On-Tissue Derivatization of Lipopolysaccharide for Detection of Lipid A Using MALDI-MSI

Abstract

We developed a method to directly detect and map the Gram-negative bacterial virulence factor lipid A derived from lipopolysaccharide (LPS) by coupling acid hydrolysis with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). As the structure of lipid A (endotoxin) determines the innate immune outcome during infection, the ability to map its location within an infected organ or animal is needed to understand localized inflammatory responses that results during host-pathogen interactions. We previously demonstrated detection of free lipid A from infected tissue; however detection of lipid A derived from intact (smooth) LPS from host-pathogen MSI studies, proved elusive. Here, we detected LPS-derived lipid A from the Gram-negative pathogens, Escherichia coli (Ec, m/z 1797) and Pseudomonas aeruginosa (Pa, m/z 1446) using on-tissue acid hydrolysis to cleave the glycosidic linkage between the polysaccharide (core and O-antigen) and lipid A moieties of LPS. Using accurate mass methods, the ion corresponding to the major Ec and Pa lipid A species (m/z 1797 and 1446, respectively) were unambiguously discriminated from complex tissue substrates. Further, we evaluated potential delocalization and signal loss of other tissue lipids and found no evidence for either, making this LPS-to-Lipid A-MSI (LLA-MSI) method, compatible with simultaneous host-pathogen lipid imaging following acid hydrolysis. This spatially sensitive technique is the first step in mapping host-influenced de novo lipid A modifications, such as those associated with antimicrobial resistance phenotypes, during Gram-negative bacterial infection and will advance our understanding of the host-pathogen interface.

Figure 1.

MALDI ion map and spectra of bacterial culture spotted on mouse kidney tissue. (a, b) Ec and (c, d) Pa. (a) Labels 1, 2, 3, and 4 have an OD₆₀₀ value of 1.7, 1.0, 0.5, and 0.3, respectively. Label 5 is no deposition of Ec on tissue. (b) Average spectra for (a) with Ec lipid A m/z 1797 (e). (c) Labels 1 and 5, 2 and 6, and 3 have an OD₆₀₀ value of 1.7, 1.0, and 0.1, respectively. Labels 7 and 8 have an OD₆₀₀ value 0.5 and 0.25, respectively. Label 4 is no deposition of Pa on the tissue. Ion map at m/z 1446 (f) represents on-tissue (–8) derivatization or from ITO surface (–3). (d) Average spectra for (c). (a–d) MALDI-TOF MSI, normalized, TIC (total ion current).

Figure 2.

Differentiation of the Pa lipid A ion from neighboring cardiolipin by accurate mass assignment on-tissue. (a) Spectrum of summed area in box (inset) showing low signal for Pa lipid A without LLA-MSI treatment. (b) Spectrum of summed area in red box (inset) showing Pa lipid A ion (m/z 1445.8624) with LLA-MSI, separated from neighboring cardiolipin peak (m/z 1445.9521). Tissue thicknesses 25 and 50 μm for (a) and (b), respectively. Chemical formulas given for both Pa lipid A and cardiolipin with respective mass errors. (a, b) MALDI-IT-Orbitrap MSI, negative ion mode, NRM matrix, normalized TIC, and raster width 400 μm.