Identification of unique cardiolipin and monolysocardiolipin species in Acinetobacter baumannii

Abstract

Acidic glycerophospholipids play an important role in determining the resistance of Gram-negative bacteria to stress conditions and antibiotics. Acinetobacter baumannii, an opportunistic human pathogen which is responsible for an increasing number of nosocomial infections, exhibits broad antibiotic resistances. Here lipids of A. baumannii have been analyzed by combined MALDI-TOF/MS and TLC analyses; in addition GC-MS analyses of fatty acid methyl esters released by methanolysis of membrane phospholipids have been performed. The main glycerophospholipids are phosphatidylethanolamine, phosphatidylglycerol, acyl-phosphatidylglycerol and cardiolipin together with monolysocardiolipin, a lysophospholipid only rarely detected in bacterial membranes. The major acyl chains in the phospholipids are C16:0 and C18:1, plus minor amounts of short chain fatty acids. The structures of the cardiolipin and monolysocardiolipin have been elucidated by post source decay mass spectrometry analysis. A large variety of cardiolipin and monolysocardiolipin species were found in A. baumannii. Similar lysocardiolipin levels were found in the two clinical strains A. baumannii ATCC19606^T and AYE whereas in the nonpathogenic strain Acinetobacter baylyi ADP1 lysocardiolipin levels were highly reduced.

Figure 1

MALDI-TOF/MS analysis of the total lipid extract of A. baumannii cells. Mass spectra were acquired in negative ion mode using 9-aminoacridine as matrix. Mass spectrum in the lower panel represents the MALDI-TOF/MS lipid profile in the full m/z range 600–1500. In panel (A) x-axis enlargement of the m/z range 640–800 referable to the main phospholipids (PLs); in panel (B) x-axis enlargement of the m/z range 1100–1200 referable to the monolysocardiolipins (MLCLs); in panel (C) x-axis enlargement of the m/z range 1350–1450 referable to the cardiolipins (CLs). A detailed list of detected peaks is shown in Table 1 (PLs), and Table 2 (CLs, MLCLs).

Figure 2

GC-MS analyses of FAMEs released by acid methanolysis from the total lipid extract. Carbon atoms and unsaturations of various fatty acid chains are reported.

Figure 3

MALDI-TOF/MS analyses of lipid bands #1, 3, 4 and 6, isolated from the total lipid extract of A. baumannii by TLC. The total lipid extract of A. baumannii cells was loaded on the plate (160 μg per each lane). One lane was sprayed with sulfuric acid and charred in the oven (permanent staining of all class of lipids) (i); the other lanes were stained with iodine vapors (temporary staining of all class of lipids) (ii); each band was marked with a pencil and silica was scraped in correspondence of lipid bands. The chromatography plate is shown in panel A. Lipid bands #1, 3, 4 and 6 were extracted from silica and were analyzed by MALDI-TOF/MS. The MALDI/TOF-MS (negative ion mode) spectra of the four lipid bands corresponding to Acyl-PGs, PE, PG and LPE (from the top to the bottom) are shown in panel B. The detailed list of detected peaks is shown in Table 1. The peak at m/z 763.4 labeled with star has not been included in the list because it results from TLC artifact.

Figure 4

MALDI-TOF/MS analyses of lipid bands #2 and 5, isolated from the total lipid extract of A. baumannii by TLC. The chromatography plate is shown in panel A. Lipid bands #2 and 5 were extracted from silica and were analyzed by MALDI-TOF/MS. The MALDI/TOF-MS (negative ion mode) spectra of the two lipid bands corresponding to CLs (top), MLCLs (bottom) are shown in panel B. The detailed list of detected peaks is shown in Table 2. Peaks labeled with star have been attributed to TLC artifacts.

Figure 5

Fragmentation patterns of monolysocardiolipin and cardiolipins. PSD analysis of the peaks at m/z 1183.3 (a) 1293.0 (b), 1403.9 (c) and 1165.6 (d). (a) PSD analysis of the peak at m/z 1183.3: ion fragments correspond to PA (m/z 618.9), PA (m/z 562.9), LPA-H₂O (m/z 390.7) and the fatty acid 16:0 (m/z 254.6). (b) PSD analysis of the peak at m/z 1293.0: ion fragments correspond to PGP-H₂O (m/z 808.9), PA (m/z 673.2), PA (m/z 563.0), LPA (m/z 408.9), LPA-H₂O (m/z 391.1) and fatty acids 18:1 (m/z 281.4) and 16:0 (m/z 254.6). (c) PSD analysis of the peak at m/z 1403.9: ion fragments correspond to PGP-H₂O (m/z 809.9), PG-H₂O (m/z 729.4), PA (m/z 673.6), LPA (m/z 408.9), LPA-H₂O (m/z 391.1). In the x-axis enlargement of m/z range 150–350, two peaks are referable to the fatty acids 18:1 (m/z 281.4) and 16:0 (m/z 254.5). (d) PSD analysis of the peak at m/z 1165.6: ion fragments correspond to PGP-H₂O (m/z 809.5), PG (m/z 747.4), PGP-H₂O (m/z 729.6), PA (m/z 673.7), LPA (m/z 436.3), LPA-H₂O (m/z 391.8). In the x-axis enlargement of m/z range 150–350, two peaks are referable to fatty acids 18:1 (m/z 281.5) and 16:0 (m/z 255.4).

Figure 6

MLCL and CL fingerprint of intact membranes of Acinetobacter baumannii, acquired by MALDI-TOF/MS analyses. (a) m/z range of MLCLs; (b) m/z range of CLs.

Figure 7

MLCL and CL levels of A. baumannii, AYE and A. baylyi. The lipid analyses of high and low virulence cells have been performed by combining mass spectrometry and TLC. Mass spectra were acquired in negative ion mode using 9-aminoacridine as matrix. The m/z range 1000–1500 refers to monolysocardiolipins (MLCLs) and cardiolipins (CLs). At the bottom the TLC bands of CL and MLCL in the three strains.