BIOLOGIA CELULAR E MOLECULAR Biologia celular e molecular / Coord. Carlos Azevedo. This file you can free download and review.

Introduction Cryptococcus gattii and Cryptococcus neoformans are basidiomycetous yeasts that can be found in the environment and are the etiological agents of cryptococcosis, a life-threatening disease that is associated with nearly 200,000 annual deaths worldwide (). Yeast or spores are found in diverse ecological niches, especially in trees and soil, and are able to infect different hosts (). Infection in mammalian hosts is initiated by the inhalation of airborne dehydrated yeast cells or spores that reach the lung and typically cause pneumonia or meningitis, which are driven by dissemination through the blood system (). In the lung, alveolar macrophages initiate host defense by phagocytosis of the yeast cells.

Despite the effectiveness of the host defense, Cryptococcus spp. Developed virulence factors that allow them to inhibit and escape from the immune system. The best-characterized virulence factors in cryptococcal species include the production of a polysaccharide capsule, the synthesis of melanin and the secretion of enzymes that can destroy host cells. The production of such virulence factors thus allows cryptococcal survival in host cells and fluids (). During its life cycle, C.

Gattii and its sibling species C. Neoformans can also interact with other organisms in the environment, such as amoebae and nematodes (). Free-living amoebae are protozoa that feed on both bacteria and fungi by phagocytosis (). Acanthamoeba castellanii, for instance, can phagocyte and digest C.

Neoformans in a similar mechanism that macrophages use when the latter enters a host system (). However, the yeast has also developed strategies to inhibit and escape from the amoeba antifungal repertoire. Cryptococcal cells are capable of killing amoebae, replicating inside the phagocytic vacuole and undergoing non-lytic exocytosis (). Both protozoan and mammalian phagocytes share common properties and strategies. Neoformans infection, both macrophage and amoeba cells engulf yeast cells within vacuoles, promoting the interaction of such phagosomes with other organelles and the secretion of lysosomal enzymes (;; ). The transcriptional responses of yeasts to protozoan or to macrophage ingestion are similar (). In fact, it was suggested that the antifungal mechanisms employed by free-living amoebae and macrophages are evolutionarily conserved, possibly due to a common ancestral between Metazoa and Amoebae (; ).

Furthermore, it was shown that the interaction of pathogens such as Cryptococcus spp. And Legionella pneumophila with phagocytic cells in the environment have helped them to develop a repertoire of anti-phagocytic mechanisms to subvert the action of the mammalian host immune system (). Hence, it is currently assumed that Cryptococcus spp.

Developed its virulence toolkit under environmental selection by amoebae (;; ). Several mechanisms are involved in the innate immune system of mammalian cells to avoid cryptococcal growth (). Bronica Etrsi Serial Numbers. Nutritional immunity is defined as a restriction of essential nutrients, including transition metals needed for pathogen development ().

Zinc is the second most abundant transition metal in living organisms and is required in essential roles such as enzymes cofactors and structural constituents of proteins, in particular transcription factors (). We previously described that correct zinc metabolism regulation is important for C. Gattii virulence in murine models of cryptococcosis (, ), reinforcing the importance of zinc uptake for proper cell metabolism. In addition, J774.A1 macrophages are also capable of decreasing zinc levels in response to C. Neoformans infection (). This is similar to phenotypes observed for macrophages infected with different pathogens such as Histoplasma capsulatum () and Candida albicans (; ). This suggests that zinc restriction should be considered a broad antifungal strategy ().

Based on the fact that amoebae and macrophages share similar antifungal mechanisms and on the similarity of pathogenicity and behaviors between C. Neoformans and C. Gattii inside the host (), we hypothesized that amoeboid cells could also apply nutritional immunity as an antifungal strategy. We investigated the possible use by amoebae of a nutritional immunity mechanism, specifically zinc, as an antifungal strategy against C. We found that A. Castellanii cells actively reduced zinc levels after exposure to C. Gattii, possibly by mechanisms that include the activity of zinc exporters belonging to the ZnT family.

Materials and Methods Strains and Growth Conditions The C. Gattii R265 (), the Δ zip1 mutant, and the Δ zip1:: ZIP1 complemented mutant () strains were used in this work. Yeast strains were routinely cultured in YPD medium (2% glucose, 2% peptone, and 1% yeast extract) and incubated in an orbital shaker (200 rpm) at 30°C for 18 h. Castellanii strain Neff (ATCC 30010) was cultured in PYG (2% peptone; 0.2% yeast extract; 1.8% glucose, pH 6.5) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin, and incubated at 30°C. Phagocytosis Index and Intracellular Proliferation Rate (IPR) Assays To evaluate the phagocytosis index of C. Gattii and fungal survival inside amoebae, protozoa cells were cultured in cell culture flasks, counted in a Neubauer chamber (1 × 10 5 cells) and grown in 96-well plates containing PYG for 2 h to allow adhesion. Gattii WT, Δ zip1 and Δ zip1:: ZIP1 cells were inoculated in YPD medium for 18 h at 30°C.

Cryptococcal cells were washed three times with phosphate buffered saline (PBS) and the cell density was evaluated in a Neubauer chamber. Yeast cells were inoculated at a ratio 10:1 with A. Castellanii in PYG added or not of 10 μM ZnCl 2.

The incubation was allowed to proceed for 3 and 24 h. The wells were washed three times with warm PBS to remove non-phagocytosed C. Gattii cells. Amoeba cells were lysed with 0.1% Triton X-100 (Sigma) to recover yeasts associated to amoeba cells. The intracellular proliferation rate (IPR) assay was performed using amoebae, as previously described for macrophages ().

Briefly, infection of amoebae was performed as described above. After 3 h of incubation, the amoeboid cells were washed with PBS. Amoebae of one set of wells were lysed 0.1% Triton X-100 and the number of associated yeasts was determined.

Fresh YPG medium was added to another set of wells and interaction was allowed to occur for a further 24 h. Then, amoeba cells were washed with PBS and intracellular yeast cells determined. For both phagocytosis and IPR analysis, the lysates were diluted and plated on YPD-agar to analyze the number of colony forming units (CFUs). The IPR was defined as the ratio between CFUs recovered after 24 h incubation and the initial 3 h incubation. Another set of experiments was performed to assess the effect of the presence of extracellular zinc on fungal replication and survival inside amoebae. The phagocytosis assay was allowed to proceed for 3 h under the same conditions as described above. Next, the interaction cells were washed with PBS and incubated with fresh medium containing 10 μM of zinc (ZnCl 2) for a further 24 h.

Amoeba cells were then lysed and the CFU analysis was performed in YPD solid agar to determine the IPR. In Silico Analysis Sequences from Mus musculus, A.

Castellanii, and Saccharomyces cerevisiae belonging to the SLC39 and SLC30 transporter families were collected from Uniprot () and AmoebaDB (). Such sequences were identified based on PFAM-conserved domain signatures ZIP (PF02535) and Cation_efflux (PF01545), respectively. We applied an OrthoMCL analysis () to identify orthologs of amoeba zinc transporters in yeast and mouse. Cellular localization was predicted using a Cell-PLoc server (). Phylogenetic analysis was conducted using protein sequences aligned by Clustal Omega () using the default options. The best fitting model of amino acid substitution was evaluated using ProtTest () under the BIONJ JTT assumption.

Bayesian inference was conducted using an LG+F+G+I model, while the MCMC sampling approach was used to calculate posterior probabilities. Four Markov chains were run 1,000,000 times.

The chain was sampled every 100th generation, and burn-in values were determined from the likelihood values. The final tree diagram was generated using FigTree. Dosch 3d Food Drawings.

RNA Extraction and Quantitative RT-PCR (qRT-PCR) Total RNA was extracted from A. Castellanii infected with C. Gattii WT and Δ zip1 after 3 and 24 h of interaction using Trizol ® reagent (Invitrogen) according to the manufacturer’s recommendations. RNA integrity was assessed by electrophoresis on a 1% agarose gel and RNA concentration was measured by spectrophotometry (NanoDrop 2000 spectrophotometer, Thermo Scientific).

The samples were treated with RQ DNase (Promega) to purify RNA. Reverse transcription and cDNA synthesis were performed with ImProm-II Reverse transcriptase (Promega) using oligo-dT. The relative expression of genes identified as zinc transporters by the conserved domain (PF02535 and PF01545) of their coding products in A. Castellanii were determined by qRT-PCR (StepOne Real-Time PCR System) with an initial step of 95°C for 10 min, followed by 50 cycles of 95°C for 15 s, 55°C for 15 s, and 60°C for 60 s. All experiments were performed in biological triplicate and each cDNA sample was also analyzed in technical triplicate for each primer pair.

A melting curve analysis was performed at the end of the reaction to confirm the presence of a single PCR product. The results were processed according to the 2 -ΔCt method () and relative transcript levels were normalized with actin transcript levels.

The primers are listed in Supplementary Table S1. Flow Cytometry Assay To measure zinc levels in amoebae exposed to fungal cells, protozoa cells (1 × 10 5) were grown in 12-well plates for 2 h at 30°C to allow adhesion to the surface. Gattii WT and Δ zip1 cells were then added to the amoeba culture in a 10:1 ratio.

The interaction was allowed to proceed for 24 h, after which the cells were washed three times with warm PBS. Attached cells were incubated with 20 μM of Zinpyr-1 fluorescent probe (Sigma) for 30 min at 30°C in the dark in PBS. Non-incorporated probe was removed by washing with PBS and the cells were collected from the well by cell scraper.

Free zinc levels in amoeba cells were analyzed with a Guava easyCyte Flow Cytometer (Merck Millipore) by measuring the green fluorescence of 5,000 events. Fluorescence Microscopy The lectin wheat germ agglutinin (WGA) was used to evaluate the chitin-like structures by fluorescence microscopy (). Briefly, WT and Δ zip1 C. Gattii cells were grown overnight in YPD broth, at 30°C and 200 rpm. Cells were recovered, washed with PBS and fixed with 4% paraformaldehyde in PBS for 30 min at room temperature.

Fixed yeast cells were washed with PBS and blocked with 1% bovine serum albumine (BSA) in PBS, for 1 h, at 37°C. BSA solution was removed by washes with PBS and cells were then suspended in a 5 μg/mL solution of the Alexa Fluor 594 conjugate of WGA (Molecular Probes) and incubated for 30 min, at 37°C in the dark. After three consecutive washes with PBS, yeast cells were incubated with a calcofluor White (Invitrogen) solution at 5 μg/mL final concentration for 30 min, at 37°C in the dark. Cells were washed, suspended in 100 μL of PBS and placed onto glass slides containing glycerol plus N-propyl gallate. Images were analyzed and collected using an Olympus FluoView 1000 Confocal Laser Scanning Microscope (CME – UFRGS). Statistical Analysis Data were expressed as mean ± standard deviation (SD) of replicates. All assays were performed in three experiment conditions, with technical triplicate repetitions.

The Student’s t-test was employed to test for significance between values. P-values ≤ 0.05 were considered statistically significant. Results Zinc Uptake Is Important for C. Gattii Survival in A. Castellanii We recently provided evidence that C.

Neoformans cells experience zinc deprivation inside macrophages (). The main mechanism by which cryptococcal cells acquire zinc is through activity of the Zip1 protein (; ).

As null mutants of the ZIP1 gene display severe growth impairment in zinc-limiting conditions, we hypothesized that C. Gattii Δzip1 strains could be used as biosensors to evaluate the modulation of zinc concentrations in A. Castellanii cells exposed to C. The interaction between A. Castellanii and C. Gattii WT, Δ zip1 mutant, and Δ zip1:: ZIP1 complemented strains showed that the mutant strain was more easily associated with amoebae compared to WT and complemented strain, independent of the extent of incubation (Figure ).

We then sought to determine if the addition of zinc to the medium would alter the association of cryptococcal Δ zip1 mutant strain to amoeba cells. We performed an interaction of amoebae and cryptococcal strains in the presence of zinc for 24 h, a period that would allow the cryptococcal Δ zip1 mutant strain to properly acquire zinc. We could not detect a reversal of the higher number of Δ zip1 mutant cells associated to amoeba compared to WT and complemented strains (Figure ). This suggest that zinc sufficiency is not responsible for alterations that led to this phenotype. In order to evaluate possible molecules associated with the higher phagocytosis sensitivity of cell lacking the ZIP1 gene, we evaluated the distribution of chitin-like oligomers in the cryptococcal cells surface.

These structures were show to be involved in the association of C. Neoformans with murine phagocytes (). Confocal fluorescent microscopy analysis revealed no differences in the staining pattern of chitin-like oligomers in WT or Δ zip1 mutant strains (Figure ). This data suggest that other molecules present in cell surface could be affected in the absence of Zip1, which would increase the association of such cells with phagocytes. Absence of zinc transporter influences the outcome of Cryptococcus gattii from Acanthamoeba castellanii. (A) Cells of A.

Castellanii (1 × 10 5) were incubated with WT, Δ zip1 or Δ zip1:: ZIP1 C. Gattii strains (1 × 10 6 cells) in PYG medium for 3 or 24 h in 96-well plates to allow phagocytosis.

The wells were washed with PBS and A. Castellanii cells were lysed.

Yeast CFU count was assessed in YPD agar. The phagocytosis index was calculated using the total number of CFUs by the number of protozoan cells.

Data are shown as mean ± SD. The asterisks denote statistically significant differences between the Δ zip1 and the WT or Δ zip1:: ZIP1 conditions ( ∗ p. We then analyzed whether the reduced capability of acquiring zinc from the extracellular environment could influence cryptococcal replication or survival inside phagocytic amoeba.

IPR experiments were performed using WT, Δ zip1 mutant, and Δ zip1:: ZIP1 complemented strains. We observed that the ability of C. Gattii to survive and replicate inside amoebae was affected by the absence of ZIP1 gene, as Δ zip1 mutant strain showed reduced recovery from amoebae when compared to WT and complemented strains (Figure ). This led us to hypothesize that engulfed cryptococcal cells experience a reduced zinc bioavailability. We first performed a growth curve analysis to rule out that the observed phenotype could be associated with a lower fitness of C. Gattii Δzip1 in PYG medium compared to the WT and complemented strains.

No differences in growth were detected for such strains in PYG medium (data not shown). The IPR assay was repeated by including 10 μM of ZnCl 2 in the interaction system, a concentration that did not alter the viability of the amoebae nor the cryptococcal cells according to MTT assays (data not shown). We were able to recover more CFUs from amoebae infected by Δzip1 (Figure ). This suggests that addition of zinc to the media alters zinc bioavailability and promotes intracellular survival of C. Gattii cells lacking the ZIP1 gene. Gattii Presence Alters Zinc Levels Inside Amoebae To investigate the reduction of zinc bioavailability to cryptococcal cells inside amoebae, we explored the labile zinc levels using the cell permeable fluorescent zinc probe ZinPyr-1. Despite the use of this probe to measure intracellular zinc levels in mammalian cells (), little is known about zinc quantification in amoeboid cells.

We therefore first validated our method by measuring ZinPyr-1 fluorescence in A. Castellanii cells recovered from 2 h-cultures in PYG, PYG supplemented with 50 μM of ZnCl 2 or PYG containing the zinc chelator TPEN (10 μM). Cytometry analysis revealed that ZinPyr-1 fluorescence was reduced when amoeba cells were exposed to TPEN and increased by the presence of ZnCl 2, confirming the zinc-dependent fluorescence emission, as well the sensitivity of the assay (Figure ). We next evaluated whether intracellular zinc levels were reduced in amoeba cells exposed to C.

Gattii cells for distinct periods (3 and 24 h). Irrespective of the cryptococcal genotype used (WT or Δ zip1), a decrease in the number of cells in the gated region (M1) could be observed in amoeba cells incubated with C. Gattii cells for 24 h, but not for 3 h (Figure ). We also noted that for Δ zip1 mutant cells the reduction in zinc concentration was even more pronounced compared to that observed in amoeba cells exposed to WT cryptococcal cells, which could be due to higher Δ zip1 mutant cells associated to amoebae. These results suggest that exposure of amoeba cells to C. Gattii results in reduced intracellular zinc levels in the former.

Acanthamoeba castellanii cells reduce intracellular zinc levels in the presence of C. (A) Cytometry histogram of ZinPyr-1 fluorescence A. Castellanii cells cultured in PYG (Control), PYG plus 10 μM zinc chelator TPEN (TPEN) and PYG plus 50 μM ZnCl 2.

Castellanii (1 × 10 5 cells) and C. Gattii WT or zip1Δ (1 × 10 6 cells) were incubated at 1:10 ratio in PYG medium for 3 and 24 h at 30°C. The wells were washed with PBS and then incubated with Zinpyr-1 cell-permeable fluorescent probe for 30 min. After, washes with PBS were performed and the cells were collected for flow cytometry analysis. Data are shown as the mean ± SD from three experimental replicates per condition. The asterisks denote statistically significant differences between the conditions, as evaluated by Student’s t-test ( ∗ p. Identification and Function Assignment of ZIP and ZnT Proteins from A.

Castellanii The SLC30 (ZnT) and SLC39 (ZIP) group of proteins are responsible for the maintenance of proper zinc levels inside cells (). We conducted a sequence comparison analysis in order to assign a possible function to the Zinc-transporting proteins coded by the amoeba genome. Analysis of the A. Castellanii-predicted proteome for a PFAM-domain ZIP zinc transporter (PF02535) and Cation efflux family (PF01545) revealed the presence of 14 and 7 different proteins, respectively (Table ).

Analysis employing the OrthoMCL database revealed that both families of proteins (ZIP and ZnT) could be assigned to five different orthologous groups. In addition, the predicted subcellular localization analysis suggests that ZIP and ZnT proteins can occupy a range of different cell compartments (Table ). In order to strengthen the OrthoMCL analysis, we verified the phylogenetically conserved level of these transporters by comparing them to ZIP and ZnT transporters from model organisms.

We collected the zinc transporters from predicted proteomes of the yeast Saccharomyces cerevisiae (4 ZIPs and 5 ZnTs) and from the rodent Mus musculus (14 ZIPs and 10 ZnTs). The phylogenetic analysis of ZIP protein sequences showed that, with some exceptions, each amoeba protein has a close relationship with some proteins from the different analyzed organisms (Figure ). We observed a division of such sequences into three major groups, which indicates a divergence in the evolution paths of these transporters. In the three clusters observed, it was possible to associate amoeba-specific zinc transporters with similar proteins from mouse or S. These relations allow us to suggest that different sets of amoeba zinc transporters are closely related to transporters from different organisms.

For instance, there are four A. Castellanii proteins (ACA1_093920, ACA1_222780, ACA1_069540, and ACA1_368320) related to the main mammalian zinc importers (ZIP1 and ZIP2), as well with S. Cerevisiae zinc importers (Zrt1p and Zrt2p). Castellanii ZIP proteins (ACA1_065010, ACA1_157200, ACA1_154170, ACA1_364600, ACA1_148440) cluster with the mammalian ZIP11 transporter. The remaining A. Castellanii ZIP proteins are related to several mammalian ZIP transporters (Figure ). Phylogenetic reconstruction of ZIP zinc proteins from mouse, amoeba, and Saccharomyces cerevisiae.

Sequences retrieved from Uniprot and AmoebaDB based on the presence of the Pfam domain ZIP (PF02535) were aligned with Clustal Omega, and the best evolutionary model was selected based on ProtTest. Bayesian analysis was conducted and the tree was drawn using FigTree. The percentage of replicate trees in which the associated taxa is shown next to the branches. The scale bar represents substitutions of amino acids per site. A more complex pattern was observed for the phylogenetic analysis of ZnT protein sequences (Figure ). Castellanii proteins ACA1_038150 and ACA1_106270 cluster with the S. Cerevisiae mitochondrial iron transporters Mmt1 and Mmt2 ().

In addition, ACA1_107270 from amoeba cluster with mammalian ZnT5 and ZnT7 Golgi-associated transporters (; ). The ACA1_191570 protein from amoeba is related to mammalian ZnT6, a protein that associates with ZnT5 and locates to the components of the early secretory pathway (). The transporter ACA1_271600 clusters with mammalian transporters ZnT2, ZnT3, ZnT4 and Znt8, proteins with multiple intracellular localizations (; ). Phylogenetic reconstruction of ZnT zinc proteins from mouse, amoeba, and S. Sequences retrieved from Uniprot and AmoebaDB based on the presence of Pfam domain ZnT (PF01545) were aligned with Clustal Omega, and the best evolutionary model was selected based on ProtTest. Bayesian analysis was conducted and the tree was drawn using FigTree.

The percentage of replicate trees in which the associated taxa is shown next to the branches. The scale bar represents substitutions of amino acids per site. Trancriptional Profiling of ZnT and ZIP Transporters Coding Genes in A.

Castellanii during Interaction with C. Gattii We next analyzed the transcriptional profiling of 18 genes (14 ZIPs and 4 ZnTs) using qRT-PCR. We performed such experiments in order to evaluate whether alterations in zinc concentrations inside amoebae in response to cryptococcal presence was associated with the activity of zinc transporters. CDNA was synthesized from RNA samples collected from amoeba cells co-incubated or not with C. Gattii WT for 3 and 24 h. Irrespective of the condition analyzed, we detected transcripts from ZIP transporter-coding genes.

However, only ACA1_222780 was found to be differentially expressed when compared amoebae exposed or not to cryptococcal cells. This increase in expression could only be detected after 24 h of co-incubation (Figure ). Cryptococcus gattii presence alters zinc transporter expression in A. Amoebae (1 × 10 5 cells) were incubated in PYG medium with C. Gattii WT or zip1Δ (1 × 10 6 cells) in 12-well plates for 3 and 24 h, at 30°C. The cells were washed with PBS and RNA from amoebae was isolated and extraction was performed, followed by cDNA synthesis. The measured quantity of zinc transporter mRNA from ZIP coding genes (A) or ZnT coding genes (B) in each sample was normalized using the threshold cycle values obtained for the Actin gene.

Data are shown as the mean ± standard deviation from three experimental replicates of three biological replicates. Statistical analysis by t-student comparing control and fungal presence groups for each transporter ( ∗ p.