Major Facilitator Research Articles

MFSD2A-associated primary microcephaly - Expanding the clinical and mutational spectrum of this ultra-rare disease

MFSD2A, a member of the major facilitator superfamily (MFS), is a transmembrane transporter responsible for the uptake of specific essential fatty acids through the blood-brain barrier (BBB) to the brain. The transporter is crucial for early embryonic brain development and a major factor in the formation and maintenance of the BBB. Mfsd2a-knockout mice show a leakage of the BBB in early embryonic stages and develop a phenotype characterized by microcephaly, cognitive impairment, and anxiety. So far, homozygous or compound heterozygous MFSD2A mutations in humans have only been reported in 13 different families with a total of 28 affected individuals. The phenotypical spectrum of patients with MFSD2A variants is rather broad but all patients present with microcephaly and severe intellectual disability, absent or limited speech, and walking difficulties. Severely affected patients develop seizures and show brain malformations and have, above all, a profound developmental delay hardly reaching any developmental motor milestones. Here, we report on two unrelated individuals with novel homozygous variants in the MFSD2A gene, presenting with severe primary microcephaly, brain malformations, profound developmental delay, and epilepsy, including hypsarrhythmia.Our findings extend the mutational spectrum of the bi-allelic MFSD2A variants causing autosomal recessive primary microcephaly type 15 and broaden the phenotypic spectrum associated with these pathogenic variants emphasizing the role of MFSD2A in early brain development.

Major Facilitator Superfamily Transporter Gene FgMFS1 Is Essential for Fusarium graminearum to Deal with Salicylic Acid Stress and for Its Pathogenicity towards Wheat.

Wheat is a major staple food crop worldwide, due to its total yield and unique processing quality. Its grain yield and quality are threatened by Fusarium head blight (FHB), which is mainly caused by Fusarium graminearum. Salicylic acid (SA) has a strong and toxic effect on F. graminearum and is a hopeful target for sustainable control of FHB. F. graminearum is capable of efficientdealing with SA stress. However, the underlying mechanisms remain unclear. Here, we characterized FgMFS1 (FGSG_03725), a major facilitator superfamily (MFS) transporter gene in F. graminearum. FgMFS1 was highly expressed during infection and was upregulated by SA. The predicted three-dimensional structure of the FgMFS1 protein was consistent with the schematic for the antiporter. The subcellular localization experiment indicated that FgMFS1 was usually expressed in the vacuole of hyphae, but was alternatively distributed in the cell membrane under SA treatment, indicating an element of F. graminearum in response to SA. ΔFgMFS1 (loss of function mutant of FgMFS1) showed enhanced sensitivity to SA, less pathogenicity towards wheat, and reduced DON production under SA stress. Re-introduction of a functional FgMFS1 gene into ∆FgMFS1 recovered the mutant phenotypes. Wheat spikes inoculated with ΔFgMFS1 accumulated more SA when compared to those inoculated with the wild-type strain. Ecotopic expression of FgMFS1 in yeast enhanced its tolerance to SA as expected, further demonstrating that FgMFS1 functions as an SA exporter. In conclusion, FgMFS1 encodes an SA exporter in F. graminearum, which is critical for its response to wheat endogenous SA and pathogenicity towards wheat.

P–546 Exome sequencing and preimplantation genetic testing for unexplained recurrent fetal malformations

Abstract Study question Could patient suffering unexplained recurrent fetal malformations be benefit of PGT-M by exome sequencing mutations identification? Summary answer Patients suffering unexplained recurrent fetal malformations could be benefit of the use of exome sequencing in combination to PGT-M to have a healthy live birth. What is known already Fetal malformations account for approximately 3% of live births and causes include: chromosomal abnormalities, exposure to toxic substances or teratogens and infections. Recently, studies have shown that several monogenic diseases are linked to fetal abnormalities. However, because of the large number of potential genes, genetic testing is challenging. Exome sequencing is widely used to detect genetic mutations and has emerged as a useful tool for finding the genetic cause of fetal abnormalities. The aim of this study was to show how exome sequencing in patients suffering unexplained recurrent fetal malformations in combination to PGT-M could lead to successful healthy newborn. Study design, size, duration Case report of a non-consanguineous couple with unexplained, recurrent fetal malformations. Couple were recruited during clinical consultation for unexplained recurrent fetal malformations at a private reproductive medicine clinic. The couple had two malformed fetus with the same congenital abnormalities: hydrocephalus, cerebellar vermis agenesis, cerebellar hypoplasia and enlarged cisterna magna. Patients signed written informed consent regarding to exome testing. For fetal sample, informed consent was obtained from parents. Participants/materials, setting, methods Sample of the affected fetus were provided. Parental genomic DNA was extracted from peripheral blood. Exome sequencing was performed using TrusightOne (Illumina®). FASTAQ data were processed through BWA and GATK algorithm. VCF files were analysed using Variant Interpreter software. After genetic counselling, PGT-M was performed using linkage polymorphic markers analysis and mutation sequencing. Embryo biopsy was carried at blastocyst stage. Embryos were vitrified and one healthy embryo was thaw and transfer in a subsequent cycle. Main results and the role of chance An homozygous novel pathogenic mutation c.641 C>T (p.Ala214Val) in FVLCR2 gene was found. The parents were heterozygous carriers revealing that the detected variant follow an autosomal recessive pattern. The FLVCR2 (14q24.3) gene encodes a transmembrane protein that belongs to the major facilitator superfamily of secondary carriers that transport small solutes in response to chemiosmosis ion gradients, such as calcium. Mutations in this gene are related to fetal central nervous system defects. This disorder is diagnosed prenatally and is lethal. PGT-M was recommended during genetic counselling. After control ovarian stimulation 14 oocytes were retrieved and finally 4 embryos were suitable for embryo biopsy at blastocyst stage. One embryo was diagnosed as healthy, two affected and one heterozygous carrier. The healthy embryo was thaw and transferred and a healthy male baby was born. Limitations, reasons for caution Exome sequencing has technical limitations: only covers mutations in coding regions and does not cover noncoding regions of the genome. It also cannot reliably detect copy-number variants at single gene level. Wider implications of the findings: This study offers strong evidence of exome-sequencing as a new diagnostic strategy and powerful tool discovering the underlying etiology of recurrent fetal malformations and identifying new genes important for human development. Using this strategy in combination with PGT-M, clinicians can help couples with recurrent fetal malformations to have healthy newborns. Trial registration number Not applicable

Immunoinformatic approach employing modeling and simulation to design a novel vaccine construct targeting MDR efflux pumps to confer wide protection against typhoidal Salmonella serovars

Overcoming multi drug resistance is one of the crucial challenges to control enteric typhoid fever caused by Salmonella typhi and Salmonella paratyphi. Overexpression of efflux pumps predominantly causes drug resistance in microorganisms. Therefore, immunotherapy targeting the various efflux pumps antigens could be a promising strategy to increase the success of vaccines. An immunoinformatic approach was employed to design a Salmonellosis multi-epitope subunit vaccine peptide consisting of linear B-cell and T-cell epitopes of multidrug resistance protein families including ATP Binding Cassette (ABC), major facilitator superfamily (MFS), resistance nodulation cell division (RND), small multidrug resistance (SMR), and multidrug and toxin extrusion (MATE). The selected epitopes exhibited conservation in both S. typhi and S. paratyphi and thus could be helpful for cross-protection. Further, the final vaccine construct encompassing the peptides, adjuvants and specific linker sequences showed high immunogenicity, solubility, non-allergenic, nontoxic, and wide population coverage due to strong binding affinity to maximum HLA alleles. The three-dimensional structure was predicted, and validated using various structure validation tools. Additionally, protein-protein docking of the chimeric vaccine construct with the TLR-2 protein and molecular dynamics demonstrated stable and efficient binding. Conclusively, the immunoinformatic study showed that the novel multi epitopic vaccine construct can simulate the both T-cell and B-cell immune responses in typhoidal Salmonella serovars and could potentially be used for prophylactic or therapeutic applications. Communicated by Ramaswamy H. Sarma

X-ray crystallography reveals molecular recognition mechanism for sugar binding in a melibiose transporter MelB

Major facilitator superfamily_2 transporters are widely found from bacteria to mammals. The melibiose transporter MelB, which catalyzes melibiose symport with either Na+, Li+, or H+, is a prototype of the Na+-coupled MFS transporters, but its sugar recognition mechanism has been a long-unsolved puzzle. Two high-resolution X-ray crystal structures of a Salmonella typhimurium MelB mutant with a bound ligand, either nitrophenyl-α-d-galactoside or dodecyl-β-d-melibioside, were refined to a resolution of 3.05 or 3.15 Å, respectively. In the substrate-binding site, the interaction of both galactosyl moieties on the two ligands with MelBSt are virturally same, so the sugar specificity determinant pocket can be recognized, and hence the molecular recognition mechanism for sugar binding in MelB has been deciphered. The conserved cation-binding pocket is also proposed, which directly connects to the sugar specificity pocket. These key structural findings have laid a solid foundation for our understanding of the cooperative binding and symport mechanisms in Na+-coupled MFS transporters, including eukaryotic transporters such as MFSD2A.

Whole Genome Analysis of Extensively Drug-Resistant Acinetobacter baumannii Clinical Isolates in Thailand.

Acinetobacter baumannii is recognized as a majority opportunistic nosocomial pathogen causing hospital-acquired infection worldwide. The increasing prevalence of extensively drug-resistant Acinetobacter baumannii (XDRAB) has become a rising concern in healthcare facilities and has impeded public health due to limitation of therapeutic options and are associated with high morbidity and mortality as well as longer hospitalization. Whole-genome sequencing of highly multidrug resistant A. baumannii will increase the understanding of resistant mechanisms, the emergence of novel resistance, genetic relationships among the isolates, source tracking, and treatment decisions in selected patients. This study revealed the genomic analysis to explore blaOXA-23 harboring XDRAB isolates in Thailand. Whole-genome sequencing of the two XDRAB isolates was carried out on a HiSeq2000 Illumina platform and susceptibility on antimicrobials was conducted. Both isolates revealed sequence types of international, clone II-carrying, multiple antimicrobial- resistant genes-ST195 and ST451. They were resistant to antimicrobial agents in all drug classes tested for Acinetobacter spp. They carried 18 antimicrobial-resistant genes comprising of 4 β-lactamase genes (blaOXA-23, blaOXA-66, blaTEM-1D, blaADC-25), 4 aminoglycoside-resistant genes (armA, aph(3')-Ia, aph(3")-Ib, aph(6)-Id), 3 macrolide-resistant genes (amvA, mphE, msrE), 1 sulfonamide- resistant gene (sul-2), 2 tetracycline-resistant genes (tetB, tetR), 1 resistant-nodulation-cell division (RND) antibiotic efflux pump gene cluster, 2 major facilitator superfamily (MFS) antibiotic efflux pump genes (abaF, abaQ), and 1 small multidrug-resistant (SMR) antibiotic efflux pump gene (abeS). Mutation of gyrA (S81L) occurred in both isolates. Whole-genome sequencing revealed both blaOXA-23 harboring XDRAB isolates were clustered under international clone II with different STs and carrying multiple antimicrobial-resistant genes conferred their resistance to antimicrobial agents. Inactivation of antimicrobials and target modification by enzymes and pumping antibiotics by efflux pump are mainly resistance mechanism of the XDRAB in this study.

The role of MfsR, a TetR-type transcriptional regulator, in adaptive protection of Stenotrophomonas maltophilia against benzalkonium chloride via the regulation of mfsQ.

A gene encoding the TetR-type transcriptional regulator mfsR is located immediately downstream of mfsQ and is transcribed in the same transcriptional unit. mfsQ encodes a major facilitator superfamily (MFS) efflux transporter contributing to the resistance of Stenotrophomonas maltophilia towards disinfectants belonging to quaternary ammonium compounds (QACs), which include benzalkonium chloride (BAC). Phylogenetic analysis revealed that MfsR is closely related to CgmR, a QAC-responsive transcriptional regulator belonging to the TetR family. MfsR regulated the expression of the mfsQR operon in a QAC-inducible manner. The constitutively high transcript level of mfsQ in an mfsR mutant indicated that MfsR functions as a transcriptional repressor of the mfsQR operon. Electrophoretic mobility shift assays showed that purified MfsR specifically bound to the putative promoter region of mfsQR, and in vitro treatments with QACs led to the release of MfsR from binding complexes. DNase I protection assays revealed that the MfsR binding box comprises inverted palindromic sequences located between motifs -35 and -10 of the putative mfsQR promoter. BAC-induced adaptive protection was abolished in the mfsR mutant and was restored in the complemented mutant. Overall, MfsR is a QACs-sensing regulator that controls the expression of mfsQ. In the absence of QACs, MfsR binds to the box located in the mfsQR promoter and represses its transcription. The presence of QACs derepresses MfsR activity, allowing RNA polymerase binding and transcription of mfsQR. This MfsR-MsfQ system enables S. maltophilia to withstand high levels of QACs.

Host - Bacterial Pathogen Communication: The Wily Role of the Multidrug Efflux Pumps of the MFS Family.

Bacterial pathogens are able to survive within diverse habitats. The dynamic adaptation to the surroundings depends on their ability to sense environmental variations and to respond in an appropriate manner. This involves, among others, the activation of various cell-to-cell communication strategies. The capability of the bacterial cells to rapidly and co-ordinately set up an interplay with the host cells and/or with other bacteria facilitates their survival in the new niche. Efflux pumps are ubiquitous transmembrane transporters, able to extrude a large set of different molecules. They are strongly implicated in antibiotic resistance since they are able to efficiently expel most of the clinically relevant antibiotics from the bacterial cytoplasm. Besides antibiotic resistance, multidrug efflux pumps take part in several important processes of bacterial cell physiology, including cell to cell communication, and contribute to increase the virulence potential of several bacterial pathogens. Here, we focus on the structural and functional role of multidrug efflux pumps belonging to the Major Facilitator Superfamily (MFS), the largest family of transporters, highlighting their involvement in the colonization of host cells, in virulence and in biofilm formation. We will offer an overview on how MFS multidrug transporters contribute to bacterial survival, adaptation and pathogenicity through the export of diverse molecules. This will be done by presenting the functions of several relevant MFS multidrug efflux pumps in human life-threatening bacterial pathogens as Staphylococcus aureus, Listeria monocytogenes, Klebsiella pneumoniae, Shigella/E. coli, Acinetobacter baumannii.

Cloning and expression pattern of phosphate transporter 1;1 cDNA sequence from Spirodela polyrrhiza

Spirodela polyrrhiza is a floating plant widely used in biomass utilization and eutrophication phytoremediation. It becomes a common aquatic plant everywhere with the increasingly serious eutrophication. It has been reported that S. polyrrhiza has a good effect on the remediation of eutrophication water. In order to study the absorption and transportation of phosphorus in S. polyrrhiza, we extracted RNA from S. polyrrhiza and then reverse transcribed it into cDNA, which was used as a template to amplify a specific fragment. The full-length sequence of the open reading frame (ORF) was 1 620 bp, encoding 539 amino acids, named SpPHT1;1, and the accession number in GenBank was MN720003. Bioinformatical analysis showed that SpPHT1;1 had no intron. The protein it encoded was a stable, hydrophobic protein with 11 transmembrane domains. SpPHT1;1 structure was similar to that of major facilitator superfamily (MFS) superfamily members. The cluster analysis showed that SpPHT1;1 was closely related to ZMPHT2 in maize and SBPHT1-8 in sorghum. So, it might belong to plant PHT1 family. The expression of SpPHT1;1 in leaf was significantly more than that of root under normal phosphorus condition. Low phosphorus condition could promote gene expression, and the relative expression level of SpPHT1;1 arrived at the peak at 48 h both in root and leaf. High phosphorus condition could inhibit gene expression. These results indicated that SpPHT1;1 expression would be affected by external phosphorus concentration. The results of this study are helpful for further research on the function of phosphate transporter. It also can provide theoretical basis for further development and utilization of S. polyrrhiza.

Constitutive and differential expression of transport protein genes in Parascaris univalens larvae and adult tissues after in vitro exposure to anthelmintic drugs

The equine roundworm Parascaris univalens has developed resistance to the three anthelmintic substances most commonly used in horses. The mechanisms responsible for resistance are believed to be multi-genic, and transport proteins such as the P-glycoprotein (Pgp) family have been suggested to be involved in resistance in several parasites including P. univlaens. To facilitate further research into the mechanisms behind drug metabolism and resistance development in P. univalens we aimed to develop an in vitro model based on larvae. We developed a fast and easy protocol for hatching P. univalens larvae for in vitro studies, resulting in a hatching rate of 92 %. The expression of transport protein genes pgp-2, pgp-9, pgp-11.1, pgp-16.1 and major facilitator superfamily (MFS) genes PgR006_g137 and PgR015_g078 were studied in hatched larvae exposed to the anthelmintic drugs ivermecin (IVM) 10−9 M, pyrantel citrate (PYR) 10−6 M and thiabendazole (TBZ) 10−5 M for 24 h. In comparison, the expression of these transport protein genes was studied in the anterior end and intestinal tissues of adult worms in vitro exposed to IVM, TBZ and PYR, at the same concentrations as larvae, for 3 h, 10 h and 24 h. Larval exposure to sub-lethal doses of IVM for 24 h did not affect the expression levels of any of the investigated genes, however larvae exposed to PYR and TBZ for 24 h showed significantly increased expression of pgp-9. In vitro drug exposure of adult worms did not result in any significant increases in expression of transport protein genes. Comparisons of constitutive expression between larvae and adult worm tissues showed that pgp-9, pgp-11.1, pgp-16.1 and MFS gene PgR015_g078 were expressed at lower levels in larvae than in adult tissues, while pgp-2 and MFS gene PgR006_g137 had similar expression levels in larvae and adult worms. All investigated transport protein genes were expressed at higher rates in the intestine than in the anterior end of adult worms, except pgp-11.1 where the expression was similar between the two tissues. This high constitutive expression in the intestine suggests that this is an important site for xenobiotic efflux in P. univalens. Despite the fact that the results of this study show differences in expression of transport protein genes between larvae and adult tissues, we believe that the larval assay system described here will be an important tool for further research into the molecular mechanisms behind anthelmintic resistance development and for other in vitro studies.

Untargeted Metabolomics Uncovers the Essential Lysine Transporter in Toxoplasma gondii.

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.

Implication of the Type III Effector RipS1 in the Cool-Virulence of Ralstonia solanacearum Strain UW551.

Members of the Ralstonia solanacearum species complex cause a variety of wilting diseases across a wide range of hosts by colonizing and blocking xylem vessels. Of great concern are race 3 biovar 2 strains of R. solanacearum capable of causing brown rot of potato at cool temperatures, which are select agents in the United States. To gain a better understanding of cool-virulence mechanisms, we generated libraries of transposon mutants in the cool-virulent R. solanacearum strain UW551 and screened 10,000 mutants using our seedling assay for significantly reduced virulence at 20°C. We found several mutants that exhibited reduced virulence at 28 and 20°C and also mutants that were only affected at the cooler temperature. One mutant of the latter chosen for further study had the transposon inserted in an intergenic region between a type III secretion system effector gene ripS1 and a major facilitator superfamily (MFS) protein gene. Gene expression analysis showed that expression of ripS1 was altered by the transposon insertion, but not the MFS protein gene. An independent mutant with this insertion upstream of ripS1 was generated and used to confirm virulence and gene expression phenotypes. The effector, RipS1, has unknown function and is part of a family of effectors belonging to the largest known type III effectors. The functional connection between RipS1 and cool-virulence of R. solanacearum UW551 suggests that RipS1 (and/or its upstream promoter element) may serve as a potential target for development of cool-virulence-specific diagnostic tools to differentiate the highly regulated cool-virulent strains from non-cool-virulent strains of R. solanacearum. Our results provide important information for continued work toward a better understanding of cool-virulence of R. solanacearum and development of proper control strategies to combat this important plant pathogen.

Integrated analysis of transcriptome and proteome for exploring the mechanism of guaiacol production by Alicyclobacillus acidoterrestris

Alicyclobacillus spp. can cause commercially pasteurized fruit juices/beverages to spoil and the spoilage is characterized by the formation of a distinct medicinal or antiseptic off-odor attributed to guaiacol. The aim of this study was to reveal the mechanism of guaiacol production in A. acidoterrestris by combining transcriptomic and proteomic approaches. RNA-sequencing and iTRAQ analyses were conducted to investigate differences in expression levels of genes and proteins in A. acidoterrestris when producing (with 500 μM vanillic acid) and not producing (without vanillic acid) guaiacol. A total of 225 differentially expressed genes and 77 differentially expressed proteins were identified. The transcription of genes vdcBCD encoding subunits of vanillic acid decarboxylase were 626.47, 185.01 and 52.81-fold up-regulated, respectively; they were the most up-regulated genes involved in guaiacol production. Expressions of the benzoate membrane transport protein, fusaric acid resistance protein, resistance-nodulation- division transporter, some ATP-binding cassette transporters and major facilitator superfamily transporters were increased at either mRNA, protein or both levels, indicating that they participated in the uptake of vanillic acid and extrusion of guaiacol. In the metabolic process of vanillic acid to guaiacol in A. acidoterrestris, genes related to the pathway of tricarboxylic acid cycle and ribosome were up-regulated, while the expression of some genes associated with valine, leucine and isoleucine biosynthesis was decreased. These findings provide novel insight to understand the mechanism of guaiacol production in A. acidoterrestris, which will serve as an important guide for developing strategies for the control of A. acidoterrestris problems in the fruit juice industry.

The membrane transporter lactose permease increases lipid bilayer bending rigidity

Cellular life relies on membranes, which provide a resilient and adaptive cell boundary. Many essential processes depend upon the ease with which the membrane is able to deform and bend, features that can be characterized by the bending rigidity. Quantitative investigations of such mechanical properties of biological membranes have primarily been undertaken in solely lipid bilayers and frequently in the absence of buffers. In contrast, much less is known about the influence of integral membrane proteins on bending rigidity under physiological conditions. We focus on an exemplar member of the ubiquitous major facilitator superfamily of transporters and assess the influence of lactose permease on the bending rigidity of lipid bilayers. Fluctuation analysis of giant unilamellar vesicles (GUVs) is a useful means to measure bending rigidity. We find that using a hydrogel substrate produces GUVs that are well suited to fluctuation analysis. Moreover, the hydrogel method is amenable to both physiological salt concentrations and anionic lipids, which are important to mimic key aspects of the native lactose permease membrane. Varying the fraction of the anionic lipid in the lipid mixture DOPC/DOPE/DOPG allows us to assess the dependence of membrane bending rigidity on the topology and concentration of an integral membrane protein in the lipid bilayer of GUVs. The bending rigidity gradually increases with the incorporation of lactose permease, but there is no further increase with greater amounts of the protein in the membrane.

Antifungal Siderophore Conjugates for Theranostic Applications in Invasive Pulmonary Aspergillosis Using Low-Molecular TAFC Scaffolds.

Invasive pulmonary aspergillosis (IPA) is a life-threatening form of fungal infection, primarily in immunocompromised patients and associated with significant mortality. Diagnostic procedures are often invasive and/or time consuming and existing antifungals can be constrained by dose-limiting toxicity and drug interaction. In this study, we modified triacetylfusarinine C (TAFC), the main siderophore produced by the opportunistic pathogen Aspergillus fumigatus (A. fumigatus), with antifungal molecules to perform antifungal susceptibility tests and molecular imaging. A variation of small organic molecules (eflornithine, fludioxonil, thiomersal, fluoroorotic acid (FOA), cyanine 5 (Cy5) with antifungal activity were coupled to diacetylfusarinine C (DAFC), resulting in a “Trojan horse” to deliver antifungal compounds specifically into A. fumigatus hyphae by the major facilitator transporter MirB. Radioactive labeling with gallium-68 allowed us to perform in vitro characterization (distribution coefficient, stability, uptake assay) as well as biodistribution experiments and PET/CT imaging in an IPA rat infection model. Compounds chelated with stable gallium were used for antifungal susceptibility tests. [Ga]DAFC-fludioxonil, -FOA, and -Cy5 revealed a MirB-dependent active uptake with fungal growth inhibition at 16 µg/mL after 24 h. Visualization of an A. fumigatus infection in lungs of a rat was possible with gallium-68-labeled compounds using PET/CT. Heterogeneous biodistribution patterns revealed the immense influence of the antifungal moiety conjugated to DAFC. Overall, novel antifungal siderophore conjugates with promising fungal growth inhibition and the possibility to perform PET imaging combine both therapeutic and diagnostic potential in a theranostic compound for IPA caused by A. fumigatus.

Transcriptome analysis to elucidate the toxicity mechanisms of fenvalerate, sulfide gatifloxacin, and ridomil on the hepatopancreas of Procambarus clarkii

Most antibiotics, insecticides, and other chemicals used in agricultural and fishery production tend to persist in the environment. Fenvalerate, sulfide gatifloxacin, and ridomil are widely used in aquaculture as antibacterial, antifungal, and antiparasitic drugs; however, their toxicity mechanism remains unclear. Thus, we herein analyzed the effects of these three drugs on the hepatopancreas of Procambarus clarkii at the transcriptome level. Twelve normalized cDNA libraries were constructed using RNA extracted from P. clarkii after treatment with fenvalerate, sulfide gatifloxacin, or ridomil and from an untreated control group, followed by Kyoto Encyclopedia of Genes and Genomes pathway analysis. In the control vs fenvalerate and control vs sulfide gatifloxacin groups, 14 and seven pathways were significantly enriched, respectively. Further, the effects of fenvalerate and sulfide gatifloxacin were similar on the hepatopancreas of P. clarkii. We also found that the expression level of genes encoding senescence marker protein-30 and arylsulfatase A was downregulated in the sulfide gatifloxacin group, indicating that sulfide gatifloxacin accelerated the apoptosis of hepatopancreatocytes. The expression level of major facilitator superfamily domain containing 10 was downregulated, implying that it interferes with the ability of the hepatopancreas to metabolize drugs. Interestingly, we found that Niemann pick type C1 and glucosylceramidase-β potentially interact with each other, consequently decreasing the antioxidant capacity of P. clarkii hepatopancreas. In the fenvalerate group, the downregulation of the expression level of xanthine dehydrogenase indicated that fenvalerate affected the immune system of P. clarkii; moreover, the upregulation of the expression level of pancreatitis-associated protein-2 and cathepsin C indicated that fenvalerate caused possible inflammatory pathological injury to P. clarkii hepatopancreas. In the ridomil group, no pathway was significantly enriched. In total, 21 genes showed significant differences in all three groups. To conclude, although there appears to be some overlap in the toxicity mechanisms of fenvalerate, sulfide gatifloxacin, and ridomil, further studies are warranted.

MFS1, a Pleiotropic Transporter in Dermatophytes That Plays a Key Role in Their Intrinsic Resistance to Chloramphenicol and Fluconazole.

A recently identified Trichophyton rubrum major facilitator superfamily (MFS)-type transporter (TruMFS1) has been shown to give resistance to azole compounds and cycloheximide (CYH) when overexpressed in Saccharomyces cerevisiae. We investigated the roles of MFS1 in the intrinsic resistance of dermatophytes to CYH and chloramphenicol (CHL), which are commonly used to isolate these fungi, and to what extent MFS1 affects the susceptibility to azole antifungals. Susceptibility to antibiotics and azoles was tested in S. cerevisiae overexpressing MFS1 and ΔMFS1 mutants of Trichophyton benhamiae, a dermatophyte that is closely related to T. rubrum. We found that TruMFS1 functions as an efflux pump for CHL in addition to CYH and azoles in S. cerevisiae. In contrast, the growth of T. benhamiae ΔMFS1 mutants was not reduced in the presence of CYH but was severely impaired in the presence of CHL and thiamphenicol, a CHL analog. The suppression of MFS1 in T. benhamiae also increased the sensitivity of the fungus to fluconazole and miconazole. Our experiments revealed a key role of MFS1 in the resistance of dermatophytes to CHL and their high minimum inhibitory concentration for fluconazole. Suppression of MFS1 did not affect the sensitivity to CYH, suggesting that another mechanism was involved in resistance to CYH in dermatophytes.

Comparative genomic analysis of Escherichia coli isolates from cases of bovine clinical mastitis identifies nine specific pathotype marker genes.

Escherichia coli is a major causative agent of environmental bovine mastitis and this disease causes significant economic losses for the dairy industry. There is still debate in the literature as to whether mammary pathogenic E. coli (MPEC) is indeed a unique E. coli pathotype, or whether this infection is merely an opportunistic infection caused by any E. coli isolate being displaced from the bovine gastrointestinal tract to the environment and, then, into the udder. In this study, we conducted a thorough genomic analysis of 113 novel MPEC isolates from clinical mastitis cases and 100 bovine commensal E. coli isolates. A phylogenomic analysis indicated that MPEC and commensal E. coli isolates formed clades based on common sequence types and O antigens, but did not cluster based on mammary pathogenicity. A comparative genomic analysis of MPEC and commensal isolates led to the identification of nine genes that were part of either the core or the soft-core MPEC genome, but were not found in any bovine commensal isolates. These apparent MPEC marker genes were genes involved with nutrient intake and metabolism [adeQ, adenine permease; nifJ, pyruvate-flavodoxin oxidoreductase; and yhjX, putative major facilitator superfamily (MFS)-type transporter], included fitness and virulence factors commonly seen in uropathogenic E. coli (pqqL, zinc metallopeptidase, and fdeC, intimin-like adhesin, respectively), and putative proteins [yfiE, uncharacterized helix-turn-helix-type transcriptional activator; ygjI, putative inner membrane transporter; and ygjJ, putative periplasmic protein]. Further characterization of these highly conserved MPEC genes may be critical to understanding the pathobiology of MPEC.

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

Transporters 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.

Capturing the Rest: Inpatient Mammography for Nonadherent Hospitalized Women

More than a third of hospitalized women are overdue or nonadherent to breast cancer screening guidelines, and almost a third of them are also at high risk for developing breast cancer. The purpose of this study is to evaluate the feasibility of coordinating inpatient breast cancer screening mammography for these women before their discharge from the hospital. A prospective intervention study was conducted among 101 nonadherent women aged 50-74 years who were hospitalized to a general medicine service. Sociodemographic, reproductive history, family history of breast cancer, and medical comorbidities data were collected for all patients from January 2015 to October 2016. The data were analyzed in March 2018. Fisher's exact tests and unpaired t-tests were utilized to compare the characteristics of the study population. Of the 101 women enrolled who were nonadherent to breast cancer screening recommendations, their mean age was 59.3 (SD=6) years, the mean 5-year Gail risk score was 1.63 (SD=0.69), and 29% of the women were African American. Almost 80% (n=79) underwent inpatient screening mammography. All women who underwent screening mammography during their inpatient stay were extremely satisfied with the experience. The convenience of having screening mammography while hospitalized was reported to be a major facilitator of completing the overdue screening. All nurses (100%) taking care of these women believed that this practice should become part of the standard of care, and most hospitalist physicians (66%) agreed that this practice is feasible. This study shows that it is possible to coordinate mammography for hospitalized women who were overdue for screening and at high risk for developing breast cancer. This study is registered at www.clinicaltrials.gov NCT04164251.