关键词: 分枝杆菌,结核, 胆固醇, 营养需要, 能量摄取, 免疫蛋白质类

Abstract:

Objective: To investigate whether Mce4C protein is involved in the uptake and utilization of cholesterol by Mycobacterium tuberculosis (MTB). Methods: The impact of different cholesterol concentrations (cholesterol-free group, 0.01% cholesterol group, and 0.1% cholesterol group) on the expression of MTB mce4C gene was analyzed by quantitative polymerase chain reaction. Growth curves plotted by measuring A₆₀₀ value of cultured bacterial solutions with an ultraviolet-visible spectrophotometer and supplemented by scanning electron microscopy, were used to analyze the differences in growth rate and morphology among the wild-type MTB H37Rv reference strain (WT), mce4C knockout strain (Δmce4C), and its complemented strain (Δmce4C+mce4C). To determine whether Mce4C was involved in the uptake of cholesterol by MTB, the changes of cholesterol levels in bacteria, bacterial medium and lysates of infected cells were measured either by using testing kits to quantitatively measure cholesterol content or by determining the fluorescence value of NBD-cholesterol. The subcellular localization of the Mce4C protein was determined through immunoblotting experiments on separation and combination of different MTB components. Results: The expression level of mce4C gene increased with elevation of cholesterol concentration in Sauton medium. After one week’s culture, the mce4C relative expression levels were 1.000±0.588, 1.390±0.162, and 3.622±1.031 respectively for cholesterol-free group, 0.01% cholesterol group, and 0.1% cholesterol group, with statistically significant difference (F=28.200, P=0.001). As the culture time in cholesterol-containing Sauton medium extended (20, 40, 60, 70, 80 days), the decrease of A₆₀₀ value of Δmce4C became increasingly more pronounced compared to WT and Δmce4C+mce4C. By the 80th day, the A₆₀₀ value of Δmce4C (0.913±0.017) was significantly lower than that of WT (1.245±0.011) and Δmce4C+mce4C (1.246±0.029), with all differences being statistically significant (t=28.182, P<0.001; t=17.140, P<0.001). When the three strains were cultured in cholesterol-containing Sauton medium, the cholesterol content in Δmce4C bacteria was lower than that in WT and Δmce4C+mce4C, while the cholesterol content in its culture supernatant was higher. By the 21st day, the total cholesterol content in Δmce4C bacteria ((1.058±0.012) μg/ml) was significantly lower than that in WT ((1.347±0.087) μg/ml) and Δmce4C+mce4C ((1.505±0.021) μg/ml), and the cholesterol content in the culture supernatant ((16.371±0.753) μg/ml) was significantly higher than that in WT ((7.740±0.422) μg/ml) and Δmce4C+mce4C ((7.274±0.131) μg/ml). All these differences were statistically significant (t=4.621, P=0.044; t=25.679, P=0.002; t=-14.135, P=0.005; t=-16.827, P=0.004). At different time points (4, 24, 48, 72 h) after infecting THP-1 cells, the cholesterol content in the lysates of cells infected with Δmce4C was significantly higher than that in cells infected with WT and Δmce4C+mce4C. Even at the lowest point at 4 h post-infection, the cholesterol content in the lysates of cells infected with Δmce4C ((7.749±0.017) μg/ml) was higher than that in cells infected with WT ((7.180±0.173) μg/ml) and Δmce4C+mce4C ((6.725±0.288) μg/ml), and the differences were statistically significant (t=-6.556, P=0.001; t=-7.106, P<0.001). Conclusion: The expression of mce4C gene increases with increasing cholesterol concentration in the culture medium. Knockout of mce4C gene reduces the growth rate of MTB in cholesterol-containing Sauton medium and the cholesterol content of MTB bacteria, increases the cholesterol content of the culture medium and lysates of infected cells, indicating that Mce4c is involved in the uptake and utilization of cholesterol by MTB from the external environment. Deletion of its coding gene can cause MTB to grow significantly defectively when cholesterol is the only carbon source, which may be an important factor for MTB to survive in human body for long time and be pathogenic.

Key words: Mycobacterium tuberculosis, Cholesterol, Nutritional requirements, Energy intake, Immunoproteins