Changes in cell surface and metabolism associated with strains of Listeria monocytogenes displaying different sensitivities to class IIa bacteriocins

[摘要] ENGLISH ABSTRACT:The possible use of the bacterially produced antimicrobial peptides, and in particular class IIabacteriocins as food preservatives is a motivating factor in studies on resistance to them byfood-borne pathogens like Listeria monocytogenes. The high frequencies of resistance to classHa bacteriocins have however sparked concern regarding their adequacy as potential biopreservatives.Activity of these cationic peptides was reported to occur by membranepermeabilisation due to pore formation, which results in the leakage of the intracellularcontents followed by cell death. The cell envelope (cell wall and cell membrane) is thereforeenvisaged as a key site of modification in suscepti bility of bacteria to class Ha bacteriocins.Mutants of the L. monocytogenes 873 isolate, resistant to the class IIa bacteriocin, leucocin A,were generated at the start of the study to complement the existing array of L. monocytageneswild-type and resistant isolates obtained from other sources. The fifty percent inhibitoryconcentrations using a highly sensitive and reproducible bioassay were determined. Thisallowed categorisation of the mutants into intermediate and highly resistant phenotypes.Analysis of the growth patterns of all these strains showed decreased growth rates and highergrowth yields for all the resistant strains in general. This provided evidence for possibleeffects of membrane adaptation and metabolic changes in the resistant strains and promptedfurther investigation. The major focus of the study on the class Ha resistant mutants were: (1)analysis of membrane compositional changes and factors influencing cell surface charge; (2)assessment of physical changes in the membrane and bacteriocin itself using circulardichroism and fourier transform infrared spectroscopy; (3) and, determination of changes inglucose metabolism.Electrospray mass spectrometry analysis of the major listerial phospholipid,phosphatidylglycerol, revealed that membranes of resistant strains had increased levels ofunsaturated and short-acyl-chain phosphatidylglycerol molecular species, indicating morefluid membranes. In addition, treatment with a desaturase inhibitor resulted in increasedsensitivity of only the intermediate resistant strains to the class na bacteriocin, leucocin A.This indicated the influence of membrane adaptation in only lower levels of resistance. It isconceivable that more fluid membranes could also impact on decreased stability of poreformation by the bacteriocin.Complementary biophysical studies using fourier transform infrared spectroscopy indicatedthe possible occurrence of greater membrane fluidity of resistant cells, by the notable shift in the anti symmetric CH2 stretching vibration from 2921 cm-I to 2922 cm-I. Additionally,circular dichroism revealed a decreased a-helical and increased random structure of leucocinA in the presence of listerial liposomes derived from highly resistant cell membrane extracts.It is possible that this may result in reduced activity of the bacteriocin in resistant cellmembranes as a-helical stucture is a critical feature for membrane insertion of cationicantimicrobial peptides.Cell surface charge was determined by quantification of alanine and lysine esterification ofthe anionic cell surface polymer, teichoic acid, and membrane phospholipids respectively.Increased D-alanine, which causes neutralisation of the cell surface, was observed in allresistant cells. A tendency for greater lysine content in membrane phospholipids, which alsoimpacts on neutralisation of the anionic phospholipid of listerial membranes, was observed inhighly resistant strains only. This neutralisation of the negative charge of the cell surface mayinterfere with initial electrostatic interaction of bacteriocin with the cell, and subsequentinteractions required for permeabilisation of the cell membrane. These differences in alanineand lysine esterification were not the result of increased expression of certain associated genes(d/tA and /mo1695) and may be the result of post-transcriptional regulation. It was, however,found that all resistant L. monocytogenes strains, including the intermediate resistant strains,exhibited decreased expression of a putative docking molecule, the mannose-specificphosphotransferase system EIIAB subunit (EIlABMan).A clear correlation existed between thelevels of resistance and EIIABMandown-regulation.Finally, analysis of the glucose metabolism in highly resistant and wild-type strains, indicateda more efficient metabolism with regards to higher growth yields and ATP yield, in contrast toa lower specific growth rate in a spontaneous and genetically defined (EIlABMan inactivated)highly resistant mutant. The switch in metabolic end-product observed, was attributed to theloss of the glucose transporter, EIlABMan,and may cast doubts on the feasibility of the use ofclass Ha bacteriocins as food preservatives in light of a stable and efficient resistantphenotype.

[发布机构] Stellenbosch University