The two-component regulatory system mtrAB is required for morphotypic multidrug resistance in Mycobacterium avium

Abstract

Clinical isolates of the opportunistic pathogen Mycobacterium avium complex (MAC) undergo a reversible switch between red and white colony morphotypes on agar plates containing the lipoprotein stain Congo red. Compared to their isogenic red counterparts, white morphotypic variants are more virulent and more resistant to multiple antibiotics. This report shows that the two-component regulatory system mtrAB is required for the red-to-white switch as well as for other morphotypic switches of MAC. A mutant with a transposon insertion in the histidine protein kinase gene mtrB was isolated from a morphotypically white parent clone. The mutant resembled a naturally occurring red morphotypic variant in that it stained with Congo red, was sensitive to multiple antibiotics, and was permeable by a fluorescent DNA stain. However, it differed from a red variant in that it could not switch to the white or transparent morphotype, and it could not survive intracellularly within macrophage-like cells. Transcomplementation with a cloned wild-type mtrB gene restored to the mutant the ability to form impermeable, drug-resistant white and transparent variants. Quantitative reverse transcriptase PCR showed that mtrB was required for the normal expression of cell surface Mce proteins, some of which are up-regulated in the red-to-white switch. The results indicate that mtrAB functions in regulating the composition and permeability of mycobacterial cell walls and plays a role in the reversible colony type switches of MAC.

FIG. 1.

Map of the mtrAB region in the genome of M. avium subsp. avium strain 104. M. avium subsp. avium ORFs are numbered according to a recent annotation of the draft sequence (28). For reference, orthologous ORF numbers in the finished genome sequence of M. avium subsp. paratuberculosis strain K10 are also shown. The positions of the transposon insertion in mutant WR8.1409 and the cloned region are shown.

FIG. 2.

Survival of WO, RO, and mtrB mutant clones within THP1 macrophage-like cells. The parent strain HMC02-WO (diamonds), a naturally occurring RO isolate from the same strain (squares), and the mtrB mutant WR8.1409 (triangles) were inoculated into THP1 cells and their intracellular survival was tracked as described in Materials and Methods. All data points are the means and standard deviations of triplicate measurements. All data points after 24 h are statistically different from each other (P < 0.01); however, the WO and RO curves have similar slopes and therefore may not represent truly different phenotypes under these conditions.

FIG. 3.

Permeation by SYTO16. Bacteria were exposed to SYTO16, and staining was quantified as described in Materials and Methods. Results obtained with the parent strain HMC02-WO, a naturally occurring RO isolate from the same strain, the mtrB mutant 8.1409, and a back-complemented clone of mutant 8.1409 are shown. (A) Images of typical fields under visible and fluorescence microscopy. (B) Percent labeling quantified and normalized to the WO control in each of two independent experiments. Values are means and standard deviations of three measurements made within each experiment.

FIG. 4.

Resistance to azithromycin in Etests. The mtrB mutant WR8.1409 (left), a vector-only control (center), and a clone carrying the wild-type mtrAB insert (right) were challenged with azithromycin Etest strips. A second clone of WR8.1409 (pMH416::mtrAB) yielded similar results, as did both clones when challenged with ciprofloxacin Etest strips.

FIG. 5.

Spontaneous WO colonies formed by the back-complemented mtrB mutant. The white colonies formed by the mutant carrying pMH416::mtrAB are visible in the plate at left. Over the course of multiple transfers on the same medium, the original mutant (right) and the vector-only control (not shown) never formed such colonies.

FIG. 6.

Down-regulation of mce mRNA in RO and mtrB mutant cells relative to WO cells. Q-RT-PCR analysis was conducted as described in the text. HMC02-WO cells were grown and harvested under the same conditions as HMC02-RO cells (A) and the mtrB mutant (B). Values are down-regulation (n-fold) of each locus relative to HMC02-WO cells. Each data point is the mean and standard deviation of triplicate Q-RT-PCRs. For each comparison, RNA extracts were diluted 1:1, 1:10, and 1:100, and the dilution that yielded the smallest standard deviation is shown.