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.Mycobacterium tuberculosis, the causative agent of tuberculosis, is an important infectious agent that globally causes more than three million new infections each year (8). Recent years have seen an increase in the number of M. tuberculosis strains that are resistant to one or more antituberculosis drugs, and this has highlighted the need for the development of a new generation of antimicrobial agents. One hallmark of the M. tuberculosis life cycle is that it exists in two metabolically distinct growth states: an active replicative state and a nonproliferative persistent state where the bacterium survives without any increase in the bacterial burden on the host. Physiological studies carried out by Wayne and colleagues indicate that M. tuberculosis cells in the hypoxiainduced nonreplicative persistent state are blocked at the cell division stage after completing DNA replication and undergo a round of cell division prior to initiation of a new round of DNA replication (40,41). This latter process is also referred to as reactivation. Development of antimycobacterial agents targeting the cell division process could potentially prevent the multiplication and subsequent proliferation of the pathogen in active, as well as reactivation, growth states.FtsZ, a bacterial homolog of tubulin, is a key player in cell division and is essential for initiation of this process (22, 32). FtsZ protein catalyzes the formation of distinct structures, referred to as FtsZ rings (Z rings), at the midcell site and sets up a scaffold for ordered assembly of other cell division proteins. The combined action of multiple cell division proteins results in septation (22,32)....

The pleiotropic transcriptional response of Mycobacterium tuberculosis to vitamin C is robust and overlaps with the bacterial response to multiple intracellular stresses

“…Full-length devS (1737 bp) and dosT (1722 bp) were amplified from Mtb H37Rv genomic DNA using primer pairs: devSNdeIF and devSXbaIR for devS amplification, and dosTNdeIF and dosTXbaIR for dosT amplification (Table S1, available in the online Supplementary Material). The amplified products were cloned in pJFR19, an Mtb integrative plasmid (Chauhan et al, 2006), downstream of the constitutive acetamidase promoter. The constructs were electroporated individually into the Mtb DdevSDdosT mutant strain to generate single-sensor kinase complements expressing DosT and DevS.…”

Characterization of CrgA, a New Partner of the Mycobacterium tuberculosis Peptidoglycan Polymerization Complexes

“…Cell lysates were prepared from exponentially grown cultures (20). Anti-SigA was used as a loading control since SigA levels do not change during growth (7). Blots were probed simultaneously with anti-FtsZ MT and anti-SigA MT antibodies.…”

“…Despite this fact, the slow polymerization and weak GTP hydrolysis activities of M. tuberculosis FtsZ combined with the presence of novel proteins such as ChiZ and FipA, which indirectly or directly modulate FtsZ ring assembly, impart uniqueness to the mycobacterial cell division machinery (6,7,45,52). Interaction of FtsZ with phosphorylated FipA is thought to be necessary for productive cell division under oxidative stress (45).…”

“…Interaction of FtsZ with phosphorylated FipA is thought to be necessary for productive cell division under oxidative stress (45). ChiZ, an M. tuberculosis cell wall hydrolase, is induced upon DNA damage and regulates midcell FtsZ assembly (7). M. tuberculosis FtsZ interacts with FtsW, and this interaction is implicated in providing stability to the FtsZ ring (13,42).…”

Characterization of the mycobacterial chromosome segregation protein ParB and identification of its target in Mycobacterium smegmatis

“…Knowledge regarding the steps of the mycobacterial cell cycle (replication, chromosome segregation and cell division) seems to be critical for understanding the mechanisms that are responsible for the transition from an active to a non-replicative persistent state (and vice versa) of pathogenic mycobacteria, particularly M. tuberculosis. While initiation of chromosome replication Qin et al, 1999;Rajagopalan et al, 1995;Zawilak et al, 2004) and cell division (FtsZ ring formation) (Chauhan et al, 2006; Dziadek et al, 2002Dziadek et al, , 2003Huang et al, 2007;Rajagopalan et al, 2005) are relatively well studied in mycobacteria, nothing is known about the segregation of chromosomes in these bacteria.Bacterial chromosome segregation has been recently found to be an active and complex process closely coupled with replication (see Bartosik & Jagura-Burdzy, 2005;Errington et al, 2005;Hayes & Barilla, 2006; Leonard et al, 2005 for reviews). In bacteria studied to date, the newly synthesized origin (oriC) regions undergo a symmetric or asymmetric segregation process; two copies of the duplicated oriC regions migrate from the cell centre toward opposite cell poles, i.e.…”