Deletion of the Mycobacterium tuberculosis resuscitation-promoting factor Rv1009 gene results in delayed reactivation from chronic tuberculosis

Abstract

Approximately one-third of the human population is latently infected with Mycobacterium tuberculosis, comprising a critical reservoir for disease reactivation. Despite the importance of latency in maintaining M. tuberculosis in the human population, little is known about the mycobacterial factors that regulate persistence and reactivation. Previous in vitro studies have implicated a family of five related M. tuberculosis proteins, called resuscitation promoting factors (Rpfs), in regulating mycobacterial growth. We studied the in vivo role of M. tuberculosis rpf genes in an established mouse model of M. tuberculosis persistence and reactivation. After an aerosol infection with the M. tuberculosis Erdman wild type (Erdman) or single-deletion rpf mutants to establish chronic infections in mice, reactivation was induced by administration of the nitric oxide (NO) synthase inhibitor aminoguanidine. Of the five rpf deletion mutants tested, one (deltaRv1009) exhibited a delayed reactivation phenotype, manifested by delayed postreactivation growth kinetics and prolonged median survival times among infected animals. Immunophenotypic analysis suggested differences in pulmonary B-cell responses between Erdman- and deltaRv1009-infected mice at advanced stages of reactivation. Analysis of rpf gene expression in the lungs of Erdman-infected mice revealed that relative expression of four of the five rpf-like genes was diminished at late times following reactivation, when bacterial numbers had increased substantially, suggesting that rpf gene expression may be regulated in a growth phase-dependent manner. To our knowledge, deltaRv1009 is the first M. tuberculosis mutant to have a specific defect in reactivation without accompanying growth defects in vitro or during acute infection in vivo.

FIG. 1.

The ΔRv1009 rpf mutant is reactivation deficient. (A) C57BL/6 mice were infected by aerosol as described in Materials and Methods with initial CFU of ∼400 to 900 (values for each strain are provided in Table 1), of M. tuberculosis Erdman wild type (black circles), ΔRv1009 (white triangles), ΔRv2389c (black diamonds), ΔRv2450c (white squares), ΔRv1884c (gray circles), or ΔRv0867c (gray triangles). At 16 weeks postinfection during the chronic persistent phase of infection, 2.5% AG was administered ad libitum in the drinking water. Mice were observed daily for signs of illness and sacrificed when death was imminent. Data represent results for 12 mice per group. (B) C57BL/6 mice were infected with ∼500 to 900 CFU of M. tuberculosis Erdman wild type (black circles) or ΔRv1009 (white triangles), and the mortality experiment was carried out as described for the data shown in panel A, with AG again administered at 16 weeks postinfection. Each group consisted of nine mice.

FIG. 2.

Complementation of the ΔRv1009 mutant restores reactivation kinetics toward the wild type. Mice were infected by aerosol with ∼250 to 450 CFU of M. tuberculosis Erdman wild type (black circles), ΔRv1009 (white triangles), ΔRv1009 attB::P_hsp60 Rv1009 (complemented strain with a single copy of the Rv1009 gene integrated at the attB site, gray squares; abbreviated ΔRv1009::Rv1009 in the key), or ΔRv1009 attB::P_hsp60 Rv1010 (complemented strain with a single copy of the Rv1010 gene integrated at the attB site, white circles; abbreviated ΔRv1009::Rv1010 in the key). AG at 2.5% was administered ad libitum in the drinking water at 18 weeks postinfection. Mice were observed daily for signs of illness and sacrificed when moribund. There were 12 mice per group for M. tuberculosis Erdman and 9 mice per group for the remaining strains.

FIG. 3.

ΔRv1009 also exhibits delayed kinetics of mortality following low-dose aerosol infection. C57BL/6 mice were infected by aerosol with ∼50 to 100 CFU of the M. tuberculosis Erdman wild type (black circles) or ΔRv1009 (white triangles) (actual CFU values are given in Table 1), reactivation was induced at 20 weeks postinfection by administration of AG, and mice were observed for mortality as described in the legend to Fig. 1. Each group consisted of 12 mice.

FIG. 4.

Organ bacterial burdens after infection with M. tuberculosis Erdman, ΔRv1009, or ΔRv1009 attB::P_hsp60 Rv1009-complemented strain. C57BL/6 mice were infected as described in the legend to Fig. 3 with ∼50 to 100 CFU of the indicated strains: Erdman wild type (black circles), ΔRv1009 (white triangles), or ΔRv1009 attB::P_hsp60 Rv1009 (abbreviated ΔRv1009::Rv1009 in the key; gray squares). AG at 2.5% was provided in the drinking water starting at 20 weeks postinfection (arrow). Organ homogenates were plated to determine bacterial numbers at various times postinfection, as described in Materials and Methods. Results are shown as means ± standard errors. Data represent results for three mice per time point, with the exception of M. tuberculosis Erdman at 4 weeks postinfection and at 11 weeks post-AG, with data for two mice, and ΔRv1009 at the final 19-week post-AG time point, in which the one remaining mouse was sacrificed.

FIG. 5.

Lung histopathology following AG-induced reactivation. C57BL/6 mice were infected by aerosol with ∼50 to 100 CFU of the M. tuberculosis Erdman wild type (A), the ΔRv1009 deletion mutant (B and D), or the ΔRv1009 attB::P_hsp60 Rv1009-complemented strain (C) and treated with AG at 20 weeks postinfection. Lungs were procured, formalin fixed, and paraffin embedded, and 6-μm sections were stained with hematoxylin and eosin as described in Materials and Methods. Sections A to C were obtained at 9 weeks after AG treatment was initiated, while section D was obtained at 11 weeks of AG treatment. All panels were acquired at 100× magnification. Arrows (black) indicate regions of significant necrosis.

FIG. 6.

B-cell populations in lungs of M. tuberculosis Erdman wild type- and ΔRv1009-infected mice following AG-induced reactivation. Single-cell suspensions of lung cells from mice infected with the M. tuberculosis Erdman wild type (top) or ΔRv1009 (bottom) were prepared after 9 weeks of AG treatment, and CD45⁺ cells were analyzed for the presence of the CD19 surface marker as described in Materials and Methods. The FACS analysis dot plots are shown, with each plot representing an individual mouse.

FIG. 7.

Relative expression of the M. tuberculosis rpf-like genes pre- and postreactivation. RNA was prepared from lung tissue harvested from M. tuberculosis Erdman-infected mice prereactivation (19 weeks postinfection; time zero) and at 4, 9, or 11 weeks after AG treatment and subjected to real-time RT-PCR as described in Materials and Methods. Shown are the ratios of expression at the time points indicated on the x axis versus expression in a “prereactivation” mouse arbitrarily set at a value of 1, with all rpf genes normalized to the amount of 16S cDNA in each sample. The relative expression levels of Rv0867c (rpfA; black circles), Rv1009 (rpfB; light-gray circles), Rv2389c (rpfD; dark-gray triangles), and Rv2450c (rpfE; light-gray triangles) are indicated for two mice per time point, except for 11 weeks post-AG, where data for one mouse per time point are shown. An exception is the Rv0867c gene, which was undetectable in one of the two prereactivation lung samples and in both 4-week post-AG samples but was detected in all 9- and 11-week post-AG samples. As noted in Results, Rv1884c was not included in the analysis, as it was not reliably detected in the prereactivation samples.