Mycobacterium tuberculosis cells growing in macrophages are filamentous and deficient in FtsZ rings

Abstract

FtsZ, a bacterial homolog of tubulin, forms a structural element called the FtsZ ring (Z ring) at the predivisional midcell site and sets up a scaffold for the assembly of other cell division proteins. The genetic aspects of FtsZ-catalyzed cell division and its assembly dynamics in Mycobacterium tuberculosis are unknown. Here, with an M. tuberculosis strain containing FtsZ(TB) tagged with green fluorescent protein as the sole source of FtsZ, we examined FtsZ structures under various growth conditions. We found that midcell Z rings are present in approximately 11% of actively growing cells, suggesting that the low frequency of Z rings is reflective of their slow growth rate. Next, we showed that SRI-3072, a reported FtsZ(TB) inhibitor, disrupted Z-ring assembly and inhibited cell division and growth of M. tuberculosis. We also showed that M. tuberculosis cells grown in macrophages are filamentous and that only a small fraction had midcell Z rings. The majority of filamentous cells contained nonring, spiral-like FtsZ structures along their entire length. The levels of FtsZ in bacteria grown in macrophages or in broth were comparable, suggesting that Z-ring formation at midcell sites was compromised during intracellular growth. Our results suggest that the intraphagosomal milieu alters the expression of M. tuberculosis genes affecting Z-ring formation and thereby cell division.

FIG. 1.

Schematic for construction of M. tuberculosis 41. Plasmids are described in Table 1, and details for creating M. tuberculosis 41 are described in the text. Gray box, ftsZ_TB coding region; black box, deleted region in ftsZ_TB; white box, 5′ and 3′ flanking regions of ftsZ_TB. SCO, single-crossover; Mtb, M. tuberculosis.

FIG. 2.

M. tuberculosis ftsZ gene can be replaced with ftsZ-gfp. (A and B) Southern hybridization profiles of M. tuberculosis 41 and wild-type (WT) M. tuberculosis DNAs. Wild-type M. tuberculosis or M. tuberculosis 41 genomic DNA was digested with NotI, electrophoretically resolved on agarose gels, transferred to nitrocellulose membranes, and probed with a ³²P-labeled ftsZ (A) or gfp probe (B). NotI-digested pJFR41 plasmid DNA was used as a positive control. Lanes: 1, pJFR41; 2, wild-type M. tuberculosis; 3, M. tuberculosis 41. Bands corresponding to a chromosomal copy of ftsZ (wild-type copy), an integrated copy of ftsZ-gfp (Integ.copy), and a mutant copy are indicated. Only the wild-type copy of ftsZ can be seen in M. tuberculosis. The arrowhead indicates the position of the ftsZ-gfp integrated copy. (C and D) Verification of M. tuberculosis 41 by immunoblotting. One microgram of total cell lysate each from wild-type M. tuberculosis or M. tuberculosis 41 was resolved on a 12% SDS-PA gel, transferred to nitrocellulose membrane, and probed with either anti-FtsZ (C) or anti-GFP (D) specific antibodies. Positions of FtsZ and FtsZ-GFP are marked. Lanes: M, markers; 1, M. tuberculosis lysate; 2, M. tuberculosis 41 lysate.

FIG. 3.

M. tuberculosis 41 needs acetamide for growth. (A) Viability of M. tuberculosis 41. Actively growing cultures of wild-type (WT) M. tuberculosis or M. tuberculosis 41 were plated on 7H10 Middlebrook agar plates with or without 0.2% acetamide. Colony counts obtained after 3 weeks of incubation at 37°C are shown. Means and standard errors from three separate experiments are shown. (B) Growth of M. tuberculosis 41 in the presence or absence of acetamide. Exponentially growing cultures of M. tuberculosis 41 were washed two times with medium lacking acetamide, followed by growth in medium with (squares) or without (triangles) acetamide (acet.). For comparison, wild-type M. tuberculosis H37Ra was also grown (circles). Cultures were grown with shaking at 37°C, and their optical density at 600 nm (O.D. 600) was measured at the indicated times. Mtb, M. tuberculosis.

FIG. 4.

Microscopy of M. tuberculosis 41. Actively growing cultures of M. tuberculosis 41 grown with 0.2% acetamide were examined by fluorescence (a and c) and bright-field (b and d) microscopy. Images were selected to show the shape, size, and FtsZ structures of as many cells as possible and therefore do not reflect the actual frequency of the various FtsZ structures seen (Table 3). Arrowheads and arrows indicate midcell FtsZ-GFP rings and polar FtsZ-GFP localization, respectively.

FIG. 5.

SRI-3072 inhibits cell division and growth of M. tuberculosis 41. (A) Effect of SRI-3072 on growth of M. tuberculosis 41. Exponentially growing cultures of M. tuberculosis 41 were diluted to an optical density at 600 nm [OD (600 nm)] of 0.2 and grown in the presence or absence of 0.56 μM SRI-3072. The culture optical density at 600 nm was measured for up to 6 days and plotted. (B) Z-ring formation is inhibited by SRI-3072. M. tuberculosis 41 was grown in the presence of acetamide and 0.56 μM SRI-3072 for various periods of time and examined by fluorescence (a, c, e, and g) and bright-field (b, d, f, and h) microscopy. Images were captured, analyzed, and processed as described in Materials and Methods. Parts: a and b, no treatment; c and d, 24 h; e and f, 48 h; g and h, 120 h. (C) SRI-3072 inhibits cell division. Cell length measurements were made for untreated (M. tuberculosis wild type [WT], M. tuberculosis 41) and SRI-3072-treated M. tuberculosis 41 cells (M. tuberculosis 41/D1, M. tuberculosis 41/D2, and M. tuberculosis 41/D5). D1, D2, and D5 indicate 24, 48, and 120 h of treatment. At least 100 cells for each time point were measured with the Metamorph 6.2 software. Mtb, M. tuberculosis.

FIG. 6.

Growth of M. tuberculosis in macrophages leads to filamentation. Wild-type M. tuberculosis or M. tuberculosis 41 was used to infect monolayers of gamma interferon-activated THP-1 macrophages at a multiplicity of infection of 1:10. After 3 h of incubation, unattached bacteria were washed off and macrophages were cultured for 72 h. Macrophages were then lysed and bacteria collected by centrifugation and examined by fluorescence and bright-field microscopy. (A) Macrophage-grown wild-type M. tuberculosis. Bright-field images of broth (i)- and macrophage (ii)-grown M. tuberculosis are shown. (B) Lengths of intracellular M. tuberculosis cells. Cell length measurements were made for broth-grown (RV Broth) and intracellular wild-type M. tuberculosis after 3 days (Rv.D3) of growth in THP-I cells. (C) Broth- and macrophage-grown M. tuberculosis 41. Fluorescence (i and iii) and bright-field (ii and iv) images of broth (i and ii)- and macrophage (iii and iv)-grown M. tuberculosis 41 are shown. Arrowheads indicate either bud-like structures or Z rings (Z). (D) Macrophage-grown M. tuberculosis cells show non-midcell localization of FtsZ. Fluorescence images of macrophage-grown M. tuberculosis 41 bacteria were manipulated with the FFT processing function of the Metamorph 6.2 software (see Materials and Methods). This revealed the presence of almost spiral-like structures of FtsZ-GFP along the length of the cells (arrows in parts iii and vi). Parts i and iv and parts ii and v are respective bright-field and fluorescence images. Images in parts iii and vi are FFT processed. Images in panel D are slightly enlarged to show the FtsZ-GFP structures more clearly.

FIG. 7.

FtsZ levels in macrophage-grown M. tuberculosis. Levels of FtsZ_TB and FtsZ_TB-GFP in broth- and macrophage-grown bacteria were determined by immunoblotting. Cellular lysates of broth- and macrophage-grown bacteria were prepared as described in Materials and Methods. Bacterial pellet was lysed by bead beating, resolved by SDS-PA gel electrophoresis, transferred to nitrocellulose membranes, probed with anti-FtsZ_TB and monoclonal anti-E. coli sigma 70 antibodies, and processed as previously described, with the ECF Western blotting kit from Amersham (12). In some cases, macrophages containing bacteria were pelleted and directly lysed by beat beating (lane 5) and processed as described above. Lanes: 1, recombinant FtsZ_TB protein; 2, lysate from broth-grown M. tuberculosis 41; 3, lysate from macrophage-isolated M. tuberculosis 41; 4, lysate from broth-grown wild-type M. tuberculosis; 5, lysate from macrophage-grown wild-type M. tuberculosis. Note that lysates in lane 5 were obtained by bead beating macrophages containing wild-type M. tuberculosis, whereas for lane 3, bacteria were first recovered from macrophages by gentle lysis and then cellular lysates were prepared by bead beating. Although the former approach (lane 5) resulted in a slightly higher background level compared to the one in lane 3, the ratio of SigA to FtsZ was unaffected. Positions of SigA, FtsZ_TB, and FtsZ_TB-GFP are marked.