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. 2008 Jun;190(11):3955-61.
doi: 10.1128/JB.01476-07. Epub 2008 Apr 4.

Glycine betaine uptake by the ProXVWZ ABC transporter contributes to the ability of Mycobacterium tuberculosis to initiate growth in human macrophages

Christopher T D Price  1 , Archana Bukka, Michael Cynamon, James E Graham
Affiliations

Glycine betaine uptake by the ProXVWZ ABC transporter contributes to the ability of Mycobacterium tuberculosis to initiate growth in human macrophages

Christopher T D Price et al. J Bacteriol. 2008 Jun.

Abstract

Mycobacterium tuberculosis maintains a large genetic capacity necessary for growth in different environments during infection and survival upon aerosol transmission to new hosts. Screening for bacterial RNAs produced in response to host interactions produced candidate lists where we noted proXVWZ, annotated as encoding a putative glycine betaine or proline transporter. As high surface-to-volume ratios make bacterial cells particularly vulnerable to changes in water availability, we investigated the contributions of this transporter to the ability of M. tuberculosis to colonize macrophages. An H37Rv proXVWZ mutant was impaired for initial survival and intracellular growth and exhibited reduced growth at elevated medium osmolarity. This defect could be complemented by restoring proXVWZ and was attributable to a failure to accumulate the compatible solute glycine betaine. We then demonstrated that ProXVWZ allows M. tuberculosis to obtain betaine from host macrophages and thereby contributes to early steps in colonizing this niche.

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Figures

FIG. 1.
FIG. 1.
proV mRNA levels in intracellular M. tuberculosis. THP-1 cells (A) or human primary macrophages (B to D) containing M. tuberculosis H37Rv were lysed at various time points postinfection to determine steady-state transcript levels relative to those in bacteria at mid-logarithmic phase in broth culture. Levels were normalized relative to a sigA mRNA internal standard as previously described (21, 37) and reported as determined by the method of reference , as described in the text (P < 0.05).
FIG. 2.
FIG. 2.
Accumulation of betaine in response to osmotic stress. Closed squares indicate [methyl-14C]betaine accumulation in H37Rv, open squares indicate accumulation by H37Rv in medium with 0.25 M NaCl, closed circles indicate accumulation by H37RvΔproXVWZ, and open circles indicate accumulation by H37RvΔproXVWZ in medium with 0.25 M NaCl. Data points are the means of three individual samples, and error bars indicate standard deviations.
FIG. 3.
FIG. 3.
Reduced osmotolerance in an M. tuberculosis H37Rv ΔproXVWZ mutant. Bacteria were inoculated into M7H9 medium either with or without supplemental 0.25 M NaCl. Closed squares indicate OD600 values for H37Rv in M7H9, open squares for H37Rv in M7H9 plus 0.25 M NaCl, closed circles for H37RvΔproXVWZ in M7H9, and open circles for H37RvΔproXVWZ in M7H9 plus 0.25 M NaCl. Results for a representative experiment are shown.
FIG. 4.
FIG. 4.
Intracellular growth of M. tuberculosis ΔproXVWZ and complemented strains. THP-1 monolayers were infected with H37Rv, H37RvΔproXVWZ, or the complemented mutant at a final MOI of 1:50 as described in the text. Macrophage monolayers in wells were lysed every 24 h for 5 days, and lysates were plated for CFU determination. Squares are for H37Rv, circles for H37RvΔproXVWZ, and triangles for the complemented strain. Time points indicate the mean numbers of CFU for three individual wells, and error bars indicate standard deviations. Results for three independent experiments are shown. Reduced survival of the mutant relative to that of the isogenic parent in all experiments was significant (P < 0.005) at 48 h as determined by an unpaired t test.
FIG. 5.
FIG. 5.
Accumulation of host betaine by intracellular M. tuberculosis. Acquisition of betaine by bacteria at 24 h postinfection was determined as described in Materials and Methods. Results for two representative experiments are shown, and each data point is the mean of three individual samples, with error bars indicating standard deviations.
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