Early evolutionary loss of the lipid A modifying enzyme PagP resulting in innate immune evasion in Yersinia pestis

Chandler CE, Harberts EM, Pelletier MR, et al. Proc Natl Acad Sci U S A 2020 Sep 15.

Immune evasion through membrane remodeling is a hallmark of Yersinia pestis pathogenesis. Yersinia remodels its membrane during its life cycle as it alternates between mammalian hosts (37 °C) and ambient (21 °C to 26 °C) temperatures of the arthropod transmission vector or external environment. This shift in growth temperature induces changes in number and length of acyl groups on the lipid A portion of lipopolysaccharide (LPS) for the enteric pathogens Yersinia pseudotuberculosis (Ypt) and Yersinia enterocolitica (Ye), as well as the causative agent of plague, Yersinia pestis (Yp). Addition of a C16 fatty acid (palmitate) to lipid A by the outer membrane acyltransferase enzyme PagP occurs in immunostimulatory Ypt and Ye strains, but not in immune-evasive Yp Analysis of Yp pagP gene sequences identified a single-nucleotide polymorphism that results in a premature stop in translation, yielding a truncated, nonfunctional enzyme. Upon repair of this polymorphism to the sequence present in Ypt and Ye, lipid A isolated from a Yp pagP+ strain synthesized two structures with the C16 fatty acids located in acyloxyacyl linkage at the 2′ and 3′ positions of the diglucosamine backbone. Structural modifications were confirmed by mass spectrometry and gas chromatography. With the genotypic restoration of PagP enzymatic activity in Yp, a significant increase in lipid A endotoxicity mediated through the MyD88 and TRIF/TRAM arms of the TLR4-signaling pathway was observed. Discovery and repair of an evolutionarily lost lipid A modifying enzyme provides evidence of lipid A as a crucial determinant in Yp infectivity, pathogenesis, and host innate immune evasion. … Continue readingEarly evolutionary loss of the lipid A modifying enzyme PagP resulting in innate immune evasion in Yersinia pestis

Streamlined Analysis of Cardiolipins in Prokaryotic and Eukaryotic Samples Using a Norharmane Matrix by MALDI-MSI

Yang H, Jackson SN, Woods AS, et al. J Am Soc Mass Spectrom. 2020 Oct 5.

Cardiolipins (CLs) are an important, regulated lipid class both in prokaryotic and eukaryotic cells, yet they remain largely unexplored by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) in tissues. To date, no in-depth optimization studies of label-free visualization of CLs in complex biological samples have been reported. Here we report a streamlined modification to our previously reported MALDI-MSI method for detection of endogenous CLs in prokaryotic and eukaryotic cells based on preparation with norharmane (NRM) matrix. Notably, the use of NRM matrix permitted sensitive detection (4.7 pg/mm2) of spotted CL synthetic standards. By contrast, four other MALDI matrices commonly used for lipid analysis failed to generate CL ions. Using this NRM-based method, endogenous CLs were detected from two types of complex biological samples: dried bacterial arrays and mouse tissue sections. In both cases, using NRM resulted in a better signal/noise for CL ions than the other matrices. Furthermore, inclusion of a washing step improved CL detection from tissue and this combined tissue preparation method (washing and NRM matrix) was used to profile normal mouse lung. Mouse lung yielded 26 unique CLs that were mapped and identified. Consistent with previous findings, CLs containing polyunsaturated fatty acids (PUFAs) were found in abundance in the airway and vascular features of the lung. This work represents a comprehensive investigation of detection conditions for CL using MALDI-MSI in complex biological samples that resulted in a streamlined method that enables future studies of the biological role(s) of CL in tissue. … Continue readingStreamlined Analysis of Cardiolipins in Prokaryotic and Eukaryotic Samples Using a Norharmane Matrix by MALDI-MSI