[Profiles of intracellular expression, distribution, and activation of phospholipase A2 in host macrophages infected with Mycobacterium avium complex].

Our recent studies have shown that type IV cytosolic phospholipase A2 (cPLA2) plays an important role in the expression of macrophage (MPh) antimicrobial activity against the Mycobacterium avium complex (MAC). To clarify the modes of cPLA2 participation in MPh anti-MAC antimicrobial function in detail, we studied intracellular profiles of phospholilase A2, focusing on cPLA2, in MAC-infected MPhs, with the following findings: In murine peritoneal MPhs, cPLA2 was constitutively expressed even in uninfected MPhs, and MAC infection did not increase intramacrophage cPLA2 expression. In an RAW264.7 mouse MPh cell line (RAW264.7 MPhs) infected with MAC, a portion of intracellular cPLA2 was concentrated in MAC organisms residing within MPh phagosomes. MAC infection upregulated intramacrophage cPLA2 expression and induced its phosphorylation. These findings suggest that MAC infection of RAW264.7 MPhs may induce the activation of intracellular cPLA2, translocating it to phagosomes engulfing infected MAC organisms.

Combined effects of ATP on the therapeutic efficacy of antimicrobial drug regimens against Mycobacterium avium complex infection in mice and roles of cytosolic phospholipase A2-dependent mechanisms in the ATP-mediated potentiation of antimycobacterial host resistance.

ATP, which serves as a mediator of intramacrophage signaling pathways through purinoceptors, is known to potentiate macrophage antimycobacterial activity. In this study we examined the effects of ATP in potentiating host resistance to Mycobacterium avium complex (MAC) infection in mice undergoing treatment with a drug regimen using clarithromycin and rifamycin and obtained the following findings. First, the administration of ATP in combination with the clarithromycin and rifamycin regimen accelerated bacterial elimination in MAC-infected mice without causing changes in the histopathological features or the mRNA expression of pro- or anti-inflammatory cytokines from those in the mice not given ATP. Second, ATP potentiated the anti-MAC bactericidal activity of macrophages cultivated in the presence of clarithromycin and rifamycin. This effect of ATP was closely related to intracellular Ca2+ mobilization and was specifically blocked by a cytosolic phospholipase A2 (cPLA2) inhibitor, arachidonyl trifluoromethylketone. Third, intramacrophage translocation of membranous arachidonic acid molecules to MAC-containing phagosomes was also specifically blocked by arachidonyl trifluoromethylketone. In the confocal microscopic observation of MAC-infected macrophages, ATP enhanced the intracellular translocation of cPLA2 into MAC-containing phagosomes. These findings suggest that ATP increases the host anti-MAC resistance by potentiating the antimycobacterial activity of host macrophages and that the cPLA2-dependent generation of arachidonic acid from the phagosomal membrane is essential for such a phenomenon.

Mycobacterium avium binds to mouse intestinal mucus aldolase.

SETTING: Mycobacterium avium complex (MAC) is known to colonize the gastrointestinal tract of human immunodeficiency virus (HIV) infected patients before causing bacteremia and disseminated disease. However, the mechanism involved in the gastrointestinal colonization is not known. OBJECTIVE: To identify putative intestinal mucus receptors which serve as anchor for MAC colonization. DESIGN: C57BL/6 mouse intestinal mucus was subjected to single and two-dimensional electrophoresis and blotted on nitrocellulose membranes. MAC specific mucus proteins were identified by probing the mucus western blots with biotinylated proteins derived from M.avium strain 101 (MAC101). RESULTS: Biotinylated MAC 101 proteins recognized a 39 kDa intestinal mucus glycoprotein. The protein displaying an isoelectric point (pI) of 9.0, was found to be periodate sensitive but resistant to sialidase, heparinase I and chondroitinase ABC. The internal amino acid sequence of the 39 kDa protein displayed homology with fructose-1-6-bisphosphate aldolase B (aldolase). The proclivity between MAC adhesins and aldolase was confirmed by probing rabbit muscle aldolase with MAC proteins. Furthermore, both 25 and 31 kDa MAC adhesins, superoxide dismutase and heparin binding protein, respectively, were found to bind to aldolase. CONCLUSIONS: MAC binds to intestinal mucus aldolase, conceivably facilitating intestinal colonization of the organism.