There is increasing epidemiologic evidence implying a role for chronic infection in atherosclerosis and that microbial TLR agonists may contribute to this disease. Mycoplasma arthritidis is an agent of acute and chronic inflammatory disease in rodents, and has been used extensively as a model for defining the mechanisms involved in arthritis and other inflammatory diseases. We have purified a 28-kDa, apolipoprotein A-1 (apoA-1)-like TLR2-dependent macrophage-activating moiety from a culture of a virulent strain of M. arthritidis. ApoA-1 similarly isolated from uninoculated mycoplasma medium was without bioactivity. The activity of the mycoplasma-derived molecule was resistant to heat and to digestion with proteinase K, but was susceptible to alkaline hydrolysis and H(2)O(2) oxidation. Infrared profiles of normal apoA-1 and that derived from mycoplasma were distinct. Unlike the activity of other mycoplasmal TLR2 agonists such as macrophage-activating lipopeptide-2, activity of the M. arthritidis-derived 28-kDa component was dependent upon CD14, a coreceptor for LPS. Finally, we showed that bioactive lipopeptides prepared from M. arthritidis grown in serum-free medium and also from a 41-kDa known bioactive lipoprotein of M. arthritidis, avidly bound to purified apoA-1 that separated out by SDS-PAGE, induced TNF-alpha and IL-12p40 both in vitro and in vivo. ApoA-1 is a key functional component of the high-density lipoprotein cholesterol complex by scavenging and removing unwanted lipids. Our finding that this molecule can acquire macrophage-activating properties from microbial TLR2-dependent agonists suggests a novel mechanism whereby some microbial agents might reverse the protective role of apoA-1, thus contributing to the genesis of atherosclerosis.