Hypervirulence of a rough variant of the Mycobacterium abscessus type strain

Abstract

We isolated a rough variant of Mycobacterium abscessus CIP 104536T during experimental infection of mice. We show that this variant has lost the ability to produce glycopeptidolipids, is hyperlethal for C57BL/6 mice infected intravenously, and induces a strong tumor necrosis factor-alpha response by murine monocyte-derived macrophages.

FIG. 1.

Lack of GPLs in the rough variants of M. abscessus. (A) Colonial morphotypes of CIP 104536T (right) and CIP 104536T-R (left) after 4 days of incubation at 37°C on Trypticase soy agar; (B) thin-layer chromatography analysis of the crude lipid extracts of CIP 104536T, CIP 104536T-R, 390S, and 390R. Sugar-containing lipids were visualized by anthrone staining. The positions of the GPLs are indicated on the right. Abbreviations: E, exponential phase; S, stationary phase; T, CIP 104536T; T-R, CIP 104536T-R.

FIG. 2.

Hyperlethality of R variants. Survival analysis was done with C57/BL6 mice injected i.v. with 10⁷ CFU per animal (12 mice per group).

FIG. 3.

Increased TNF-α release by BMMs infected with R variants. BMMs were infected for 1 h with S and R strains at a multiplicity of infection of 5:1, washed twice, and further incubated for 24 h in fresh medium containing 40 mg/liter of amikacin. TNF-α was measured in culture supernatants at 12 and 24 h postinfection. CIP 104536T-R versus CIP 104536T at 12 and 24 h, P = 0.014 and 0.017, respectively. 390R versus 390S at 12 and 24 h, P = 0.041 and 0.0043, respectively.

FIG. 4.

TNF-α release by BMMs infected with R variants is unaffected by GPL supplementation. CIP 104536T-R and CIP 104536T were incubated for 1 h with low (1-fold) or high (100-fold) concentrations of “native” or “deacetylated” GPLs purified from CIP 104536T prior to infection. Infection of BMMs was carried out as described in the legend to Fig. 3, and TNF-α was measured in culture supernatants at 12 (open bars) and 24 (hatched bars) h postinfection. The addition of native or deacetylated GPL did not significantly alter TNF-α production.