Growth within macrophages increases the efficiency of Mycobacterium avium in invading other macrophages by a complement receptor-independent pathway

Abstract

Infections caused by organisms of the Mycobacterium avium complex occur in approximately 50 to 60% of patients with AIDS. M. avium is an intracellular pathogen that survives and multiplies within mononuclear phagocytes. In this study, we investigated the uptake of M. avium grown within macrophages (intracellular growth M. avium [IG]) by a second macrophage compared with M. avium cultured in broth (extracellular growth M. avium [EG]). The results showed that IG was six-to eightfold more efficient than EG in entering macrophages. In addition, while an anti-CR3 antibody was able to inhibit approximately 60% of EG uptake by macrophages, it failed to inhibit the entry of IG. In contrast to EG, IG uptake into macrophages was significantly inhibited in the presence of anti-␤1-integrin and anti-transferrin receptor antibodies. Entry into macrophages by alternate receptors was associated with resistance to tumor necrosis factor alpha (TNF-␣) stimulation. While stimulation with TNF-␣ resulted in inhibition of the growth of EG, it was not associated with inhibition of intracellular growth of IG. Investigation of the reason why M. avium is able to sense the changes in the intracellular environment triggering a change to the invasive phenotype suggests a direct relationship with macrophage apoptosis. These results suggest that intracellular growth is associated with novel mechanisms of M. avium uptake of macrophages and that those mechanisms appear to offer advantages to the bacteria in escaping the host defense.

Intracellular phenotype of Mycobacterium avium enters macrophages primarily by a macropinocytosis‐like mechanism and survives in a compartment that differs from that with extracellular phenotype

“…avium strain 101, isolated from a patient with AIDS, and Mycobacterium smegmatis mc 2 155, kindly provided by Dr. William Jacobs Jr., were cultured onto 7H10 Middlebrook agar, as previously reported (Bermudez et al, 1997(Bermudez et al, , 1999. EG and IG phenotypes were obtained as described (Bermudez et al, 1997). Briefly, IG bacteria were isolated after 3 days of infection of human monocyte-derived macrophages lysed in sterile water, as described (Bermudez et al, 1997).…”

“…EG and IG phenotypes were obtained as described (Bermudez et al, 1997). Briefly, IG bacteria were isolated after 3 days of infection of human monocyte-derived macrophages lysed in sterile water, as described (Bermudez et al, 1997). The lysate was differentially centrifuged, as described (Bermudez et al, 1997), to separate macrophage debris from bacteria, and was resuspended in Hanks balanced salt solution (HBSS).…”

“…Human monocyte-derived macrophages were purified from blood, as described (Bermudez et al, 1997), and allowed to differentiate into macrophages for 4 days prior to infection (Bermudez et al, 1997). The monolayers were monitored for cell number and viability, as described (Bermudez et al, 1997).…”

“…Human monocyte-derived macrophages were purified from blood, as described (Bermudez et al, 1997), and allowed to differentiate into macrophages for 4 days prior to infection (Bermudez et al, 1997). The monolayers were monitored for cell number and viability, as described (Bermudez et al, 1997). Macrophages cultured in RPMI-1640 supplemented with 10% endotoxinfree fetal bovine serum (FBS) (Sigma Chemicals, MO, USA) were incubated with 1!10 5 or 1!10 6 M. avium IG or EG (ratio 1:1 and 1:10 of macrophage to bacterium, respectively) in the presence or absence of Texas Red-labeled dextran (dextran 70,000, Molecular Probes Inc., OR, USA) at 37 (C and 5% CO 2 .…”

“…Then, intracellular bacteria multiply and subsequently the infection spreads to other cells. It has been shown that M. avium, after leaving macrophages (intracellular growth, IG bacterium), has a phenotype different from that of M. avium grown in laboratory media (extracellular growth, EG bacterium) and is capable of invading other, uninfected macrophages with an 8-to 10-fold increase in efficiency compared to the EG phenotype (Bermudez et al, 1997). In addition, internalization of the IG phenotype by macrophages, in contrast to the uptake of the EG phenotype, is complement receptor-independent, as previously shown in in vitro (Bermudez et al, 1997) and in vivo (Bermudez et al, 1999) studies.…”