scholarly journals Identification of Genes Encoding Exported Mycobacterium tuberculosis Proteins Using a Tn552′phoA In Vitro Transposition System

2000 ◽  
Vol 182 (10) ◽  
pp. 2732-2740 ◽  
Author(s):  
Miriam Braunstein ◽  
Thomas J. Griffin ◽  
Jordan I. Kriakov ◽  
Sarah T. Friedman ◽  
Nigel D. F. Grindley ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Protective Immunity ◽  
Cell Envelope ◽  
Open Reading Frames ◽  
Signal Peptides ◽  
Genomic Libraries ◽  
Target Dna ◽  
Genes Encoding ◽  
Associated Proteins

ABSTRACT Secreted and cell envelope-associated proteins are important to both Mycobacterium tuberculosis pathogenesis and the generation of protective immunity to M. tuberculosis. We used an in vitro Tn552′phoA transposition system to identify exported proteins of M. tuberculosis. The system is simple and efficient, and the transposon inserts randomly into target DNA. M. tuberculosis genomic libraries were targeted with Tn552′phoA transposons, and these libraries were screened in M. smegmatis for active PhoA translational fusions. Thirty-two different M. tuberculosis open reading frames were identified; eight contain standard signal peptides, six contain lipoprotein signal peptides, and seventeen contain one or more transmembrane domains. Four of these proteins had not yet been assigned as exported proteins in the M. tuberculosisdatabases. This collection of exported proteins includes factors that are known to participate in the immune response of M. tuberculosis and proteins with homologies, suggesting a role in pathogenesis. Nine of the proteins appear to be unique to mycobacteria and represent promising candidates for factors that participate in protective immunity and virulence. This technology of creating comprehensive fusion libraries should be applicable to other organisms.

scholarly journals Compartmentalized Cell Envelope Biosynthesis in Mycobacterium tuberculosis

2022 ◽  
Author(s):  
Julia Puffal ◽  
Ian L. Sparks ◽  
James R. Brenner ◽  
Xuni Li ◽  
John D. Leszyk ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Proteomic Analysis ◽  
Fluorescent Protein ◽  
Cell Envelope ◽  
Membrane Domain ◽  
Protein Fusion ◽  
Final Maturation ◽  
Associated Proteins ◽  
Gradient Fractionation ◽  
Membrane Compartmentalization

The intracellular membrane domain (IMD) is a metabolically active and laterally discrete membrane domain initially discovered in Mycobacterium smegmatis. The IMD correlates both temporally and spatially with the polar cell envelope elongation in M. smegmatis. Whether or not a similar membrane domain exists in pathogenic species remains unknown. Here we show that the IMD is a conserved membrane structure found in Mycobacterium tuberculosis. We used two independent approaches, density gradient fractionation of membrane domains and visualization of IMD-associated proteins through fluorescence microscopy, to determine the characteristics of the plasma membrane compartmentalization in M. tuberculosis. Proteomic analysis revealed that the IMD is enriched in metabolic enzymes that are involved in the synthesis of conserved cell envelope components such as peptidoglycan, arabinogalactan, and phosphatidylinositol mannosides. Using a fluorescent protein fusion of IMD-associated proteins, we demonstrated that this domain is concentrated in the polar region of the rod-shaped cells, where active cell envelope biosynthesis is taking place. Proteomic analysis further revealed the enrichment of enzymes involved in synthesis of phthiocerol dimycocerosates and phenolic glycolipids in the IMD. We validated the IMD association of two enzymes, α1,3-fucosyltransferase and fucosyl 4-O-methyltransferase, which are involved in the final maturation steps of phenolic glycolipid biosynthesis. Taken together, these data indicate that functional compartmentalization of membrane is an evolutionarily conserved feature found in both M. tuberculosis and M. smegmatis, and M. tuberculosis utilizes this membrane location for the synthesis of its surface-exposed lipid virulence factors.

scholarly journals Virulence Potential of Ehrlichia chaffeensis Strains of Distinct Genome Sequences

2007 ◽  
Vol 75 (7) ◽  
pp. 3604-3613 ◽  
Author(s):  
Koshiro Miura ◽  
Yasuko Rikihisa
Keyword(s):  
Cell Envelope ◽  
Severe Combined Immunodeficiency ◽  
Clinical Signs ◽  
Granulomatous Inflammation ◽  
Open Reading Frames ◽  
Comparative Genome Hybridization ◽  
Ehrlichia Chaffeensis ◽  
Genome Sequences ◽  
Genes Encoding ◽  
Ankyrin Repeat Proteins

ABSTRACT Human monocytic ehrlichiosis, one of the most frequent life-threatening tick-borne zoonoses, is caused by Ehrlichia chaffeensis that lacks endotoxin and peptidoglycan. While sequence polymorphisms in several genes in E. chaffeensis strains have been reported, global genomic divergence and biological differences among strains are unknown. The objectives of the present study were to compare the genome sequences of strains of E. chaffeensis and to examine the virulence potentials of the strains with defined genome sequences. Genomic DNA was extracted from purified E. chaffeensis strains Wakulla and Liberty, and comparative genome hybridization was performed using a densely tiled microarray of 147,027 chromosome positions of the E. chaffeensis strain Arkansas genome. The results revealed that 4,663 and 5,325 positions in the chromosomes of strains Wakulla and Liberty, respectively, were different from those in the chromosome of strain Arkansas, including three common major polymorphic chromosomal regions. Of various functional categories, the differences were most concentrated in genes predicted to encode cell envelope proteins. Of all the open reading frames (ORFs), 21 omp-1 (p28 gene) paralogs, nine genes encoding hypothetical proteins, two genes encoding ankyrin repeat proteins, and hemE contained the most differences. Several highly polymorphic ORFs were confirmed by sequencing. When the E. chaffeensis strains were inoculated into severe combined immunodeficiency mice, the order of the severity of clinical signs and the bacterial burden detected in mice was Wakulla > Liberty > Arkansas. Severe diffuse inflammation and granulomatous inflammation were evident in the livers of mice infected with strains Wakulla and Arkansas, respectively, but not in the livers of mice infected with strain Liberty. These results revealed distinct virulence phenotypes of E. chaffeensis strains with defined genome sequences.

scholarly journals Therv1184cLocus Encodes Chp2, an Acyltransferase in Mycobacterium tuberculosis Polyacyltrehalose Lipid Biosynthesis

2014 ◽  
Vol 197 (1) ◽  
pp. 201-210 ◽  
Author(s):  
Megan H. Touchette ◽  
Cynthia M. Holsclaw ◽  
Mary L. Previti ◽  
Viven C. Solomon ◽  
Julie A. Leary ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cell Envelope ◽  
Selective Inhibition ◽  
Serine Hydrolase ◽  
Acyltransferase Activity ◽  
Content Type ◽  
Regulatory Interactions ◽  
Similar Phenotype ◽  
Lipid Transporter

Trehalose glycolipids are found in many bacteria in the suborderCorynebacterineae, but methyl-branched acyltrehaloses are exclusive to virulent species such as the human pathogenMycobacterium tuberculosis. InM. tuberculosis, the acyltransferase PapA3 catalyzes the formation of diacyltrehalose (DAT), but the enzymes responsible for downstream reactions leading to the final product, polyacyltrehalose (PAT), have not been identified. The PAT biosynthetic gene locus is similar to that of another trehalose glycolipid, sulfolipid 1. Recently, Chp1 was characterized as the terminal acyltransferase in sulfolipid 1 biosynthesis. Here we provide evidence that the homologue Chp2 (Rv1184c) is essential for the final steps of PAT biosynthesis. Disruption ofchp2led to the loss of PAT and a novel tetraacyltrehalose species, TetraAT, as well as the accumulation of DAT, implicating Chp2 as an acyltransferase downstream of PapA3. Disruption of the putative lipid transporter MmpL10 resulted in a similar phenotype. Chp2 activity thus appears to be regulated by MmpL10 in a relationship similar to that between Chp1 and MmpL8 in sulfolipid 1 biosynthesis. Chp2 is localized to the cell envelope fraction, consistent with its role in DAT modification and possible regulatory interactions with MmpL10. Labeling of purified Chp2 by an activity-based probe was dependent on the presence of the predicted catalytic residue Ser141 and was inhibited by the lipase inhibitor tetrahydrolipstatin (THL). THL treatment ofM. tuberculosisresulted in selective inhibition of Chp2 over PapA3, confirming Chp2 as a member of the serine hydrolase superfamily. Efforts to producein vitroreconstitution of acyltransferase activity using straight-chain analogues were unsuccessful, suggesting that Chp2 has specificity for native methyl-branched substrates.

scholarly journals Defining Daptomycin Resistance Prevention Exposures in Vancomycin-Resistant Enterococcus faecium and E. faecalis

2014 ◽  
Vol 58 (9) ◽  
pp. 5253-5261 ◽  
Author(s):  
B. J. Werth ◽  
M. E. Steed ◽  
C. E. Ireland ◽  
T. T. Tran ◽  
P. Nonejuie ◽  
...  
Keyword(s):  
Enterococcus Faecium ◽  
Cell Envelope ◽  
Fatty Acid Synthetase ◽  
Phospholipid Metabolism ◽  
Cyclopropane Fatty Acid ◽  
Time Curve ◽  
Content Type ◽  
Genes Encoding ◽  
Enterococcal Infections

ABSTRACTDaptomycin is used off-label for enterococcal infections; however, dosing targets for resistance prevention remain undefined. Doses of 4 to 6 mg/kg of body weight/day approved for staphylococci are likely inadequate against enterococci due to reduced susceptibility. We modeled daptomycin regimensin vitroto determine the minimum exposure to prevent daptomycin resistance (Dapr) in enterococci. Daptomycin simulations of 4 to 12 mg/kg/day (maximum concentration of drug in serum [Cmax] of 57.8, 93.9, 123.3, 141.1, and 183.7 mg/liter; half-life [t1/2] of 8 h) were tested against oneEnterococcus faeciumstrain (S447) and oneEnterococcus faecalisstrain (S613) in a simulated endocardial vegetation pharmacokinetic/pharmacodynamic model over 14 days. Samples were plated on media containing 3× the MIC of daptomycin to detect Dapr. Mutations in genes encoding proteins associated with cell envelope homeostasis (yycFGandliaFSR) and phospholipid metabolism (cardiolipin synthase [cls] and cyclopropane fatty acid synthetase [cfa]) were investigated in Daprderivatives. Daprderivatives were assessed for changes in susceptibility, surface charge, membrane depolarization, cell wall thickness (CWT), and growth rate. Strains S447 and S613 developed Daprafter simulations of 4 to 8 mg/kg/day but not 10 to 12 mg/kg/day. MICs for Daprstrains ranged from 8 to 256 mg/liter. Some S613 derivatives developed mutations inliaForcls. S447 derivatives lacked mutations in these genes. Daprderivatives from both strains exhibited lowered growth rates, up to a 72% reduction in daptomycin-induced depolarization and up to 6-nm increases in CWT (P< 0.01). Peak/MIC and AUC0–24/MIC ratios (AUC0–24is the area under the concentration-time curve from 0 to 24 h) associated with Daprprevention were 72.1 and 780 for S447 and 144 and 1561 for S613, respectively. Daptomycin doses of 10 mg/kg/day may be required to prevent Daprin serious enterococcal infections.

scholarly journals Comprehensive Essentiality Analysis of the Mycobacterium tuberculosis Genome via Saturating Transposon Mutagenesis

mBio ◽  
2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Michael A. DeJesus ◽  
Elias R. Gerrick ◽  
Weizhen Xu ◽  
Sae Woong Park ◽  
Jarukit E. Long ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Noncoding Rnas ◽  
Open Reading Frames ◽  
Insertion Mutant ◽  
Statistical Interpretation ◽  
Genomic Features ◽  
Small Noncoding Rnas ◽  
Transposon Insertion ◽  
Biological Selection

ABSTRACT   For decades, identifying the regions of a bacterial chromosome that are necessary for viability has relied on mapping integration sites in libraries of random transposon mutants to find loci that are unable to sustain insertion. To date, these studies have analyzed subsaturated libraries, necessitating the application of statistical methods to estimate the likelihood that a gap in transposon coverage is the result of biological selection and not the stochasticity of insertion. As a result, the essentiality of many genomic features, particularly small ones, could not be reliably assessed. We sought to overcome this limitation by creating a completely saturated transposon library in Mycobacterium tuberculosis . In assessing the composition of this highly saturated library by deep sequencing, we discovered that a previously unknown sequence bias of the Himar1 element rendered approximately 9% of potential TA dinucleotide insertion sites less permissible for insertion. We used a hidden Markov model of essentiality that accounted for this unanticipated bias, allowing us to confidently evaluate the essentiality of features that contained as few as 2 TA sites, including open reading frames (ORF), experimentally identified noncoding RNAs, methylation sites, and promoters. In addition, several essential regions that did not correspond to known features were identified, suggesting uncharacterized functions that are necessary for growth. This work provides an authoritative catalog of essential regions of the M. tuberculosis genome and a statistical framework for applying saturating mutagenesis to other bacteria. IMPORTANCE Sequencing of transposon-insertion mutant libraries has become a widely used tool for probing the functions of genes under various conditions. The Himar1 transposon is generally believed to insert with equal probabilities at all TA dinucleotides, and therefore its absence in a mutant library is taken to indicate biological selection against the corresponding mutant. Through sequencing of a saturated Himar1 library, we found evidence that TA dinucleotides are not equally permissive for insertion. The insertion bias was observed in multiple prokaryotes and influences the statistical interpretation of transposon insertion (TnSeq) data and characterization of essential genomic regions. Using these insights, we analyzed a fully saturated TnSeq library for M. tuberculosis , enabling us to generate a comprehensive catalog of in vitro essentiality, including ORFs smaller than those found in any previous study, small (noncoding) RNAs (sRNAs), promoters, and other genomic features.

scholarly journals Ribonucleotide Reduction in Mycobacterium tuberculosis: Function and Expression of Genes Encoding Class Ib and Class II Ribonucleotide Reductases

2003 ◽  
Vol 71 (11) ◽  
pp. 6124-6131 ◽  
Author(s):  
Stephanie S. Dawes ◽  
Digby F. Warner ◽  
Liana Tsenova ◽  
Juliano Timm ◽  
John D. McKinney ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Class Ii ◽  
Growth Conditions ◽  
Small Subunit ◽  
Reverse Transcription Pcr ◽  
Hypoxic Conditions ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Class Ib

ABSTRACT Mycobacterium tuberculosis, the causative agent of tuberculosis, possesses a class Ib ribonucleotide reductase (RNR), encoded by the nrdE and nrdF2 genes, in addition to a putative class II RNR, encoded by nrdZ. In this study we probed the relative contributions of these RNRs to the growth and persistence of M. tuberculosis. We found that targeted knockout of the nrdF2 gene could be achieved only in the presence of a complementing allele, confirming that this gene is essential under normal, in vitro growth conditions. This observation also implied that the alternate class Ib small subunit encoded by the nrdF1 gene is unable to substitute for nrdF2 and that the class II RNR, NrdZ, cannot substitute for the class Ib enzyme, NrdEF2. Conversely, a ΔnrdZ null mutant of M. tuberculosis was readily obtained by allelic exchange mutagenesis. Quantification of levels of nrdE, nrdF2, nrdF1, and nrdZ gene expression by real-time, quantitative reverse transcription-PCR with molecular beacons by using mRNA from aerobic and O2-limited cultures showed that nrdZ was significantly induced under microaerophilic conditions, in contrast to the other genes, whose expression was reduced by O2 restriction. However, survival of the ΔnrdZ mutant strain was not impaired under hypoxic conditions in vitro. Moreover, the lungs of B6D2/F1 mice infected with the ΔnrdZ mutant had bacterial loads comparable to those of lungs infected with the parental wild-type strain, which argues against the hypothesis that nrdZ plays a significant role in the virulence of M. tuberculosis in this mouse model.

scholarly journals Glucan Is a Component of the Mycobacterium tuberculosis Surface That Is Expressed In Vitro and In Vivo

2002 ◽  
Vol 70 (5) ◽  
pp. 2566-2575 ◽  
Author(s):  
J. Reid Schwebach ◽  
Aharona Glatman-Freedman ◽  
Leslie Gunther-Cummins ◽  
Zhongdong Dai ◽  
John B. Robbins ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cell Envelope ◽  
Tween 80 ◽  
Enzyme Linked Immunosorbent Assay ◽  
In Vitro Growth ◽  
Comprehensive Picture ◽  
The Media ◽  
Surface Polysaccharide

ABSTRACT The outermost layer of Mycobacterium tuberculosis is composed primarily of two polysaccharides, glucan (GC) and arabinomannan. To analyze the surface polysaccharide composition of M. tuberculosis, we generated a monoclonal antibody (MAb) that binds M. tuberculosis GC and is known as MAb 24c5. Immunofluorescence and whole-mount immunoelectron microscopy indicated that GC is on the outermost portion of the bacteria. M. tuberculosis strains Erdman and CDC 1551 were analyzed for their ability to bind MAb 24c5 after in vitro growth in media with and without the detergent Tween 80. MAb 24c5 bound to Erdman and CDC 1551 at all culture times with only slightly greater apparent affinity after extended culture in the absence of Tween 80, indicating that a stable amount of GC polysaccharide antigen is associated with the cell surface of M. tuberculosis. An enzyme-linked immunosorbent assay indicated that GC is antigenically similar to glycogen, and the amount of GC antigen increased in the media of M. tuberculosis cultures grown either with or without the detergent Tween 80. Other nontuberculosis mycobacteria have antigenically similar GCs on their surfaces after in vitro growth. Inoculation of mice with live bacilli but not inoculation with dead bacilli elicited a strong antibody response to GC consistent with production of this antigen in vivo. Our results provide a more comprehensive picture of the M. tuberculosis cell envelope and the conditions that allow expression of M. tuberculosis GC.

scholarly journals Rv0180c contributes to Mycobacterium tuberculosis cell shape and to infectivity in mice and macrophages

2021 ◽  
Vol 17 (11) ◽  
pp. e1010020
Author(s):  
Delphine Payros ◽  
Henar Alonso ◽  
Wladimir Malaga ◽  
Arnaud Volle ◽  
Serge Mazères ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cell Shape ◽  
Cell Envelope ◽  
Membrane Receptors ◽  
Host Cells ◽  
Wild Type ◽  
Genes Encoding ◽  
The Difference ◽  
Complement Receptor 3 ◽  
New Perspective

Mycobacterium tuberculosis, the main causative agent of human tuberculosis, is transmitted from person to person via small droplets containing very few bacteria. Optimizing the chance to seed in the lungs is therefore a major adaptation to favor survival and dissemination in the human population. Here we used TnSeq to identify genes important for the early events leading to bacterial seeding in the lungs. Beside several genes encoding known virulence factors, we found three new candidates not previously described: rv0180c, rv1779c and rv1592c. We focused on the gene, rv0180c, of unknown function. First, we found that deletion of rv0180c in M. tuberculosis substantially reduced the initiation of infection in the lungs of mice. Next, we established that Rv0180c enhances entry into macrophages through the use of complement-receptor 3 (CR3), a major phagocytic receptor for M. tuberculosis. Silencing CR3 or blocking the CR3 lectin site abolished the difference in entry between the wild-type parental strain and the Δrv0180c::km mutant. However, we detected no difference in the production of both CR3-known carbohydrate ligands (glucan, arabinomannan, mannan), CR3-modulating lipids (phthiocerol dimycocerosate), or proteins in the capsule of the Δrv0180c::km mutant in comparison to the wild-type or complemented strains. By contrast, we established that Rv0180c contributes to the functionality of the bacterial cell envelope regarding resistance to toxic molecule attack and cell shape. This alteration of bacterial shape could impair the engagement of membrane receptors that M. tuberculosis uses to invade host cells, and open a new perspective on the modulation of bacterial infectivity.

scholarly journals Definition of novel cell envelope associated proteins in Triton X-114 extracts of Mycobacterium tuberculosis H37Rv

2010 ◽  
Vol 10 (1) ◽  
pp. 132 ◽  
Author(s):  
Hiwa Målen ◽  
Sharad Pathak ◽  
Tina Søfteland ◽  
Gustavo A de Souza ◽  
Harald G Wiker
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cell Envelope ◽  
Tuberculosis H37rv ◽  
Mycobacterium Tuberculosis H37rv ◽  
Associated Proteins ◽  
Definition Of ◽  
Triton X

scholarly journals The cell-envelope proteome of Bifidobacterium longum in an in vitro bile environment

Microbiology ◽  
2009 ◽  
Vol 155 (3) ◽  
pp. 957-967 ◽  
Author(s):  
Lorena Ruiz ◽  
Yohann Couté ◽  
Borja Sánchez ◽  
Clara G. de los Reyes-Gavilán ◽  
Jean-Charles Sanchez ◽  
...  
Keyword(s):  
Membrane Fraction ◽  
Ribosomal Proteins ◽  
Soluble Fraction ◽  
Cell Envelope ◽  
Bifidobacterium Longum ◽  
Cell Wall Proteins ◽  
Stable Isotope Labelling ◽  
Cytoplasmic Proteins ◽  
Associated Proteins

Host–bacteria interactions are often mediated via surface-associated proteins. The identification of these proteins is an important goal of bacterial proteomics. To address how bile can influence the cell-envelope proteome of Bifidobacterium longum biotype longum NCIMB 8809, we analysed its membrane protein fraction using stable isotope labelling of amino acids in cell culture (SILAC). We were able to identify 141 proteins in the membrane fraction, including a large percentage of the theoretical transporters of this species. Moreover, the envelope-associated soluble fraction was analysed using different subfractionation techniques and differential in-gel fluorescence electrophoresis (DIGE). This approach identified 128 different proteins. Some of them were well-known cell wall proteins, but others were highly conserved cytoplasmic proteins probably displaying a ‘moonlighting’ function. We were able to identify 11 proteins in the membrane fraction and 6 proteins in the envelope-associated soluble fraction whose concentration varied in the presence of bile. Bile promoted changes in the levels of proteins with important biological functions, such as some ribosomal proteins and enolase. Also, oligopeptide-binding proteins were accumulated on the cell surface, which was reflected in a different tripeptide transport rate in the cells grown with bile. The data reported here will provide the first cell-envelope proteome map for B. longum, and may contribute to understanding the bile tolerance of these bacteria.

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