scholarly journals Characterization of LtsA from Rhodococcus erythropolis, an Enzyme with Glutamine Amidotransferase Activity

2005 ◽  
Vol 187 (8) ◽  
pp. 2582-2591 ◽  
Author(s):  
Yasuo Mitani ◽  
XianYing Meng ◽  
Yoichi Kamagata ◽  
Tomohiro Tamura
Keyword(s):  
Cell Wall ◽  
Culture Media ◽  
Rhodococcus Erythropolis ◽  
Mycolic Acid ◽  
Site Directed Mutagenesis ◽  
Host Cells ◽  
Wall Structure ◽  
Synthetase Activity ◽  
Glutamine Amidotransferase ◽  
Glutaminase Activity

ABSTRACT The nocardioform actinomycete Rhodococcus erythropolis has a characteristic cell wall structure. The cell wall is composed of arabinogalactan and mycolic acid and is highly resistant to the cell wall-lytic activity of lysozyme (muramidase). In order to improve the isolation of recombinant proteins from R. erythropolis host cells (N. Nakashima and T. Tamura, Biotechnol. Bioeng. 86:136-148, 2004), we isolated two mutants, L-65 and L-88, which are susceptible to lysozyme treatment. The lysozyme sensitivity of the mutants was complemented by expression of Corynebacterium glutamicum ltsA, which codes for an enzyme with glutamine amidotransferase activity that results from coupling of two reactions (a glutaminase activity and a synthetase activity). The lysozyme sensitivity of the mutants was also complemented by ltsA homologues from Bacillus subtilis and Mycobacterium tuberculosis, but the homologues from Streptomyces coelicolor and Escherichia coli did not complement the sensitivity. This result suggests that only certain LtsA homologues can confer lysozyme resistance. Wild-type recombinant LtsA from R. erythropolis showed glutaminase activity, but the LtsA enzymes from the L-88 and L-65 mutants displayed drastically reduced activity. Interestingly, an ltsA disruptant mutant, which expressed the mutated LtsA, changed from lysozyme sensitive to lysozyme resistant when NH4Cl was added into the culture media. The glutaminase activity of the LtsA mutants inactivated by site-directed mutagenesis was also restored by addition of NH4Cl, indicating that NH3 can be used as an amide donor molecule. Taken together, these results suggest that LtsA is critically involved in mediating lysozyme resistance in R. erythropolis cells.

scholarly journals Carbon Source-Induced Modifications in the Mycolic Acid Content and Cell Wall Permeability of Rhodococcus erythropolis E1

2003 ◽  
Vol 69 (12) ◽  
pp. 7019-7027 ◽  
Author(s):  
Ivana Sokolovská ◽  
Raoul Rozenberg ◽  
Christophe Riez ◽  
Paul G. Rouxhet ◽  
Spiros N. Agathos ◽  
...  
Keyword(s):  
Cell Wall ◽  
Carbon Source ◽  
Acid Content ◽  
Rhodococcus Erythropolis ◽  
Mycolic Acid ◽  
Mycolic Acids ◽  
Carbon Chains ◽  
Linear Alkanes ◽  
Wall Permeability ◽  
Cell Wall Permeability

ABSTRACT The influence of the carbon source on cell wall properties was analyzed in an efficient alkane-degrading strain of Rhodococcus erythropolis (strain E1), with particular focus on the mycolic acid content. A clear correlation was observed between the carbon source and the mycolic acid profiles as estimated by high-performance liquid chromatography and mass spectrometry. Two types of mycolic acid patterns were observed after growth either on saturated linear alkanes or on short-chain alkanoates. One type of pattern was characterized by the lack of odd-numbered carbon chains and resulted from growth on linear alkanes with even numbers of carbon atoms. The second type of pattern was characterized by mycolic acids with both even- and odd-numbered carbon chains and resulted from growth on compounds with odd-numbered carbon chains, on branched alkanes, or on mixtures of different compounds. Cellular short-chain fatty acids were twice as abundant during growth on a branched alkane (pristane) as during growth on acetate, while equal amounts of mycolic acids were found under both conditions. More hydrocarbon-like compounds and less polysaccharide were exposed at the cell wall surface during growth on alkanes. Whatever the substrate, the cells had the same affinity for aqueous-nonaqueous solvent interfaces. By contrast, bacteria displayed completely opposite susceptibilities to hydrophilic and hydrophobic antibiotics and were found to be strongly stained by hydrophobic dyes after growth on pristane but not after growth on acetate. Taken together, these data show that the cell wall composition of R. erythropolis E1 is influenced by the nutritional regimen and that the most marked effect is a radical change in cell wall permeability.

scholarly journals The aceE involves in mycolic acid synthesis and biofilm formation in Mycobacterium smegmatis

2020 ◽  
Author(s):  
Suting Chen ◽  
Tianlu Teng ◽  
Shuan Wen ◽  
Tingting Zhang ◽  
Hairong Huang
Keyword(s):  
Cell Wall ◽  
Biofilm Formation ◽  
Morphological Changes ◽  
Acid Synthesis ◽  
Mycolic Acid ◽  
Colony Morphology ◽  
Wall Structure ◽  
Wild Type ◽  
Mycobacterial Cell Wall

Abstract Background: The integrity of cell wall structure is highly significant for the in vivo survival for mycobacteria. However, the mechanisms underlying the biosynthesis of mycobacterial cell wall remain poorly understood. aceE encodes the E1 component of pyruvate dehydrogenase (PDH)complex. This study aimed to know the functional role of aceE gene in cell wall biosynthesis in M. smegmatis.Results: We observed that the colony morphology of aceE-deficient mutants(aceE-mut)was quite different from the wild-type(WT) strain during the transposon library screening of M.smegmatis, smaller and smoother on the solid culture medium. Notably, the aceE-mut lost its ability of growing aggregately and biofilm forming, which are two very important features of mycobacteria.The morphological changes of the aceE-mut strain were further confirmed by electron microscopy that presented shorter, smoother and thinner images in contrast withWT strain.Additionally, the analysis of mycolic acid(MA)using LC-MS indicated deficiency of alpha-MA and epoxy-MA in aceE-mut strain whereas complementation of the aceE-mut with a wild-type aceEgene restored the composition of MA. Conclusions: Overall, this study indicates that aceE gene plays a significant role in the mycolic acid synthesis and affects the colony morphology and biofilm formation of M.smegmatis.

scholarly journals Biosynthesis and Virulent Behavior of Lipids Produced by Mycobacterium tuberculosis: LAM and Cord Factor: An Overview

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Rajni ◽  
Nisha Rao ◽  
Laxman S. Meena
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Host Cell ◽  
Phosphatidyl Inositol ◽  
Polymorphonuclear Neutrophils ◽  
Host Cells ◽  
Wall Structure ◽  
Trehalose Dimycolate ◽  
Cell Immunity ◽  
Cord Factor

Mycobacterium tuberculosis is the causative agent of tuberculosis disease, which has developed a myriad of exceptional features contributing to its survival within the hostile environment of host cell. Unique cell wall structure with high lipid content plays an imperative role in the pathogenicity of mycobacteria. Cell wall components of MTB such as lipoarabinomannan and Trehalose dimycolate (cord factor) are virulent in nature apart from its virulence genes. Virulent effect of these factors on host cells reduces host cell immunity. LAM has been known to inhibit phagosome maturation by inhibiting the Ca2+/calmodulin phosphatidyl inositol-3-kinase hvps34 pathways. Moreover, TDM (Trehalose dimycolate) also inhibits fusion between phospholipid vesicles and migration of polymorphonuclear neutrophils. The objective of this paper is to understand the virulence of LAM and cord factor on host cell which might be helpful to design an effective drug against tuberculosis.

scholarly journals FbpA-Dependent Biosynthesis of Trehalose Dimycolate Is Required for the Intrinsic Multidrug Resistance, Cell Wall Structure, and Colonial Morphology of Mycobacterium smegmatis

2005 ◽  
Vol 187 (19) ◽  
pp. 6603-6611 ◽  
Author(s):  
Liem Nguyen ◽  
Satheesh Chinnapapagari ◽  
Charles J. Thompson
Keyword(s):  
Cell Wall ◽  
Antibiotic Resistance ◽  
Mycobacterium Smegmatis ◽  
Mycolic Acid ◽  
Wall Structure ◽  
Single Mutation ◽  
Mycolic Acids ◽  
Trehalose Dimycolate ◽  
Colonial Morphology ◽  
Mycobacterial Cell Wall

ABSTRACT Ligation of mycolic acids to structural components of the mycobacterial cell wall generates a hydrophobic, impermeable barrier that provides resistance to toxic compounds such as antibiotics. Secreted proteins FbpA, FbpB, and FbpC attach mycolic acids to arabinogalactan, generating mycolic acid methyl esters (MAME) or trehalose, generating α,α′-trehalose dimycolate (TDM; also called cord factor). Our studies of Mycobacterium smegmatis showed that disruption of fbpA did not affect MAME levels but resulted in a 45% reduction of TDM. The fbpA mutant displayed increased sensitivity to both front-line tuberculosis-targeted drugs as well as other broad-spectrum antibiotics widely used for antibacterial chemotherapy. The irregular, hydrophobic surface of wild-type M. smegmatis colonies became hydrophilic and smooth in the mutant. While expression of M. smegmatis fbpA restored defects of the mutant, heterologous expression of the Mycobacterium tuberculosis fbpA gene was less effective. A single mutation in the M. smegmatis FbpA esterase domain inactivated its ability to provide antibiotic resistance. These data show that production of TDM by FbpA is essential for the intrinsic antibiotic resistance and normal colonial morphology of some mycobacteria and support the concept that FbpA-specific inhibitors, alone or in combination with other antibiotics, could provide an effective treatment to tuberculosis and other mycobacterial diseases.

scholarly journals An Inhibitor of Exported Mycobacterium tuberculosis Glutamine Synthetase Selectively Blocks the Growth of Pathogenic Mycobacteria in Axenic Culture and in Human Monocytes: Extracellular Proteins as Potential Novel Drug Targets

1999 ◽  
Vol 189 (9) ◽  
pp. 1425-1436 ◽  
Author(s):  
Günter Harth ◽  
Marcus A. Horwitz
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Glutamine Synthetase ◽  
Extracellular Proteins ◽  
Mononuclear Phagocytes ◽  
Cell Wall Structure ◽  
Host Cells ◽  
Wall Structure ◽  
Mycobacterial Cell ◽  
Mycobacterial Cell Wall

Mycobacterium tuberculosis and other pathogenic mycobacteria export abundant quantities of proteins into their extracellular milieu when growing either axenically or within phagosomes of host cells. One major extracellular protein, the enzyme glutamine synthetase, is of particular interest because of its link to pathogenicity. Pathogenic mycobacteria, but not nonpathogenic mycobacteria, export large amounts of this protein. Interestingly, export of the enzyme is associated with the presence of a poly-l-glutamate/glutamine structure in the mycobacterial cell wall. In this study, we investigated the influence of glutamine synthetase inhibitors on the growth of pathogenic and nonpathogenic mycobacteria and on the poly-l-glutamate/glutamine cell wall structure. The inhibitor l-methionine-S-sulfoximine rapidly inactivated purified M. tuberculosis glutamine synthetase, which was 100-fold more sensitive to this inhibitor than a representative mammalian glutamine synthetase. Added to cultures of pathogenic mycobacteria, l-methionine- S-sulfoximine rapidly inhibited extracellular glutamine synthetase in a concentration-dependent manner but had only a minimal effect on cellular glutamine synthetase, a finding consistent with failure of the drug to cross the mycobacterial cell wall. Remarkably, the inhibitor selectively blocked the growth of pathogenic mycobacteria, all of which release glutamine synthetase extracellularly, but had no effect on nonpathogenic mycobacteria or nonmycobacterial microorganisms, none of which release glutamine synthetase extracellularly. The inhibitor was also bacteriostatic for M. tuberculosis in human mononuclear phagocytes (THP-1 cells), the pathogen's primary host cells. Paralleling and perhaps underlying its bacteriostatic effect, the inhibitor markedly reduced the amount of poly-l-glutamate/glutamine cell wall structure in M. tuberculosis. Although it is possible that glutamine synthetase inhibitors interact with additional extracellular proteins or structures, our findings support the concept that extracellular proteins of M. tuberculosis and other pathogenic mycobacteria are worthy targets for new antibiotics. Such proteins constitute readily accessible targets of these relatively impermeable organisms, which are rapidly developing resistance to conventional antibiotics.

scholarly journals Synthesis of a homologous series of galactofuranose-containing mycobacterial arabinogalactan fragments

2016 ◽  
Vol 94 (11) ◽  
pp. 976-988 ◽  
Author(s):  
Maju Joe ◽  
Todd L. Lowary
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Chemical Synthesis ◽  
Mycolic Acid ◽  
Wall Structure ◽  
Complex Cell ◽  
Human Pathogen ◽  
Structural Element ◽  
Target Molecules ◽  
Mycobacterial Cell Wall

Mycobacteria, including the human pathogen Mycobacterium tuberculosis, the causative agent of tuberculosis, produce a complex cell wall structure made of carbohydrates and lipids. The major structural element of the mycobacterial cell wall is a glycoconjugate called the mycolic acid – arabinogalactan – peptidoglycan (mAGP) complex. Inhibition of mAGP biosynthesis is a proven strategy for developing anti-mycobacterial drugs, and thus, understanding the pathways and enzymes involved in the assembly of this molecule is of interest. In this paper, we describe the chemical synthesis of a panel of nine oligosaccharide fragments (4–12) of the galactan domain of the mAGP complex designed as biosynthetic probes. These structures, ranging in size from a hexasaccharide to a tetradecasaccharide, are potential substrates for two biosynthetic enzymes, GlfT2 and AftA, and represent the largest mycobacterial galactan fragments synthesized to date. The route developed was iterative and provided multimilligram quantities of the target molecules 4–12 in good overall yield.

scholarly journals An essential mycolate remodeling program for mycobacterial adaptation in host cells

2018 ◽  
Author(s):  
Eliza J.R. Peterson ◽  
Rebeca Bailo ◽  
Alissa C. Rothchild ◽  
Mario Arrieta-Ortiz ◽  
Amardeep Kaur ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
New Technology ◽  
Mycolic Acid ◽  
Host Cells ◽  
Macrophage Infection ◽  
Cell Wall Remodeling ◽  
Mycobacterial Cell Wall

AbstractThe success of Mycobacterium tuberculosis (MTB) stems from its ability to remain hidden from the immune system within macrophages. Here, we report a new technology (Path-seq) to sequence miniscule amounts of MTB transcripts within up to million-fold excess host RNA. Using Path-seq we have discovered a novel transcriptional program for in vivo mycobacterial cell wall remodeling when the pathogen infects alveolar macrophages in mice. We have discovered that MadR transcriptionally modulates two mycolic acid desaturases desA1/A2 to initially promote cell wall remodeling upon in vitro macrophage infection and, subsequently, reduces mycolate biosynthesis upon entering dormancy. We demonstrate that disrupting MadR program is lethal to diverse mycobacteria making this evolutionarily conserved regulator a prime antitubercular target for both early and late stages of infection.One Sentence SummaryNovel technology (Path-seq) discovers cell wall remodeling program during Mycobacterium tuberculosis infection of macrophages

scholarly journals The aceE involves in mycolic acid synthesis and biofilm formation in Mycobacterium smegmatis

2020 ◽  
Author(s):  
Suting Chen ◽  
Tianlu Teng ◽  
Shuan Wen ◽  
Tingting Zhang ◽  
Hairong Huang
Keyword(s):  
Cell Wall ◽  
Mycobacterium Smegmatis ◽  
Biofilm Formation ◽  
Morphological Changes ◽  
Acid Synthesis ◽  
Mycolic Acid ◽  
Colony Morphology ◽  
Wall Structure ◽  
Wild Type

Abstract Background: The integrity of cell wall structure is highly significant for the in vivo survival of mycobacteria. We hypothesized that changes in morphology may indicate changes in cell wall metabolism and identified an aceE gene mutant ( aceE -mut) which presented a deficient colony morphology on 7H10 agar by screening transposon mutagenesis in Mycolicibacterium smegmatis , basonym Mycobacterium smegmatis ( M. smegmatis ). This study aimed to identify the functional role of aceE gene in cell wall biosynthesis in M. smegmatis. Results: We observed that the colony morphology of aceE -mut was quite different, smaller and smoother on the solid culture medium than the wild-type (WT) strain during the transposon library screening of M. smegmatis . Notably, in contrast with the WT, which aggregates and forms biofilm, the aceE -mut lost its ability of growing aggregately and biofilm formation, which are two very important features of mycobacteria. The morphological changes in the aceE -mut strain were further confirmed by electron microscopy which indicated smoother and thinner cell envelope images in contrast with the rough morphology of WT strains. Additionally, the aceE -mut was more fragile to acidic stress and exhibited a pronounced defects in entering the macrophages as compared to the WT. The analysis of mycolic acid (MA) using LC-MS indicated deficiency of alpha-MA and epoxy-MA in aceE -mut strain whereas complementation of the aceE -mut with a wild-type aceE gene restored the composition of MA. Conclusions: Over all, this study indicates that aceE gene plays a significant role in the mycolic acid synthesis and affects the colony morphology, biofilm formation of M. smegmatis and bacteria invasion of macrophage.

scholarly journals Inhibition of Respiration of Candida albicans by Small Molecules Increases Phagocytosis Efficacy by Macrophages

mSphere ◽  
2020 ◽  
Vol 5 (2) ◽  
Author(s):  
Shuna Cui ◽  
Minghui Li ◽  
Rabeay Y. A. Hassan ◽  
Anna Heintz-Buschart ◽  
Junsong Wang ◽  
...  
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Plasma Membrane ◽  
Energy Metabolism ◽  
Unsaturated Fatty Acids ◽  
Immune Defense ◽  
Host Cells ◽  
Wall Structure ◽  
Content Type ◽  
Metabolic Studies

ABSTRACT Candida albicans adapts to various conditions in different body niches by regulating gene expression, protein synthesis, and metabolic pathways. These adaptive reactions not only allow survival but also influence the interaction with host cells, which is governed by the composition and structure of the fungal cell wall. Numerous studies had shown linkages between mitochondrial functionality, cell wall integrity and structure, and pathogenicity. Thus, we decided to inhibit single complexes of the respiratory chain of C. albicans and to analyze the resultant interaction with macrophages via their phagocytic activity. Remarkably, inhibition of the fungal bc1 complex by antimycin A increased phagocytosis, which correlated with an increased accessibility of β-glucans. To contribute to mechanistic insights, we performed metabolic studies, which highlighted significant changes in the abundance of constituents of the plasma membrane. Collectively, our results reinforce the strong linkage between fungal energy metabolism and other components of fungal physiology, which also determine the vulnerability to immune defense reactions. IMPORTANCE The yeast Candida albicans is one of the major fungal human pathogens, for which new therapeutic approaches are required. We aimed at enhancements of the phagocytosis efficacy of macrophages by targeting the cell wall structure of C. albicans, as the coverage of the β-glucan layer by mannans is one of the immune escape mechanisms of the fungus. We unambiguously show that inhibition of the fungal bc1 complex correlates with increased accessibilities of β-glucans and improved phagocytosis efficiency. Metabolic studies proved not only the known direct effects on reactive oxygen species (ROS) production and fermentative pathways but also the clear downregulation of the ergosterol pathway and upregulation of unsaturated fatty acids. The changed composition of the plasma membrane could also influence the interaction with the overlying cell wall. Thus, our work highlights the far-reaching relevance of energy metabolism, indirectly also for host-pathogen interactions, without affecting viability.

Sign in / Sign up

Export Citation Format

Share Document