names in red boxes were (Continued on next page)