Identification of a methyltransferase from Mycobacterium smegmatis involved in glycopeptidolipid synthesis

Glycopeptidolipids (GPLs) are major components of the cell walls of several species of mycobacteria. We have isolated a transposon mutant of Mycobacterium smegmatis that is unable to synthesize mature GPLs and that displays a rough colony morphology. The disrupted gene, mtf1, shares a high degree of homology with several S-adenosylmethionine-dependent methyltransferases. The enzyme encoded by mtf1 is required for the methylation of a single rhamnose residue that forms part of the conserved GPL core structure. This conclusion is supported by the finding that (a) the mutant synthesized only GPLs with undermethylated (either mono- or nonmethylated instead of di- or trimethylated) rhamnose residues; (b) complementation of the mutant with a wild-type copy of mtf1 restored high levels of synthesis of GPLs containing di- and trimethylated rhamnose; and (c) S-adenosylmethionine-dependent methylation of rhamnosylated GPLs could be detected in cell lysates of wild-type cells and mtf1-complemented mutant cells, but not in mutant cells lacking intact mtf1. Structural analysis of wild-type and mutant GPLs suggests that disruption of mtf1 specifically inhibits addition of O-methyl groups to the 3 (or 2)-position of the rhamnose. In the absence of 3-O-methylation, further methylation of GPL rhamnose is apparently inhibited, and overall GPL synthesis is down-regulated by 90%.     

Mutation of environmental mycobacteria to resist silver nanoparticles also confers resistance to a common antibiotic

Non-tuberculous mycobacteria are a threat to human health, gaining entry to the body through contaminated water systems, where they form persistent biofilms despite extensive attempts at disinfection. Silver is a natural antibacterial agent and in nanoparticle form activity is increased by a high surface area. Silver nanoparticles (AgNPs) have been used as alternative disinfectants in circulating water systems, washing machines and even clothing. However, nanoparticles, like any other antibiotic that has a pervasive durable presence, carry the risk of creating a resistant population. In this study Mycobacterium smegmatis strain mc²155 was cultured in AgNP enriched agar such that only a small population survived. Surviving cultures were isolated and re-exposed to AgNPs and AgNO₃ and resistance to silver was compared to a negative control. After only a single exposure, mutant M. smegmatis populations were resistant to AgNPs and AgNO₃. Further, the silver resistant mutants were exposed to antibiotics to determine if general resistance had been conferred. The minimum inhibitory concentration of isoniazid was four times higher for silver resistant mutants than for strain mc²155. However, core resistance was not conferred to other toxic metal ions. The mutants had lower resistance to CuSO₄ and ZnSO₄ than the mc²155 strain.     

Proteomic Analysis of Drug-Resistant Mycobacteria: Co-Evolution of Copper and INH Resistance

Tuberculosis, caused by the pathogen Mycobacterium tuberculosis, is a worldwide public health threat. Mycobacterium tuberculosis is capable of resisting various stresses in host cells, including high levels of ROS and copper ions. To better understand the resistance mechanisms of mycobacteria to copper, we generated a copper-resistant strain of Mycobacterium smegmatis, mc²155-Cu from the selection of copper sulfate treated-bacteria. The mc²155-Cu strain has a 5-fold higher resistance to copper sulfate and a 2-fold higher resistance to isoniazid (INH) than its parental strain mc²155, respectively. Quantitative proteomics was carried out to find differentially expressed proteins between mc²155 and mc²155-Cu. Among 345 differentially expressed proteins, copper-translocating P-type ATPase was up-regulated, while all other ABC transporters were down-regulated in mc²155-Cu, suggesting copper-translocating P-type ATPase plays a crucial role in copper resistance. Results also indicated that the down-regulation of metabolic enzymes and decreases in cellular NAD, FAD, mycothiol, and glutamine levels in mc²155-Cu were responsible for its slowing growth rate as compared to mc²155. Down-regulation of KatG2 expression in both protein and mRNA levels indicates the co-evolution of copper and INH resistance in copper resistance bacteria, and provides new evidence to understanding of the molecular mechanisms of survival of mycobacteria under stress conditions.     

The Mycobacterium avium complex gtfTB gene encodes a glucosyltransferase required for the biosynthesis of serovar 8-specific glycopeptidolipid

Mycobacterium avium complex (MAC) is one of the most common opportunistic pathogens widely distributed in the natural environment. The 28 serovars of MAC are defined by variable oligosaccharide portions of glycopeptidolipids (GPLs) that are abundant on the surface of the cell envelope. These GPLs are also known to contribute to the virulence of MAC. Serovar 8 is one of the dominant serovars isolated from AIDS patients, but the biosynthesis of serovar 8-specific GPL remains unknown. To clarify this, we compared gene clusters involved in the biosynthesis of several serovar-specific GPLs and identified the genomic region predicted to be responsible for GPL biosynthesis in a serovar 8 strain. Sequencing of this region revealed the presence of four open reading frames, three unnamed genes and gtfTB, the function of which has not been elucidated. The simultaneous expression of gtfTB and two downstream genes in a recombinant Mycobacterium smegmatis strain genetically modified to produce serovar 1-specific GPL resulted in the appearance of 4,6-O-(1-carboxyethylidene)-3-O-methyl-glucose, which is unique to serovar 8-specific GPL, suggesting that these three genes participate in its biosynthesis. Furthermore, functional analyses of gtfTB indicated that it encodes a glucosyltransferase that transfers a glucose residue via 1→3 linkage to a rhamnose residue of serovar 1-specific GPL, which is critical to the formation of the oligosaccharide portion of serovar 8-specific GPL. Our findings might provide a clue to understanding the biosynthetic regulation that modulates the biological functions of GPLs in MAC.     

Glycopeptidolipid Acetylation Affects Sliding Motility and Biofilm Formation in Mycobacterium smegmatis

The absence of glycopeptidolipids (GPLs) abolishes the ability of mycobacteria both to slide over the surface of motility plates and to form biofilms on polyvinyl chloride. In a screen for biofilm-defective mutants of Mycobacterium smegmatis mc(2)155, a new mutant was obtained that resulted in partial inhibition of both processes and also showed an intermediate rough colony morphology. The mariner transposon insertion mapped to a GPL biosynthesis gene (atf1) which encodes a putative acetyltranferase involved in the transfer of acetyl groups to the glycopeptide core. Physical characterization of the GPLs from the atf1 mutant demonstrated that they were not acetylated.     

Modulation of isoniazid susceptibility by flavonoids in Mycobacterium

In the course of a project to identify plant natural products which modulate the susceptibility of different strains of fast-growing mycobacteria to the first-line antituberculotic isoniazid (INH), several flavonoids without significant antimycobacterial activities at the tested concentrations were screened for their ability to decrease the minimum inhibitory concentrations (MICs) of INH. Flavonoids with different substitution patterns, namely epicatechin, isorhamnetin, kaempferol, luteolin, myricetin, quercetin, rutin and taxifolin were tested to examine structure–activity relationships (SARs) of these compounds. Different mycobacterial strains, i.e. Mycobacterium smegmatis (ATCC 14468), M. smegmatis mc²155 (ATCC 700084), M. smegmatis mc²2700, M. phlei (ATCC 11758) and M. fortuitum (ATCC 6841) were used. The strongest synergistic effects were observed in M. smegmatis mc²155 followed by M. phlei, whereas the tendency of INH potentiation by certain flavonoids remained the same within each strain. Myricetin was the most efficient intensifier of INH susceptibility in all tested strains causing a decrease of the MIC of INH up to 64-fold at 16 μg/ml, followed by quercetin. Structure–activity relationships of flavonoids as intensifiers of INH susceptibility in mycobacteria indicate that they overlap with SARs for their radical-scavenging properties, however the potentiation of INH activity cannot only be explained by their radical-scavenging activity alone.     

SmoXYB1C1Z of Mycobacterium sp. Strain NBB4: a Soluble Methane Monooxygenase (sMMO)-Like Enzyme,Active on C2 to C4 Alkanes and Alkenes

Monooxygenase (MO) enzymes initiate the aerobic oxidation of alkanes and alkenes in bacteria. A cluster of MO genes (smoXYB1C1Z) of thus-far-unknown function was found previously in the genomes of two Mycobacterium strains (NBB3 and NBB4) which grow on hydrocarbons. The predicted Smo enzymes have only moderate amino acid identity (30 to 60%) to their closest homologs, the soluble methane and butane MOs (sMMO and sBMO), and the smo gene cluster has a different organization from those of sMMO and sBMO. The smoXYB1C1Z genes of NBB4 were cloned into pMycoFos to make pSmo, which was transformed into Mycobacterium smegmatis mc²-155. Cells of mc²-155(pSmo) metabolized C₂ to C₄ alkanes, alkenes, and chlorinated hydrocarbons. The activities of mc²-155(pSmo) cells were 0.94, 0.57, 0.12, and 0.04 nmol/min/mg of protein with ethene, ethane, propane, and butane as substrates, respectively. The mc²-155(pSmo) cells made epoxides from ethene, propene, and 1-butene, confirming that Smo was an oxygenase. Epoxides were not produced from larger alkenes (1-octene and styrene). Vinyl chloride and 1,2-dichloroethane were biodegraded by cells expressing Smo, with production of inorganic chloride. This study shows that Smo is a functional oxygenase which is active against small hydrocarbons. M. smegmatis mc²-155(pSmo) provides a new model for studying sMMO-like monooxygenases.     

Mycobacterium smegmatis is a suitable cell factory for the production of steroidic synthons

A number of pharmaceutical steroid synthons are currently produced through the microbial side‐chain cleavage of natural sterols as an alternative to multi‐step chemical synthesis. Industrially, these synthons have been usually produced through fermentative processes using environmental isolated microorganisms or their conventional mutants. Mycobacterium smegmatis mc²155 is a model organism for tuberculosis studies which uses cholesterol as the sole carbon and energy source for growth, as other mycobacterial strains. Nevertheless, this property has not been exploited for the industrial production of steroidic synthons. Taking advantage of our knowledge on the cholesterol degradation pathway of M. smegmatis mc²155 we have demonstrated that the MSMEG_6039 (kshB1) and MSMEG_5941 (kstD1) genes encoding a reductase component of the 3‐ketosteroid 9α‐hydroxylase (KshAB) and a ketosteroid Δ¹‐dehydrogenase (KstD), respectively, are indispensable enzymes for the central metabolism of cholesterol. Therefore, we have constructed a MSMEG_6039 (kshB1) gene deletion mutant of M. smegmatis MS6039 that transforms efficiently natural sterols (e.g. cholesterol and phytosterols) into 1,4‐androstadiene‐3,17‐dione. In addition, we have demonstrated that a double deletion mutant M. smegmatis MS6039‐5941 [ΔMSMEG_6039 (ΔkshB1) and ΔMSMEG_5941 (ΔkstD1)] transforms natural sterols into 4‐androstene‐3,17‐dione with high yields. These findings suggest that the catabolism of cholesterol in M. smegmatis mc²155 is easy to handle and equally efficient for sterol transformation than other industrial strains, paving the way for valuating this strain as a suitable industrial cell factory to develop à la carte metabolic engineering strategies for the industrial production of pharmaceutical steroids.     

Prime–Boost with Mycobacterium smegmatis Recombinant Vaccine Improves Protection in Mice Infected with Mycobacterium tuberculosis

The development of a new vaccine as a substitute for Bacillus Calmette–Guerin or to improve its efficacy is one of the many World Health Organization goals to control tuberculosis. Mycobacterial vectors have been used successfully in the development of vaccines against tuberculosis. To enhance the potential utility of Mycobacterium smegmatis as a vaccine, it was transformed with a recombinant plasmid containing the partial sequences of the genes Ag85c, MPT51, and HspX (CMX) from M. tuberculosis. The newly generated recombinant strain mc²-CMX was tested in a murine model of infection. The recombinant vaccine induced specific IgG1 or IgG2a responses to CMX. CD4⁺ and CD8⁺ T cells from the lungs and spleen responded ex vivo to CMX, producing IFN-γ, IL17, TNF-α, and IL2. The vaccine thus induced a significant immune response in mice. Mice vaccinated with mc²-CMX and challenged with M. tuberculosis showed better protection than mice immunized with wild-type M. smegmatis or BCG. To increase the safety and immunogenicity of the CMX antigens, we used a recombinant strain of M. smegmatis, IKE (immune killing evasion), to express CMX. The recombinant vaccine IKE-CMX induced a better protective response than mc²-CMX. The data presented here suggest that the expression of CMX antigens improves the immune response and the protection induced in mice when M. smegmatis is used as vaccine against tuberculosis.     

Roles for cell wall glycopeptidolipid in surface adherence and planktonic dispersal of Mycobacterium avium

The opportunistic pathogen Mycobacterium avium is a significant inhabitant of biofilms in drinking water distribution systems. M. avium expresses on its cell surface serovar-specific glycopeptidolipids (ssGPLs). Studies have implicated the core GPL in biofilm formation by M. avium and by other Mycobacterium species. In order to test this hypothesis in a directed fashion, three model systems were used to examine biofilm formation by mutants of M. avium with transposon insertions into pstAB (also known as nrp and mps). pstAB encodes the nonribosomal peptide synthetase that catalyzes the synthesis of the core GPL. The mutants did not adhere to polyvinyl chloride plates; however, they adhered well to plastic and glass chamber slide surfaces, albeit with different morphologies from the parent strain. In a model that quantified surface adherence under recirculating water, wild-type and pstAB mutant cells accumulated on stainless steel surfaces in equal numbers. Unexpectedly, pstAB mutant cells were >10-fold less abundant in the recirculating-water phase than parent strain cells. These observations show that GPLs are directly or indirectly required for colonization of some, but by no means all, surfaces. Under some conditions, GPLs may play an entirely different role by facilitating the survival or dispersal of nonadherent M. avium cells in circulating water. Such a function could contribute to waterborne M. avium infection.     

The Novel Responses of Ethambutol Against <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis> mc<Superscript>2</Superscript>155 Revealed by Proteomics Analysis

Ethambutol (EMB), one of the effective anti-mycobacterial drugs, inhibits the biosynthesis of mycobacterium cell wall. To elucidate the molecular mechanism of EMB against tuberculosis (TB), Mycobacterium smegmatis mc²155 was employed as a model of mycobacterial system in this study. We compared the protein profiles on M. smegmatis mc²155 treated by EMB and untreated using fluorescence difference two-dimensional gel electrophoresis (2-D DIGE). A total of 40 differential protein spots were selected and 22 proteins were identified by HPLC-nano ESI–MS/MS analysis, including 16 over-expressed proteins and 6 under-expressed proteins. These proteins mainly affected energy metabolism, as well as synthesis and modification of macromolecules. The expressions of correspondent genes were confirmed by RT-PCR. This investigation provided some clues for searching potential drug targets.     

Regulation of the ahpC Gene Encoding Alkyl Hydroperoxide Reductase in Mycobacterium smegmatis

The ahpC (MSMEG_4891) gene encodes alkyl hydroperoxide reductase C in Mycobacterium smegmatis mc²155 and its expression is induced under oxidative stress conditions. Two well-defined inverted repeat sequences (IR1 and IR2) were identified in the upstream region of ahpC. Using a crp (cAMP receptor protein: MSMEG_6189) mutant and in vitro DNA-binding assay, it was demonstrated that the IR1 sequence serves as a Crp-binding site and that Crp functions as an activator in the regulation of ahpC expression. The expression level of ahpC was shown to be proportional to intracellular cAMP levels. Intracellular levels of cAMP were increased in M. smegmatis, when it was treated with oxidative stress inducers. The IR2 sequence is very similar to the known consensus sequence of FurA-binding sites and involved in the negative regulation of ahpC expression. Taken together, these results suggest that the induction of ahpC expression under oxidative stress conditions probably results from a combinatory effect of both inactivation of FurA by oxidative stress and activation of Crp in response to increased levels of cAMP.     

Absence of YhdP,TamB, and YdbH leads to defects in glycerophospholipid transport and cell morphology in Gram-negative bacteria

The outer membrane (OM) of Gram-negative bacteria provides the cell with a formidable barrier that excludes external threats. The two major constituents of this asymmetric barrier are lipopolysaccharide (LPS) found in the outer leaflet, and glycerophospholipids (GPLs) in the inner leaflet. Maintaining the asymmetric nature and balance of LPS to GPLs in the OM is critical for bacterial viability. The biosynthetic pathways of LPS and GPLs are well characterized, but unlike LPS transport, how GPLs are translocated to the OM remains enigmatic. Understanding this aspect of cell envelope biology could provide a foundation for new antibacterial therapies. Here, we report that YhdP and its homologues, TamB and YdbH, members of the “AsmA-like” family, are critical for OM integrity and necessary for proper GPL transport to the OM. The absence of the two largest AsmA-like proteins (YhdP and TamB) leads to cell lysis and antibiotic sensitivity, phenotypes that are rescued by reducing LPS synthesis. We also find that yhdP, tamB double mutants shed excess LPS through outer membrane vesicles, presumably to maintain OM homeostasis when normal anterograde GPL transport is disrupted. Moreover, a yhdP, tamB, ydbH triple mutant is synthetically lethal, but if GPL transport is partially restored by overexpression of YhdP, the cell shape adjusts to accommodate increased membrane content as the cell accumulates GPLs in the IM. Our results therefore suggest a model in which “AsmA-like” proteins transport GPLs to the OM, and when hindered, changes in cell shape and shedding of excess LPS aids in maintaining OM asymmetry.     

Roles for Cell Wall Glycopeptidolipid in Surface Adherence and Planktonic Dispersal of Mycobacterium avium

The opportunistic pathogen Mycobacterium avium is a significant inhabitant of biofilms in drinking water distribution systems. M. avium expresses on its cell surface serovar-specific glycopeptidolipids (ssGPLs). Studies have implicated the core GPL in biofilm formation by M. avium and by other Mycobacterium species. In order to test this hypothesis in a directed fashion, three model systems were used to examine biofilm formation by mutants of M. avium with transposon insertions into pstAB (also known as nrp and mps). pstAB encodes the nonribosomal peptide synthetase that catalyzes the synthesis of the core GPL. The mutants did not adhere to polyvinyl chloride plates; however, they adhered well to plastic and glass chamber slide surfaces, albeit with different morphologies from the parent strain. In a model that quantified surface adherence under recirculating water, wild-type and pstAB mutant cells accumulated on stainless steel surfaces in equal numbers. Unexpectedly, pstAB mutant cells were >10-fold less abundant in the recirculating-water phase than parent strain cells. These observations show that GPLs are directly or indirectly required for colonization of some, but by no means all, surfaces. Under some conditions, GPLs may play an entirely different role by facilitating the survival or dispersal of nonadherent M. avium cells in circulating water. Such a function could contribute to waterborne M. avium infection.     

Acanthamoeba polyphaga-enhanced growth of Mycobacterium smegmatis

Background

Mycobacterium smegmatis is a rapidly-growing mycobacterium causing rare opportunistic infections in human patients. It is present in soil and water environments where free-living amoeba also reside, but data regarding M. smegmatis-amoeba relationships have been contradictory from mycobacteria destruction to mycobacteria survival.

Methodology/Principal Findings

Using optic and electron microscopy and culture-based microbial enumeration we investigated the ability of M. smegmatis mc² 155, M. smegmatis ATCC 19420^T and M. smegmatis ATCC 27204 organisms to survive into Acanthamoeba polyphaga trophozoites and cysts. We observed that M. smegmatis mycobacteria penetrated and survived in A. polyphaga trophozoites over five-day co-culture resulting in amoeba lysis and the release of viable M. smegmatis mycobacteria without amoebal cyst formation. We further observed that amoeba-co-culture, and lysed amoeba and supernatant and pellet, significantly increased five-day growth of the three tested M. smegmatis strains, including a four-fold increase in intra-amoebal growth.

Conclusions/Significance

Amoebal co-culture increases the growth of M. smegmatis resulting in amoeba killing by replicating M. smegmatis mycobacteria. This amoeba-M. smegmatis co-culture system illustrates an unusual paradigm in the mycobacteria-amoeba interactions as mycobacteria have been mainly regarded as amoeba-resistant organisms. Using these model organisms, this co-culture system could be used as a simple and rapid model to probe mycobacterial factors implicated in the intracellular growth of mycobacteria.     

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.     

Cluster J Mycobacteriophages: Intron Splicing in Capsid and Tail Genes

Bacteriophages isolated on Mycobacterium smegmatis mc²155 represent many distinct genomes sharing little or no DNA sequence similarity. The genomes are architecturally mosaic and are replete with genes of unknown function. A new group of genomes sharing substantial nucleotide sequences constitute Cluster J. The six mycobacteriophages forming Cluster J are morphologically members of the Siphoviridae, but have unusually long genomes ranging from 106.3 to 117 kbp. Reconstruction of the capsid by cryo-electron microscopy of mycobacteriophage BAKA reveals an icosahedral structure with a triangulation number of 13. All six phages are temperate and homoimmune, and prophage establishment involves integration into a tRNA-Leu gene not previously identified as a mycobacterial attB site for phage integration. The Cluster J genomes provide two examples of intron splicing within the virion structural genes, one in a major capsid subunit gene, and one in a tail gene. These genomes also contain numerous free-standing HNH homing endonuclease, and comparative analysis reveals how these could contribute to genome mosaicism. The unusual Cluster J genomes provide new insights into phage genome architecture, gene function, capsid structure, gene mobility, intron splicing, and evolution.     

Analysis of the Exochelin Locus in Mycobacterium smegmatis: Biosynthesis Genes Have Homology with Genes of the Peptide Synthetase Family

Mycobacteria secrete the siderophore exochelin when grown under iron-limiting conditions. In order to understand iron uptake mechanisms in mycobacteria, we have taken a genetic approach to identify those genes involved in exochelin biosynthesis and transport in Mycobacterium smegmatis. Of the 6,000 chemically mutagenized clones of M. smegmatis mc²155 screened on agar plates containing chrome azural S, 19 mutants that had lost the ability to produce or secrete exochelin were identified. Thirteen of these mutants were complemented by a single M. smegmatis cosmid. Sequence analysis of this cosmid revealed nine open reading frames, three of which are homologous to genes encoding transporter proteins, which are likely involved in exochelin transport. Complementation and Tn10 mutagenesis analysis identified two new genes, fxbB and fxbC, which are required for exochelin biosynthesis. The fxbB and fxbC genes encode large proteins of 257 and 497 kDa, respectively, which are highly homologous to peptide synthetases, indicating that exochelin biosynthesis occurs by a nonribosomal mechanism.