1887

Abstract

Pathogenic mycobacteria possess two homologous chaperones encoded by and . Cpn60.2 is essential for survival, providing the basic chaperone function, while Cpn60.1 is not. In the present study, we show that inactivation of the BCG () gene does not significantly affect bacterial growth in 7H9 broth, but that this knockout mutant (Δ) forms smaller colonies on solid 7H11 medium than the parental and complemented strains. When growing on Sauton medium, the Δ mutant exhibits a thinner surface pellicle and is associated with higher culture filtrate protein content and, coincidentally, with less protein in its outermost cell envelope in comparison with the parental and complemented strains. Interestingly, in this culture condition, the Δ mutant is devoid of phthiocerol dimycocerosates, and its mycolates are two carbon atoms longer than those of the wild-type, a phenotype that is fully reversed by complementation. In addition, Δ bacteria are more sensitive to stress induced by HO but not by SDS, high temperature or acidic pH. Taken together, these data indicate that the cell wall of the Δ mutant is impaired. Analysis by 2D gel electrophoresis and MS reveals the upregulation of a few proteins such as FadA2 and isocitrate lyase in the cell extract of the mutant, whereas more profound differences are found in the composition of the mycobacterial culture filtrate, e.g. the well-known Hsp65 chaperonin Cpn60.2 is particularly abundant and increases about 200-fold in the filtrate of the Δ mutant. In mice, the Δ mutant is less persistent in lungs and, to a lesser extent, in spleen, but it induces a comparable mycobacteria-specific gamma interferon production and protection against H37Rv challenge as do the parental and complemented BCG strains. Thus, by inactivating the gene in BCG we show that Cpn60.1 is necessary for the integrity of the bacterial cell wall, is involved in resistance to HO-induced stress but is not essential for its vaccine potential.

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2011-04-01
2019-12-11
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Description of the proteins whose amount was modified in culture filtrate of the BCG Δ mutant. Proteins of the three BCG strains identified by 2-DE/MS. Methods and table are available as a single PDF(263 KB).

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Growth of the WT BCG, Δ and Δ Comp strains in liquid 7H9-ADC Middlebrook medium. Growth of the WT BCG, Δ and Δ Comp strains on Sauton medium as a surface pellicle for 11 days. Bacterial replication of luminescent H37Rv in spleen and lungs of unvaccinated BALB.B10 mice or BALB.B10 mice vaccinated 3 months before with the WT BCG, Δ and Δ Comp strains, tested at 4 and 12 weeks after challenge by luminometry. All figures are available as a single PDF(146 KB)

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