Characterization of an interplay between a Mycobacterium?tuberculosis MazF homolog,Rv1495 and its sole DNA topoisomerase I

The MazEF systems are thought to contribute to the capacity for long-term dormancy observed in the human pathogen, Mycobacterium tuberculosis. However, except for their functions as mRNA interferases, little is known regarding any additional cellular functions of these systems in the pathogen. In the present study, we observed a negative interplay between MazF protein Rv1495 and the sole M. tuberculosis DNA topoisomerase I (MtbTopA) with respect to protein functions. Through its C-terminal domain, MtbTopA physically interacted with and inhibited the mRNA cleavage activity of Rv1495. Rv1495, in turn, inhibited the DNA cleavage activity of MtbTopA as well as its function of relaxation of supercoiled DNA. An N-terminus fragment of Rv1495, designated Rv1495-N(29-56), lost mRNA cleavage activity, but retained a significant physical interaction and inhibitory effect on TopA proteins from both M. tuberculosis and M. smegmatis. This fragment, although less effective than the full-length protein, was able to inhibit mycobacterial growth when expressed through a recombinant plasmid in M. smegmatis. The Rv1495 physically interacted with the M. smegmatis TopA both in vitro and in vivo. Our findings imply that MazEF systems can affect bacterial survival by a novel mechanism that allows direct modulation of M. tuberculosis topoisomerase I.     

One‐plasmid tunable coexpression for mycobacterial protein–protein interaction studies

A single plasmid that allows controlled coexpression has been developed for use in mycobacteria. The tetracycline inducible promoter, PtetO, was used to provide tetracycline‐dependent induction of one gene, while the Psmyc, Pimyc, or Phsp promoters were used to provide three different levels of constitutive expression of a second gene. The functions of these four individual promoters were established using green fluorescent protein (GFP) and a newly identified red fluorescence inducible protein from Geobacillus sterothermophilus strain G1.13 (RFIP) as reporters. The tandem use of GFP and RFIP as reporter genes allowed optimization of the tunable coexpression in Mycobacterium smegmatis; either time at a fixed inducer concentration or changes in inducer concentration could be used to control the protein:protein ratio. This single vector system was used to coexpress the two‐protein Mycobacterium tuberculosis stearoyl‐CoA Δ⁹ desaturase complex (integral membrane desaturase Rv3229c and NADPH oxidoreductase Rv3230c) in M. smegmatis. The catalytic activity was found to increase in a manner corresponding to increasing the level of Rv3230c relative to a fixed level of Rv3229c. This system, which can yield finely tuned coexpression of the fatty acid desaturase complex in mycobacteria, may be useful for study of other multicomponent complexes. Furthermore, the tunable coexpression strategy used herein should also be applicable in other species with minor modifications.     

Cloning and characterization of Rv0621 gene related to surfactant stress tolerance in <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

To understand how Mycobacterium tuberculosis (M. tuberculosis) could survive in human lung, Genomic expression library of M. tuberculosis in Escherichia coli (E. coli) had been prepared. Taking advantage of the genetic simplicity of E. coli and the functional conservation of some prokaryote proteins, a surfactant stress resistant gene Rv0621 was identified, which encodes a 37 kDa putative membrane protein. The E. coli colony with the partial Rv0621 gene insert, named S1, was able to grow in medium containing 0.4% sodium dodecyl sulfate, while the strain carried empty vector was unable to grow. The full length of the Rv0621 gene was then cloned into plasmid pET32a (+) expressed in E. coli BL21 (DE3). Using gas chromatographic–mass spectrometric (GC–MS), the fatty acid composition of the E. coli BL21 (DE3) carrying Rv0621–pET32a (+) and the E. coli BL21 (DE3) carrying empty vector pET32a (+) were compared. E. coli BL21 (DE3) carrying Rv0621–pET32a (+) contained more oleic acid. This suggests the gene may be involved in regulation of fatty acid synthesis and M. tuberculosis resistance to the surfactant defense of its host.     

Viability,morphology, and proteome of Mycobacterium smegmatis MSMEG_0319 knockout strain

Mycobacterium tuberculosis Rv0228, a membrane protein, is predicted as a drug target through computational methods. MSMEG_0319 (MS0319) in Mycobacterium smegmatis mc²155 is the ortholog of Rv0228. To study the effect of MS0319 protein on M. smegmatis, an MS0319 gene knockout strain (ΔMS0319) was generated via a homologous recombination technique in this study. The results showed that the lack of MS0319 protein in mc²155 cells led to the loss of viability at nonpermissive temperature. Scanning electron microscopy and transmission electron microscopy observations showed drastic changes in cellular shape especially cell wall disruption in ΔMS0319 cells. Proteomic analysis of ΔMS0319 cells through LC‐MS/MS revealed that 462 proteins had changes in their expressions by lacking MS0319 protein. The M. tuberculosis orthologs of these 462 proteins were found through BLASTp search and functional clustering and metabolic pathway enrichment were performed on the orthologs. The results revealed that most of them were enzymes involved in metabolism of carbohydrates and amino acids, indicating that Rv0228 played an important role in cellular metabolism. All these results suggested Rv0228 as a potential target for development of antituberculosis drugs.     

Mammalian heterotrimeric G-protein-like proteins in mycobacteria: implications for cell signalling and survival in eukaryotic host cells

Mammalian heterotrimeric GTP-binding proteins (G proteins) are involved in transmembrane signalling that couples a number of receptors to effectors mediating various physiological processes in mammalian cells. We demonstrate that bacterial proteins such as a Ras-like protein from Pseudomonas aeruginosa or a 65 kDa protein from Mycobacterium smegmatis can form complexes with human or yeast nucleoside diphosphate kinase (Ndk) to modulate their nucleoside triphosphate synthesizing specificity to GTP or UTP. In addition, we demonstrate that bacteria such as M. smegmatis or Mycobacterium tuberculosis harbour proteins that cross react with antibodies against the α-, β- or the γ-subunits of heterotrimeric G proteins. Such antibodies also alter the GTP synthesizing ability of specific membrane fractions isolated from glycerol gradients of such cells, suggesting that a membrane-associated Ndk–G-protein homologue complex is responsible for part of GTP synthesis in these bacteria. Indeed, purified Ndk from human erythrocytes and M. tuberculosis showed extensive complex formation with the purified mammalian α and β G-protein subunits and allowed specific GTP synthesis, suggesting that such complexes may participate in transmembrane signalling in the eukaryotic host. We have purified the α-, β- and γ-subunit homologues from M. tuberculosis and we present their internal amino acid sequences as well as their putative homologies with mammalian subunits and the localization of their genes on the M. tuberculosis genome. Using oligonucleotide probes from the conserved regions of the α- and γ-subunit of M. tuberculosis G-protein homologue, we demonstrate hybridization of these probes with the genomic digest of M. tuberculosis H37Rv but not with that of M. smegmatis, suggesting that M. smegmatis might lack the genes present in M. tuberculosis H37Rv. Interestingly, the avirulent strain H37Ra showed weak hybridization with these two probes, suggesting that these genes might have been deleted in the avirulent strain or are present in limited copy numbers as opposed to those in the virulent strain H37Rv.     

Mycobacterium avium Genes Associated with the Ability To Form a Biofilm

Mycobacterium avium is widely distributed in the environment, and it is chiefly found in water and soil. M. avium, as well as Mycobacterium smegmatis, has been recognized to produce a biofilm or biofilm-like structure. We screened an M. avium green fluorescent protein (GFP) promoter library in M. smegmatis for genes involved in biofilm formation on polyvinyl chloride (PVC) plates. Clones associated with increased GFP expression ≥2.0-fold over the baseline were sequenced. Seventeen genes, most encoding proteins of the tricarboxylic acid (TCA) cycle and GDP-mannose and fatty acid biosynthesis, were identified. Their regulation in M. avium was confirmed by examining the expression of a set of genes by real-time PCR after incubation on PVC plates. In addition, screening of 2,000 clones of a transposon mutant bank constructed using M. avium strain A5, a mycobacterial strain with the ability to produce large amounts of biofilm, revealed four mutants with an impaired ability to form biofilm. Genes interrupted by transposons were homologues of M. tuberculosis 6-oxodehydrogenase (sucA), enzymes of the TCA cycle, protein synthetase (pstB), enzymes of glycopeptidolipid (GPL) synthesis, and Rv1565c (a hypothetical membrane protein). In conclusion, it appears that GPL biosynthesis, including the GDP-mannose biosynthesis pathway, is the most important pathway involved in the production of M. avium biofilm.     

Function of a mycobacterial major facilitator superfamily pump requires a membrane-associated lipoprotein

The lprG-Rv1410c operon is critical for the survival of Mycobacterium tuberculosis during infection, but very little is known about the functions of its proteins. LprG is a lipoprotein, and Rv1410c encodes the major facilitator superfamily small molecule transporter P55. P55 likely exports small molecules outside of the bacterial cell, but the function of LprG is unclear. A deletion of the homologous operon in Mycobacterium smegmatis is more susceptible to ethidium bromide, and drug resistance is restored by the intact operon from M. tuberculosis. The multidrug resistance pump inhibitor reserpine inhibits resistance to ethidium bromide in both wild-type M. smegmatis and the complemented mutant, suggesting that P55-mediated transport is responsible for drug resistance and that ethidium bromide is a novel substrate for P55. In addition to hypersensitivity to ethidium bromide, cells that lack the lprG-Rv1410c operon display abnormal colony morphology and are defective for sliding motility, properties that suggest an alteration of cell wall composition. Strikingly, both ethidium bromide transport and normal cell surface properties require functional P55 and LprG, as neither alone is sufficient to restore function to the deletion mutant. Thus, P55 requires the cell surface lipoprotein for normal function.     

A Mycobacterial Cyclic AMP Phosphodiesterase That Moonlights as a Modifier of Cell Wall Permeability

Mycobacterium tuberculosis utilizes many mechanisms to establish itself within the macrophage, and bacterially derived cAMP is important in modulating the host cellular response. Although the genome of M. tuberculosis is endowed with a number of mammalian-like adenylyl cyclases, only a single cAMP phosphodiesterase has been identified that can decrease levels of cAMP produced by the bacterium. We present the crystal structure of the full-length and sole cAMP phosphodiesterase, Rv0805, found in M. tuberculosis, whose orthologs are present only in the genomes of slow growing and pathogenic mycobacteria. The dimeric core catalytic domain of Rv0805 adopts a metallophosphoesterase-fold, and the C-terminal region builds the active site and contributes to multiple substrate utilization. Localization of Rv0805 to the cell wall is dependent on its C terminus, and expression of either wild type or mutationally inactivated Rv0805 in M. smegmatis alters cell permeability to hydrophobic cytotoxic compounds. Rv0805 may therefore play a key role in the pathogenicity of mycobacteria, not only by hydrolyzing bacterial cAMP, but also by moonlighting as a protein that can alter cell wall functioning.     

Tetrahydrolipstatin Inhibition,Functional Analyses,and Three-dimensional Structure of a Lipase Essential for Mycobacterial Viability

The highly complex and unique mycobacterial cell wall is critical to the survival of Mycobacteria in host cells. However, the biosynthetic pathways responsible for its synthesis are, in general, incompletely characterized. Rv3802c from Mycobacterium tuberculosis is a partially characterized phospholipase/thioesterase encoded within a genetic cluster dedicated to the synthesis of core structures of the mycobacterial cell wall, including mycolic acids and arabinogalactan. Enzymatic assays performed with purified recombinant proteins Rv3802c and its close homologs from Mycobacterium smegmatis (MSMEG_6394) and Corynebacterium glutamicum (NCgl2775) show that they all have significant lipase activities that are inhibited by tetrahydrolipstatin, an anti-obesity drug that coincidently inhibits mycobacterial cell wall biosynthesis. The crystal structure of MSMEG_6394, solved to 2.9 Å resolution, revealed an α/β hydrolase fold and a catalytic triad typically present in esterases and lipases. Furthermore, we demonstrate direct evidence of gene essentiality in M. smegmatis and show the structural consequences of loss of MSMEG_6394 function on the cellular integrity of the organism. These findings, combined with the predicted essentiality of Rv3802c in M. tuberculosis, indicate that the Rv3802c family performs a fundamental and indispensable lipase-associated function in mycobacteria.     

A lipoprotein modulates activity of the MtrAB two‐component system to provide intrinsic multidrug resistance,cytokinetic control and cell wall homeostasis in Mycobacterium

The MtrAB signal transduction system, which participates in multiple cellular processes related to growth and cell wall homeostasis, is the only two‐component system known to be essential in Mycobacterium. In a screen for antibiotic resistance determinants in Mycobacterium smegmatis, we identified a multidrug‐sensitive mutant with a transposon insertion in lpqB, the gene located immediately downstream of mtrA–mtrB. The lpqB mutant exhibited increased cell–cell aggregation and severe defects in surface motility and biofilm growth. lpqB cells displayed hyphal growth and polyploidism, reminiscent of the morphology of Streptomyces, a related group of filamentous Actinobacteria. Heterologous expression of M. tuberculosis LpqB restored wild‐type characteristics to the lpqB mutant. LpqB interacts with the extracellular domain of MtrB, and influences MtrA phosphorylation and promoter activity of dnaA, an MtrA‐regulated gene that affects cell division. Furthermore, in trans expression of the non‐phosphorylated, inactive form of MtrA in wild‐type M. smegmatis resulted in phenotypes similar to those of lpqB deletion, whereas expression of the constitutively active form of MtrA restored wild‐type characteristics to the lpqB mutant. These results support a model in which LpqB, MtrB and MtrA form a three‐component system that co‐ordinates cytokinetic and cell wall homeostatic processes.     

Transformation of distinct mycobacterial species by shuttle vectors derived from theMycobacterium fortuitum pAL5000 plasmid

Mycobacterium tuberculosis H37Ra,M. smegmatisATCC 607,M. smegmatis MC²155,M. aurum A +,M. aurum A11, and one representative strain ofM. flavescens were transformed by electroporation with plasmid pMY10 and cosmid pDC100. Plasmid pMY 10 contained the origin of replication of pAL5000, the origin of replication of pBR322, a kanamycin resistance gene, and the origin of transfer of the Inc plasmid RK2; the cosmid pDC100 contained the pHC79 SS cosmid, the origin of replication of pAL5000, and a kanamycin resistance gene. The efficiency of transformation varied with the recipient cells used and was in decreasing order: 7×10⁵ forM. smegmatis MC²155, 6×10³ forM. tuberculosis H37Ra, 10³ forM. aurum, 50 forM. smegmatis ATCC 607, and 5 forM. flavescens. A rapid protocol for plasmid extraction from mycobacteria was developed.The satisfactory transformation of the nonvirulentM. tuberculosis strain H37Ra was of interest for future studies on cloning of virulence genes, while the satisfactory transformation ofM. aurum was of interest for future studies on the genetics of drug resistance because these bacteria are sensitive to drugs specifically used in the treatment of tuberculosis and leprosy. However, neither vector was stably maintained inM. smegmatis, indicating that further investigations are still necessary to resolve this difficulty.     

Identification of two Mycobacterium tuberculosis H37Rv ORFs involved in resistance to killing by human macrophages

Background  

The ability of Mycobacterium tuberculosis to survive and replicate in macrophages is crucial for the mycobacterium's ability to infect the host and cause tuberculosis. To identify Mycobacterium tuberculosis genes involved in survival in macrophages, a library of non-pathogenic Mycobacterium smegmatis bacteria, each carrying an individual integrated cosmid containing M. tuberculosis H37Rv genomic DNA, was passed through THP-1 human macrophages three times.     

Crystal structure of the Mycobacterium tuberculosis phosphate binding protein PstS3

Mycobacterium tuberculosis evades host immune responses by colonizing macrophages. Intraphagosomal M. tuberculosis is exposed to environmental stresses such as reactive oxygen and nitrogen intermediates as well as acid shock and inorganic phosphate (Pi) depletion. Experimental evidence suggests that expression levels of mycobacterial protein PstS3 (Rv0928) are significantly increased when M. tuberculosis bacilli are exposed to Pi starvation. Hence, PstS3 may be important for survival of Mtb in conditions where there is limited supply of Pi. We report here the structure of PstS3 from M. tuberculosis at 2.3‐Å resolution. The protein presents a structure typical for ABC phosphate transfer receptors. Comparison with its cognate receptor PstS1 showed a different pattern distribution of surface charges in proximity to the Pi recognition site, suggesting complementary roles of the two proteins in Pi uptake. Proteins 2014; 82:2268–2274. © 2014 Wiley Periodicals, Inc.     

Aptamer inhibits <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> (H37Rv) invasion of macrophage

There is an urgent need to develop new anti-tuberculosis drugs due to the rising tendency in tuberculosis (TB) around the world. It is known that Mycobacterium tuberculosis (M. tuberculosis) generally infects mammalian host via aerosol route. The pathogenic process has been fully studied that it can initially invade alveolar macrophage, then established stable residence within those phagocytic cells, suggesting that one of the possible ways to prevent this pathogen is to inhibit its invasion and growth in the macrophage. Aptamers from SELEX (Systematic Evolution of Ligands by Exponential Enrichment) have been used to rival virulent M. tuberculosis (H37Rv) in our previous work, and the materials to which aptamers bound were proved to be some outer membrane proteins of H37Rv. In the present study, the interaction between M. tuberculosis and macrophage in the presence of aptamers was investigated in more details. The results suggested that the selective aptamers significantly inhibited H37Rv invasion of macrophage in vitro, and the effect correspond to the binding affinity of these aptamers to H37Rv. The values of equilibrium dissociation constant (Kd) was calculated by flow cytometry, all in the nanomolar range, showed much higher affinity to H37Rv than M. bovis Bacillus Guerin (BCG). Moreover, the aptamer-treated H37Rv can stimulate IFN-γ, IL-15 and IL-17 secretion of macrophages compared with H37Rv (no treated). In summary, our data indicated that the NK2 aptamer not only acted as anti-tuberculosis agent by inhibiting virulent M. tuberculosis (H37Rv) invasion of macrophage, but also might be used as molecular probe for exploring the interaction between the outer membrane of M. tuberculosis and macrophage.     

Fullertubes inhibit mycobacterial viability and prevent biofilm formation by disrupting the cell wall

Mycobacterium tuberculosis and nontuberculous mycobacteria such as Mycobacterium abscessus cause diseases that are becoming increasingly difficult to treat due to emerging antibiotic resistance. The development of new antimicrobial molecules is vital for combating these pathogens. Carbon nanomaterials (CNMs) are a class of carbon-containing nanoparticles with promising antimicrobial effects. Fullertubes (C₉₀) are novel carbon allotropes with a structure unique among CNMs. The effects of fullertubes on any living cell have not been studied. In this study, we demonstrate that pristine fullertube dispersions show antimicrobial effects on Mycobacterium smegmatis and M. abscessus. Using scanning electron microscopy, light microscopy, and molecular probes, we investigated the effects of these CNMs on mycobacterial cell viability, cellular integrity, and biofilm formation. C₉₀ fullertubes at 1 µM inhibited mycobacterial viability by 97%. Scanning electron microscopy revealed that the cell wall structure of M. smegmatis and M. abscessus was severely damaged within 24 h of exposure to fullertubes. Additionally, exposure to fullertubes nearly abrogated the acid-fast staining property of M. smegmatis. Using SYTO-9 and propidium iodide, we show that exposure to the novel fullertubes compromises the integrity of the mycobacterial cell. We also show that the permeability of the mycobacterial cell wall was increased after exposure to fullertubes from our assays utilizing the molecular probe dichlorofluorescein and ethidium bromide transport. C₉₀ fullertubes at 0.37 µM and C₆₀ fullerenes at 0.56 µM inhibited pellicle biofilm formation by 70% and 90%, respectively. This is the first report on the antimycobacterial activities of fullertubes and fullerenes.     

Molecular Cloning and Characterization of Tap, a Putative Multidrug Efflux Pump Present in Mycobacterium fortuitum and Mycobacterium tuberculosis

A recombinant plasmid isolated from a Mycobacterium fortuitum genomic library by selection for gentamicin and 2-N′-ethylnetilmicin resistance conferred low-level aminoglycoside and tetracycline resistance when introduced into M. smegmatis. Further characterization of this plasmid allowed the identification of the M. fortuitum tap gene. A homologous gene in the M. tuberculosis H37Rv genome has been identified. The M. tuberculosis tap gene (Rv1258 in the annotated sequence of the M. tuberculosis genome) was cloned and conferred low-level resistance to tetracycline when introduced into M. smegmatis. The sequences of the putative Tap proteins showed 20 to 30% amino acid identity to membrane efflux pumps of the major facilitator superfamily (MFS), mainly tetracycline and macrolide efflux pumps, and to other proteins of unknown function but with similar antibiotic resistance patterns. Approximately 12 transmembrane regions and different sequence motifs characteristic of the MFS proteins also were detected. In the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), the levels of resistance to antibiotics conferred by plasmids containing the tap genes were decreased. When tetracycline accumulation experiments were carried out with the M. fortuitum tap gene, the level of tetracycline accumulation was lower than that in control cells but was independent of the presence of CCCP. We conclude that the Tap proteins of the opportunistic organism M. fortuitum and the important pathogen M. tuberculosis are probably proton-dependent efflux pumps, although we cannot exclude the possibility that they act as regulatory proteins.     

Chemical shift assignments for Rv0577, a putative glyoxylase associated with virulence from Mycobacterium tuberculosis

Approximately one-third of mankind has been exposed to Mycobacterium tuberculosis, the etiological agent responsible for tuberculosis (TB). As part of an effort to develop a new generation of anti-TB agents, the chemical shifts for the 261-residue, virulence-associated protein Rv0577 from M. tuberculosis has been extensively assigned.