Construction and characterization of a Mycobacterium tuberculosis mutant lacking the alternate sigma factor gene, sigF

Abstract

The alternate RNA polymerase sigma factor gene, sigF, which is expressed in stationary phase and under stress conditions in vitro, has been deleted in the virulent CDC1551 strain of Mycobacterium tuberculosis. The growth rate of the DeltasigF mutant was identical to that of the isogenic wild-type strain in exponential phase, although in stationary phase the mutant achieved a higher density than the wild type. The mutant showed increased susceptibility to rifampin and rifapentine. Additionally, the DeltasigF mutant displayed diminished uptake of chenodeoxycholate, and this effect was reversed by complementation with a wild-type sigF gene. No differences in short-term intracellular growth between mutant and wild-type organisms within human monocytes were observed. Similarly, the organisms did not differ in their susceptibilities to lymphocyte-mediated inhibition of intracellular growth. However, mice infected with the DeltasigF mutant showed a median time to death of 246 days compared with 161 days for wild-type strain-infected animals (P < 0.001). These data indicate that M. tuberculosis sigF is a nonessential alternate sigma factor both in axenic culture and for survival in macrophages in vitro. While the DeltasigF mutant produces a lethal infection of mice, it is less virulent than its wild-type counterpart by time-to-death analysis.

FIG. 1

(A) Cartoon representation of the strategy used to interrupt the sigF gene using pPC47. The location and spacing of BamHI sites are shown. (B) BamHI-restricted chromosomal DNA from the recombinant M. tuberculosis strains was analyzed by Southern blotting using the four probes shown in panel A. The sizes of the hybridizing bands are indicated at the left margin.

FIG. 2

In vitro growth rates of M. tuberculosis CDC1551 (wild type) and the isogenic ΔsigF mutant agitated at 37°C in Middlebrook 7H9 broth supplemented with glycerol, 10% ADC, and Tween 80 (19). Each 100-ml culture was started by inoculation with 1 ml of a declumped suspension from freshly grown colonies. (Main panel) Results of a 22-day growth curve in which aliquots were sampled every 1 to 2 days and the bacterial density was determined by plate counts; (inset) display of the same data for the first 5 days plotted on an expanded scale to show differences in the lag phase between the strains. This experiment was performed twice by both plating dilutions and optical density determinations, each producing similar results in lag and stationary phases.

FIG. 3

[¹⁴C]chenodeoxycholate uptake by wild-type (WT) M. tuberculosis, the ΔsigF mutant, and the complemented mutant. Measured values for counts per minute taken up were normalized to the dry weight of bacterial pellets. Each value represents the mean of at least three determinations. Standard deviations were less than 5%. KO, knockout.

FIG. 4

Intracellular survival of M. tuberculosis CDC1551 (wild type [wt]) and the isogenic ΔsigF mutant within monocytes (MN) or within MN plus PBL (nonadherent cells [NAC]) from four unrelated, tuberculin-positive, healthy human subjects. Each patient's cells were tested in triplicate under each of the conditions. Data points represent the surviving mycobacterial CFU per 10⁶ MN plus 1 standard error.

FIG. 5

Characteristics of infection by M. tuberculosis CDC1551 (wild type) versus the M. tuberculosis ΔsigF mutant in mice. Results are from a long-term infection model using 6- to 8-week-old BALB/c mice infected with wild-type (n = 12) and mutant (n = 11) M. tuberculosis, respectively. (A) Individual weights were determined biweekly and are plotted as mean weight per group ± 1 standard error. Asterisks, times at which the weight differences achieved statistical significance. (B) Survival data are shown as a Kaplan-Meier plot. The median time to death was 161 days for wild-type infection and 246 days for infection with the ΔsigF mutant.