The major facilitator superfamily-type protein LbtC promotes the utilization of the legiobactin siderophore by Legionella pneumophila

ABSTRACTIn response to environmental fluctuations or stresses, bacteria can activate transcriptional and phenotypic programs to coordinate an adaptive response. The intracellular pathogenLegionella pneumophilaconverts from a noninfectious replicative form to an infectious transmissive form when the bacterium encounters alterations in either amino acid concentrations or fatty acid biosynthesis. Here, we report thatL. pneumophiladifferentiation is also triggered by nicotinic acid, a precursor of the central metabolite NAD+. In particular, when replicativeL. pneumophilaare treated with 5 mM nicotinic acid, the bacteria induce numerous transmissive-phase phenotypes, including motility, cytotoxicity toward macrophages, sodium sensitivity, and lysosome avoidance. Transcriptional profile analysis determined that nicotinic acid induces the expression of a panel of genes characteristic of transmissive-phaseL. pneumophila. Moreover, an additional 213 genes specific to nicotinic acid treatment were altered. Although nearly 25% of these genes lack an assigned function, the gene most highly induced by nicotinic acid treatment encodes a putative major facilitator superfamily transporter, Lpg0273. Indeed,lpg0273protectsL. pneumophilafrom toxic concentrations of nicotinic acid as judged by analyzing the growth of the corresponding mutant. The broad utility of the nicotinic acid pathway to couple central metabolism and cell fate is underscored by this small metabolite's modulation of gene expression by diverse microbes, includingCandida glabrata,Bordetella pertussis,Escherichia coli, andL. pneumophila.

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ThephtC-phtDLocus Equips Legionella pneumophila for Thymidine Salvage and Replication in Macrophages

Infection and Immunity ◽

10.1128/iai.01043-13 ◽

2013 ◽

Vol 82 (2) ◽

pp. 720-730 ◽

Cited By ~ 13

Author(s):

Maris V. Fonseca ◽

John-Demian Sauer ◽

Sebastien Crepin ◽

Brenda Byrne ◽

Michele S. Swanson

Keyword(s):

Escherichia Coli ◽

Life Cycle ◽

Thymidylate Synthase ◽

Legionella Pneumophila ◽

Opportunistic Pathogen ◽

Nucleoside Transporters ◽

Major Facilitator Superfamily ◽

Pyrimidine Nucleoside ◽

Content Type ◽

Major Facilitator

ABSTRACTThe phagosomal transporter (Pht) family of the major facilitator superfamily (MFS) is encoded by phylogenetically related intracellular gammaproteobacteria, including the opportunistic pathogenLegionella pneumophila. The location of thephtgenes between the putative thymidine kinase (tdk) and phosphopentomutase (deoB) genes suggested that thephtCandphtDloci contribute to thymidine salvage inL. pneumophila. Indeed, aphtC+allele intransrestored pyrimidine uptake to anEscherichia colimutant that lacked all known nucleoside transporters, whereas aphtD+allele did not. The results of phenotypic analyses ofL. pneumophilastrains lackingphtCorphtDstrongly indicate thatL. pneumophilarequires PhtC and PhtD function under conditions where sustained dTMP synthesis is compromised. First, in broth cultures that mimicked thymidine limitation or starvation,L. pneumophilaexhibited a marked requirement for PhtC function. Conversely, mutation ofphtDconferred a survival advantage. Second, in medium that lacked thymidine, multicopyphtC+orphtD+alleles enhanced the survival ofL. pneumophilathymidylate synthase (thyA)-deficient strains, which cannot synthesize dTMP endogenously. Third, under conditions in which transport of the pyrimidine nucleoside analog 5-fluorodeoxyuridine (FUdR) would inhibit growth, PhtC and PhtD conferred a growth advantage toL. pneumophilathyA+strains. Finally, when cultured in macrophages,L. pneumophilarequired thephtC-phtDlocus to replicate. Accordingly, we propose that PhtC and PhtD contribute to protectL. pneumophilafrom dTMP starvation during its intracellular life cycle.

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lbtA and lbtB Are Required for Production of the Legionella pneumophila Siderophore Legiobactin

Journal of Bacteriology ◽

10.1128/jb.188.4.1351-1363.2006 ◽

2006 ◽

Vol 188 (4) ◽

pp. 1351-1363 ◽

Cited By ~ 50

Author(s):

Kimberly A. Allard ◽

V. K. Viswanathan ◽

Nicholas P. Cianciotto

Keyword(s):

Legionella Pneumophila ◽

Iron Chelator ◽

Defined Medium ◽

Major Facilitator Superfamily ◽

Wild Type ◽

Multidrug Efflux ◽

Multidrug Efflux Pumps ◽

Cas Assay ◽

Major Facilitator ◽

Optimized Conditions

ABSTRACT Under iron stress, Legionella pneumophila secretes legiobactin, a nonclassical siderophore that is reactive in the chrome azurol S (CAS) assay. Here, we have optimized conditions for legiobactin expression, shown its biological activity, and identified two genes, lbtA and lbtB, which are involved in legiobactin production. lbtA appears to be iron repressed and encodes a protein that has significant homology with siderophore synthetases, and FrgA, a previously described iron-regulated protein of L. pneumophila. lbtB encodes a protein homologous with members of the major facilitator superfamily of multidrug efflux pumps. Mutants lacking lbtA or lbtB were defective for legiobactin, producing 40 to 70% less CAS reactivity in deferrated chemically defined medium (CDM). In bioassays, mutant CDM culture supernatants, unlike those of the wild type, did not support growth of iron-limited wild-type bacteria in 2′,2′-dipyridyl-containing buffered charcoal yeast extract (BCYE) agar and a ferrous iron transport mutant on BCYE agar without added iron. The lbtA mutant was modestly defective for growth in deferrated CDM containing the iron chelator citrate, indicating that legiobactin is required in conditions of severe iron limitation. Complementation of the lbt mutants restored both siderophore expression, as measured by the CAS assay and bioassays, and bacterial growth in deferrated, citrate-containing media. The lbtA mutant replicated as the wild type did in macrophages, amoebae, and the lungs of mice. However, L. pneumophila expresses lbtA in the macrophage, suggesting that legiobactin, though not required, may play a dispensable role in intracellular growth. The discovery of lbtAB represents the first identification of genes required for L. pneumophila siderophore expression.

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Untargeted Metabolomics Uncovers the Essential Lysine Transporter in Toxoplasma gondii

Metabolites ◽

10.3390/metabo11080476 ◽

2021 ◽

Vol 11 (8) ◽

pp. 476

Author(s):

Joachim Kloehn ◽

Matteo Lunghi ◽

Emmanuel Varesio ◽

David Dubois ◽

Dominique Soldati-Favre

Keyword(s):

Amino Acids ◽

Toxoplasma Gondii ◽

Rna Degradation ◽

Major Facilitator Superfamily ◽

Untargeted Metabolomics ◽

Apicomplexan Parasites ◽

Obligate Intracellular ◽

Intracellular Parasites ◽

Major Facilitator ◽

Plasma Membrane Transporter

Apicomplexan parasites are responsible for devastating diseases, including malaria, toxoplasmosis, and cryptosporidiosis. Current treatments are limited by emerging resistance to, as well as the high cost and toxicity of existing drugs. As obligate intracellular parasites, apicomplexans rely on the uptake of many essential metabolites from their host. Toxoplasma gondii, the causative agent of toxoplasmosis, is auxotrophic for several metabolites, including sugars (e.g., myo-inositol), amino acids (e.g., tyrosine), lipidic compounds and lipid precursors (cholesterol, choline), vitamins, cofactors (thiamine) and others. To date, only few apicomplexan metabolite transporters have been characterized and assigned a substrate. Here, we set out to investigate whether untargeted metabolomics can be used to identify the substrate of an uncharacterized transporter. Based on existing genome- and proteome-wide datasets, we have identified an essential plasma membrane transporter of the major facilitator superfamily in T. gondii—previously termed TgApiAT6-1. Using an inducible system based on RNA degradation, TgApiAT6-1 was depleted, and the mutant parasite’s metabolome was compared to that of non-depleted parasites. The most significantly reduced metabolite in parasites depleted in TgApiAT6-1 was identified as the amino acid lysine, for which T. gondii is predicted to be auxotrophic. Using stable isotope-labeled amino acids, we confirmed that TgApiAT6-1 is required for efficient lysine uptake. Our findings highlight untargeted metabolomics as a powerful tool to identify the substrate of orphan transporters.

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Structural basis of inhibition of a transporter from Staphylococcus aureus, NorC, through a single-domain camelid antibody

Communications Biology ◽

10.1038/s42003-021-02357-x ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Sushant Kumar ◽

Arunabh Athreya ◽

Ashutosh Gulati ◽

Rahul Mony Nair ◽

Ithayaraja Mahendran ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Pathogenic Bacteria ◽

Major Facilitator Superfamily ◽

Single Domain ◽

Fluoroquinolone Resistance ◽

Structural Basis ◽

Antibody Complex ◽

Complementarity Determining Regions ◽

Major Facilitator ◽

Alternating Access

AbstractTransporters play vital roles in acquiring antimicrobial resistance among pathogenic bacteria. In this study, we report the X-ray structure of NorC, a 14-transmembrane major facilitator superfamily member that is implicated in fluoroquinolone resistance in drug-resistant Staphylococcus aureus strains, at a resolution of 3.6 Å. The NorC structure was determined in complex with a single-domain camelid antibody that interacts at the extracellular face of the transporter and stabilizes it in an outward-open conformation. The complementarity determining regions of the antibody enter and block solvent access to the interior of the vestibule, thereby inhibiting alternating-access. NorC specifically interacts with an organic cation, tetraphenylphosphonium, although it does not demonstrate an ability to transport it. The interaction is compromised in the presence of NorC-antibody complex, consequently establishing a strategy to detect and block NorC and related transporters through the use of single-domain camelid antibodies.

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calderon encodes an organic cation transporter of the major facilitator superfamily required for cell growth and proliferation of Drosophila tissues

Development ◽

10.1242/dev.02436 ◽

2006 ◽

Vol 133 (14) ◽

pp. 2617-2625 ◽

Cited By ~ 10

Author(s):

H. Herranz

Keyword(s):

Cell Growth ◽

Organic Cation ◽

Organic Cation Transporter ◽

Major Facilitator Superfamily ◽

Major Facilitator ◽

Cell Growth And Proliferation

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Establishment of a Genetic Transformation System in Guanophilic Fungus Amphichorda guana

Journal of Fungi ◽

10.3390/jof7020138 ◽

2021 ◽

Vol 7 (2) ◽

pp. 138

Author(s):

Min Liang ◽

Wei Li ◽

Landa Qi ◽

Guocan Chen ◽

Lei Cai ◽

...

Keyword(s):

Genetic Transformation ◽

Genetic Manipulation ◽

Major Facilitator Superfamily ◽

Protoplast Regeneration ◽

Transformation System ◽

Regeneration Rate ◽

Bioactive Natural Products ◽

Genetics Research ◽

Genetic Transformation System ◽

Major Facilitator

Fungi from unique environments exhibit special physiological characters and plenty of bioactive natural products. However, the recalcitrant genetics or poor transformation efficiencies prevent scientists from systematically studying molecular biological mechanisms and exploiting their metabolites. In this study, we targeted a guanophilic fungus Amphichorda guana LC5815 and developed a genetic transformation system. We firstly established an efficient protoplast preparing method by conditional optimization of sporulation and protoplast regeneration. The regeneration rate of the protoplast is up to about 34.6% with 0.8 M sucrose as the osmotic pressure stabilizer. To develop the genetic transformation, we used the polyethylene glycol-mediated protoplast transformation, and the testing gene AG04914 encoding a major facilitator superfamily transporter was deleted in strain LC5815, which proves the feasibility of this genetic manipulation system. Furthermore, a uridine/uracil auxotrophic strain was created by using a positive screening protocol with 5-fluoroorotic acid as a selective reagent. Finally, the genetic transformation system was successfully established in the guanophilic fungus strain LC5815, which lays the foundation for the molecular genetics research and will facilitate the exploitation of bioactive secondary metabolites in fungi.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00853-09 ◽

2009 ◽

Vol 53 (11) ◽

pp. 4673-4677 ◽

Cited By ~ 24

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.

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A genomic strategy for cloning, expressing and purifying efflux proteins of the major facilitator superfamily

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkm036 ◽

2007 ◽

Vol 59 (6) ◽

pp. 1265-1270 ◽

Cited By ~ 13

Author(s):

Gerda Szakonyi ◽

Dong Leng ◽

Pikyee Ma ◽

Kim E. Bettaney ◽

Massoud Saidijam ◽

...

Keyword(s):

Major Facilitator Superfamily ◽

Major Facilitator ◽

Efflux Proteins

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Characteristics of 29 novel atypical solute carriers of major facilitator superfamily type: evolutionary conservation, predicted structure and neuronal co-expression

Open Biology ◽

10.1098/rsob.170142 ◽

2017 ◽

Vol 7 (9) ◽

pp. 170142 ◽

Cited By ~ 16

Author(s):

Emelie Perland ◽

Sonchita Bagchi ◽

Axel Klaesson ◽

Robert Fredriksson

Keyword(s):

Lipid Bilayers ◽

Markov Models ◽

Phylogenetic Analyses ◽

Molecular Transport ◽

Major Facilitator Superfamily ◽

Proximity Ligation Assay ◽

Sequence Comparisons ◽

Altered Expression ◽

Solute Carriers ◽

Major Facilitator

Solute carriers (SLCs) are vital as they are responsible for a major part of the molecular transport over lipid bilayers. At present, there are 430 identified SLCs, of which 28 are called atypical SLCs of major facilitator superfamily (MFS) type. These are MFSD1, 2A, 2B, 3, 4A, 4B, 5, 6, 6 L, 7, 8, 9, 10, 11, 12, 13A, 14A and 14B; SV2A, SV2B and SV2C; SVOP and SVOPL; SPNS1, SPNS2 and SPNS3; and UNC93A and UNC93B1. We studied their fundamental properties, and we also included CLN3, an atypical SLC not yet belonging to any protein family (Pfam) clan, because its involvement in the same neuronal degenerative disorders as MFSD8. With phylogenetic analyses and bioinformatic sequence comparisons, the proteins were divided into 15 families, denoted atypical MFS transporter families (AMTF1-15). Hidden Markov models were used to identify orthologues from human to Drosophila melanogaster and Caenorhabditis elegans . Topology predictions revealed 12 transmembrane segments (for all except CLN3), corresponding to the common MFS structure. With single-cell RNA sequencing and in situ proximity ligation assay on brain cells, co-expressions of several atypical SLCs were identified. Finally, the transcription levels of all genes were analysed in the hypothalamic N25/2 cell line after complete amino acid starvation, showing altered expression levels for several atypical SLCs.

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