Mycobacterium tuberculosis catalase and peroxidase activities and resistance to oxidative killing in human monocytes in vitro - PubMed (original) (raw)

Mycobacterium tuberculosis catalase and peroxidase activities and resistance to oxidative killing in human monocytes in vitro

C Manca et al. Infect Immun. 1999 Jan.

Abstract

Mycobacterium tuberculosis has a relatively high resistance to killing by hydrogen peroxide and organic peroxides. Resistance may be mediated by mycobacterial catalase-peroxidase (KatG) and possibly by alkyl hydroperoxide reductase (AhpC). To determine the interrelationship between sensitivity to H2O2, catalase and peroxidase activities, and bacillary growth rates measured both intracellularly in human monocytes and in culture medium, we examined one laboratory strain, two clinical isolates, and three recombinant strains of M. tuberculosis with differing levels of KatG and AhpC. Five of the mycobacterial strains had intracellular doubling times of 27 to 32 h, while one KatG-deficient clinical isolate (ATCC 35825) doubled in approximately 76 h. Killing of mycobacteria by exogenously added H2O2 was more pronounced for intracellular bacilli than for those bacilli derived from disrupted monocytes. Strains with no detectable KatG expression or catalase activity were relatively sensitive to killing (43 to 67% killing) by exogenous H2O2. However, once even minimal catalase activity was present, mycobacterial catalase activity over a 10-fold range (0.56 to 6.2 U/mg) was associated with survival of 85% of the bacilli. Peroxidase activity levels correlated significantly with resistance of the mycobacterial strains to H2O2-mediated killing. An endogenous oxidative burst induction by 4beta-phorbol 12beta-myristate 13alpha-acetate treatment of infected monocytes reduced the viability of the KatG null strain (H37Rv Inhr) but not the KatG-overexpressing strain [H37Rv(pMH59)]. These results suggest that mycobacterial resistance to oxidative metabolites (including H2O2 and other peroxides) may be an important mechanism of bacillary survival within the host phagocyte.

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Figures

FIG. 1

Effect of exogenous H2O2 on mycobacterial viability in cell-free Middlebrook 7H9 medium. H2O2 (at the concentrations shown) was added to growing bacteria, and the number of CFU was determined as described in Materials and Methods. Results are expressed as percent survival relative to baseline ± SEM and are from one representative experiment with six replicate cultures. The numbers of CFU used for the experiment (and expressed as 100% in the figure) were 4.7 × 105 for H37Rv, 3.2 × 105 for H37Rv Inhr, and 2.8 × 105 for H37Rv(pMH59).

FIG. 2

(A) Killing of mycobacteria following treatment with exogenous H2O2. Fresh human monocytes were infected with M. tuberculosis, and 10 mM H2O2 was added to intact monolayers. After 6 h of treatment, numbers of CFU were determined (intracellular killing) (closed bars). Alternatively, the infected monocytes were disrupted by sonication before 6 h of treatment with H2O2. Cultures were then harvested for the CFU assay (extracellular killing) (open bars). Results are expressed as percent of mycobacteria killed and are the mean of three to six experiments, each done in triplicate, plus 1 SEM. (B) Mycobacterial catalase and peroxidase activities. Mycobacteria in log phase were harvested and homogenized, and the enzymatic activities were determined as described in Materials and Methods. The enzymatic activities are expressed as units per milligram. Results are means of one representative experiment carried out in quadruplicate.

FIG. 3

PMA-induced killing of intracellular mycobacteria. Monocytes were infected with H37Rv Inhr (circles) or H37Rv(pMH59) (squares). Intracellular bacilli (solid lines) and cell free bacilli (broken lines) were treated with PMA at a final concentration of 100 ng/ml. The open symbols indicate controls not treated with PMA. Results are expressed as mean CFU ± standard deviation of three experiments, each done in triplicate. Data were analyzed with a paired t test to compare control and PMA-treated cells. Significantly different results for controls versus corresponding PMA-treated cells are indicated by ∗ (P = 0.02) and ∗∗ (P = 0.04).

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Mycobacterium tuberculosis catalase and peroxidase activities and resistance to oxidative killing in human monocytes in vitro - PubMed (original) (raw)