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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The electron micrograph shows some typically pleomorph mycoplasmas, in this case M. hyorhinis

(courtesy of Dr. H. Lünsdorf, GBF)