Brucella canis, is the primary cause of reproductive failure in dogs and represents a public health concern due to its zoonotic nature. Cases in dogs in the United States have been increasing due to the persistent nature of the bacterium, deficiencies in current diagnostic testing, and, most importantly, the lack of a protective vaccine. Current estimates place the seroprevalence of B. canis in the southern United States at 7% to 8%, but with the unprecedented rates of animals moving across state and international borders and the lack of federal regulations in regard to testing, the true seroprevalence of B. canis in the United States may very well be higher. Vaccination represents the most effective method of brucellosis control and, in response to the demand for a vaccine against B. canis, we have developed the live attenuated B. canis RM6/66 ΔvjbR vaccine strain capable of protecting mice against challenge.Canine brucellosis, caused by  Brucella canis is a Gram-negative, facultative intracellular bacterium and the causative agent of canine brucellosis, a highly contagious disease of dogs that can be transmitted to humans. Unfortunately, no vaccine is available to prevent infection. We recently characterized the kinetics of B. canis infection in the mouse model, establishing the required dose necessary to achieve systemic infection. The objective of this study was to investigate the utility of the mouse model in assessing canine brucellosis vaccine candidates and to subsequently investigate the safety and efficacy of a live attenuated vaccine, the B. canis RM6/66 ΔvjbR strain. Mice vaccinated with a dose of 109 CFU of the vaccine strain by both intraperitoneal and subcutaneous routes were afforded significant protection against organ colonization and development of histopathologic lesions following intraperitoneal challenge. Addition of an adjuvant or a booster dose 2 weeks following initial vaccination did not alter protection levels. Vaccination also resulted in a robust humoral immune response in mice, and B. canis RM6/66 ΔvjbR was capable of activating canine dendritic cells in vitro. These data demonstrate that the B. canis RM6/66 ΔvjbR strain shows promise as a vaccine for canine brucellosis and validates the mouse model for future vaccine efficacy studies.IMPORTANCE Canine brucellosis, caused by Brucella canis, is the primary cause of reproductive failure in dogs and represents a public health concern due to its zoonotic nature. Cases in dogs in the United States have been increasing due to the persistent nature of the bacterium, deficiencies in current diagnostic testing, and, most importantly, the lack of a protective vaccine. Current estimates place the seroprevalence of B. canis in the southern United States at 7% to 8%, but with the unprecedented rates of animals moving across state and international borders and the lack of federal regulations in regard to testing, the true seroprevalence of B. canis in the United States may very well be higher. Vaccination represents the most effective method of brucellosis control and, in response to the demand for a vaccine against B. canis, we have developed the live attenuated B. canis RM6/66 ΔvjbR vaccine strain capable of protecting mice against challenge. Brucella, a facultative intracellular bacterium, is the causative agent of brucellosis, a devastating disease in multiple domestic and wild mammalian species and the most commonly reported zoonotic disease in humans worldwide  and verified following PCR and sequencing , merodiploid, and mutant colonies. Clone 7 (C7) in lane 4 and clone 9 (C9) in lane 6 were selected as B. canis RM6/66 ΔvjbR mutants for use in vaccination studies. Successful production of B. canis RM6/66 ΔvjbR resulted in deletion of a 753-bp fragment, resulting in a final product size of 193 bp. Download FIG S1, TIF file, 0.5 MB.Confirmation and selection of Copyright © 2020 Stranahan et al.2020Stranahan et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the B. canis RM6/66 ΔvjbR in vivo to this aim, dose titration studies were carried out in mice via intraperitoneal (i.p.) infection at various doses  was compared  group. Mice in the high-dose group also exhibited scattered microgranulomas (70- to 150-μm diameter) composed of macrophages by 2 weeks postvaccination, a change not observed in the other dose groups  were vaccinated i.p. with 105, 107, or 109 CFU of B. canis RM6/66 ΔvjbR or PBS and euthanized at weekly intervals through 4 weeks postvaccination. H&E staining of the liver at 2 weeks postvaccination at low magnification (left) and high magnification (right). Vaccination with a high dose of 109 CFU resulted in the formation of multifocal microgranulomas (asterisks) as well as small foci of extramedullary hematopoiesis (EMH) (arrows) adjacent to portal and central veins. Bars, 200 μm (left) and 50 μm (right). Download FIG S2, PDF file, 0.7 MB.Vaccination of mice with 10Copyright © 2020 Stranahan et al.2020Stranahan et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 7 CFU of B. canis RM6/66 (9 CFU of B. canis RM6/66 ΔvjbR exhibited significantly lower colonization in the spleen (3.092-log reduction), liver (2.543-log reduction), and lung (2.365-log reduction), with one mouse exhibiting no detectable colonization in the spleen and 2 mice demonstrating the same effect in the lung  and liver (3.033-log reduction)  . Interescination .5 and 107 CFU   . Vaccina10.1128/mSphere.00172-20.3FIG S3B. canis RM6/66 ΔvjbR protects against histiocytic inflammation in the liver following B. canis challenge. Female C57BL/6 mice were vaccinated with 109 CFU of B. canis RM6/66 ΔvjbR s.q., with some mice receiving the adjuvant Quil-A, a lysate of the vaccine strain, and/or a booster vaccination 2 weeks following the first vaccination. One group of mice was vaccinated with PBS as a negative control. Mice were challenged at 8 weeks postvaccination with 107 CFU of B. canis RM6/66 i.p., and livers were evaluated histopathologically at 2 weeks postchallenge. (A) H&E staining of the liver following challenge. Note the multifocal foci of histiocytic inflammation, or microgranulomas (arrows), within the red pulp, most numerous in mice in the first 5 groups. Bars, 100 μm. (B) The number of microgranulomas per liver section at 2 weeks postchallenge was counted and compared between groups. Data are expressed as the means ± standard deviations. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (Tukey’s multiple-comparison test). Download FIG S3, PDF file, 0.4 MB.Subcutaneous vaccination of mice with Copyright © 2020 Stranahan et al.2020Stranahan et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 9 CFU B. canis RM6/66 ΔvjbR with a 2-week booster were also afforded high levels of protection, with reductions in colonization of 1.91 log in the liver, 3.03 log in the spleen, and 2.62 log in the lung. The level of organ colonization between mice vaccinated with a single dose (group 7) versus those that received a booster dose (group 6) was not significantly different. Unexpectedly, addition of the adjuvant, Quil-A, and/or a lysate of the vaccine strain did not improve protection against colonization. Rather, log reduction in organ colonization in these groups was inferior to that achieved by the vaccine alone, either in single dose or booster format  and group 7 (single vaccine dose) demonstrating the greatest reduction , although the change was not significant  which received two doses and a single dose of B. canis RM6/66 ΔvjbR, respectively. Further demonstrating the TH1 skew to the immune response in groups 6 and 7 are the low IgG1/IgG2a optical density (OD) ratios of 0.098 and 0.041, respectively, compared to ratios of up to 2.06 for group 2.To further investigate the mechanism behind vaccination-induced protection, the levels of anti-controls . InteresB. canis RM6/66 ΔvjbR to induce a cell-mediated immune response in the natural canine host was investigated in vitro. Dendritic cells were selected as they are key antigen-presenting cells that play a critical role in the initial immune response to foreign antigens, including those associated with vaccines. Stimulation of mononuclear derived dendritic cells from healthy dogs with both wild-type B. canis RM6/66 and B. canis RM6/66 ΔvjbR resulted in a significantly higher percentage of cells expressing both HLA-DR, an MHC class II surface receptor, and CD86, a costimulatory protein for T lymphocytes, changes which were also observed in cells stimulated with the Escherichia coli lipopolysaccharide (LPS) positive control , or production of blood cells outside the bone marrow in response to increased demand  and canine blood collection protocols (protocol 2018-0457 CA) were reviewed and approved by the Texas A&M University Laboratory Animal Care and Use Committee. All protocols were approved and were in accordance with the Institutional Animal Care and use Committee (IACUC) policies of Texas A&M University. Texas A&M is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, International (AAALAC).ad libitum access to food and filtered water. Mice were monitored daily for signs of pain or distress according to the guidelines of the Animal Research Advisory Committee published by the National Institutes of Health. For generation of dendritic cells in vitro, fresh blood was collected from healthy client-owned dogs presenting to Texas A&M University Small Animal Hospital following informed client consent. All dogs were up to date on routine vaccinations  and had no systemic inflammatory, infectious, or neoplastic conditions. Blood (20 to 25 ml) was collected by licensed veterinary technicians from nonanesthetized dogs via the saphenous vein.Female C57BL/6J (6 to 8 weeks old) were obtained from the Texas A&M Institute for Genomic Medicine and housed in microisolator caging in biosafety level 2 and 3 facilities at Texas A&M College of Veterinary Medicine. All mice were acclimated to the facility for 5 days prior to vaccination or infection and were maintained on a 12-h/12-h light-dark cycle with B. canis ATCC RM6/66 was used for these studies. Bacterial stocks were stored at −80°C in 10% glycerol and were routinely grown on tryptic soy agar (TSA) plates or in standard tryptic soy broth (TSB). Bacteria were harvested from plates using phosphate-buffered saline (PBS), pH 7.2 (Gibco), and adjusted to a final concentration of either 105, 107, or 109 CFU/0.1 ml using a Klett colorimeter meter reading against a standard curve. Viable counts were retrospectively confirmed by serial dilution and plating onto TSA plates.Brucella canis RM6/66 ΔvjbR was generated using the kanamycin-resistant pUC19 suicide plasmid and the ampicillin-resistant pCVD442 plasmid. Briefly, a 650-bp upstream fragment and a 350-bp downstream fragment of the vjbR gene (BCAN_RS10670) were amplified. The upstream fragment was amplified using forward primer (CCCCCGAGCTCTTATCGGCCAGTTGGAAAAG) and reverse primer (CCCCCGGATCCGAGATCAAGACTCATTGGAAATATCC). The downstream fragment was amplified using forward primer (CCCCCGGATCCCATCTCGTCTGATCAACATGG) and reverse primer (CCCCCTCTAGAATTTCTATCCCGGCACACTG). A BamHI restriction site was placed at the 5′ end and an Xbal restriction at the 3′ end for both PCR products. Following PCR amplification and restriction digestion, upstream and downstream gene segments were separately cloned into pUC19 plasmids. Upstream and downstream gene segments were joined using crisscross reactions, and positive clones were selected, with proper orientation confirmed by PCR amplification and sequencing. E. coli BL21 cells were used to amplify the plasmid, and transformed clones were selected based on kanamycin resistance.vjbR deletion cassette from the pUC19 plasmid was restriction digested and ligated into the pCVD442 plasmid. Ligated products were transformed into E. coli β2155 using heat shock, and successful transformation was confirmed following sequencing. The pCVD442 plasmid was transformed into B. canis RM6/66 via electroporation. Positive clones were selected based on ampicillin susceptibility (100 μg/ml) and sucrose resistance (10%). Successful deletion of the vjbR gene (753 bp) was confirmed using PCR amplification and sequencing.To remove antibiotic resistance, the B. canis RM6/66 and B. canis RM6/66 ΔvjbR were grown on TSA plates for 3 days and then subcultured in 5 ml of TSB in 50-ml, sterile conical plastic tubes. Liquid cultures were maintained at 37°C and 200 rpm for 16 to 20 h. Cells were infected with a multiplicity of infection (MOI) of 100, and all inocula were serially diluted and plated on TSA to confirm inoculation dose. Plates containing infected cells were centrifuged at 1,000 rpm at 22°C, incubated for 30 min at 37°C and 5% CO2, and washed with warm culture medium. Extracellular bacteria were eliminated by addition of 50 μg/ml of gentamicin for 1 h. After 1, 24, and 48 h of incubation, plates were washed with phosphate-buffered saline (PBS), and cells were lysed by treatment with 0.5% Tween 20 for 5 min with vigorous scraping. Aliquots were serially diluted, plated on TSA, and incubated at 37°C for 3 days for CFU determination. For determination of cell death, lactate dehydrogenase (LDH) released into cell culture supernatants of infected and uninfected cells was determined at all time points using the CytoTox 96 nonradioactive cytotoxicity assay kit  according to the manufacturer’s instructions. Cell cytotoxicity was expressed as the percentage of LDH release, which was calculated using the following formula: percentage of LDH release = 100 × (test LDS release − spontaneous release)/(maximum release − spontaneous release).Cellular infections for estimation of bacterial invasion and replication were performed using canine DH82 macrophage-like cells (ATCC CRL-10389). Cells were grown to confluence in 24-well tissue culture plates using Eagle’s minimum essential medium (EMEM) plus 10% fetal bovine serum. n = 5). For assessment of virulence and kinetics of organ colonization of the vaccine strain, mice were vaccinated intraperitoneally (i.p.) with either PBS or 105, 107, or 109 CFU of B. canis RM6/66 ΔvjbR. Mice were sacrificed by CO2 asphyxiation and cervical dislocation at weekly intervals for 4 weeks. For dose titration and preliminary investigation of protective efficacy, mice vaccinated i.p. with the aforementioned doses of B. canis RM6/66 ΔvjbR were challenged i.p. with 107 CFU of wild-type B. canis RM6/66 in 100 μl of PBS at 4 weeks postvaccination and euthanized at 1 and 2 weeks postchallenge.For all experiments, mice were randomly divided into groups . Lysates were prepared following 3 passages through a French press. The components of the vaccine per group and dosing scheduled are highlighted in 7 CFU of B. canis RM6/66 by i.p. inoculation, with euthanasia following 2 weeks later.In a second protection study, mice were vaccinated subcutaneously (s.q.) with 100 μl PBS containing 1010.1128/mSphere.00172-20.4FIG S4n = 5) were vaccinated subcutaneously with 109 CFU B. canis RM6/66 ΔvjbR, with some mice receiving additional components of the adjuvant Quil-A (15 μg) or a lysate of the vaccine strain (50 μg). Three groups of mice received a booster vaccination 2 weeks following the first vaccination. Blood was collected from the tail vein every 2 weeks until 10 weeks postvaccination. Mice were challenged i.p. with 107 CFU of B. canis RM6/66 and euthanized 2 weeks postchallenge. Download FIG S4, TIF file, 0.2 MB.Schematic of experimental design and vaccine composition by group. Female C57BL/6 mice  of the log number of CFU per gram of tissue. Spleens were weighed at necropsy, and the aforementioned tissues, in addition to mesenteric lymph nodes and heart, were collected at each time point and fixed in 10% neutral buffered formalin for routine histopathologic evaluation. Tissues were routinely processed and embedded, sectioned at 4 μm, and stained with hematoxylin and eosin. Sections from the spleen following challenge were graded for severity of granulomatous inflammation using QuPath Bioimage analysis v. 0.1.2   and incubated at 37°C. Bacterial colonies were enumerated after 72 h to quantify tissue colonization. Levels of infection were expressed as mean values and standard deviations  . Foci ofB. canis-specific immunoglobulin G (IgG) indirect enzyme-linked immunosorbent assay (iELISA). Briefly, 96-well plates  were coated with 250 ng/well of B. canis RM6/66 heat-killed lysate in coating buffer  at 4°C overnight. Plates were washed three times with PBS containing 0.05% Tween 20 (PBST), and nonspecific binding was blocked with 100 μl of 3% skim milk in PBST at room temperature for 2 h. Following three washes, 2-fold dilutions of sera in PBST containing 1% skim milk were added and incubated at 37°C for 1 h. Plates were washed five times, and horseradish peroxidase (HRP)-labeled goat anti-mouse IgG , HRP-labeled rat anti-mouse IgG2a , or rat anti-mouse IgG1  was added, followed by incubation at 37°C for 1 h. Afterwards, OPD peroxidase substrate (Sigma-Aldrich) was added (100 μl/well) and incubated for 30 min at 37°C in the dark. The enzyme reaction was stopped by addition of 0.5 M NaOH, and absorbance was measured at 450 nm. Endpoint titers were reported as the Log10 of the highest dilution giving an OD reading higher than the mean plus 2 standard deviations of that for the baseline sera. All assays were performed in triplicates, and the results are presented as the mean reciprocal endpoint titer.Mice were bled by ventral tail vein puncture prior to the start of each experiment, at each euthanasia time point, and at biweekly intervals following s.q. vaccination. Blood was centrifuged at 3,000 rpm for 5 min, and serum was collected for anti-g at room temperature with no brake. Afterwards, the interface layer consisting of peripheral blood mononuclear cells (PBMCs) was collected and transferred to a new tube. Cells were incubated with red blood cell lysis buffer (Sigma-Aldrich) for 5 min at room temperature, followed by dilution in PBS and centrifugation at 500 × g for 5 min at 4°C. The cell pellet was washed twice in PBS, with the pellet finally being resuspended in separation buffer  for magnetic bead purification. CD14+ monocytes were isolated from the PBMCs using anti-human CD14 monoclonal antibody (clone TÜK4) conjugated to magnetic beads  according to the manufacturer’s instructions. Cells were counted, resuspended in complete medium , and plated at a density of 5 × 105 to 1 × 106 cells/ml in 24-well plates. Culture medium was supplemented with 50 ng/ml of recombinant canine granulocyte-macrophage colony-stimulating factor (GM-CSF) (R&D Systems) and 30 ng/ml of recombinant canine interleukin 4 (IL-4) (R&D Systems) to induce differentiation to DCs. Half of the medium was replaced every 2 days, and monocytes were allowed to differentiate into DCs at 37°C and 5% CO2 for 7 days.Canine dendritic cells (DCs) were derived as previously described , 47. BriB. canis RM6/66, B. canis RM6/66 ΔvjbR, PBS as a negative control, or 1 μg/ml LPS from Escherichia coli strain 0128:B12 (Sigma-Aldrich) as a positive control. Infected cells were incubated at 37°C and 5% CO2 for 1 h, followed by a 1-h treatment with 50 μg/ml gentamicin. Cultures were maintained for 24 h, and cells were collected into microcentrifuge tubes by gentle pipetting and washed twice with PBSA (PBS plus 0.5% BSA). Following centrifugation, supernatants were collected and stored at −20°C until further use. Afterwards, cells were incubated with 20 μl of monoclonal antibodies, mouse anti-human CD86-fluorescein isothiocyanate (FITC)  and mouse anti-human HLA-DRII-allophycocyanin (APC) , per 100-μl reaction mixture of cells in PBSA for 30 min at 4°C in the dark. Cells were washed twice with PBSA and resuspended in 200 μl of 4% formaldehyde for 30 min at room temperature with addition of 10 μl of propidium iodide. Surface marker expression was assessed via flow cytometry using an BD Accuri C6 Plus flow cytometer (BD Biosciences). The amount of canine interferon γ in cell supernatants was measured via a commercial ELISA kit according to the manufacturer’s instructions (R&D Systems).After 7 days in culture, canine DCs were infected at an MOI of 100 with P values for mean comparisons. Splenic weight and histologic scores were compared using one-way ANOVA, and Tukey’s multiple-comparison test was used to generate P values. Canine dendritic cell samples were evaluated using FlowJo software, with absorbance gated for viability. Data were analyzed using fluorescence minus one (FMO) controls and were expressed as the mean ± standard deviation (SD) percentage of positive cells for each surface marker. Data were compared using one-way ANOVA, and Tukey’s multiple-comparison test was used to generate P values. In all analyses, a P value of less than 0.05 constituted statistical significance.Analysis was performed using GraphPad Prism software, version 6.0 . The CFU data were normalized by log transformation and evaluated by two-way analysis of variance (ANOVA) repeated-measures test. Tukey’s multiple-comparison test was used to generate


Aspergillus niger produces a wide spectrum of extracellular polysaccharide hydrolases that hydrolyze cellulose into soluble glucose and cellodextrins. Transporters are essential for sugar uptake, yet it is not clear whether cellodextrin transporters exist in A. niger. Here, one cellulose inducible cellodextrin transporter CtA was identified in A. niger B2. It was found that CtA not only could transport cellobiose, but also cellotriose, cellotetraose, and cellopentaose. The yeast strain YPβG-CtA, expressing cellodextrin transporter CtA and an intracellular β-glucosidase, grew on cellobiose with the cell growth rate of 0.0830 ± 0.0113 h–1 under aerobic condition. Furthermore, the engineered yeast could produce 1.1 g/L ethanol anaerobically on cellobiose in 2 days. The identification of CtA provides evidence that the cellodextrin uptake is a complementary strategy of cellulose utilization in A. niger, and the CtA could be a strong transporter candidate for constructing engineered cellodextrin-utilizing microorganisms. Aspergillus niger is ubiquitous in the environment and has been widely used in industry for the production of organic acids and extracellular enzymes  transporters, which are single polypeptide facilitating the movement of small soluble substrates in response to ion gradients . In the a crassa , and throxalicum , and theectively .A. niger showed that it contains 86 putative sugar transporters, which were classified as members of the Pfam family of “Sugar and other transporters” (PF00083)  . Until nensively , while tA. niger, to provide insight into the cellulose utilization mechanism of A. niger, and to provide potentially useful cellodextrin transporter for constructing efficient biofuel producer strains.The aim of this study was to identify and characterize the cellodextrin transporters in Aspergillus niger B2 was isolated from rotting wheat straw in our laboratory  solid medium 5 days for spores preparation. The modified Mandel’s medium for fungal spores germination and hyphal growth (growth media) contained glucose (20 g/L) and peptone (1 g/L) ( (1 g/L) . The modSaccharomyces cerevisiae YPH499 strain was grown in synthetic media (SM) contained yeast nitrogen base without amino acids and ammonium sulfate (6.7 g/L), adenine 50 mg/L, histidine (20 mg/L), lysine (30 mg/L), tryptophan (20 mg/L), leucine (100 mg/L), uracil (20 mg/L), and glucose (20 g/L) or cellodextrins (5 g/L) as the sole carbon source at 30°C and 250 rpm. S. cerevisiae YPH499 harboring plasmid pRS425 was grown in the SM without leucine (SM-Leu), and S. cerevisiae YPH499 harboring plasmid pRS425 and plasmid pRS426 was grown in the SM without leucine and uracil (SM-Leu-Ura).An12g09270, An14g01600, An13g03250, An03g05320, An08g09350, An16g06220, and An04g02790) upon the induction of cellulosic biomass, 50 mL Mandel’s growth medium was inoculated with fresh A. niger B2 spore suspension in Mandel’s salt solution (about 107 spores) and cultured at 200 rpm and 30°C for 48 h. Mycelia were harvested by vacuum filtration and washed with sterile Mandel’s salt solution three times, and then 1.5 g mycelia were transferred to 50 mL Mandel’s induction medium containing 0.5% of steam-exploded sugarcane bagasse, grown at 30°C and 200 rpm. Mycelia were harvested at 6 h, frozen in liquid nitrogen, stored in a freezer at −80°C, and then used for RNA isolation by using RNeasy Plant Mini Kit  according to the manufacturer’s protocols.To characterize the expression of potential cellodextrin transporters (® 1000 (Thermo Fisher Scientific). mRNA was then reversed transcribed to cDNA by using the ProtoScript® II Reverse Transcriptase (NEB), following the manufacturer’s menus. The primers used in RT-qPCR were listed in The extracted mRNA samples were quantified on the NanoDropAmino acid sequences of orthologs of CtA were obtained from NCBI database. Multiple sequence alignments were performed with ClustalX 2.0 , and theAn12g09270 and An16g06220 (named as ctA and ctB), from A. niger B2 were amplified by using the cDNA as the template, and the primers were listed in ctA and ctB were cloned into a pRS426 plasmid, creating plasmids pRS426-ctA and pRS426-ctB (S. cerevisiae YPH499 (MATa ura3-52 lys2-801_amber ade2-101_ochre trp1-Δ63 his3-Δ200 leu2-Δ1) was used as the host for the cellodextrin transport assay (N. crassa intracellular β-glucosidase gene (gh1-1) was transformed into the S. cerevisiae YPH499 competent cells by electroporation , creating the strain YPβG  to a final OD600 of 0.5. The mixtures were transferred into serum bottles (100 mL), sealed and incubated at 30°C and 120 rpm. Samples were taken at various time intervals.In the fermentation experiments, the YPβG-CtA strain was first grown aerobically on SM-Leu-Ura medium (50 mL) containing 20 g/L glucose for 24 h. Cells were then harvested by centrifugation and were washed 3 times with sterile water 50 mL. Cell pellets were resuspended in SM-Leu-Ura medium (50 mL) containing cellobiose (20 g/L) and CuSO4 (0.2 mM) and cellobiose (5 g/L), cellotriose (2.5 g/L), cellotetraose (2.5 g/L), or cellopentaose (2.5 g/L) was inoculated with YPβG-CtA or YPβG-CtB to the final OD600 of 0.1. Flasks were incubated at 30°C and 250 rpm. The cell density was monitored by measuring the optical density at 600 nm. An aliquot of 1 mL culture was collected and analyzed by HPLC for measuring the residual cellodextrins.In cellodextrins uptake assay, flask (250 mL) containing SM-Leu-Ura medium (50 mL) with CuSO2SO4 (5 mM) and was eluted at a flow rate of 0.6 mL/min.Concentrations of cellodextrins  and ethanol in the culture samples were analyzed using an UltiMate 3000 HPLC equipped with a refraction index detector and an Aminex HPX-87H . The eluent was HA. niger CBS 513.88 showed that it had 86 putative sugar transporters  and locus An14g01600  were annotated as MFS lactose permease and MFS hexose transporter, respectively. The others having the best hit to CDT-2 were annotated as sugar transporters  and gene An16g06220 have identity with the N. crassa cellobiose transporter cdt-1  and cdt-2 , respectively. These two genes were selected for further functional analysis, and here now were named ctA and ctB, respectively.The here identified, putative lactose permease gene (A. niger B2 and were cloned into S. cerevisiae YPβG that harbored the N. crassa-glucosidase gene gh1-1 (NCU00130). The expression of a functional cellodextrin transporter will enable the YPβG strain to grow on the medium with cellobiose as the sole carbon source, which has been demonstrated in the previous work that we participated in ctA and ctB, respectively, were constructed.To evaluate the ability of CtA and CtB to transport cellobiose, both genes were amplified from ctA gene and β-glucosidase gene could grow on cellobiose. Therefore, CtA’s function as a cellobiose transporter was confirmed. However, YPβG-CtB strain harboring the ctB gene and β-glucosidase gene could not grow in the medium, indicating that the CtB dose not have the ability to transport cellobiose into the cells. Herein, the sequence of ctA was deposited in GenBank under the accession number of MH648002. The cellodextrin transporter CtA in A. niger shares 36% identity with CDT-1  was confirmed to be a novel cellodextrin transporter. The phylogenetic analysis of the orthologs of CtA showed that it did not group with cellobiose transporter CltA from A. nidulans  of the seven predicted sugar transporters genes were up-regulated when nidulans , which in length , 4. The A. niger led us to hypothesize that A. niger could import partial hydrolysis products, cellodextrins, and utilize them intracellularly, in addition to completely hydrolyzing cellodextrins to glucose and uptaking glucose. The analysis of A. niger CBS 513.88 genome showed that the fungus contains 5 putative BGLs without signal peptide , or directly transported into the cells through cellodextrin transporter CtA followed by degradation by intracellular BGLs  enabled the strain to use cellobiose efficiently and produced ethanol under the anaerobic fermentation. The YPβG-CtA strain had the cell growth rate of 0.0830 ± 0.0113 h–1 . The sequence of CtA has been deposited in the GenBank under the accession number of HC, CT, and HL designed the study. QZ, SC, and JZ carried out the experiments. HC, HL, QZ, and SC analyzed the data. HC, HL, and ZF wrote and edited the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


CKAP2L), which plays key roles in neural progenitor cell division, has also been linked to poor prognosis in lung cancer. In the present study, we investigated the role of CKAP2L in glioma. From bioinformatics analyses of datasets from The Cancer Gene Atlas and the Chinese Glioma Genome Atlas, we found that CKAP2L expression correlates with tumor grade and overall survival. Gene set enrichment analysis (GSEA) showed that MITOTIC_SPINDLE, G2M_CHECKPOINT, and E2F_TARGETS are crucially enriched phenotypes associated with high CKAP2L expression. Using U87MG, U118MG, and LNZ308 human glioma cells, we confirmed that CKAP2L knockdown with siCKAP2L inhibits glioma cell proliferation, migration, invasion, and epithelial-mesenchymal transition. Interestingly, CKAP2L knockdown also induced cell cycle arrest at G2/M phase, which is consistent with the GSEA finding. Finally, we observed that CKAP2L knockdown led to significant increases in miR-4496. Treating cells with exogenous miR-4496 mimicked the effect of CKAP2L knockdown, and the effects of CKAP2L knockdown could be suppressed by miR-4496 inhibition. These findings suggest that CKAP2L is a vital regulator of miR-4496 activity and that CKAP2L is a potentially useful prognostic marker in glioma.Despite advances in the diagnosis and treatment of the central nervous system malignancy glioma, overall survival remains poor. Cytoskeleton-associated protein 2-like ( IDH1/2 [EGFR amplification [CDKN2A/B homozygous deletion [MGMT methylation status [Central nervous system (CNS) malignancies are responsible for significant morbidity and mortality worldwide . Among CIDH1/2 ,9, 1p19qIDH1/2 , EGFR amfication , combinefication , TERT prfication , CDKN2A/deletion , and MGMn status . For insn status ,12. Howen status ,14. HencOne group of potential targets is microRNAs (miRNAs), which are non-coding, single-stranded RNAs that contain around 21–25 nucleotides and act post-transcriptionally to influence gene expression ,16,17,18CKAP2L) gene, located on chromosome 2, encodes a protein that is vital for cell division in neural progenitor cells [CKAP2L function leads to Filippi syndrome and microcephaly [CKAP2L expression is associated with a poorer prognosis in pulmonary adenocarcinoma patients [CKAP2L in gliomas has not been addressed. In the present study, therefore, we used bioinformatic analysis, clinical validation, cell modeling, and miRNA screening to investigate the role played by CKAP2L in gliomas.Cytoskeleton-associated protein 2-like  with tumor grade (CKAP2L mRNA expression significantly (p < 0.01) differed among groups, though no difference was detected between normal brain tissue (n = 5) and Grade II glioma  and low CKAP2L  subgroups based on the median CKAP2L expression, Kaplan–Meier analysis showed that higher CKAP2L mRNA expression was significantly (p < 0.0001) associated with a poorer prognosis (CKAP2L mRNA expression correlated significantly (p < 0.0001) with the tumor grade (CKAP2L mRNA expression significantly differed among the groups (p < 0.0001). Furthermore, as with TCGA dataset, dividing the CGGA dataset into high-CKAP2L  and low-CKAP2L  expression groups based on the median expression showed that high CKAP2L expression was significantly (p < 0.0001) associated with a poor prognosis . In the grade II gliomas, the curves were separated, but only the TCGA dataset reached statistical significance (p < 0.001). In the GBM cluster, high and low CKAP2L expression groups’ curves only revealed borderline significance (p = 0.054).Statistical analysis showed a strong correlation between or grade A. When tI glioma A. This lrognosis B. Similaor grade C. Moreovrognosis D. These  grading , we founCKAP2L expression groups, we performed gene set enrichment analysis (GSEA) with TCGA and the CGGA datasets. A total of 29 gene sets were significant at False Discovery Rate (FDR) < 25%, and 12 gene sets were significantly enriched at a nominal p-value < 5%. The top up-regulated phenotype in the high-CKAP2L group from TCGA  was MITOTIC_SPINDLE, followed by DNA_REPAIR, G2M_CHECKPOINT, MTORC1_SIGNALING, and E2F_TARGETS  were E2F_TARGETS, G2M_CHECKPOINT, MITOTIC_SPINDLE, DNA_REPAIR, and MYC_TARGETS_V1. Thus, four of the top five phenotypes were the same in the two datasets  staining showed that the glioma cases contained pilocytic astrocytoma, diffuse astrocytoma with/without IDH-mutant, anaplastic astrocytomas with/without IDH-mutant, oligodendrogliomas, anaplastic oligodendrogliomas, glioblastomas with/without IDH-mutant, and diffuse midline glioma H3K27M-mutant . We obsegulation . In addi factors .CKAP2L mRNA and protein expression in glioma, we performed quantitative RT-PCR and Western blot analyses with normal brain tissue and the U87MG, U118MG, and LNZ308 human glioma cell lines. We found that CKAP2L mRNA expression was significantly higher in all three glioma cell lines than in normal brain tissue  could be suppressed by knocking down CKAP2L expression using small interfering RNA (siRNA). After transfection of siCKAP2L or siRNA (scramble siRNA) into U87MG and U118MG glioma cells, the expression of CKAP2L was substantially reduced at both 25 nM and 37.5 nM siRNA supplementation  in the TCGA dataset, as well as the CGGA dataset . These findings supported the proposal that the CKAP2L expression is correlated with proliferation ability.Given that U87MG and U118MG glioma cells exhibit higher entation A,B. In oentation C,D. To f67 [MCM6 ,27 expreCKAP2L is known to be a component of the mitotic spindle [CKAP2L on mitotic function, we compared the cell cycle phase distributions between glioma cells expressing siCKAP2L and siControl. We found that among U87MG and U118MG cells, CKAP2L knockdown led to an increase in the numbers of cells in the G2/M phase and a dramatic decrease in cells in the G1 phase  is an important paralog of CKAP2L that functions to stabilize microtubules and plays a key role in the regulation of cell division [CKAP2 gene is also upregulated in various kinds of cancer, including breast [CKAP2L may play similar roles in glioma. Interestingly, CKAP2L also appears to function during mitotic phases and to play a critical role in cell division. Furthermore, an earlier study showed that downregulation of CKAP2L triggers multipolar spindles and cell cycle arrest [At present, there is only limited information about the role of division ,29. CKAPg breast , prostatg breast ,32, stomg breast ,34, and g breast  cancers,g breast . We there arrest , which ip < 0.0001) among the tumor grades. However, the error bars  to generate expression data [Furthermore, in the Kaplan–Meier plots, it seems that the CGGA has better overall survival in both low and high er plots . From thion data  and bothion data . NeverthCKAP2L plays a vital role in regulating cell division [CKAP2L expression on cell growth, we investigated the effect of CKAP2L knockdown on the proliferation in U87MG and U118MG glioma cells. From our data, we found CKAP2L knockdown suppresses U87MG and U118MG cell growth. To further support the proposal that the decrease of cell number was due to the decline of proliferation ability, we looked at the association of the CKAP2L versus two common proliferation biomarkers in the TCGA and CGGA datasets, including MKI67 and MCM6, often used in brain tumor studies [CKAP2L expression positively correlated with MKI67 and MCM6 in the TCGA and CGGA. These findings support the proposal that the CKAP2L expression is associated with proliferation capability. Additionally, from the results of GSEA analyses, the E2F, mitotic spindle, and G2/M checkpoint are the leading phenotypes in the high CKAP2L group. To our knowledge, the E2F is the crucial regulator of the G1/S transition [In previous sections, we have introduced the proposal that division . To furt studies ,26,27. Tansition , and theCKAP2L expression on the cell cycle, we examined the effect of CKAP2L knockdown on the cell cycle profile in U87MG and U118MG glioma cells. The results showed that the cell fraction in the G2/M phase was dramatically increased in both cell lines, while the fraction in the G1 phase was decreased , which may be due to the limited number in the study sample. A larger sample size will be needed in future investigations. We also found that age, MGMT methylation, EGFRvIII, p53, NF1, AxL, p-AxL, and H3Lys27 expression were associated with CKAP2L upregulation, and Cox regression analyses showed that older age, IDH1, loss of ATRX, and positive expression of AxL, p-AxL, NUR77, H3Lys27, or PDGFRA were the independent prognostic factors. We anticipate that this information will be useful to future investigations into the pathogenesis and progression of glioma. Additionally, a few unexpected results in the multivariate survival analysis, including MGMT, EGFR, EGFR vIII, and H3K27M are not statistically significant in our data. These factors are usually described as decisive prognostic markers for gliomas [MGMT protein expression is poorly correlated with MGMT promoter methylation [EGFR and EGFR vIII, it was also found there were around 10% of cases with EGFR protein expression lacking real EGFR gene amplification [Our immunohistochemical staining showed that levels of  gliomas ,45,46,47fication . Hence, CKAP2L knockdown  and the Chinese Glioma Genome Atlas (CGGA). The samples obtained from UCSC Xena, https://xenabrowser.net/ (accessed on 2 June 2020), contained 701 cases, including 5 normal samples and 258 grade II, 271 grade III, and 167 grade IV glioma cases. Additionally, another 325 glioma samples obtained from the CGGA database, http://www.cgga.org.cn/ (accessed on 2 June 2020), included 103 grade II, 79 grade III, and 139 grade IV cases, as well as 4 cases without grading details. Statistical comparisons of CKAP2L mRNA expression among tumor grades were made using multiple t-tests and one-way ANOVA. These cases were also divided into high and low CKAP2L expression groups around the median CKAP2L expression level Kaplan–Meier plot that were then constructed to compare overall survival between the high and low CKAP2L expression groups. Furthermore, this was done to understand the regression between CKAP2L expression versus two proliferation markers, MKI67 and MCM6. We also downloaded these datasets from The Cancer Genome Atlas  for further investigation.To understand the role of https://www.gsea-msigdb.org/gsea/index.jsp, accessed on 10 June 2020) was conducted by comparing the high- and low CKAP2L expression groups in TCGA  and the CGGA  datasets. Data were analyzed against the hallmark gene sets v7.1 available in the Molecular Signatures Database. For all other parameters, the default settings were used.Gene set enrichment analysis , for immunohistochemistry (IHC). The commercial Tissue Array contained 132 cases of brain astrocytoma, 31 brain glioblastoma, and 9 brain oligodendroglioma, with 8 each of adjacent normal brain tissue. The core measured 1 mm in diameter with 5 µm tissue thickness. Immunohistochemical staining was performed using an automated immunostainer (Ventana Benchmark®XT), which ensured consistent results. Before staining, antigen retrieval was performed by heating the samples at 125 °C for 30 min in sodium citrate buffer  using a pressure cooker. We switched off the cooker when it reached full pressure, and then waited till the pressure was released. Next, we took the retrieval slides and washed 3 times for 5 min each in phosphate-buffered saline (PBS). Thereafter, the slides were uploaded to the autostainer following the manufacturer’s protocol, and the selected antibodies were evaluated by checking their binding to both positive and negative controls. Following optimization of the retrieval and the antibody concentration, robust staining of the positive, but not the negative, control was observed. The test slides were then examined and scored using a semi-quantitative scoring system, same as previous publications [The array of brain tumor tissue, clinical information, histology diagnosis, and pathology grade (GL2083b) was purchased from GenDiscovey Biotechnology Inc., ications ,53, thatp < 0.05 were considered significant.To determine whether CKAP2L IHC scores correlate with tumor grades, we used Pearson’s correlation to evaluate the relation between CKAP2L expression and other possible independent factors. We also performed univariate and multivariate analyses and Cox proportional hazards regression to assess the association between factors significantly associated with CKAP2L and overall survival. The linear regression was used to evaluate the association between CKAP2L versus MKI67 and MCM6. Values of 2 at 37 °C. For Western blot analysis of CKAP2L expression, the cells were lysed in lysis buffer, which contained 100 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, and 1% Triton X-100. GAPDH, Actin, and β-actin served as an internal control. Normal brain cell lysate purchased from Biocompare  also served as a control.The U87MG, U118MG, and LNZ308 human glioma cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin. Cultures were kept in an incubator under 5% COCKAP2L siRNA (siCKAP2L) and siControl were purchased from Dharmacon . Using DharmaFECT 1 Transfection Reagent, U87MG and U118MG cells grown in 12-well plates were transfected with 25 and 37.5 nM siCKAP2L or siControl . Cells from each group were then seeded into 12-well plates at a density of 5 × 104 cells/well and incubated overnight at 37 °C. To collect the cells, 200 µL 10% FBS DMEM and 100 µL of 0.05% trypsin were added to each well. Cell proliferation was tested 24, 48, and 72 h after transfection. After preparing a mixture of 10 µL of each cell line and 10 µL trypan blue, cells excluding the dye were counted using a TC20™ Automated Cell Counter . Cell proliferation assays were replicated three times with each cell line. Finally, Western blot analysis was performed to detect cell cycle checkpoint  expression in the cells following siCKAP2L and siControl transfection.CKAP2L, 5′-GAGCCAAAACACCAAGCCTTA-3′ (forward) and 5′-GGAGTTTAATGCTGATGGACCTT-3′ (reverse); for GAPDH, 5′-GCACCGTCAAGGCTGAGAAC-3′ (forward) and 5′-ATGGTGGTGAAGACGCCAGT-3′ (reverse).Total RNA was extracted using TRIzol™ Reagent  and reverse transcribed to single-stranded cDNA with Tetro™ Reverse Transcriptase . For qRT-PCR, the PCR reactions were carried out using a StepOne™ Real-Time PCR System  with Fast Plus EvaGreen qPCR Master Mix . Thermocycling was performed with 0.25 µM each primer (PrimerBank) and 2.5 µL of the cDNA diluent. The PCR protocol entailed denaturation for 2 min at 95 °C followed by 40 cycles of a touch-down PCR protocol . The primers used were as follows: for The glioma cell lines  were washed three times with PBS and lysed in RIPA buffer . The protein lysates  were separated by 10% SDS-PAGE and analyzed by immunoblotting with antibodies against polyclonal rabbit anti-human CKAP2L  and anti-GAPDH  antibodies and monoclonal mouse anti-ACTN . After repeating the experiments, the quantification was performed using ImageJ .CKAP2L for 72 h, harvested, fixed with 70% ethanol, briefly washed with PBS/1% FBS, and then incubated with 10 mg/mL RNAse A and 50 mg/mL propidium iodide in PBS plus 1% Tween 20 for 30 min at 37 °C in the dark. Flow cytometric analysis was performed using FACS Calibur flow cytometer . The cell fractions in the respective cell cycle phases were calculated using Cell Quest Pro software .U87MG and U118MG cells were transfected with 25 nM si4 cells/well and incubated overnight, after which they were transfected with siCKAP2L or siControl as described above. After incubation for an additional 48 h, a scratch wound was made in the cultures using a 200-µL pipet tip. The cells were then washed with PBS, and the scratched area was photographed under a microscope (0 h). One microliter of DMEM supplemented with 10% FBS was then added to each well, and after incubation for 16 h at 37 °C under 5% CO2, the medium was removed and the scratched area was photographed again.U87MG glioma cells were plated in 12-well plates supplemented with mitomycin C at a density of 5 × 105 cells/dish in 10-cm dishes and incubated for 24 h, after which they were transfected with siCKAP2L or siControl and incubated for an additional 48 h. The transfected cells were then taken up, suspended in serum-free DMEM, and aliquots containing 2 × 105 cells in 500 µL of DMEM were seeded into the upper chambers of Transwell plates, and 750 µL of DMEM supplemented with 10% FBS medium was added to the lower chambers. After incubation for 16 h, the membranes between the upper and lower chambers were fixed with formalin, stained with crystal violet, and photographed under a microscope.Cells were plated at a density of 5 × 10CKAP2L or siControl, total RNA was extracted from each sample and its quality confirmed spectrophotometrically . Samples of total RNA from the siCKAP2L or siControl transfectants were then screened using a Human miRNA OneArray®, https://www.phalanxbiotech.com/onearray-mirna-microarrays/ (accessed 13 April 2020), for analysis of differentially expressed genes encoding miRNAs. Genes were considered differentially expressed when there was a log2|Fold change| ≥ 0.585 and p < 0.05. In addition, miR-4496 was purchased from Dharmacon , after which U87MG and U118MG cells were transfected with 10 nM or 50 nM miR-4496 using DharmaFECT 1 Transfection Reagent . miR-4496 levels were then measured 24, 48, and 72 h after transfection. To assess the effect of miRNA inhibitor, U87MG and U118MG glioma cells were transfected with 5, 10, or 15 nM miR-4496 inhibitor , and cell numbers were counted 48 h later.After transfecting U87MG and U118MG cells with si


Paracoccidioides brasiliensis. In the lungs, this fungus interacts with epithelial cells, activating host cell signalling pathways, resulting in the production of inflammatory mediators. This event may be initiated through the activation of Pattern-Recognition Receptors such as Toll-like Receptors (TLRs). By interacting with cell wall components, TLR2 is frequently related to fungal infections. In this work, we show that, after 24 h post-infection with P. brasiliensis, A549 lung epithelial cells presented higher TLR2 levels, which is important for IL-8 secretion. Besides, integrins may also participate in pathogen recognition by host cells. We verified that P. brasiliensis increased α3 integrin levels in A549 cells after 5 h of infection and promoted interaction between this receptor and TLR2. However, after 24 h, surprisingly, we verified a decrease of α3 integrin levels, which was dependent on direct contact between fungi and epithelial cells. Likewise, we observed that TLR2 is important to downmodulate α3 integrin levels after 24 h of infection. Thus, P. brasiliensis can modulate the host inflammatory response by exploiting host cell receptors and cell signalling pathways.Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin America and may be caused by the species  Although this disease has been frequently related to poverty, unfortunately, it is still not recognised as a neglected tropical disease by the World Health Organisation (WHO)2. Nowadays, environmental factors are changing PCM epidemiology, which is reflected in the increasing number of patients in some regions5. One example was recently reported by Do Valle and co-workers (2017), who described the increase of PCM cases by sixfold after a highway construction in Rio de Janeiro (Brazil), possibly caused by deforestation and the removal of vast amounts of soil5.Paracoccidioidomycosis (PCM) is a deep human mycosis that is restricted to Latin AmericaParacoccidioides that present mycelial and yeast forms. Host infection may occur by inhalation of mycelial propagules, probably contained in contaminated soil, and once in the lungs, these fungi convert into yeasts1. Aiming to survive, pathogens adopt some mechanisms to exploit host cells, leading to the establishment, maintenance and dissemination of the infection.PCM is caused by dimorphic fungi from the genus 6.Epithelial cell is an important cell type of host lungs that interacts with inhaled pathogens, not only primarily forming a physical barrier against microorganisms, but also acts in the innate immune response by secreting inflammatory mediators such as cytokines and chemokines, which results in the recruitment of immune cells to the site of infectionParacoccidioides or Histoplasma capsulatum13. Our data have demonstrated that different fungal species promote cytokine secretion in lung epithelial cell in distinct manners. In fact, we verified that the stimulation mechanism of cytokine release differs even among isolates of the same species13.Over the past years, our research group has focused on studying the lung epithelial cell responses to fungal infections, especially regarding the mechanisms involved in the release of inflammatory cytokines induced by yeasts of P. brasiliensis yeasts interact with α3 and α5 integrins in A549 epithelial cells and promote the recruitment of these receptors to host cell structures known as lipid rafts. In addition, we verified that both integrins and these lipid platforms are involved in IL-8 secretion by P. brasiliensis infected-epithelial cells9.It was also observed that 15. Under particular stimuli, TLRs may also collaborate with integrins to activate signalling pathways that culminate in cytokine secretion17. Marre et al., for example, verified that the TLR2 synthetic peptide ligand PAM3CSK4 induces cooperation between the heterodimers TLR2/1 and α3β1 integrin in macrophages, which results in IL-6 secretion by these cells16.Besides integrins, other receptors such as Toll-like Receptors (TLRs) have been extensively studied in fungal infections6. In the present work, we investigated the involvement of TLR2 in IL-8 secretion that was induced by P. brasiliensis yeasts in lung epithelial cells. Moreover, we analysed whether TLR2 interacts with α3 integrin and modulates its expression in A549 cells during this fungal infection.Evidence that lung epithelial cells influence host immune responses against infections is increasing over the past decadeP. brasiliensis yeasts (isolate Pb 18), we evaluated by Western blot whether this fungus modulates the expression of TLR2. Figure P. brasiliensis interaction, we verified higher TLR2 levels (3.55-fold) than A549 cell basal levels - or with TLR2-directed small interfering RNA (siRNA), and then, infected with P. brasiliensis yeasts. After 24 h, IL-8 concentrations were measured by ELISA. As expected, this fungus induced an increase of this chemokine levels by 5.2-fold when compared to uninfected cells  of α3 integrin levels in A549 cells after 5 h incubation . Thus, the direct contact between A549 cells and P. brasiliensis is mandatory for a yeast-induced reduction of α3 integrin levels in epithelial cells. Moreover, in a different manner, secreted factors by this fungus promoted an increase of α3 integrin levels in A549 cells.As 21. In the present study, we demonstrated that P. brasiliensis modulates TLR2 expression in A549 epithelial cells and, by silencing this receptor, we also found that TLR2 participates in IL-8 secretion by these cells. In fact, some groups described the participation of TLR2 in the immune response against P. brasiliensis24. For example, the work developed by Balderramas and co-workers (2014) showed that P. brasiliensis binds to TLR2 to induce the production of IL-12 by neutrophils23. Another group, Loures and co-workers (2009), showed that TLR2 is an important receptor for the innate immune system in a P. brasiliensis infection, and also this receptor may have a protective effect in pulmonary fungal infections24.In an infection process, inside the host, a pathogen may interact with a particular cell, engaging several receptors and highjacking host cell signalling pathways in order to survive. One family of receptors extensively studied in infection models is the Toll-Like Receptor (TLR) family. In fungal infections, by promoting the secretion of cytokines and chemokines, these receptors are important for the host immune response, recognising several ligands in these pathogens, such as O-linked mannans, phospholipomannans, and unmethylated DNA with CpG motif 27. McGarry and co-workers (2015)27, for example, demonstrated that the TLR2 ligand PAM3CSK4 induces cell migration of the rheumatoid arthritis synovial fibroblast cells (RASFC), and also increases β1 integrin levels in these cells. Moreover, these authors observed that RASFC cell migration, induced by PAM3CSK4, was inhibited by β1 integrin neutralizing antibodies, demonstrating that activation of TLR2 induces RASFC migration that occurs in a β1 integrin dependent manner.In the literature, several groups have shown that TLRs and integrins pathways communicate with each other in different cell processes3CSK4 increased α3 integrin levels, which was inhibited by silencing TLR2, both data indicate that the heterodimer TLR2/1 upmodulates this integrin levels in A549 epithelial cells. However, when these epithelial cells were infected with P. brasiliensis yeasts for 24 h, we observed higher levels of TLR2 and also that α3 integrin was almost undetectable by Western blot. The latter result was not expected since, in a previous study, we verified an augmentation of the expression of α3 and α5 integrins in A549 epithelial cells infected with this fungus for 5 h9. Regarding IL-8 secretion, although we observed the decrease of α3 integrin levels after 24 h of incubation with P. brasiliensis yeasts, the upmodulation of α3 integrin levels in the first 5 h of A549 cell infection seems to be important to promote this cytokine secretion, since after α3 integrin silencing, we observed a reduction of IL-8 levels9.In the present work, we verified in A549 lung epithelial cells that the TLR2 ligand PAMP. brasiliensis yeasts induce a raise in α3 integrin levels in A549 epithelial cells, while TLR2 levels are similar to those of uninfected cells. Moreover, interaction of α3 integrin with TLR2 occurs during this early period. However, in longer periods of infection (24 h), P. brasiliensis downmodulates α3 integrin levels in direct contact- and TLR2-dependent manners. Together, these results indicate that P. brasiliensis yeasts differently modulate the activity of host cell receptors and signalling pathways during the course of the infection. Interestingly, Kim and co-workers (2009)28 described that α3β1 integrin is involved in the formation of myofibroblasts and lung fibrosis. These authors showed that mice lacking α3 integrin-pulmonary epithelial cells, upon bleomycin stimulus, presented lower accumulation of myofibroblasts and did not progress to lung fibrosis. Moreover, lack of lung fibrosis is seen in acute cases of PCM29, a severe form of this mycosis. In this manner, the reduction of α3 integrin in lung epithelial cells promoted by P. brasiliensis might contribute to the pathogenesis of PCM acute form. Therefore, a better understanding of the cellular mechanisms that promote fungal infection will lead to the development of new therapies in a scenario where there are no vaccines against fungal infections, antifungal drugs are limited, and some fungi are already developing resistance to these drugs20.In summary, this work shows that during early infection (5 h), 2 atmosphere. For P. brasiliensis-A549 interaction assays, cells were grown in 100 mm2 dishes, 6-well or 24-well plates.A549 epithelial cell line (human lung adenocarcinoma) was cultured in Dulbecco’s Modified Eagle’s Medium—DMEM  with 10% fetal bovine serum – FBS , 10 mM HEPES, 100 U/mL penicillin, and 100 μg/mL streptomycin  (complete DMEM) at 37 °C, in 5% COP. brasiliensis yeasts, Pb18 isolate, were kindly provided by Dr. Wagner Luiz Batista, from Universidade Federal de São Paulo . Fungi were cultured in PGY medium  containing 1.4 g/L asparagine and 0.1 g/L thiamine  for 5–7 days in incubator shaker at 37 °C, 120 rpm, as previously described by Ywazaki and co-workers (2011) 30. Fungal virulence was maintained by reisolating P. brasiliensis from infected B10.A mice. For epithelial cell-fungus interaction assays, single fungal mother and daughter yeasts from P. brasiliensis were obtained as previously described by Barros and co-workers (2016) 9. The resultant supernatant contained only single fungal cells, which were washed three times with DMEM.2 (starving). A549 cells were then incubated for 5 h or 24 h with: (1) 5 µg/mL of TLR2/1 ligand PAM3CSK4 ; or (2) P. brasiliensis yeasts with a multiplicity of infection (MOI) of 2.5 fungi for 1 epithelial cell. Controls without stimuli were also performed. Then, epithelial cells (or supernatant culture) were analysed by ELISA, co-immunoprecipitation or Western blot.A549 epithelial cells were seeded in tissue culture plates. After 48 h, cells were washed three times with FBS-free DMEM, and incubated in this medium overnight, at 37 °C, 5% CO3CSK4 or P. brasiliensis yeasts, A549 cell culture supernatants were collected, centrifuged to remove fungi or cell debris, and then analysed using Duo Set Kit , according to manufacturer’s instructions.IL-8 secretion of A549 cell cultures was analysed by sandwich ELISA. After 24 h of incubation with PAMP. brasiliensis-A549 cell interaction for 5 or 24 h, cells were washed with PBS, collected using a cell scraper, and incubated with RIPA lysis buffer  containing protease inhibitors as previously described by Barros and co-workers (2016) 9. After 30 min at 4 °C, samples were centrifuged, and the resultant supernatant containing proteins was quantified using QuantiPro BCA Assay kit , according to manufacturer’s instructions. Twenty μg of protein extract was solubilised in sample buffer , 8% SDS, 0.1% bromophenol blue, and 10% β-mercaptoethanol), and then subjected to SDS–PAGE (SDS–polyacrylamide gel electrophoresis) 31. After electrophoresis, proteins were transferred to PVDF (polyvinylidene difluoride) membranes 32, which in turn were blocked with 5% non-fat milk  in TBST (200 mM Tris–HCl pH 8.0 containing 0.1% Tween-20)  or with Milk Diluent Blocking solution  (for TLR2). Next, membranes were incubated at 4 °C with TBST containing 5% bovine serum albumin (BSA)  and one of the following antibodies: (1) anti-TLR2 1:1000 , (2) anti-α3 integrin 1:1000 , or (3) anti-β-actin 1:15,000 . After 16 h, membranes were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit  in 1% BSA in TBST for 1 h at room temperature. After each step, membranes were washed three times with TBST. Finally, membranes were incubated with SuperSignal West Pico Chemiluminescent Substrate  and proteins were detected by using an imaging system . Densitometric analyses were performed for protein level quantification using Image J (FIJI).After As mentioned in figure legends and/or supplementary information, in Western blot assays, β-actin was used as loading protein control for blots. For this, before incubation with primary antibodies, PVDF membranes were cut between 75 and 50 kDa markers. The upper membranes were incubated with anti-α3 integrin or -TLR2 antibodies, and the bottom, with anti-β-actin antibodies. All experiments were performed at least three times to guarantee data reproducibility and a representative Western blot was shown in this article. For clarity, Western blot images were edited and presented in the main article. Bands of the same blot with the same exposure time were cut and joined into an edited image that was shown in the main paper. The original Western blot images were presented in the supplementary information file. Different exposure times of the PVDF membranes, obtained in the UVITEC imaging system, were presented as supplementary figures. In this manner, it may be verified that the results of edited figures correspond to the original images and the differences of α3 integrin or TLR2 levels were not dependent on the exposure time of the PVDF membrane. Therefore, all authors declare the veracity/reproducibility of the data provided in this article. Besides, Western blot images were edited only for succinctness in the main paper and did not compromise the results of original blots.Aliquots containing 500 µg of proteins, obtained as described in “3CSK4 or (2) P. brasiliensis yeasts (MOI = 2.5) and then, culture supernatants were collected. IL-8 concentrations were determined by sandwich ELISA. Concomitantly, A549 cells were collected and TLR2 and α3 integrin levels were analysed by Western blot.A549 cells were grown in 6 or 24 well plates for 1 day and serum-starved for 5 h. Then, cells were transfected using a mix of Lipofectamine RNAiMAX Reagent and Silencer Select Pre-designed siRNA for TLR2  at the final concentration of 10 nM. Silencer Select Negative Control siRNA , which has a sequence that does not target any gene, was used as a negative control. After 24 h of transfection, cells were washed three times with DMEM and incubated for 24 h with: (1) 5 µg/mL of TLR2/1 ligand, PAM7. For all experiments, cell viability was greater than 95%.A549 cell viability was determined by MTT  assay, as previously described by Maza and co-workers (2012) t test through GraphPad Prism Software . Values were considered statistically significant at p ≤ 0.01.Statistical analyses were performed using ANOVA or Student’s Supplementary Information


When drugs exert their effects in the brain, linear extrapolation of doses from adults could be harmful for children as the blood–brain barrier (BBB) and blood–CSF barrier (BCSFB) function is still immature. More specifically, age-related variation in membrane transporters may impact brain disposition. As human data on brain transporter expression is scarce, age dependent  variation in immunohistochemical localization and staining intensity of the ABC transporters P-glycoprotein (Pgp), breast cancer resistance protein (BCRP), and multidrug resistance-associated proteins 1, 2, 4, and 5 (MRP1/2/4/5) was investigated. Post mortem brain cortical and ventricular tissue was derived from 23 fetuses (GA range 12.9–39 weeks), 17 neonates , 8 children (PNA range 0.1–3 years), and 4 adults who died from a wide variety of underlying conditions. In brain cortical BBB, immunostaining increased with age for Pgp and BCRP, while in contrast, MRP1 and MRP2 staining intensity appeared higher in fetuses, neonates, and children, as compared to adults. BCSFB was positively stained for Pgp, MRP1, and MRP2 and appeared stable across age, while BCRP was not detected. MRP4 and MRP5 were not detected in BBB or BCSFB. In conclusion, human BBB and BCSFB ABC membrane transporters show brain location and transporter-specific maturation.The online version of this article (10.1007/s00418-020-01884-8) contains supplementary material, which is available to authorized users. ATP-binding cassette (ABC) drug transporters form a vital part of the blood–brain barrier (BBB) and blood–cerebrospinal fluid barrier (BCSFB), where they mediate the efflux of a wide variety of chemical structures from endothelial and ependymal cells. Their specific localization and expression at the apical or basolateral plasma membrane impacts drug exposure in brain extracellular fluid (ECF) or cerebrospinal fluid (CSF) , breast cancer resistance protein (BCRP), and multidrug resistance-associated proteins (MRPs) 1, -2, -4, and -5 are expressed at the adult BBB  and human placental tissue samples from Rick Greupink, PhD . These had been collected anonymously as  waste material. For these samples, a no-objection clause permitted use for research purposes in line with the Dutch guidelines on secondary use of human tissue.The formalin fixed, paraffin-embedded tissue blocks were cut in 4 μm slices and mounted on positively charged glass slides. The slides of post mortem tissues were heated for 25 min at 55 °C, deparaffinized and rehydrated in xylene and a series of ethanol. Depending on the transporter, antigen retrieval was performed with citrate buffer (pH6): Pgp, BCRP and MRP5 (“AMF”), or Tris–EDTA buffer (pH 9): MRP1, MRP2, MRP4, and MRP5 (“M5II-54”). Slides were incubated with a solution of 2% H2O2  in phosphate buffered saline (PBS)/Tween (0.1% Tween20 (T)) for half an hour, to quench endogenous peroxidase. Nonspecific binding was blocked using a solution of 1% bovine serum albumin (BSA) in PBS/T for 1 h and slides were incubated with primary antibody , and placenta tissue (MRP5) were used as positive controls, because these tissues show expression for all transporters investigated. The same procedure as described for brain samples was used, except that kidney material was blocked with an avidin and a biotin solution for 15 min, as kidney has high endogenous biotin expression.Slides were scanned using the Slide Scanner Pannoramic 250  and visualized using Pannoramic viewer version 1.15.2. Staining intensity was assessed separately for brain microvessels in the cortex regions (BBB), choroid plexus, and ventricular ependyma (both BCSFB). Subsequently, slides were scored: negative (0), detectable (1), intermediate (2) or high (3) by two independent observers  as previously performed by Daood et al. , and scoThe data were described using standard statistics. To test for correlations between staining intensity score and postmenstrual-, postnatal-, and gestational age , a Spearman’s rank correlation coefficient was calculated using Graphpad Prism version 5.03.Post mortem brain cortex tissue derived from 23 fetuses , 17 neonates , 8 children (PNA range 0.1–3 years), and 4 adults were obtained from the Erasmus MC Tissue Bank, Rotterdam. For 15 fetuses and children ventricle/choroid plexus tissue (BCSFB) was available.BBB: An age dependent increase was observed for Pgp staining intensity score as a function of PMA, GA and PNA in cortex microvessels Fig. ; Table 2BCSFB: Positive staining was observed for choroid plexus and ventricular ependymal cells as early as 12.9 weeks PMA. No age-related differences in staining intensity were observed Fig. .Fig. 2PoBBB: BCRP-staining intensity related to age as a function of PMA and GA Fig. , Table 2BCSFB: No staining was detected in choroid plexus or ventricle ependymal cells Fig. .BBB: MRP1 staining was weakly positive in part of the brain cortical vessels in fetal and pediatric samples (between 13 and 40 weeks PMA), but not in adults. For a few samples, clear staining of vessels could be seen at a PMA between 30 and 40 weeks Figs. , 3.BCSFB: Staining was pronounced in choroid plexus and ventricular epithelium, but no age-related effects could be observed at this barrier. In ventricular epithelium, staining could be observed only at the apical membrane Figs. , 3.BBB: For most samples, staining intensity of MRP2 appeared higher until 100 weeks PMA, as compared to adult material by visual inspection . MRP4 was detected in positive control kidney cortex material using two different antibodies (Online Resource 1).Staining in brain cortex microvessels was only positive in erythrocytes in these vessels. Staining in microvessel endothelial cells, choroid plexus, or ventricle was not detected (data not shown). MRP5 was stained in placental tissue used as positive control with two different antibodies (Online Resource 1).Online Resources 2 and 3 present the staining intensity of the brain cortex BBB transporters BCRP, MRP1 and MRP2 and BCSFB transporters Pgp, MRP1, and MRP2 in relation to postnatal and gestational age.Online Resource 4 presents the staining intensity of the brain cortex BBB transporters Pgp, BCRP, MRP1, and MRP2 in the age groups defined previously.Our results show both location as well as transporter-specific variation in ABC transporter expression in young children as compared to adults. Pgp, BCRP, MRP1, and MRP2 were detected in the cortical BBB, although not across the complete age range. Staining intensity was lower for Pgp and BCRP in brain cortex microvessels from fetuses and neonates, as compared to adults. In contrast, MRP1 and MRP2 staining intensity appeared higher in fetuses, neonates and children, but a correlation with age was not statistically significant. Choroid plexus and ventricle wall ependymal cells were positively stained for Pgp, MRP1, and MRP2 and intensity appeared stable. Transporter expression detected in ventricle was present at the apical side of ependymal cells for Pgp, MRP1 and MRP2, whereas plasma membrane localization could not be specified in microvessels and choroid plexus. MRP4 and MRP5 could not be detected in brain cortex microvessels, ventricular epithelium, and choroid plexus.Pgp expression has previously been described in brain cortex microvessels forming the BBB in fetal, pediatric, and adult animals and humans  may have influenced the identified age-related variation , b (M4I-10), and MRP5 expression in placental tissue c (M5II-54), d (AMF). Arrows indicate localization of the protein of interest. Magnification is 400x (TIFF 38049 kb)ESM_2 Postnatal age versus staining intensity score in BBB for a: BCRP, b: MRP1, c: MRP2, and postnatal age versus staining intensity score in BCSFB for d: Pgp, e: MRP1, and f: MRP2. In panel d black dots indicate scoring of ventricle ependymal cells and gray dots scoring of choroid plexus. wks indicates age in weeks (EPS 1337 kb)ESM_3 Gestational age versus staining intensity score in BBB for a: BCRP, b: MRP1, c: MRP2, and gestational age versus staining intensity score in BCSFB for d: Pgp, e: MRP1, and f: MRP2. In panel d black dots indicate scoring of ventricle ependymal cells and gray dots scoring of choroid plexus. wks indicates age in weeks (EPS 1305 kb)ESM_4 Staining intensity (mean and SEM) of the brain cortex BBB transporters a: Pgp, b: BCRP, c: MRP1, and d: MRP2 in the age groups: preterm newborns , newborns (PNA 0–28 days), infants/children (1 month–12 years), and adults (> 16 years) (EPS 1070 kb)Below is the link to the electronic supplementary material.