Microaerophilic induction of the alpha-crystallin chaperone protein homologue (hspX) mRNA of Mycobacterium tuberculosis

Abstract

Among the products that are expressed when Mycobacterium tuberculosis undergoes hypoxic shiftdown to nonreplicating persistence (NRP) is the alpha-crystallin chaperone protein homologue (Acr). This expression coincides with the previously reported appearance of a respiratory type of nitrate reductase activity, the increase in glycine dehydrogenase activity, and the production of a unique antigen, URB-1. In a timed sampling study, using a slowly stirred oxygen depletion culture model, we have demonstrated that the hspX mRNA that codes for Acr protein as well as the protein itself is induced just as the bacilli enter the microaerophilic NRP stage 1 (NRP-1). In contrast to the induction observed for hspX mRNA, levels of 16S rRNA, fbpB mRNA (encoding the 85B alpha antigen), and aroB mRNA (encoding dehydroquinate synthase) demonstrate relatively small to no change upon entering NRP-1. Acr protein was shown to be identical to URB-1 by Western analysis with anti-URB-1 antibody. The fact that antibody to Acr is found in a high percentage of tuberculosis patients suggests that the hypoxic shiftdown of tubercle bacilli to the NRP state that occurs in vitro, resulting in production of the alpha-crystallin protein, occurs in vivo as well. Simultaneous abrupt increases in hspX mRNA and Acr protein suggest that Acr protein expression is controlled at the level of transcription.

FIG. 1

Optical absorbance (A₅₈₀) and DNA content (genomes per milliliter) of cultures of M. tuberculosis H37Rv in the slowly stirred 0.5 HSR model. Aerobic growth occurs for approximately 60 h, after which the bacilli shift down to the microaerophilic NRP-1 stage until oxygen is completely depleted, when the culture is about 160 h old and bacilli shift down to the anaerobic NRP-2 stage. DNA synthesis stops abruptly as the bacilli enter NRP-1. This figure represents one of two independent experiments that gave comparable results. Symbols: A₅₈₀, solid circles; DNA, solid squares.

FIG. 2

Amounts of selected RNA species in cultures of M. tuberculosis H37Rv in the slowly stirred 0.5 HSR model. (A) RNA expressed as number of molecules per genome. (B) mRNA expressed as number of molecules (×10,000) per molecule of 16S rRNA (mRNA/rRNA). The ×10,000 factor was used to reflect the approximate numbers of transcripts per cell equivalent.

FIG. 3

Protein analysis of M. tuberculosis. (A) SDS-PAGE analysis of protein extracts from M. tuberculosis stained with Coomassie blue. Cultures were incubated for the indicated times in slowly stirred sealed tubes. AG indicates shaking aerobic incubation for 80 h. The alpha-crystallin homologue is indicated. (B) Immunoblot analysis of the Acr protein. Proteins from either aerobic (AG) or 141-h-old hypoxic cultures were probed with either monoclonal anti-Acr (CS49) or polyclonal anti-URB-1 (RS88-13078) antibody.