scholarly journals Requirement of the mymA Operon for Appropriate Cell Wall Ultrastructure and Persistence of Mycobacterium tuberculosis in the Spleens of Guinea Pigs

2005 ◽  
Vol 187 (12) ◽  
pp. 4173-4186 ◽  
Author(s):  
Amit Singh ◽  
Radhika Gupta ◽  
R. A. Vishwakarma ◽  
P. R. Narayanan ◽  
C. N. Paramasivan ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Parental Strain ◽  
Guinea Pigs ◽  
Cell Envelope ◽  
Sodium Dodecyl ◽  
Phenotypic Traits ◽  
Antitubercular Drugs ◽  
Mycolic Acids ◽  
Increased Sensitivity

ABSTRACT We had recently reported that the mymA operon (Rv3083 to Rv3089) of Mycobacterium tuberculosis is regulated by AraC/XylS transcriptional regulator VirS (Rv3082c) and is important for the cell envelope of M. tuberculosis. In this study, we further show that a virS mutant (MtbΔvirS) and a mymA mutant (Mtbmym::hyg) of M. tuberculosis exhibit reduced contents and altered composition of mycolic acids along with the accumulation of saturated C24 and C26 fatty acids compared to the parental strain. These mutants were markedly more susceptible to major antitubercular drugs at acidic pH and also showed increased sensitivity to detergent (sodium dodecyl sulfate) and to acidic stress than the parental strain. We show that disruption of virS and mymA genes impairs the ability of M. tuberculosis to survive in activated macrophages, but not in resting macrophages, suggesting the importance of the mymA operon in protecting the bacterium against harsher conditions. Infection of guinea pigs with MtbΔvirS, Mtbmym::hyg, and the parental strain resulted in an ∼800-fold-reduced bacillary load of the mutant strains compared with the parental strain in spleens, but not in the lungs, of animals at 20 weeks postinfection. Phenotypic traits were fully complemented upon reintroduction of the virS gene into MtbΔvirS. These observations show the important role of the mymA operon in the pathogenesis of M. tuberculosis at later stages of the disease.

scholarly journals Redundant Function of cmaA2 and mmaA2 in Mycobacterium tuberculosis cis Cyclopropanation of Oxygenated Mycolates

2010 ◽  
Vol 192 (14) ◽  
pp. 3661-3668 ◽  
Author(s):  
Daniel Barkan ◽  
Vivek Rao ◽  
George D. Sukenick ◽  
Michael S. Glickman
Keyword(s):  
Fatty Acids ◽  
Mycobacterium Tuberculosis ◽  
Genetic Analysis ◽  
Biosynthetic Pathway ◽  
Cell Envelope ◽  
Mycolic Acid ◽  
Acid Modification ◽  
Mycolic Acids ◽  
Powerful Approach ◽  
Long Chain

ABSTRACT The Mycobacterium tuberculosis cell envelope contains a wide variety of lipids and glycolipids, including mycolic acids, long-chain branched fatty acids that are decorated by cyclopropane rings. Genetic analysis of the mycolate methyltransferase family has been a powerful approach to assign functions to each of these enzymes but has failed to reveal the origin of cis cyclopropanation of the oxygenated mycolates. Here we examine potential redundancy between mycolic acid methyltransferases by generating and analyzing M. tuberculosis strains lacking mmaA2 and cmaA2, mmaA2 and cmaA1, or mmaA1 alone. M. tuberculosis lacking both cmaA2 and mmaA2 cannot cis cyclopropanate methoxymycolates or ketomycolates, phenotypes not shared by the mmaA2 and cmaA2 single mutants. In contrast, a combined loss of cmaA1 and mmaA2 had no effect on mycolic acid modification compared to results with a loss of mmaA2 alone. Deletion of mmaA1 from M. tuberculosis abolishes trans cyclopropanation without accumulation of trans-unsaturated oxygenated mycolates, placing MmaA1 in the biosynthetic pathway for trans-cyclopropanated oxygenated mycolates before CmaA2. These results define new functions for the mycolic acid methyltransferases of M. tuberculosis and indicate a substantial redundancy of function for MmaA2 and CmaA2, the latter of which can function as both a cis and trans cyclopropane synthase for the oxygenated mycolates.

scholarly journals Generation of Attenuated Mycobacterium bovis Strains by Signature-Tagged Mutagenesis for Discovery of Novel Vaccine Candidates

2005 ◽  
Vol 73 (4) ◽  
pp. 2379-2386 ◽  
Author(s):  
Desmond M. Collins ◽  
Bronwyn Skou ◽  
Stefan White ◽  
Shalome Bassett ◽  
Lauren Collins ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Bovis ◽  
Guinea Pigs ◽  
Essential Role ◽  
Immunoblot Analysis ◽  
Infection Model ◽  
Vaccine Candidates ◽  
Mouse Infection Model ◽  
Potential Vaccine

ABSTRACT Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex, has a particularly wide host range and causes tuberculosis in most mammals, including humans. A signature tag mutagenesis approach, which employed illegitimate recombination and infection of guinea pigs, was applied to M. bovis to discover genes important for virulence and to find potential vaccine candidates. Fifteen attenuated mutants were identified, four of which produced no lesions when inoculated separately into guinea pigs. One of these four mutants had nine deleted genes including mmpL4 and sigK and, in guinea pigs with aerosol challenge, provided protection against tuberculosis at least equal to that of M. bovis BCG. Seven mutants had mutations near the esxA (esat-6) locus, and immunoblot analysis of these confirmed the essential role of other genes at this locus in the secretion of EsxA (ESAT-6) and EsxB (CFP10). Mutations in the eight other attenuated mutants were widely spread through the chromosome and included pks1, which is naturally inactivated in clinical strains of M. tuberculosis. Many genes identified were different from those found by signature tag mutagenesis of M. tuberculosis by use of a mouse infection model and illustrate how the use of different approaches enables identification of a wider range of attenuating mutants.

scholarly journals Lipoprotein Processing Is Essential for Resistance of Mycobacterium tuberculosis to Malachite Green

2009 ◽  
Vol 53 (9) ◽  
pp. 3799-3802 ◽  
Author(s):  
Niaz Banaei ◽  
Eleanor Z. Kincaid ◽  
S.-Y. Grace Lin ◽  
Edward Desmond ◽  
William R. Jacobs ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Malachite Green ◽  
Cell Envelope ◽  
Sodium Dodecyl ◽  
Signal Peptidase ◽  
Wild Type ◽  
First Line ◽  
Green Is ◽  
Antituberculosis Drugs ◽  
Forming Efficiency

ABSTRACT Malachite green, a synthetic antimicrobial dye, has been used for over 50 years in mycobacterial culture medium to inhibit the growth of contaminants. The molecular basis of mycobacterial resistance to malachite green is unknown, although the presence of malachite green-reducing enzymes in the cell envelope has been suggested. The objective of this study was to investigate the role of lipoproteins in resistance of Mycobacterium tuberculosis to malachite green. The replication of an M. tuberculosis lipoprotein signal peptidase II (lspA) mutant (ΔlspA::lspA mut) on Middlebrook agar with and without 1 mg/liter malachite green was investigated. The lspA mutant was also compared with wild-type M. tuberculosis in the decolorization rate of malachite green and sensitivity to sodium dodecyl sulfate (SDS) detergent and first-line antituberculosis drugs. The lspA mutant has a 104-fold reduction in CFU-forming efficiency on Middlebrook agar with malachite green. Malachite green is decolorized faster in the presence of the lspA mutant than wild-type bacteria. The lspA mutant is hypersensitive to SDS detergent and shows increased sensitivity to first-line antituberculosis drugs. In summary, lipoprotein processing by LspA is essential for resistance of M. tuberculosis to malachite green. A cell wall permeability defect is likely responsible for the hypersensitivity of lspA mutant to malachite green.

scholarly journals Impact of lgt mutation on lipoprotein biosynthesis and in vitro phenotypes of Streptococcus agalactiae

Microbiology ◽  
2009 ◽  
Vol 155 (5) ◽  
pp. 1451-1458 ◽  
Author(s):  
Beverley A. Bray ◽  
Iain C. Sutcliffe ◽  
Dean J. Harrington
Keyword(s):  
Oxidative Stress ◽  
Streptococcus Agalactiae ◽  
Molecular Basis ◽  
Cell Envelope ◽  
Host Cells ◽  
Human Endothelial Cells ◽  
Increased Sensitivity ◽  
Group B

Although Streptococcus agalactiae, the group B Streptococcus, is a leading cause of invasive neonatal disease worldwide the molecular basis of its virulence is still poorly understood. To investigate the role of lipoproteins in the physiology and interaction of this pathogen with host cells, we generated a mutant S. agalactiae strain (A909ΔLgt) deficient in the Lgt enzyme and thus unable to lipidate lipoprotein precursors (pro-lipoproteins). The loss of pro-lipoprotein lipidation did not affect the viability of S. agalactiae or its growth in several different media, including cation-depleted media. The processing of two well-characterized lipoproteins, but not a non-lipoprotein, was clearly shown to be aberrant in A909ΔLgt. The mutant strain was shown to be more sensitive to oxidative stress in vitro although the molecular basis of this increased sensitivity was not apparent. The inactivation of Lgt also resulted in changes to the bacterial cell envelope, as demonstrated by reduced retention of both the group B carbohydrate and the polysaccharide capsule and a statistically significant reduction (P=0.0079) in A909ΔLgt adherence to human endothelial cells of fetal origin. These data confirm that failure to process lipoproteins correctly has pleiotropic effects that may be of significance to S. agalactiae colonization and pathogenesis.

scholarly journals Two Accessory Proteins Govern MmpL3 Mycolic Acid Transport in Mycobacteria

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Allison Fay ◽  
Nadine Czudnochowski ◽  
Jeremy M. Rock ◽  
Jeffrey R. Johnson ◽  
Nevan J. Krogan ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Cell Envelope ◽  
Complex Structure ◽  
Mycolic Acid ◽  
Accessory Proteins ◽  
Acid Transport ◽  
Content Type ◽  
Mycolic Acids ◽  
Antibiotic Target

ABSTRACT Mycolic acids are the signature lipid of mycobacteria and constitute an important physical component of the cell wall, a target of mycobacterium-specific antibiotics and a mediator of Mycobacterium tuberculosis pathogenesis. Mycolic acids are synthesized in the cytoplasm and are thought to be transported to the cell wall as a trehalose ester by the MmpL3 transporter, an antibiotic target for M. tuberculosis. However, the mechanism by which mycolate synthesis is coupled to transport, and the full MmpL3 transport machinery, is unknown. Here, we identify two new components of the MmpL3 transport machinery in mycobacteria. The protein encoded by MSMEG_0736/Rv0383c is essential for growth of Mycobacterium smegmatis and M. tuberculosis and is anchored to the cytoplasmic membrane, physically interacts with and colocalizes with MmpL3 in growing cells, and is required for trehalose monomycolate (TMM) transport to the cell wall. In light of these findings, we propose MSMEG_0736/Rv0383c be named “TMM transport factor A”, TtfA. The protein encoded by MSMEG_5308 also interacts with the MmpL3 complex but is nonessential for growth or TMM transport. However, MSMEG_5308 accumulates with inhibition of MmpL3-mediated TMM transport and stabilizes the MmpL3/TtfA complex, indicating that it may stabilize the transport system during stress. These studies identify two new components of the mycobacterial mycolate transport machinery, an emerging antibiotic target in M. tuberculosis. IMPORTANCE The cell envelope of Mycobacterium tuberculosis, the bacterium that causes the disease tuberculosis, is a complex structure composed of abundant lipids and glycolipids, including the signature lipid of these bacteria, mycolic acids. In this study, we identified two new components of the transport machinery that constructs this complex cell wall. These two accessory proteins are in a complex with the MmpL3 transporter. One of these proteins, TtfA, is required for mycolic acid transport and cell viability, whereas the other stabilizes the MmpL3 complex. These studies identify two new components of the essential cell envelope biosynthetic machinery in mycobacteria.

scholarly journals Role of Metal-Dependent Regulation of ESX-3 Secretion in Intracellular Survival of Mycobacterium tuberculosis

2016 ◽  
Vol 84 (8) ◽  
pp. 2255-2263 ◽  
Author(s):  
Emir Tinaztepe ◽  
Jun-Rong Wei ◽  
Jenelle Raynowska ◽  
Cynthia Portal-Celhay ◽  
Victor Thompson ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cell Envelope ◽  
Bacterial Pathogen ◽  
Transcriptional Response ◽  
Secretion Systems ◽  
Content Type ◽  
Type Vii Secretion ◽  
Significant Difference ◽  
Iron And Zinc

More people die every year fromMycobacterium tuberculosisinfection than from infection by any other bacterial pathogen. Type VII secretion systems (T7SS) are used by both environmental and pathogenic mycobacteria to secrete proteins across their complex cell envelope. In the nonpathogenMycobacterium smegmatis, the ESX-1 T7SS plays a role in conjugation, and the ESX-3 T7SS is involved in metal homeostasis. InM. tuberculosis, these secretion systems have taken on roles in virulence, and they also are targets of the host immune response. ESX-3 secretes a heterodimer composed of EsxG (TB9.8) and EsxH (TB10.4), which impairs phagosome maturation in macrophages and is essential for virulence in mice. Given the importance of EsxG and EsxH during infection, we examined their regulation. WithM. tuberculosis, the secretion of EsxG and EsxH was regulated in response to iron and zinc, in accordance with the previously described transcriptional response of theesx-3locus to these metals. While iron regulated theesx-3expression in bothM. tuberculosisandM. smegmatis, there is a significant difference in the dynamics of this regulation. InM. smegmatis, theesx-3locus behaved like other iron-regulated genes such asmbtB. InM. tuberculosis, both iron and zinc modestly repressedesx-3expression. Diminished secretion of EsxG and EsxH in response to these metals altered the interaction ofM. tuberculosiswith macrophages, leading to impaired intracellularM. tuberculosissurvival. Our findings detail the regulatory differences ofesx-3inM. tuberculosisandM. smegmatisand demonstrate the importance of metal-dependent regulation of ESX-3 for virulence inM. tuberculosis.

scholarly journals Massive Gene Duplication Event among Clinical Isolates of the Mycobacterium tuberculosis W/Beijing Family

2010 ◽  
Vol 192 (18) ◽  
pp. 4562-4570 ◽  
Author(s):  
Pilar Domenech ◽  
Gaëlle S. Kolly ◽  
Lizbel Leon-Solis ◽  
Ashley Fallow ◽  
Michael B. Reed
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Duplication Event ◽  
The Novel ◽  
Beijing Family ◽  
Antitubercular Drugs ◽  
Sister Chromatids ◽  
Sequences Analysis ◽  
Overexpression Phenotype

ABSTRACT As part of our effort to uncover the molecular basis for the phenotypic variation among clinical Mycobacterium tuberculosis isolates, we have previously reported that isolates belonging to the W/Beijing lineage constitutively overexpress the DosR-regulated transcriptional program. While generating dosR knockouts in two independent W/Beijing sublineages, we were surprised to discover that they possess two copies of dosR. This dosR amplification is part of a massive genomic duplication spanning 350 kb and encompassing >300 genes. In total, this equates to 8% of the genome being present as two copies. The presence of IS6110 elements at both ends of the region of duplication, and in the novel junction region, suggests that it arose through unequal homologous recombination of sister chromatids at the IS6110 sequences. Analysis of isolates representing the major M. tuberculosis lineages has revealed that the 350-kb duplication is restricted to the most recently evolved sublineages of the W/Beijing family. Within these isolates, the duplication is partly responsible for the constitutive dosR overexpression phenotype. Although the nature of the selection event giving rise to the duplication remains unresolved, its evolution is almost certainly the result of specific selective pressure(s) encountered inside the host. A preliminary in vitro screen has failed to reveal a role of the duplication in conferring resistance to common antitubercular drugs, a trait frequently associated with W/Beijing isolates. Nevertheless, this first description of a genetic remodeling event of this nature for M. tuberculosis further highlights the potential for the evolution of diversity in this important global pathogen.

scholarly journals Proteome remodeling in the Mycobacterium tuberculosis PknG knockout: molecular evidence for the role of this kinase in cell envelope biogenesis and hypoxia response.

2021 ◽  
Author(s):  
Analía Lima ◽  
Alejandro Leyva ◽  
Bernardina Rivera ◽  
María Magdalena Portela ◽  
Magdalena Gil ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Molecular Mechanisms ◽  
Cell Envelope ◽  
Molecular Evidence ◽  
Bacterial Survival ◽  
Human Pathogens ◽  
Hypoxic Conditions ◽  
Parallel Reaction Monitoring ◽  
Mutant Derivative

Mycobacterium tuberculosis, the ethiological agent of tuberculosis, is among the deadliest human pathogens. One of M. tuberculosis pathogenic hallmarks is its ability to persist in a dormant state in the host for long periods, reinitiating the infectious cycle when favorable environmental conditions are found. Thus, it is not surprising that this pathogen has developed different mechanisms to withstand the stressful conditions found in the host. In particular, the Ser/Thr protein kinase PknG has gained special relevance since it regulates nitrogen metabolism and facilitates bacterial survival inside macrophages. Nevertheless, the molecular mechanisms underlying these effects are far from being elucidated. To further investigate these issues, we performed quantitative proteomics analyses of protein extracts from M. tuberculosis H37Rv and a mutant derivative lacking pknG. Our results showed that in the absence of PknG the mycobacterial proteome was remodeled since 5.7% of the proteins encoded by M. tuberculosis presented significant changes in its relative abundance when compared to the wild-type strain. The main biological processes affected by pknG deletion were the biosynthesis of cell envelope components and the response to hypoxic conditions. As many as 13 DosR-regulated proteins were underrepresented in the pknG deletion mutant, including the distinctive Hrp-1, which was found to be 12-fold decreased according to Parallel Reaction Monitoring experiments. Altogether, the results presented here allow us to postulate that PknG regulation of bacterial adaptation to stress conditions might be an important mechanism underlying its reported effect on intracellular bacterial survival.

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