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Volume 157, Issue 4

Biochemical and immunological characterization of a knockout mutant of BCG Free

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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Keyword(s): , 2-DE, 2D gel electrophoresis , CE, cell extract , CF, culture filtrate , ICL, isocitrate lyase , IFN-γ, gamma interferon , Mtb, M. tuberculosis , OCEM, outermost cell envelope material and RLU, relative light units
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Biochemical and immunological characterization of a cpn60.1 knockout mutant of Mycobacterium bovis BCG
Microbiology 157, 1205 (2011); https://doi.org/10.1099/mic.0.045120-0
/content/journal/micro/10.1099/mic.0.045120-0
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