MASS SPECTRAL ANALYSIS OF NOVEL BACTERIAL LIPOPOLYSACCHARIDES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Lipopolysaccharides (LPS) and their lipid A (LA) anchors are known endotoxins due to their important role in the origin of infectious diseases. Marine bacterial LPS are involved in their own environment adaptation processes. We are investigating whether the low endotoxicity of LAs from marine bacteria may result from remarkable differences in their structures, compared to those of pathogenic congeners. It is also expected that determination of unusual and unknown patterns of marine lipid A structures could facilitate improvements in protocol for structural characterization of lipids A of pathogenic stains. In this study, a combination of mass spectrometric dissociation and chemical degradation techniques are used, taking as the examples two strains from the Pseudoalteromonas and Marinamonas genus. Crude LAs were obtained from LPS suspension by gentle acidic hydrolysis in acetate buffer, cooling and centrifugation of treated suspension, and extraction with chloroform. More harsh acidic treatment was used to obtain the partially de-O-acylated LA ladder. Preparative layer chromatography (PLC) was used isolate phosphate-acylated types and was followed mass spectral analysis. MS methods for dissociation of gaseous cationic and anioinic LA species included the following: ESI ITMSn; PSD MALDI-TOF MS and NSCD ESI-, SORI CAD VC-MALDI- and ESI IRMPD- FTMS, as well as NMR. These methods were utilized to obtain data for determination of the molecular structures marine lipids A. The lipids A of M. vaga and P. haloplanktis strains were found to have mono- and di-phosphate/ penta-acyl homogeneous pattern, respectively, on a common lipid A backbone. However, the profiles of the obtained ESI and MALDI spectra were very complex, especially in the case of the strains of the Pseudoalteromonas genus. More than 5 species of the last genus were screened by MS methods. Two single homogeneous initial lipid A type samples, which were recovered from the Pseudoalteromonas genus, were represented in the profiles by ion clusters that extended over a range of 80 Da. However, lipid A molecules carried only four fatty acids [OH10:0; OH11:0; OH12:0; OH13:0] on five positions. Such a pattern may include at least 64 molecular types arising from precursors in a fatty acid pool instead of a single acid. MS analyses indicated that the phosphate/acyl heterogeneity of crude lipids A results from chemical treatments during the isolation from LPS. Subsequent experiments have explored the use of electron capture dissociation and LC/MS/MS for characterization of lipid A. The initial lipid A is rather homogeneous in vivo so long as the bacteria do not suddenly encounter a new environment. Mutant lipids A were found in the LPS of strain M. vaga when its cells were transferred to the modified medium for the first time. With support from a COBASE grant for exchange of scientists from the former Soviet Union and the US, this collaboration between the BUSM Resource and the Far East Branch of the Russian Academy of Sciences was initiated with a visit of Dr. Yelkine to BUSM. Prof. Costello later visited Vladivostok and Dr. Yelkine returned to join the Resource staff to pursue this investigation. The project continues as data interpretation is carried out and several manuscripts are being prepared.