Rickettsia Lipid A Biosynthesis Utilizes the Late Acyltransferase LpxJ for Secondary Fatty Acid Addition

Abstract

Members of the Rickettsia genus are obligate intracellular, Gram-negative coccobacilli that infect mammalian and arthropod hosts. Several rickettsial species are human pathogens and are transmitted by blood-feeding arthropods. In Gram-negative parasites, the outer membrane (OM) sits at the nexus of the host-pathogen interaction and is rich in lipopolysaccharide (LPS). The lipid A component of LPS anchors the molecule to the bacterial surface and is an endotoxic agonist of Toll-like receptor 4 (TLR4). Despite the apparent importance of lipid A in maintaining OM integrity, as well as its inflammatory potential during infection, this molecule is poorly characterized in Rickettsia pathogens. In this work, we have identified and characterized new members of the recently discovered LpxJ family of lipid A acyltransferases in both Rickettsia typhi and Rickettsia rickettsii, the etiological agents of murine typhus and Rocky Mountain spotted fever, respectively. Our results demonstrate that these enzymes catalyze the addition of a secondary acyl chain (C₁₄/C₁₆) to the 3'-linked primary acyl chain of the lipid A moiety in the final steps of the Raetz pathway of lipid A biosynthesis. Since lipid A architecture is fundamental to bacterial OM integrity, we believe that rickettsial LpxJ may be important in maintaining membrane dynamics to facilitate molecular interactions at the host-pathogen interface that are required for adhesion and invasion of mammalian cells. This work contributes to our understanding of rickettsial outer membrane physiology and sets a foundation for further exploration of the envelope and its role in pathogenesis.IMPORTANCE Lipopolysaccharide (LPS) triggers an inflammatory response through the TLR4-MD2 receptor complex and inflammatory caspases, a process mediated by the lipid A moiety of LPS. Species of Rickettsia directly engage both extracellular and intracellular immunosurveillance, yet little is known about rickettsial lipid A. Here, we demonstrate that the alternative lipid A acyltransferase, LpxJ, from Rickettsia typhi and R. rickettsii catalyzes the addition of C₁₆ fatty acid chains into the lipid A 3'-linked primary acyl chain, accounting for major structural differences relative to the highly inflammatory lipid A of Escherichia coli.

Keywords: LPS; LpxJ; Rickettsia; lipid A; outer membrane; pathogenesis.

FIG 1

Lipid A structures of Escherichia coli and Rickettsia typhi.

FIG 2

LpxJ^Rt and LpxJ^Rr complement the loss of LpxM in E. coli and restore a hexa-acylated lipid A phenotype. (A) E. coli mutant strain MLK1067 (ΔlpxM) produces mostly penta-acylated lipid A, corresponding to a major ion peak at m/z 1,587. (B and C) Expression of LpxJ^Rt (B) or LpxJ^Rr (C) restores a hexa-acylated lipid A phenotype by addition of C₁₄ or C₁₆ fatty acid corresponding to the molecular ion at m/z 1,797 or m/z 1,825, respectively.

FIG 3

LpxJ transfers secondary C₁₄ or C₁₆ to the hydroxymyristate at the 3′ position. The fatty acid proportions present in the LPS isolated from E. coli strain MLK1067 carrying an empty vector or expressing lpxJ of R. typhi are given as percentages of the total identified fatty acids. A 4-fold increase of C₁₄ and C₁₆ addition was observed in the E. coli mutant complemented with lpxJ^Rt. Analysis was run in biological triplicate from three isolated transformants and values were plotted ± standard deviations. *, P < 0.05, as determined by Student's t test.

FIG 4

Acylation of lipid A by LpxJ^Rt does not depend upon prior secondary acylation. MALDI-TOF MS analysis was performed of lipid A from E. coli strain MKV15b (ΔlpxM ΔlpxL ΔlpxP) (35) transformed with empty vector (A) or expressing LpxJ^Rt (B). The tetra-acylated major lipid A ion of the parental strain (m/z 1,404) is acylated by LpxJ, which catalyzes the addition of C₁₄ (m/z 1,614) or C₁₆ (m/z 1,642). The minor ion peak at m/z 1,642 shown in panel A is likely the result of minimal constitutive PagP activity in this strain. A complete list of peaks and their raw measurements are reported in Table S1 in the supplemental material.

FIG 5

Structural and mutational analysis of LpxJ homologs. (A) Multiple sequence alignment of Rickettsia typhi LpxJ and three Epsilonproteobacteria LpxJ homologs that were previously characterized as lipid A late acyltransferases (31). NCBI protein accession numbers are in parentheses. Only the four most conserved regions of the alignment are shown (numbered 1 to 4 above the sequences). Amino acid coloring is as follows: black, hydrophobic; red, negatively charged; green, hydrophilic; purple, aromatic; blue, positively charged. Black and light blue circles below indicate critical and noncritical residues, respectively, as determined by mutagenesis, shown in panel B. Below each conserved region is a sequence logo (57) depicting conservation across 2,842 compiled LpxJ homologs. Asterisks denote the four residues invariant across all LpxJ homologs. (B) The histidine, aspartic acid, or tryptophan residue at the indicated position in the primary sequence of LpxJ was mutated to alanine, and each construct was individually expressed in MLK1067. Loss of these highly conserved residues abolished the acyltransferase activity of the enzyme, reverting the lipid A phenotype to that of the background strain (penta-acyl, m/z 1,587). Mutation of histidine at position 84 to alanine or serine had no effect on enzymatic activity (data not shown). (C) Comparison of R. typhi LpxJ to four divergent lipid acyltransferases. Proteins with associated structures were obtained from the Protein Data Bank: Mycobacterium smegmatis PatA (PDB code 5F34), Acinetobacter baumannii LpxM (5KNK), Cucurbita moschata PlsB (1IUQ), and Thermotoga maritima PlsC (5KYM). R. typhi LpxJ was modeled to all four acyltransferase structures using Phyre2 (58) and fitted to an existing structural alignment template (37), which follows the convention established for naming conserved blocks within GPAT, LPAAT, DHAPAT, and LPEAT acyltransferases (59, 60). Active-site residues for each structure are colored. For LpxJ, Asp132 is proposed to participate in the active site charge relay system with His61 (36, 61).

FIG 6

Late acyltransferase activity for R. typhi. The bidirectional additions of the final two fatty acids to complete the biosynthesis of lipid A at the conclusion of the Raetz pathway are shown. Blue arrows indicate LpxJ-mediated acylation, and red arrows indicate LpxL-mediated acylation.