Mycobacterial Cell Division Research Articles

Septum site placement in Mycobacteria - identification and characterisation of mycobacterial homologues of Escherichia coli MinD.

A major virulence trait of Mycobacterium tuberculosis (M. tb) is its ability to enter a dormant state within its human host. Since cell division is intimately linked to metabolic shut down, understanding the mechanism of septum formation and its integration with other events in the division pathway is likely to offer clues to the molecular basis of dormancy. The M. tb genome lacks obvious homologues of several conserved cell division proteins, and this study was aimed at identifying and functionally characterising mycobacterial homologues of the E. coli septum site specification protein MinD (Ec MinD). Sequence homology based analyses suggested that the genomes of both M. tb and the saprophyte Mycobacterium smegmatis (M. smegmatis) encode two putative Ec MinD homologues - Rv1708/MSMEG_3743 and Rv3660c/ MSMEG_6171. Of these, Rv1708/MSMEG_3743 were found to be the true homologues, through complementation of the E. coli ∆minDE mutant HL1, overexpression studies, and structural comparisons. Rv1708 and MSMEG_3743 fully complemented the mini-cell phenotype of HL1, and over-expression of MSMEG_3743 in M. smegmatis led to cell elongation and a drastic decrease in c.f.u. counts, indicating its essentiality in cell-division. MSMEG_3743 displayed ATPase activity, consistent with its containing a conserved Walker A motif. Interaction of Rv1708 with the chromosome associated proteins ScpA and ParB, implied a link between its septum formation role, and chromosome segregation. Comparative structural analyses showed Rv1708 to be closer in similarity to Ec MinD than Rv3660c. In summary we identify Rv1708 and MSMEG_3743 to be homologues of Ec MinD, adding a critical missing piece to the mycobacterial cell division puzzle.

Retraction:Novel Role of Phosphorylation-Dependent Interaction between FtsZ and FipA in Mycobacterial Cell Division.

Retraction:Novel Role of Phosphorylation-Dependent Interaction between FtsZ and FipA in Mycobacterial Cell Division.

Rv0954 Is a Member of the Mycobacterial Cell Division Complex.

Proper control of cell division in the intracellular pathogen Mycobacterium tuberculosis is central to its growth, survival, pathogenesis, and resistance to antibiotics. Nevertheless, the divisome components and mechanisms by which mycobacteria regulate their cell cycle are not entirely understood. Here we demonstrate that the previously uncharacterized Rv0954 protein localizes to the mid-cell during cell division and interacts with the division-related proteins LamA, PbpA, and PknH. Deletion of rv0954 did not result in alterations in cell morphology or sensitivity to cell wall-targeting antibiotics but transposon mutagenesis demonstrated genetic interactions with genes related to cell division. This work suggests that Rv0954 participates in cell division and reveals potential components of the mycobacterial divisome for future investigation.

Effects of Mycobacterium tuberculosis Rv1096 on mycobacterial cell division and modulation on macrophages

Mycobacterium tuberculosis is capable of escaping the clearance of immune system mainly due to its complex constituents of cell wall. Certain studies show that glycoproteins are involved in immune evasion and act as virulence factors. Peptidoglycan deacetylase Rv1096 is a member of mannosylated proteins. Previously, we reported Rv1096 protein contributed to the resistance of Mycobacterium smegmatis (M. smegmatis) to lysozyme, but more characterization of this protein is required where further intracellular function is unknown. Here, Rv1096 was heterologously over-expressed in the fast-growing and nonpathogenic M. smegmatis (named as M. smegmatis/Rv1096). We observed the morphological alterations in M. smegmatis/Rv1096 including an elongated rod-like shape and increased amounts of Z-rings, which implied that Rv1096 facilitated the cell growth and division. Moreover, a series of assays concerning the interaction between M. smegmatis/Rv1096 and host were carried out. The results showed that M. smegmatis/Rv1096 evaded the killing of macrophages due to the inhibition of phagosome-lysosome fusion, nicotinamide adenine dinucleotide phosphate oxidase activity and reactive oxygen species production. The secretion of interleukin-12 and tumor necrosis factor-α was also impaired by Rv1096. In addition, five putative interaction partners of Rv1096 were identified, which possibly cooperated with Rv1096 in cell division and immune regulation. These results suggested that Rv1096 had effects on mycobacterial division and might act as a virulence factor to mediate the immune evasion in macrophage during mycobacterial infection.

Overlapping and essential roles for molecular and mechanical mechanisms in mycobacterial cell division.

IntroductoryMechanisms to control cell division are essential for cell proliferation and survival 1. Bacterial cell growth and division require the coordinated activity of peptidoglycan synthases and hydrolytic enzymes 2–4 to maintain mechanical integrity of the cell wall 5. Recent studies suggest that cell separation is governed by mechanical forces 6,7. How mechanical forces interact with molecular mechanisms to control bacterial cell division in space and time is poorly understood. Here, we use a combination of atomic force microscope (AFM) imaging, nanomechanical mapping, and nanomanipulation to show that enzymatic activity and mechanical forces serve overlapping and essential roles in mycobacterial cell division. We find that mechanical stress gradually accumulates in the cell wall concentrated at the future division site, culminating in rapid (millisecond) cleavage of nascent sibling cells. Inhibiting cell wall hydrolysis delays cleavage; conversely, locally increasing cell wall stress causes instantaneous and premature cleavage. Cells deficient in peptidoglycan hydrolytic activity fail to locally decrease their cell wall strength and undergo natural cleavage, instead forming chains of non-growing cells. Cleavage of these cells can be mechanically induced by local application of stress with AFM. These findings establish a direct link between actively controlled molecular mechanisms and passively controlled mechanical forces in bacterial cell division.

Lack of MSMEG_6281, a peptidoglycan amidase, affects cell wall integrity and virulence of Mycobacterium smegmatis

Mycolyl-arabinogalactan-peptidoglycan (mAGP) is the major content of the mycobacterium cell wall structure and essential for mycobacterial survival. Peptidoglycan (PG) plays an important role in maintenance of cell division, cell wall integrity and pathogenesis. Mycobacterium smegmatis MSMEG_6281, a peptidoglycan amidase, is vital for mycobacterial cell division. However, the effects of MSMEG_6281on cell wall integrity and mycobacterial virulence remain unknown. In the current study, we demonstrate that MSMEG_6281gene knockout in M.smegmatis alters the microbiological characteristics. Our results revealed that MSMEG_6281gene knockout bacteria (M. sm-ΔM_6281) lost their acid-fastness, increased their sensitivity to lipophilic compounds and presented an abnormal morphology. Our results revealed that MSMEG_6281was related to maintaining the cell wall integrity. Furthermore, we investigated the effects of MSMEG_6281 inactivation on mycobacterial virulence using mice models infected by different M.smegmatis strains. MSMEG_6281 inactivation in the M sm-ΔM_6281 infected group caused less mycobacterial colonization, reduced pathological signs, decreased the anti-microbial enzymes production including iNOS and β-defensins in mouse lungs. Moreover, IL-1β and TLR2 expression were significantly down-regulated, while the production of IFN-γ and TNF-α was up-regulated. These findings indicated the diversity of host immune responses induced by different strains of M.smegmatis, suggesting that MSMEG_6281 inactivation impact mycobacterial virulence. In conclusion, the MSMEG_6281 protein plays important roles in maintaining cell wall integrity and mycobacterial virulence.

A Symmetric Molecule Produced by Mycobacteria Generates Cell-Length Asymmetry during Cell-Division and Thereby Cell-Length Heterogeneity.

Diadenosine polyphosphates, Ap(2-7)A, which contain two adenosines in a 5',5' linkage through phosphodiester bonds involving 2-7 phosphates, regulate diverse cellular functions in all organisms, from bacteria to humans, under normal and stress conditions. We had earlier reported consistent occurrence of asymmetric constriction during division (ACD) in 20-30% of dividing mycobacterial cells in culture, irrespective of different growth media, implying exogenous action of some factor of mycobacterial origin. Consistent with this premise, concentrated culture supernatant (CCS), but not the equivalent volume-wise concentrated unused medium, dramatically enhanced the ACD proportion to 70-90%. Mass spectrometry and biochemical analyses of the bioactive fraction from CCS revealed the ACD-effecting factor to be Ap6A. Synthetic Ap6A showed a mass spectrometry profile, biochemical characteristics, and bioactivity identical to native Ap6A in the CCS. Thus, the present work reveals a novel role for Ap6A in generating cell-length asymmetry during mycobacterial cell-division and thereby cell-length heterogeneity in the population.

Rv0004 is a new essential member of the mycobacterial DNA replication machinery.

DNA replication is fundamental for life, yet a detailed understanding of bacterial DNA replication is limited outside the organisms Escherichia coli and Bacillus subtilis. Many bacteria, including mycobacteria, encode no identified homologs of helicase loaders or regulators of the initiator protein DnaA, despite these factors being essential for DNA replication in E. coli and B. subtilis. In this study we discover that a previously uncharacterized protein, Rv0004, from the human pathogen Mycobacterium tuberculosis is essential for bacterial viability and that depletion of Rv0004 leads to a block in cell cycle progression. Using a combination of genetic and biochemical approaches, we found that Rv0004 has a role in DNA replication, interacts with DNA and the replicative helicase DnaB, and affects DnaB-DnaA complex formation. We also identify a conserved domain in Rv0004 that is predicted to structurally resemble the N-terminal protein-protein interaction domain of DnaA. Mutation of a single conserved tryptophan within Rv0004’s DnaA N-terminal-like domain leads to phenotypes similar to those observed upon Rv0004 depletion and can affect the association of Rv0004 with DnaB. In addition, using live cell imaging during depletion of Rv0004, we have uncovered a previously unappreciated role for DNA replication in coordinating mycobacterial cell division and cell size. Together, our data support that Rv0004 encodes a homolog of the recently identified DciA family of proteins found in most bacteria that lack the DnaC-DnaI helicase loaders in E. coli and B. subtilis. Therefore, the mechanisms of Rv0004 elucidated here likely apply to other DciA homologs and reveal insight into the diversity of bacterial strategies in even the most conserved biological processes.

Mycobacterial DNA Replication as a Target for Antituberculosis Drug Discovery.

Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, is a leading infectious disease organism, causing millions of deaths each year. This serious pathogen has been greatly spread worldwide and recent years have observed an increase in the number of multi-drug resistant and totally drug resistant M. tuberculosis strains (WHO report, 2014). The danger of tuberculosis becoming an incurable disease has emphasized the need for the discovery of a new generation of antimicrobial agents. The development of novel alternative medical strategies, new drugs and the search for optimal drug targets are top priority areas of tuberculosis research. Key characteristics of mycobacteria include: slow growth, the ability to transform into a metabolically silent - latent state, intrinsic drug resistance and the relatively rapid development of acquired drug resistance. These factors make finding an ideal antituberculosis drug enormously challenging, even if it is designed to treat drug sensitive tuberculosis strains. A vast majority of canonical antibiotics including antituberculosis agents target bacterial cell wall biosynthesis or DNA/RNA processing. Novel therapeutic approaches are being tested to target mycobacterial cell division, twocomponent regulatory factors, lipid synthesis and the transition between the latent and actively growing states. This review discusses the choice of cellular targets for an antituberculosis therapy, describes putative drug targets evaluated in the recent literature and summarizes potential candidates under clinical and pre-clinical development. We focus on the key cellular process of DNA replication, as a prominent target for future antituberculosis therapy. We describe two main pathways: the biosynthesis of nucleic acids precursors - the nucleotides, and the synthesis of DNA molecules. We summarize data regarding replication associated proteins that are critical for nucleotide synthesis, initiation, unwinding and elongation of the DNA during the replication process. They are pivotal processes required for successful multiplication of the bacterial cells and hence they are extensively investigated for the development of antituberculosis drugs. Finally, we summarize the most potent inhibitors of DNA synthesis and provide an up to date report on their status in the clinical trials.

An Amidase_3 domain-containing N-acetylmuramyl-L-alanine amidase is required for mycobacterial cell division

Mycobacteria possess a multi-layered cell wall that requires extensive remodelling during cell division. We investigated the role of an amidase_3 domain-containing N-acetylmuramyl-L-alanine amidase, a peptidoglycan remodelling enzyme implicated in cell division. We demonstrated that deletion of MSMEG_6281 (Ami1) in Mycobacterium smegmatis resulted in the formation of cellular chains, illustrative of cells that were unable to complete division. Suprisingly, viability in the Δami1 mutant was maintained through atypical lateral branching, the products of which proceeded to form viable daughter cells. We showed that these lateral buds resulted from mislocalization of DivIVA, a major determinant in facilitating polar elongation in mycobacterial cells. Failure of Δami1 mutant cells to separate also led to dysregulation of FtsZ ring bundling. Loss of Ami1 resulted in defects in septal peptidoglycan turnover with release of excess cell wall material from the septum or newly born cell poles. We noted signficant accumulation of 3-3 crosslinked muropeptides in the Δami1 mutant. We further demonstrated that deletion of ami1 leads to increased cell wall permeability and enhanced susceptiblity to cell wall targeting antibiotics. Collectively, these data provide novel insight on cell division in actinobacteria and highlights a new class of potential drug targets for mycobacterial diseases.

WhmD Promotes the Assembly of Mycobacterium Smegmatis FtsZ: A Possible Role of WhmD in Bacterial Cell Division

WhmD, a Mycobacterium smegmatis cell division protein was identified in search of potential Mycobacterial cell division regulatory genes. The viability of Mycobacterium smegmatis cells got significantly reduced upon the deletion or overexpression of WhmD indicating its importance in the division process of M. smegmatis cells. In this study, the deletion of WhmD was found to perturb medially located septum and to produce filamentation in the M. smegmatis 628 - 53 strains. The heterologous overexpression of WhmD induced elongation and perturbed the Z-ring formation in Escherichia coli cells indicating that WhmD may influence the assembly of FtsZ. Therefore, we determined the effect of WhmD on the assembly of MsFtsZ in vitro. Purified WhmD increased the rate and extent of the assembly of MsFtsZ in a concentration-dependent manner. WhmD increased the amount of polymerized MsFtsZ. Under the polymerizing conditions, MsFtsZ formed thin filaments while thick filaments were formed in the presence of WhmD suggesting WhmD enhanced the bundling of MsFtsZ filaments. In addition, WhmD increased the GTPase activity of Mycobacterium smegmatis FtsZ. WhmD bound to FtsZ with a high affinity. The evidence suggested that WhmD may play an important role in regulating the assembly of FtsZ during the division process of M. smegmatis cells.

Identification and functional annotation of mycobacterial septum formation genes using cell division mutants of Escherichia coli

The major virulence trait of Mycobacterium tuberculosis is its ability to enter a latent state in the face of robust host immunity. Clues to the molecular basis of latency can emerge from understanding the mechanism of cell division, beginning with identification of proteins involved in this process. Using complementation of Escherichia coli mutants, we functionally annotated M. tuberculosis and Mycobacterium smegmatis homologs of divisome proteins FtsW and AmiC. Our results demonstrate that E. coli can be used as a surrogate model to discover mycobacterial cell division genes, and should prove invaluable in delineating the mechanisms of this fundamental process in mycobacteria.

The external PASTA domain of the essential serine/threonine protein kinase PknB regulates mycobacterial growth.

PknB is an essential serine/threonine protein kinase required for mycobacterial cell division and cell-wall biosynthesis. Here we demonstrate that overexpression of the external PknB_PASTA domain in mycobacteria results in delayed regrowth, accumulation of elongated bacteria and increased sensitivity to β-lactam antibiotics. These changes are accompanied by altered production of certain enzymes involved in cell-wall biosynthesis as revealed by proteomics studies. The growth inhibition caused by overexpression of the PknB_PASTA domain is completely abolished by enhanced concentration of magnesium ions, but not muropeptides. Finally, we show that the addition of recombinant PASTA domain could prevent regrowth of Mycobacterium tuberculosis, and therefore offers an alternative opportunity to control replication of this pathogen. These results suggest that the PknB_PASTA domain is involved in regulation of peptidoglycan biosynthesis and maintenance of cell-wall architecture.

The essential role of SepF in mycobacterial division.

Mycobacteria lack several of the components that are essential in model systems as Escherichia coli or Bacillus subtilis for the formation of the divisome, a ring-like structure assembling at the division site to initiate bacterial cytokinesis. Divisome assembly depends on the correct placement of the FtsZ protein into a structure called the Z ring. Notably, early division proteins that assist in the localisation of the Z ring to the cytoplasmic membrane and modulate its structure are missing in the so far known mycobacterial cell division machinery. To find mycobacterium-relevant components of the divisome that might act at the level of FtsZ, a yeast two-hybrid screening was performed with FtsZ from Mycobacterium tuberculosis. We identified the SepF homolog as a new interaction partner of mycobacterial FtsZ. Depending on the presence of FtsZ, SepF-GFP fusions localised in ring-like structures at potential division sites. Alteration of SepF levels in Mycobacterium smegmatis led to filamentous cells, indicating a division defect. Depletion of SepF resulted in a complete block of division. The sepF gene is highly conserved in the M. tuberculosis complex members. We therefore propose that SepF is an essential part of the core division machinery in the genus Mycobacterium.

Disruption of an M. tuberculosis membrane protein causes a magnesium-dependent cell division defect and failure to persist in mice.

The identification of Mycobacterium tuberculosis genes necessary for persistence in vivo provides insight into bacterial biology as well as host defense strategies. We show that disruption of M. tuberculosis membrane protein PerM (Rv0955) resulted in an IFN-γ-dependent persistence defect in chronic mouse infection despite the mutant’s near normal growth during acute infection. The perM mutant required increased magnesium for replication and survival; incubation in low magnesium media resulted in cell elongation and lysis. Transcriptome analysis of the perM mutant grown in reduced magnesium revealed upregulation of cell division and cell wall biosynthesis genes, and live cell imaging showed PerM accumulation at the division septa in M. smegmatis. The mutant was acutely sensitive to β-lactam antibiotics, including specific inhibitors of cell division-associated peptidoglycan transpeptidase FtsI. Together, these data implicate PerM as a novel player in mycobacterial cell division and pathogenesis, and are consistent with the hypothesis that immune activation deprives M. tuberculosis of magnesium.

Novel Role of Phosphorylation-Dependent Interaction between FtsZ and FipA in Mycobacterial Cell Division

The bacterial divisome is a multiprotein complex. Specific protein-protein interactions specify whether cell division occurs optimally, or whether division is arrested. Little is known about these protein-protein interactions and their regulation in mycobacteria. We have investigated the interrelationship between the products of the Mycobacterium tuberculosis gene cluster Rv0014c-Rv0019c, namely PknA (encoded by Rv0014c) and FtsZ-interacting protein A, FipA (encoded by Rv0019c) and the products of the division cell wall (dcw) cluster, namely FtsZ and FtsQ. M. smegmatis strains depleted in components of the two gene clusters have been complemented with orthologs of the respective genes of M. tuberculosis. Here we identify FipA as an interacting partner of FtsZ and FtsQ and establish that PknA-dependent phosphorylation of FipA on T77 and FtsZ on T343 is required for cell division under oxidative stress. A fipA knockout strain of M. smegmatis is less capable of withstanding oxidative stress than the wild type and showed elongation of cells due to a defect in septum formation. Localization of FtsQ, FtsZ and FipA at mid-cell was also compromised. Growth and survival defects under oxidative stress could be functionally complemented by fipA of M. tuberculosis but not its T77A mutant. Merodiploid strains of M. smegmatis expressing the FtsZ(T343A) showed inhibition of FtsZ-FipA interaction and Z ring formation under oxidative stress. Knockdown of FipA led to elongation of M. tuberculosis cells grown in macrophages and reduced intramacrophage growth. These data reveal a novel role of phosphorylation-dependent protein-protein interactions involving FipA, in the sustenance of mycobacterial cell division under oxidative stress.

Mycobacterium tuberculosis Senses Host-Derived Carbon Monoxide during Macrophage Infection

Mycobacterium tuberculosis (MTB) expresses a set of genes known as the dormancy regulon in vivo. These genes are expressed in vitro in response to nitric oxide (NO) or hypoxia, conditions used to model MTB persistence in latent infection. Although NO, a macrophage product that inhibits respiration, and hypoxia are likely triggers in vivo, additional cues could activate the dormancy regulon during infection. Here, we show that MTB infection stimulates expression of heme oxygenase (HO-1) by macrophages and that the gaseous product of this enzyme, carbon monoxide (CO), activates expression of the dormancy regulon. Deletion of macrophage HO-1 reduced expression of the dormancy regulon. Furthermore, we show that the MTB DosS/DosT/DosR two-component sensory relay system is required for the response to CO. Together, these findings demonstrate that MTB senses CO during macrophage infection. CO may represent a general cue used by pathogens to sense and adapt to the host environment.

The Mycobacterium tuberculosis serine/threonine kinase PknL phosphorylates Rv2175c: Mass spectrometric profiling of the activation loop phosphorylation sites and their role in the recruitment of Rv2175c

Although Mycobacterium tuberculosis (M. tb) comprises 11 serine/threonine protein kinases, the mechanisms of regulation of these kinases and the nature of their endogenous substrates remain largely unknown. Herein, we characterized the M. tb kinase PknL by demonstrating that it expresses autophosphorylation activity and phosphorylates Rv2175c. On-target dephosphorylation/MALDI-TOF for identification of phosphorylated peptides was used in combination with LC-ESI/MS/MS for localization of phosphorylation sites. By doing so, five phosphorylated threonine residues were identified in PknL. Among them, we showed that the activation loop phosphorylated residues Thr173 and Thr175 were essential for the autophosphorylation activity of PknL. Phosphorylation of the activation loop Thr173 residue is also required for optimal PknL-mediated phosphorylation of Rv2175c. Together, our results indicate that phosphorylation of the PknL activation loop Thr residues not only controls PknL kinase activity but is also required for recruitment and phosphorylation of its substrate. Rv2175c was found to be phosphorylated when overexpressed and purified from Mycobacterium smegmatis as 2-DE indicated the presence of different phosphorylated isoforms. Given the presence of the dcw gene cluster in the close vicinity of the pknL/Rv2175c locus, and its conservation in all mycobacterial species, we propose that PknL/Rv2175c may represent a functional pair in the regulation of mycobacterial cell division and cell envelope biosynthesis.

Expression of mycobacterial cell division protein, FtsZ, and dormancy proteins, DevR and Acr, within lung granulomas throughout guinea pig infection

The ability of Mycobacterium tuberculosis to persist in a dormant state is a hallmark of tuberculosis. An insight into the expression of mycobacterial proteins will contribute to our understanding of bacterial physiology in vivo. To this end, the expression of FtsZ, Acr and DevR was assessed in the lung granulomas of guinea pigs infected with M. tuberculosis. Antigen immunostaining was then compared with the detection of acid-fast bacilli (AFB) and mycobacterial DNA. Surprisingly, immunostaining for all three antigens was observed throughout the course of infection; maximum expression of all antigens was noted at 20 weeks of infection. The intensity of immunostaining correlated well with the presence of intact bacteria, suggesting that mycobacterial antigens in the extracellular fraction have a short half-life; in contrast to protein, extracellular bacterial DNA was found to be more stable. Immunostaining for bacterial division and dormancy markers could not clearly distinguish between replicating and non-replicating organisms during the course of infection. The detection of Acr and DevR from 4 weeks onwards indicates that the dormancy proteins are expressed from early on in infection. Both antigen staining and DNA detection from intact bacilli were useful for detecting intact mycobacteria in the absence of AFB.