scholarly journals ABCI3 Is a New Mitochondrial ABC Transporter from Leishmania major Involved in Susceptibility to Antimonials and Infectivity

2017 ◽  
Vol 61 (12) ◽  
Author(s):  
Talia Arcari ◽  
José Ignacio Manzano ◽  
Francisco Gamarro
Keyword(s):  
Abc Transporter ◽  
Leishmania Major ◽  
Essential Gene ◽  
Wild Type ◽  
Double Knockout ◽  
Content Type ◽  
Mitochondrial Superoxide ◽  
Dual Localization ◽  
Pentavalent Antimony ◽  
Digitonin Permeabilization

ABSTRACT We have identified and characterized ABCI3 as a new mitochondrial ABC transporter from Leishmania major. Localization studies using confocal microscopy, a surface biotinylation assay, and trypsin digestion after digitonin permeabilization suggested that ABCI3 presents a dual localization in both mitochondria and the plasma membrane. From studies using parasites with a single knockout of ABCI3 (ABCI3 +/−), we provide evidence that ABCI3 is directly involved in susceptibility to the trivalent form of antimony (SbIII) and metal ions. Attempts to obtain parasites with a double knockout of ABCI3 were unsuccessful, suggesting that ABCI3 could be an essential gene in L. major. ABCI3 +/− promastigotes were 5-fold more resistant to SbIII than the wild type, while ABCI3 +/− amastigotes were approximately 2-fold more resistant to pentavalent antimony (SbV). This resistance phenotype was associated with decreased SbIII accumulation due to decreased SbIII uptake. ABCI3 +/− parasites presented higher ATP levels and generated less mitochondrial superoxide after SbIII incubation. Finally, we observed that ABCI3 +/− parasites showed a slightly higher infection capacity than wild-type and add-back ABCI3 +/−::3×FABCI3 parasites; however, after 72 h the number of ABCI3 +/− intracellular parasites per macrophage increased significantly. Our results show that ABCI3 is responsible for SbIII transport inside mitochondria, where it contributes to enhancement of the general toxic effects caused by SbIII. To our knowledge, ABCI3 is the first ABC transporter which is involved in susceptibility toward antimony, conferring SbIII resistance to parasites when it is partially deleted.

scholarly journals Leishmania-Derived Trimannose Modulates the Inflammatory Response To Significantly Reduce Leishmania major-Induced Lesions

2017 ◽  
Vol 86 (1) ◽  
Author(s):  
Tara L. Grinnage-Pulley ◽  
Daniel E. K. Kabotso ◽  
Chelsea L. Rintelmann ◽  
Rajarshi Roychoudhury ◽  
Robert G. Schaut ◽  
...  
Keyword(s):  
Leishmania Major ◽  
Parasite Load ◽  
Mannose Receptor ◽  
Lesion Size ◽  
Wild Type ◽  
Cutaneous Lesions ◽  
Content Type ◽  
Latex Beads

ABSTRACTLeishmanialipophosphoglycan (LPG) is a key virulence factor, initiating inflammation resulting in cutaneous lesions. LPG is capped by various oligosaccharides. How these glycans are recognized and how they alter the course ofLeishmaniainfection are poorly understood. Previous studies synthesized α-1,2-trimannose cap sugars on latex beads and demonstrated that C57BL/6 mice coinoculated withLeishmania majorand trimannose-coated beads produced significantly higher levels of interleukin-12p40 (IL-12p40) and other proinflammatory, type 1 cytokines than mice inoculated withL. majoralone within the first 48 h of infection. However, asL. majorinfection typically progress over weeks to months, the role of trimannose in altering disease progression over the course of infection was unknown. Wild-type mice were inoculated with either trimannose-coated or carrier (uncoated) beads, infected withL. majoralone, coinoculated with carrier beads andL. major, or coinoculated with trimannose-coated beads andL. major. Trimannose treatment ofL. major-infected mice decreased the parasite load and significantly decreased the lesion size at 14 days postinfection (p.i.) compared to results for nontreated, infected mice. Infected, trimannose-treated mice had decreased IL-12p40 and IL-10 secretion and increased interferon gamma secretion at 14 days p.i. Mannose receptor knockout (MR−/−) mice lack the ability to detect trimannose. When MR−/−mice were infected withL. majorand treated with trimannose beads, they did not have decreased lesion size.Leishmania-derived trimannose represents a novel immunomodulator that provides early type 1-skewed cytokine production to control the parasite load and alter the course of cutaneous leishmaniasis.

scholarly journals Role of the Zinc Uptake ABC Transporter of Moraxella catarrhalis in Persistence in the Respiratory Tract

2013 ◽  
Vol 81 (9) ◽  
pp. 3406-3413 ◽  
Author(s):  
Timothy F. Murphy ◽  
Aimee L. Brauer ◽  
Charmaine Kirkham ◽  
Antoinette Johnson ◽  
Mary Koszelak-Rosenblum ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Respiratory Tract ◽  
Abc Transporter ◽  
Divalent Cations ◽  
Zinc Uptake ◽  
Wild Type ◽  
Human Respiratory Tract ◽  
Respiratory Epithelial Cells ◽  
Content Type ◽  
Respiratory Epithelial

ABSTRACTMoraxella catarrhalisis a human respiratory tract pathogen that causes otitis media in children and lower respiratory tract infections in adults with chronic obstructive pulmonary disease. We have identified and characterized a zinc uptake ABC transporter that is present in all strains ofM. catarrhalistested. A mutant in which theznugene cluster is knocked out shows markedly impaired growth compared to the wild type in medium that contains trace zinc; growth is restored to wild-type levels by supplementing medium with zinc but not with other divalent cations. Thermal-shift assays showed that the purified recombinant substrate binding protein ZnuA binds zinc but does not bind other divalent cations. Invasion assays with human respiratory epithelial cells demonstrated that the zinc ABC transporter ofM. catarrhalisis critical for invasion of respiratory epithelial cells, an observation that is especially relevant because an intracellular reservoir ofM. catarrhalisis present in the human respiratory tract and this reservoir is important for persistence. Theznuknockout mutant showed marked impairment in its capacity to persist in the respiratory tract compared to the wild type in a mouse pulmonary clearance model. We conclude that the zinc uptake ABC transporter mediates uptake of zinc in environments with very low zinc concentrations and is critical for full virulence ofM. catarrhalisin the respiratory tract in facilitating intracellular invasion of epithelial cells and persistence in the respiratory tract.

scholarly journals The Vps/VacJ ABC Transporter Is Required for Intercellular Spread of Shigella flexneri

2013 ◽  
Vol 82 (2) ◽  
pp. 660-669 ◽  
Author(s):  
Chandra D. Carpenter ◽  
Benjamin J. Cooley ◽  
Brittany D. Needham ◽  
Carolyn R. Fisher ◽  
M. Stephen Trent ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Outer Membrane ◽  
Abc Transporter ◽  
Shigella Flexneri ◽  
Dual Function ◽  
Wild Type ◽  
Lipid Asymmetry ◽  
Content Type ◽  
Intercellular Spread ◽  
Phospholipid Profile

ABSTRACTThe Vps/VacJ ABC transporter system is proposed to function in maintaining the lipid asymmetry of the outer membrane. Mutations invpsorvacJinShigella flexneriresulted in increased sensitivity to lysis by the detergent sodium dodecyl sulfate (SDS), and thevpsCmutant showed minor differences in its phospholipid profile compared to the wild type.vpsCmutants were unable to form plaques in cultured epithelial cells, but this was not due to a failure to invade, to replicate intracellularly, or to polymerize actin via IcsA for movement within epithelial cells. The addition of the outer membrane phospholipase genepldAon a multicopy plasmid in avpsCorvacJmutant restored its resistance to SDS, suggesting a restoration of lipid asymmetry to the outer membrane. However, thepldAplasmid did not restore the mutant's ability to form plaques in tissue culture cells. Increased PldA levels also failed to restore the mutant's phospholipid profile to that of the wild type. We propose a dual function of the Vps/VacJ ABC transporter system inS. flexneriin both the maintenance of lipid asymmetry in the outer membrane and the intercellular spread of the bacteria between adjacent epithelial cells.

scholarly journals A New ABC Half-Transporter in Leishmania major Is Involved in Resistance to Antimony

2013 ◽  
Vol 57 (8) ◽  
pp. 3719-3730 ◽  
Author(s):  
J. I. Manzano ◽  
R. García-Hernández ◽  
S. Castanys ◽  
F. Gamarro
Keyword(s):  
Leishmania Major ◽  
Fluorescent Protein ◽  
Peritoneal Macrophages ◽  
Zinc Protoporphyrin ◽  
Content Type ◽  
Dual Localization ◽  
Green Fluorescent ◽  
Species Production ◽  
Mouse Peritoneal Macrophages

ABSTRACTThe characterization of ABCI4, a new intracellular ATP-binding cassette (ABC) half-transporter inLeishmania major, is described. We show that ABCI4 is involved in heavy metal export, thereby conferring resistance to Pentostam, to Sb(III), and to As(III) and Cd(II). Parasites overexpressing ABCI4 showed a lower mitochondrial toxic effect of antimony by decreasing reactive oxygen species production and maintained higher values of both the mitochondrial electrochemical potential and total ATP levels with respect to controls. The ABCI4 half-transporter forms homodimers as determined by a coimmunoprecipitation assay. A combination of subcellular localization studies under a confocal microscope and a surface biotinylation assay using parasites expressing green fluorescent protein- and FLAG-tagged ABCI4 suggests that the transporter presents a dual localization in both mitochondria and the plasma membrane. Parasites overexpressing ABCI4 present an increased replication in mouse peritoneal macrophages. We have determined that porphyrins are substrates for ABCI4. Consequently, the overexpression of ABCI4 confers resistance to some toxic porphyrins, such as zinc-protoporphyrin, due to the lower accumulation resulting from a significant efflux, as determined using the fluorescent zinc-mesoporphyrin, a validated heme analog. In addition, ABCI4 has a significant ability to efflux thiol after Sb(III) incubation, thus meaning that ABCI4 could be considered to be a potential thiol-X-pump that is able to recognize metal-conjugated thiols. In summary, we have shown that this new ABC transporter is involved in drug sensitivity to antimony and other compounds by efflux as conjugated thiol complexes.

scholarly journals Lipase and Protease Double-Deletion Mutant of Pseudomonas fluorescens Suitable for Extracellular Protein Production

2012 ◽  
Vol 78 (23) ◽  
pp. 8454-8462 ◽  
Author(s):  
Myunghan Son ◽  
Yuseok Moon ◽  
Mi Jin Oh ◽  
Sang Bin Han ◽  
Ki Hyun Park ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Pseudomonas Fluorescens ◽  
Abc Transporter ◽  
Recombinant Proteins ◽  
Fusion Proteins ◽  
Deletion Mutant ◽  
Protein Production ◽  
Type I ◽  
Wild Type ◽  
Content Type

ABSTRACTPseudomonas fluorescens, a widespread Gram-negative bacterium, is an ideal protein manufacturing factory (PMF) because of its safety, robust growth, and high protein production.P. fluorescenspossesses a type I secretion system (T1SS), which mediates secretion of a thermostable lipase (TliA) and a protease (PrtA) through its ATP-binding cassette (ABC) transporter. Recombinant proteins inP. fluorescensare attached to the C-terminal signal region of TliA for transport as fusion proteins to the extracellular medium. However, intrinsic TliA from theP. fluorescensgenome interferes with detection of the recombinant protein and the secreted recombinant protein is hydrolyzed, due to intrinsic PrtA, resulting in decreased efficiency of the PMF. In this research, the lipase and protease genes ofP. fluorescensSIK W1 were deleted using the targeted gene knockout method. Deletion mutantP. fluorescensΔtliAΔprtAsecreted fusion proteins without TliA or protein degradation. Using wild-typeP. fluorescensas an expression host, degradation of the recombinant protein varied depending on the type of culture media and aeration; however, degradation did not occur with theP. fluorescensΔtliAΔprtAdouble mutant irrespective of growth conditions. By homologous expression oftliAand the ABC transporter in a plasmid, TliA secreted fromP. fluorescensΔprtAandP. fluorescensΔtliAΔprtAcells was found to be intact, whereas that secreted from the wild-typeP. fluorescensandP. fluorescensΔtliAcells was found to be hydrolyzed. Our results demonstrate that theP. fluorescensΔtliAΔprtAdeletion mutant is a promising T1SS-mediated PMF that enhances production and detection of recombinant proteins in extracellular media.

scholarly journals Multiple Functions of Glutamate Uptake via Meningococcal GltT-GltM l-Glutamate ABC Transporter in Neisseria meningitidis Internalization into Human Brain Microvascular Endothelial Cells

2015 ◽  
Vol 83 (9) ◽  
pp. 3555-3567 ◽  
Author(s):  
Hideyuki Takahashi ◽  
Tatsuo Yanagisawa ◽  
Kwang Sik Kim ◽  
Shigeyuki Yokoyama ◽  
Makoto Ohnishi
Keyword(s):  
Endothelial Cells ◽  
Human Brain ◽  
Neisseria Meningitidis ◽  
Abc Transporter ◽  
Glutamate Uptake ◽  
Underlying Mechanism ◽  
Host Cells ◽  
Wild Type ◽  
Content Type ◽  
Fetal Bovine

We previously reported thatNeisseria meningitidisinternalization into human brain microvasocular endothelial cells (HBMEC) was triggered by the influx of extracellularl-glutamate via the GltT-GltMl-glutamate ABC transporter, but the underlying mechanism remained unclear. We found that the ΔgltTΔgltMinvasion defect in assay medium (AM) was alleviated in AM without 10% fetal bovine serum (FBS) [AM(−S)]. The alleviation disappeared again in AM(−S) supplemented with 500 μM glutamate. Glutamate uptake by the ΔgltTΔgltMmutant was less efficient than that by the wild-type strain, but only upon HBMEC infection. We also observed that both GltT-GltM-dependent invasion and accumulation of ezrin, a key membrane-cytoskeleton linker, were more pronounced whenN. meningitidisformed larger colonies on HBMEC under physiological glutamate conditions. These results suggested that GltT-GltM-dependent meningococcal internalization into HBMEC might be induced by the reduced environmental glutamate concentration upon infection. Furthermore, we found that the amount of glutathione within the ΔgltTΔgltMmutant was much lower than that within the wild-typeN. meningitidisstrain only upon HBMEC infection and was correlated with intracellular survival. Considering that thel-glutamate obtained via GltT-GltM is utilized as a nutrient in host cells,l-glutamate uptake via GltT-GltM plays multiple roles inN. meningitidisinternalization into HBMEC.

scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

scholarly journals The Serratia marcescens Siderophore Serratiochelin Is Necessary for Full Virulence during Bloodstream Infection

2020 ◽  
Vol 88 (8) ◽  
Author(s):  
Danelle R. Weakland ◽  
Sara N. Smith ◽  
Bailey Bell ◽  
Ashootosh Tripathi ◽  
Harry L. T. Mobley
Keyword(s):  
Serratia Marcescens ◽  
Bloodstream Infection ◽  
Iron Acquisition ◽  
Gene Clusters ◽  
Clinical Isolates ◽  
Wild Type ◽  
Serratia Plymuthica ◽  
Content Type ◽  
Cas Assay ◽  
Essential Nutrient

ABSTRACT Serratia marcescens is a bacterium frequently found in the environment, but over the last several decades it has evolved into a concerning clinical pathogen, causing fatal bacteremia. To establish such infections, pathogens require specific nutrients; one very limited but essential nutrient is iron. We sought to characterize the iron acquisition systems in S. marcescens isolate UMH9, which was recovered from a clinical bloodstream infection. Using RNA sequencing (RNA-seq), we identified two predicted siderophore gene clusters (cbs and sch) that were regulated by iron. Mutants were constructed to delete each iron acquisition locus individually and in conjunction, generating both single and double mutants for the putative siderophore systems. Mutants lacking the sch gene cluster lost their iron-chelating ability as quantified by the chrome azurol S (CAS) assay, whereas the cbs mutant retained wild-type activity. Mass spectrometry-based analysis identified the chelating siderophore to be serratiochelin, a siderophore previously identified in Serratia plymuthica. Serratiochelin-producing mutants also displayed a decreased growth rate under iron-limited conditions created by dipyridyl added to LB medium. Additionally, mutants lacking serratiochelin were significantly outcompeted during cochallenge with wild-type UMH9 in the kidneys and spleen after inoculation via the tail vein in a bacteremia mouse model. This result was further confirmed by an independent challenge, suggesting that serratiochelin is required for full S. marcescens pathogenesis in the bloodstream. Nine other clinical isolates have at least 90% protein identity to the UMH9 serratiochelin system; therefore, our results are broadly applicable to emerging clinical isolates of S. marcescens causing bacteremia.

scholarly journals Bacterial Resistance to Antisense Peptide Phosphorodiamidate Morpholino Oligomers

2012 ◽  
Vol 56 (12) ◽  
pp. 6147-6153 ◽  
Author(s):  
Susan E. Puckett ◽  
Kaleb A. Reese ◽  
Georgi M. Mitev ◽  
Valerie Mullen ◽  
Rudd C. Johnson ◽  
...  
Keyword(s):  
Bacterial Resistance ◽  
Acyl Carrier Protein ◽  
Essential Gene ◽  
Resistant Mutant ◽  
Resistant Isolate ◽  
Carrier Protein ◽  
Bacterial Gene ◽  
Content Type ◽  
Bacterial Gene Expression ◽  
Phosphorodiamidate Morpholino Oligomers

ABSTRACTPeptide phosphorodiamidate morpholino oligomers (PPMOs) are synthetic DNA mimics that bind cRNA and inhibit bacterial gene expression. The PPMO (RFF)3RXB-AcpP (where R is arginine, F, phenylalanine, X is 6-aminohexanoic acid, B is β-alanine, and AcpP is acyl carrier protein) is complementary to 11 bases of the essential geneacpP(which encodes acyl carrier protein). The MIC of (RFF)3RXB-AcpP was 2.5 μM (14 μg/ml) inEscherichia coliW3110. The rate of spontaneous resistance ofE. colito (RFF)3RXB-AcpP was 4 × 10−7mutations/cell division. A spontaneous (RFF)3RXB-AcpP-resistant mutant (PR200.1) was isolated. The MIC of (RFF)3RXB-AcpP was 40 μM (224 μg/ml) for PR200.1. The MICs of standard antibiotics for PR200.1 and W3110 were identical. The sequence ofacpPwas identical in PR200.1 and W3110. PR200.1 was also resistant to other PPMOs conjugated to (RFF)3RXB or peptides with a similar composition or pattern of cationic and nonpolar residues. Genomic sequencing of PR200.1 identified a mutation insbmA, which encodes an active transport protein. In separate experiments, a (RFF)3RXB-AcpP-resistant isolate (RR3) was selected from a transposome library, and the insertion was mapped tosbmA. Genetic complementation of PR200.1 or RR3 withsbmArestored susceptibility to (RFF)3RXB-AcpP. Deletion ofsbmAcaused resistance to (RFF)3RXB-AcpP. We conclude that resistance to (RFF)3RXB-AcpP was linked to the peptide and not the phosphorodiamidate morpholino oligomer, dependent on the composition or repeating pattern of amino acids, and caused by mutations insbmA. The data further suggest that (RFF)3R-XB PPMOs may be transported across the plasma membrane by SbmA.

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