scholarly journals Identification of CTL Epitopes on Efflux Pumps of the ATP-Binding Cassette and the Major Facilitator Superfamily of Mycobacterium tuberculosis

2021 ◽  
Vol 2021 ◽  
pp. 1-13
Author(s):  
Yan Lin ◽  
Yu Dong ◽  
Yanfeng Gao ◽  
Ranran Shi ◽  
Yubing Li ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
T Lymphocyte ◽  
Efflux Pumps ◽  
Genetically Engineered ◽  
Major Facilitator Superfamily ◽  
Atp Binding ◽  
Drug Resistant ◽  
Genetically Engineered Mice ◽  
Major Facilitator ◽  
Atp Binding Cassette

Tuberculosis is the world’s most deadly infectious disease, with 10 million people falling ill and 1.5 million people dying from the disease every year. With the increasing number of drug-resistant Mycobacterium tuberculosis (MTB) strains and prevalence of coinfection of MTB with human immunodeficiency virus, many challenges remain in the prevention and treatment of tuberculosis. Therefore, the development of safe and effective tuberculosis vaccines is an urgent issue. In this study, we identified cytotoxic T lymphocyte epitopes on drug resistance-associated membrane protein efflux pumps of MTB, the ATP-binding cassette and the major facilitator superfamilies. First, three online software were used to predict HLA-A2-restricted epitopes. Then, the candidate epitopes were confirmed with the T2A2 cell binding affinity and peptide/MHC (pMHC) complex stability assays and in vitro immune activity experiments. Two drug-resistant T lymphocyte epitopes, designated Rv1218c-p24 and Rv2477c-p182, were selected, and their immunogenic activities studied in vivo in genetically engineered mice. The immune activities of these two epitopes were improved with the help of complete Freund’s adjuvant (CFA). The epitopes identified here provide a foundation for the diagnosis and treatment of patients infected with drug resistant and the future development of a multiepitope vaccine.

scholarly journals Cysteine Is Exported from theEscherichia coliCytoplasm by CydDC, an ATP-binding Cassette-type Transporter Required for Cytochrome Assembly

2002 ◽  
Vol 277 (51) ◽  
pp. 49841-49849 ◽  
Author(s):  
Marc S. Pittman ◽  
Hazel Corker ◽  
Guanghui Wu ◽  
Marie B. Binet ◽  
Arthur J. G. Moir ◽  
...  
Keyword(s):  
Redox Homeostasis ◽  
Membrane Vesicles ◽  
Major Facilitator Superfamily ◽  
Atp Binding ◽  
Major Protein ◽  
Wild Type ◽  
Independent Manner ◽  
Periplasmic Transport ◽  
Major Facilitator ◽  
Atp Binding Cassette

Assembly ofEscherichia colicytochromebdand periplasmic cytochromes requires the ATP-binding cassette transporter CydDC, whose substrate is unknown. Two-dimensional SDS-PAGE comparison of periplasm from wild-type andcydDmutant strains revealed that the latter was deficient in several periplasmic transport binding proteins, but no single major protein was missing in thecydDperiplasm. Instead, CydDC exports from cytoplasm to periplasm the amino acid cysteine, demonstrated using everted membrane vesicles that transported radiolabeled cysteine inward in an ATP-dependent, uncoupler-independent manner. New pleiotropiccydDphenotypes are reported, including sensitivity to benzylpenicillin and dithiothreitol, and loss of motility, consistent with periplasmic defects in disulfide bond formation. Exogenous cysteine reversed these phenotypes and affected levels of periplasmicc-type cytochromes incydDand wild-type strains but did not restore cytochromed. Consistent with CydDC being a cysteine exporter,cydDmutant growth was hypersensitive to high cysteine concentrations and accumulated higher cytoplasmic cysteine levels, as did a mutant defective inorf299, encoding a transporter of the major facilitator superfamily. AcydD orf299double mutant was extremely cysteine-sensitive and had higher cytoplasmic cysteine levels, whereas CydDC overexpression conferred resistance to high extracellular cysteine concentrations. We propose that CydDC exports cysteine, crucial for redox homeostasis in the periplasm.

scholarly journals ThemelREDCAOperon Encodes a Utilization System for the Raffinose Family of Oligosaccharides inBacillus subtilis

2019 ◽  
Vol 201 (15) ◽  
Author(s):  
Kambiz Morabbi Heravi ◽  
Hildegard Watzlawick ◽  
Josef Altenbuchner
Keyword(s):  
Bacillus Subtilis ◽  
Transcriptional Start Site ◽  
Major Facilitator Superfamily ◽  
Atp Binding ◽  
Carbohydrate Utilization ◽  
Content Type ◽  
Storage Organs ◽  
Major Facilitator ◽  
Atp Binding Cassette

ABSTRACTBacillus subtilisis a heterotrophic soil bacterium that hydrolyzes different polysaccharides mainly found in the decomposed plants. These carbohydrates are mainly cellulose, hemicellulose, and the raffinose family of oligosaccharides (RFOs). RFOs are soluble α-galactosides, such as raffinose, stachyose, and verbascose, that rank second only after sucrose in abundance. Genome sequencing and transcriptome analysis ofB. subtilisindicated the presence of a putative α-galactosidase-encoding gene (melA) located in themsmRE-amyDC-melAoperon. Characterization of the MelA protein showed that it is a strictly Mn2+- and NAD+-dependent α-galactosidase able to hydrolyze melibiose, raffinose, and stachyose. Transcription of themsmER-amyDC-melAoperon is under control of a σA-type promoter located upstream ofmsmR(PmsmR), which is negatively regulated by MsmR. The activity of PmsmRwas induced in the presence of melibiose and raffinose. MsmR is a transcriptional repressor that binds to two binding sites at PmsmRlocated upstream of the −35 box and downstream of the transcriptional start site. MsmEX-AmyCD forms an ATP-binding cassette (ABC) transporter that probably transports melibiose into the cell. SincemsmRE-amyDC-melAis a melibiose utilization system, we renamed the operonmelREDCA.IMPORTANCEBacillus subtilisutilizes different polysaccharides produced by plants. These carbohydrates are primarily degraded by extracellular hydrolases, and the resulting oligo-, di-, and monosaccharides are transported into the cytosol via phosphoenolpyruvate-dependent phosphotransferase systems (PTS), major facilitator superfamily, and ATP-binding cassette (ABC) transporters. In this study, a new carbohydrate utilization system ofB. subtilisresponsible for the utilization of α-galactosides of the raffinose family of oligosaccharides (RFOs) was investigated. RFOs are synthesized from sucrose in plants and are mainly found in the storage organs of plant leaves. Our results revealed the modus operandi of a new carbohydrate utilization system inB. subtilis.

scholarly journals Efflux Pumps of Mycobacterium tuberculosis Play a Significant Role in Antituberculosis Activity of Potential Drug Candidates

2012 ◽  
Vol 56 (5) ◽  
pp. 2643-2651 ◽  
Author(s):  
Meenakshi Balganesh ◽  
Neela Dinesh ◽  
Sreevalli Sharma ◽  
Sanjana Kuruppath ◽  
Anju V. Nair ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Vital Role ◽  
Major Facilitator Superfamily ◽  
Content Type ◽  
Active Efflux ◽  
Drug Candidates ◽  
Small Multidrug Resistance ◽  
Major Facilitator

ABSTRACTActive efflux of drugs mediated by efflux pumps that confer drug resistance is one of the mechanisms developed by bacteria to counter the adverse effects of antibiotics and chemicals. To understand these efflux mechanisms inMycobacterium tuberculosis, we generated knockout (KO) mutants of four efflux pumps of the pathogen belonging to different classes. We measured the MICs and kill values of two different compound classes on the wild type (WT) and the efflux pump (EP) KO mutants in the presence and absence of the efflux inhibitors verapamil andl-phenylalanyl-l-arginyl-β-naphthylamide (PAβN). Among the pumps studied, the efflux pumps belonging to the ABC (ATP-binding cassette) class, encoded byRv1218c, and the SMR (small multidrug resistance) class, encoded byRv3065, appear to play important roles in mediating the efflux of different chemical classes and antibiotics. Efflux pumps encoded byRv0849andRv1258calso mediate the efflux of these compounds, but to a lesser extent. Increased killing is observed in WTM. tuberculosiscells by these compounds in the presence of either verapamil or PAβN. The efflux pump KO mutants were more susceptible to these compounds in the presence of efflux inhibitors. We have shown that these four efflux pumps ofM. tuberculosisplay a vital role in mediating efflux of different chemical scaffolds. Inhibitors of one or several of these efflux pumps could have a significant impact in the treatment of tuberculosis. The identification and characterization ofRv0849, a new efflux pump belonging to the MFS (major facilitator superfamily) class, are reported.

scholarly journals Two Distinct Major Facilitator Superfamily Drug Efflux Pumps Mediate Chloramphenicol Resistance in Streptomyces coelicolor

2009 ◽  
Vol 53 (11) ◽  
pp. 4673-4677 ◽  
Author(s):  
James J. Vecchione ◽  
Blair Alexander ◽  
Jason K. Sello
Keyword(s):  
Efflux Pump ◽  
Streptomyces Coelicolor ◽  
Efflux Pumps ◽  
Major Facilitator Superfamily ◽  
Close Relative ◽  
Gram Positive ◽  
Antibacterial Drugs ◽  
Chloramphenicol Resistance ◽  
Drug Activity ◽  
Major Facilitator

ABSTRACT Chloramphenicol, florfenicol, and thiamphenicol are used as antibacterial drugs in clinical and veterinary medicine. Two efflux pumps of the major facilitator superfamily encoded by the cmlR1 and cmlR2 genes mediate resistance to these antibiotics in Streptomyces coelicolor, a close relative of Mycobacterium tuberculosis. The transcription of both genes was observed by reverse transcription-PCR. Disruption of cmlR1 decreased the chloramphenicol MIC 1.6-fold, while disruption of cmlR2 lowered the MIC 16-fold. The chloramphenicol MIC of wild-type S. coelicolor decreased fourfold and eightfold in the presence of reserpine and Phe-Arg-β-naphthylamide, respectively. These compounds are known to potentiate the activity of some antibacterial drugs via efflux pump inhibition. While reserpine is known to potentiate drug activity against gram-positive bacteria, this is the first time that Phe-Arg-β-naphthylamide has been shown to potentiate drug activity against a gram-positive bacterium.

scholarly journals The Multidrug Transporters Belonging to Major Facilitator Superfamily (MFS) in Mycobacterium tuberculosis

2002 ◽  
Vol 8 (11) ◽  
pp. 714-724 ◽  
Author(s):  
Edda De Rossi ◽  
Patrizio Arrigo ◽  
Marco Bellinzoni ◽  
Pedro E. A. Silva ◽  
Carlos Martin ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Major Facilitator Superfamily ◽  
Multidrug Transporters ◽  
Major Facilitator
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