The disease listeriosis is caused by Listeria monocytogenes. This common food-borne
disease has been responsible for about 0.1 to 10 cases per million inhabitants per year.
However, this disease is serious with its high fatality rates of 20% - 30%, and 40% of all
cases reported have been in pregnant women suffered from a foetal abortion. Recently the
organism has acquired resistance to antibiotic treatment and the development of an
alternative treatment is necessary. Class IIa bacteriocins such as leucocin A have been
shown to be active against L. monocytogenes. However, the leucocin A receptor molecule
responsible for growth inhibition within L. monocytogenes remains unclear. Various
studies have implicated the mannose PTS permease (EIIt
Man) of L. monocytogenes as the
putative receptor for class IIa bacteriocins. The results from studies reviewed indicate that
the EIIt
Man of L. monocytogenes could be the chiral receptor needed for bacteriocin
interaction at the surface of targeted cells. Specifically, the membrane associated IIDMan
and IICMan subunits were implicated in direct interaction with class IIa bacteriocins. Our
study focused on cloning, expression and purification of the subunits of the mannose PTS
permease of L. monocytogenes EGD. Primers were designed to amplify the subunit genes
of the mptACD operon. The mptC, mptD and mptAB genes which were then successfully
cloned into pET28a expression vector and transformed into E. coli JM109(DE3) host
strain. Recombinant plasmids were screened using colony PCR. Subsequently recombinant
pET28-C, pET28-D and pET28-AB was once again transformed and expressed in the E.
coli BL21(DE3) pLysS expression host strain. After an induction at 30°C for 5 hours,
IICMan and IIDMan were found to be expressed in the cell membrane, whilst IIABMan was
expressed in the cytosol of the host expression strain. Membrane proteins His-IICMan, His-
IIDMan, and cytosol associated His-IIABMan were purified using Ni2+-NTA affinity
chromatography. Results for His-IICMan yielded a 28 kDa protein and a 55 kDa co-purified
protein. Results for His-IIDMan yielded a 31 kDa protein and a 60 kDa co-purified protein.
Results for His-IIABMan yielded a 35 kDa protein and a 68 kDa co-purified protein. A
western blot analysis revealed that all proteins purified carried an attached His-tag as
detected by an anti-mouse peroxidase conjugate anti-His-tag antibody. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/10840 |
| Date | January 2010 |
| Creators | Mia, Rizwana. |
| Contributors | Beukes, Mervyn., Watson, Gregory. |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
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