scholarly journals Iron Deprivation Affects Drug Susceptibilities of Mycobacteria Targeting Membrane Integrity

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Rahul Pal ◽  
Saif Hameed ◽  
Zeeshan Fatima
Keyword(s):  
Mycobacterium Smegmatis ◽  
Cell Envelope ◽  
Membrane Integrity ◽  
Genotoxic Stress ◽  
Drug Susceptibility ◽  
First Line ◽  
Antitubercular Drugs ◽  
Iron Deprivation ◽  
Cellular Iron ◽  
First Time

Multidrug resistance (MDR) acquired byMycobacterium tuberculosis(MTB) through continuous deployment of antitubercular drugs warrants immediate search for novel targets and mechanisms. The ability of MTB to sense and become accustomed to changes in the host is essential for survival and confers the basis of infection. A crucial condition that MTB must surmount is iron limitation, during the establishment of infection, since iron is required by both bacteria and humans. This study focuses on how iron deprivation affects drug susceptibilities of known anti-TB drugs inMycobacterium smegmatis, a “surrogate of MTB.” We showed that iron deprivation leads to enhanced potency of most commonly used first line anti-TB drugs that could be reverted upon iron supplementation. We explored that membrane homeostasis is disrupted upon iron deprivation as revealed by enhanced membrane permeability and hypersensitivity to membrane perturbing agent leading to increased passive diffusion of drug and TEM images showing detectable differences in cell envelope thickness. Furthermore, iron seems to be indispensable to sustain genotoxic stress suggesting its possible role in DNA repair machinery. Taken together, we for the first time established a link between cellular iron and drug susceptibility of mycobacteria suggesting iron as novel determinant to combat MDR.

scholarly journals Evidence for the Mycobacterial Mce4 Transporter Being a Multiprotein Complex

2021 ◽  
Vol 203 (10) ◽  
Author(s):  
Laura Rank ◽  
Laura E. Herring ◽  
Miriam Braunstein
Keyword(s):  
Mass Spectrometry ◽  
Abc Transporters ◽  
Mycobacterium Smegmatis ◽  
Cell Envelope ◽  
Cholesterol Transport ◽  
Content Type ◽  
Multiprotein Complexes ◽  
Body Of Knowledge ◽  
Unique Cell ◽  
First Time

ABSTRACT Mycobacteria possess Mce transporters that import lipids and are thought to function analogously to ATP-binding cassette (ABC) transporters. However, whereas ABC transporters import substrates using a single solute-binding protein (SBP) to deliver a substrate to permease proteins in the membrane, mycobacterial Mce transporters have a potential for six SBPs (MceA to MceF) working with a pair of permeases (YrbEA and YrbEB), a cytoplasmic ATPase (MceG), and multiple Mce-associated membrane (Mam) and orphaned Mam (Omam) proteins to transport lipids. In this study, we used the model mycobacterium Mycobacterium smegmatis to study the requirement for individual Mce, Mam, and Omam proteins in Mce4 transport of cholesterol. All of the Mce4 and Mam4 proteins we investigated were required for cholesterol uptake. However, not all Omam proteins, which are encoded by genes outside mce loci, proved to contribute to cholesterol import. OmamA and OmamB were required for cholesterol import, while OmamC, OmamD, OmamE, and OmamF were not. In the absence of any single Mce4, Mam4, or Omam protein that we tested, the abundance of Mce4A and Mce4E declined. This relationship between the levels of Mce4A and Mce4E and these additional proteins suggests a network of interactions that assemble and/or stabilize a multiprotein Mce4 transporter complex. Further support for Mce transporters being multiprotein complexes was obtained by immunoprecipitation-mass spectrometry, in which we identified every single Mce, YrbE, MceG, Mam, and Omam protein with a role in cholesterol transport as associating with Mce4A. This study represents the first time any of these Mce4 transporter proteins has been shown to associate. IMPORTANCE How lipids travel between membranes of diderm bacteria is a challenging mechanistic question because lipids, which are hydrophobic molecules, must traverse a hydrophilic periplasm. This question is even more complex for mycobacteria, which have a unique cell envelope that is highly impermeable to molecules. A growing body of knowledge identifies Mce transporters as lipid importers for mycobacteria. Here, using protein stability experiments and immunoprecipitation-mass spectrometry, we provide evidence for mycobacterial Mce transporters existing as multiprotein complexes.

scholarly journals The Mycobacteriophage Ms6 LysB N-Terminus Displays Peptidoglycan Binding Affinity

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1377
Author(s):  
Adriano M. Gigante ◽  
Francisco Olivença ◽  
Maria João Catalão ◽  
Paula Leandro ◽  
José Moniz-Pereira ◽  
...  
Keyword(s):  
Mycobacterium Smegmatis ◽  
Fluorescent Protein ◽  
Cell Envelope ◽  
Structural Similarity ◽  
Lytic Cycle ◽  
Specific Lysis ◽  
Double Stranded Dna ◽  
The Family ◽  
N Terminus ◽  
Green Fluorescent

Double-stranded DNA bacteriophages end their lytic cycle by disrupting the host cell envelope, which allows the release of the virion progeny. Each phage must synthesize lysis proteins that target each cell barrier to phage release. In addition to holins, which permeabilize the cytoplasmic membrane, and endolysins, which disrupt the peptidoglycan (PG), mycobacteriophages synthesize a specific lysis protein, LysB, capable of detaching the outer membrane from the complex cell wall of mycobacteria. The family of LysB proteins is highly diverse, with many members presenting an extended N-terminus. The N-terminal region of mycobacteriophage Ms6 LysB shows structural similarity to the PG-binding domain (PGBD) of the φKZ endolysin. A fusion of this region with enhanced green fluorescent protein (Ms6LysBPGBD-EGFP) was shown to bind to Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium bovis BGC and Mycobacterium tuberculosis H37Ra cells pretreated with SDS or Ms6 LysB. In pulldown assays, we demonstrate that Ms6 LysB and Ms6LysBPGBD-EGFP bind to purified peptidoglycan of M. smegmatis, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis, demonstrating affinity to PG of the A1γ chemotype. An infection assay with an Ms6 mutant producing a truncated version of LysB lacking the first 90 amino acids resulted in an abrupt lysis. These results clearly demonstrate that the N-terminus of Ms6 LysB binds to the PG.

scholarly journals Survival of Gastric and Enterohepatic Helicobacter spp. in Water: Implications for Transmission

2008 ◽  
Vol 74 (6) ◽  
pp. 1805-1811 ◽  
Author(s):  
N. F. Azevedo ◽  
C. Almeida ◽  
I. Fernandes ◽  
L. Cerqueira ◽  
S. Dias ◽  
...  
Keyword(s):  
Membrane Integrity ◽  
Hierarchical Cluster ◽  
Culture Method ◽  
Oral Route ◽  
Human Stomach ◽  
Aquatic Environments ◽  
Integrity Assessment ◽  
Environmental Reservoir ◽  
H Pylori ◽  
First Time

ABSTRACT Part of the reason for rejecting aquatic environments as possible vectors for the transmission of Helicobacter pylori has been the preference of this microorganism to inhabit the human stomach and hence use a direct oral-oral route for transmission. On the other hand, most enteric bacterial pathogens are well known for being able to use water as an environmental reservoir. In this work, we have exposed 13 strains of seven different Helicobacter spp. (both gastric and enterohepatic) to water and tracked their survival by standard plating methods and membrane integrity assessment. The influence of different plating media and temperatures and the presence of light on recovery was also assessed. There was good correlation between cultivability and membrane integrity results (Pearson's correlation coefficient = 0.916), confirming that the culture method could reliably estimate differences in survival among different Helicobacter spp. The species that survived the longest in water was H. pylori (>96 h in the dark at 25°C), whereas H. felis appeared to be the most sensitive to water (<6 h). A hierarchical cluster analysis demonstrated that there was no relationship between the enterohepatic nature of Helicobacter spp. and an increased time of survival in water. This work assesses for the first time the survival of multiple Helicobacter spp., such has H. mustelae, H. muridarum, H. felis, H. canadensis, H. pullorum, and H. canis, in water under several conditions and concludes that the roles of water in transmission between hosts are likely to be similar for all these species, whether enterohepatic or not.

scholarly journals Molecular architecture of theLegionellaDot/Icm type IV secretion system

2018 ◽  
Author(s):  
Debnath Ghosal ◽  
Yi-Wei Chang ◽  
Kwang Cheol Jeong ◽  
Joseph P. Vogel ◽  
Grant J. Jensen
Keyword(s):  
Legionella Pneumophila ◽  
Cell Envelope ◽  
Secretion System ◽  
Host Cells ◽  
Molecular Architecture ◽  
Intact Cells ◽  
Type Iv ◽  
Type Ivb Secretion ◽  
Electron Cryotomography ◽  
First Time

AbstractLegionella pneumophilasurvives and replicates inside host cells by secreting ~300 effectors through the Dot/Icm type IVB secretion system (T4BSS). Understanding this machine’s structure is challenging because of its large number of components (27) and integration into all layers of the cell envelope. Previously we overcame this obstacle by imaging the Dot/Icm T4BSS in its native state within intact cells through electron cryotomography. Here we extend our observations by imaging a stabilized mutant that yielded a higher resolution map. We describe for the first time the presence of a well-ordered central channel that opens up into a windowed large (~32 nm wide) secretion chamber with an unusual 13-fold symmetry. We then dissect the complex by matching proteins to densities for many components, including all those with periplasmic domains. The placement of known and predicted structures of individual proteins into the map reveals the architecture of the T4BSS and provides a roadmap for further investigation of this amazing specialized secretion system.

scholarly journals Structure of the ATP synthase from Mycobacterium smegmatis provides targets for treating tuberculosis

2021 ◽  
Vol 118 (47) ◽  
pp. e2111899118
Author(s):  
Martin G. Montgomery ◽  
Jessica Petri ◽  
Tobias E. Spikes ◽  
John E. Walker
Keyword(s):  
Atp Synthase ◽  
Mycobacterium Smegmatis ◽  
Atp Hydrolysis ◽  
Terminal Region ◽  
Catalytic Cycle ◽  
Α Subunit ◽  
Antitubercular Drugs ◽  
Safe Mechanism ◽  
Fail Safe ◽  
Α Subunits

The structure has been determined by electron cryomicroscopy of the adenosine triphosphate (ATP) synthase from Mycobacterium smegmatis. This analysis confirms features in a prior description of the structure of the enzyme, but it also describes other highly significant attributes not recognized before that are crucial for understanding the mechanism and regulation of the mycobacterial enzyme. First, we resolved not only the three main states in the catalytic cycle described before but also eight substates that portray structural and mechanistic changes occurring during a 360° catalytic cycle. Second, a mechanism of auto-inhibition of ATP hydrolysis involves not only the engagement of the C-terminal region of an α-subunit in a loop in the γ-subunit, as proposed before, but also a “fail-safe” mechanism involving the b′-subunit in the peripheral stalk that enhances engagement. A third unreported characteristic is that the fused bδ-subunit contains a duplicated domain in its N-terminal region where the two copies of the domain participate in similar modes of attachment of the two of three N-terminal regions of the α-subunits. The auto-inhibitory plus the associated “fail-safe” mechanisms and the modes of attachment of the α-subunits provide targets for development of innovative antitubercular drugs. The structure also provides support for an observation made in the bovine ATP synthase that the transmembrane proton-motive force that provides the energy to drive the rotary mechanism is delivered directly and tangentially to the rotor via a Grotthuss water chain in a polar L-shaped tunnel.

scholarly journals BacA, an ABC Transporter Involved in Maintenance of Chronic Murine Infections with Mycobacterium tuberculosis

2008 ◽  
Vol 191 (2) ◽  
pp. 477-485 ◽  
Author(s):  
Pilar Domenech ◽  
Hajime Kobayashi ◽  
Kristin LeVier ◽  
Graham C. Walker ◽  
Clifton E. Barry
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Comprehensive Evaluation ◽  
Cell Envelope ◽  
Membrane Integrity ◽  
Chronic Infections ◽  
Host Pathogen Interaction ◽  
Host Pathogen ◽  
Ultimate Outcome ◽  
Inner Membrane Protein ◽  
Related Proteins

ABSTRACT BacA is an inner membrane protein associated with maintenance of chronic infections in several diverse host-pathogen interactions. To understand the function of the bacA gene in Mycobacterium tuberculosis (Rv1819c), we insertionally inactivated this gene and analyzed the resulting mutant for a variety of phenotypes. BacA deficiency in M. tuberculosis did not affect sensitivity to detergents, acidic pH, and zinc, indicating that there was no global compromise in membrane integrity, and a comprehensive evaluation of the major lipid constituents of the cell envelope failed to reveal any significant differences. Infection of mice with this mutant revealed no impact on establishment of infection but a profound effect on maintenance of extended chronic infection and ultimate outcome. As in alphaproteobacteria, deletion of BacA in M. tuberculosis led to increased bleomycin resistance, and heterologous expression of the M. tuberculosis BacA homolog in Escherichia coli conferred sensitivity to antimicrobial peptides. These results suggest a striking conservation of function for BacA-related proteins in transport of a critical molecule that determines the outcome of the host-pathogen interaction.

Spoligotyping and Drug Sensitivity of Mycobacterium tuberculosis isolated from Pulmonary Tuberculosis Patients in the Arsi Zone of South Eastern Ethiopia

2019 ◽  
Author(s):  
Belete Haile Nega ◽  
Ketema Tafess ◽  
Aboma Zewude ◽  
Bazezew Yenew ◽  
Gilman SIU ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Sensitivity ◽  
Scientific Information ◽  
Rpob Gene ◽  
Drug Susceptibility ◽  
Strain Identification ◽  
First Line ◽  
Eastern Ethiopia ◽  
Arsi Zone ◽  
Mycobacterium Africanum

Abstract Background Tuberculosis (TB) is one of the leading disease causing morbidity and mortality in different zones of Ethiopia including the Arsi Zone. However, little or no scientific information is available on the strains of Mycobacterium tuberculosis and their drug sensitivity profiles in this Zone. This study was conducted to identify the strains of M. tuberculosis and evaluate their drug sensitivity profiles. Methodology A total of 111 clinical isolates of M. tuberculosis from patients with pulmonary TB in the Arsi Zone were used for this study. The region of difference 9 (RD 9)-based polymerase chain reaction (PCR)and spoligotyping methods were used for speciation and strain identification of Mycobacterium tuberculosis respectively.The spoligotyping patterns were compared with the international SpolDB4 (SITVIT) and Run TB-Lineage used for the identification of lineages. The phenotypic drug susceptibility patterns were confirmed by BD BactecMGIT 960 SIRE test and GenoType MTBDRplus line probe assays were used for the detection the drug resistance-conferring mutations of the isolates. Result The spoligotype patterns of 83% (92/111) of the isolates were interpretable and 56 different patterns were identified. Twenty-two of these patterns were shared types while the remaining 34 were orphans. The predominant shared types were spoligotype international type (SIT) 149 and SIT53, each consisting of 12 and 11 isolates, respectively. The lineages identified were Euro-American, East-African-Indian, Mycobacterium-africanum, and Indo-Oceanic in descending order. Phenotypically, 17.2% of the 64 tested isolates were resistant to any of the four first-line drugs while 3.1% of them were multi-drug resistant (MDR). Higher (6.2%) monoresistance was observed to Streptomycin followed by Isoniazid (3.1%) while no resistance was observed either to Rifampicin or to Ethambutol. Genotypically, five (5.4%) isolates were resistant to Isoniazid and mutated at codon S315T1 of katG. On the other hand, only 1.1% of the isolates was resistant to Rifampicin and mutated at codon S531L of rpoB gene. Conclusion The proportion of orphan strains isolated in this study was high, which could suggest the presence of new strains in the Zone. Moreover, the study showed relatively high percentage of mono-resistance to any four first-line drugs warranting for the need to strengthen the control efforts.

Sign in / Sign up

Export Citation Format

Share Document