Discovery
MALP-2 was discovered in
the culture medium of the human cell line HL 60 as an activity
that enhanced the cytolytic capacity of Concanavalin A-stimulated
mouse thymocytes. It was then found that the activity in the
culture medium correlated with a contamination of this cell
line with Mycoplasma fermentans and M. hyorhinis.
At the time the biochemical nature of this activity was unclear.
The unknown bioactive substance was named “MDHM”, for "mycoplasma-derived
high molecular weight material”, because the activity coeluted
from gel exclusion columns with high molecular weight markers.
This property could later be ascribed to membrane fragments
and vesicles containing MALP-2.
How MALP-2 generates CTL
The mechanism of "MDHM"-mediated generation of cytolytic T cells
was found to be due to a complicated sequence of events starting
with the production of the proinflammatory cytokine IL-6 from
very few MALP-2-stimulated macrophages in the
thymocyte population, which was followed by IL-6-dependent production
of IL-4 and IL-2 by T cells. This discovery initiated the establishment
of various macrophage activation tests to assay the macrophage-stimulatory
activity of MALP-2. Of these the nitric oxide
release assay turned out to be the most practical one, suitable
for the screening of large numbers of samples.
Purification and structure elucidation
A high producer M. fermentans clone
was isolated, and "MDHM" was finally purified after several
steps including detergent extraction, enzyme digestion, and
reversed phase high performance liquid chromatography. Structure
elucidation showed “MDHM” to be a lipopeptide of 2 kDa molecular
mass with two long chain fatty acids. The characteristic difference
from other bacterial lipoproteins like e.g. that from E.Coli
is the absence of the N-terminal third fatty acid. The structure
was confirmed by organic synthesis. “MDHM” was from now on
more appropriately named MALP-2, macrophage
activating lipopeptide of 2 kDa molecular mass.
Genetics and occurrence of "MALP-like" lipoproteins in other
mycoplasma species
The gene of the parent molecule of MALP-2,
the naturally occurring precursor and full size lipoprotein
MALP-404, was cloned. It turned out that many other mycoplasma
species, among them M. hyorhinis and M. salivarium,
produce and contain MALP-like lipoproteins with similar biological
activity. Genetic data from sequencing the genomes of M.
pneumoniae and M. genitalium confirmed that
the N-acyltransferase gene responsible for the biosynthesis
of “conventional” bacterial lipoprotein with three fatty acids
is lacking. The macrophage activating activity of mycoplasmas
has been independently reported by other groups. It seems
to be a general property of these microorganisms and is associated
with inflammatory reactions and other sequelae of mycoplasma
infection. MALP-like lipoproteins and -peptides appear to
be the major cause of these host reactions to a mycoplasma
infection.
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