Thesis (PhD)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: The possible use of the bacterially produced antimicrobial peptides, and in particular class IIa
bacteriocins as food preservatives is a motivating factor in studies on resistance to them by
food-borne pathogens like Listeria monocytogenes. The high frequencies of resistance to class
Ha bacteriocins have however sparked concern regarding their adequacy as potential biopreservatives.
Activity of these cationic peptides was reported to occur by membrane
permeabilisation due to pore formation, which results in the leakage of the intracellular
contents followed by cell death. The cell envelope (cell wall and cell membrane) is therefore
envisaged 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. monocytagenes
wild-type and resistant isolates obtained from other sources. The fifty percent inhibitory
concentrations using a highly sensitive and reproducible bioassay were determined. This
allowed categorisation of the mutants into intermediate and highly resistant phenotypes.
Analysis of the growth patterns of all these strains showed decreased growth rates and higher
growth yields for all the resistant strains in general. This provided evidence for possible
effects of membrane adaptation and metabolic changes in the resistant strains and prompted
further 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 circular
dichroism and fourier transform infrared spectroscopy; (3) and, determination of changes in
glucose metabolism.
Electrospray mass spectrometry analysis of the major listerial phospholipid,
phosphatidylglycerol, revealed that membranes of resistant strains had increased levels of
unsaturated and short-acyl-chain phosphatidylglycerol molecular species, indicating more
fluid membranes. In addition, treatment with a desaturase inhibitor resulted in increased
sensitivity 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 is
conceivable that more fluid membranes could also impact on decreased stability of pore
formation by the bacteriocin.
Complementary biophysical studies using fourier transform infrared spectroscopy indicated
the 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 leucocin
A 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 cell
membranes as a-helical stucture is a critical feature for membrane insertion of cationic
antimicrobial peptides.
Cell surface charge was determined by quantification of alanine and lysine esterification of
the anionic cell surface polymer, teichoic acid, and membrane phospholipids respectively.
Increased D-alanine, which causes neutralisation of the cell surface, was observed in all
resistant cells. A tendency for greater lysine content in membrane phospholipids, which also
impacts on neutralisation of the anionic phospholipid of listerial membranes, was observed in
highly resistant strains only. This neutralisation of the negative charge of the cell surface may
interfere with initial electrostatic interaction of bacteriocin with the cell, and subsequent
interactions required for permeabilisation of the cell membrane. These differences in alanine
and 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-specific
phosphotransferase system EIIAB subunit (EIlABMan).A clear correlation existed between the
levels of resistance and EIIABMandown-regulation.
Finally, analysis of the glucose metabolism in highly resistant and wild-type strains, indicated
a more efficient metabolism with regards to higher growth yields and ATP yield, in contrast to
a 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 the
loss of the glucose transporter, EIlABMan,and may cast doubts on the feasibility of the use of
class Ha bacteriocins as food preservatives in light of a stable and efficient resistant
phenotype. / AFRIKAANSE OPSOMMING:
Sien volteks vir opsomming
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/53326 |
| Date | 12 1900 |
| Creators | Vadyvaloo, Viveka |
| Contributors | Hastings, J. W., Stellenbosch University. Faculty of Science . Dept. of Biochemistry. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | [104] p. : ill. |
| Rights | Stellenbosch University |
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