Effect of metal chelators on the growth and biofilm of C. albicans.

(A) C. albicans cells were cultured in YPD media at 30 °C for 24 hrs without (cyan blue) and with the indicated concentration of EDTA (purple), MgSO4 (Green), ZnCl2 (Grey), FeCl2 (Brown), and MnCl2 (Black), EDTA+MgSO4 (Lime green), EDTA+MgSO4@6 hr (dark blue), EDTA+ZnCl2 (Maroon), EDTA+FeCl2 (Orange), and EDTA+MnCl2 (Pink). Optimal absorbance was measured at 600 nm in different intervals of incubation. (B) The cultures from the above mentioned experiment was diluted and plated on YPD plate. Colonies were counted and plotted to determine the CFU efficiency. (C) Pre-formed C. albicans biofilm was treated with EDTA and divalent metals as mentioned. Their effect on biofilm was observed post-24 h treatment by crystal violet staining and estimating at 570 nm. (D) Effect of EDTA and divalent metals on C. albicans biofilm was again observed by acridine orange staining and visualization under a 40X magnification using a CLSM. Similarly, the effect of other metal chelators like DTPA (Lime green), Aprotinin (blue), TPEN (orange) and CE (grey) on the growth (E), CFU (F), and biofilm formation of C. albicans analyzed by crystal violet staining (G) and CLSM (H). Mean values from three independent experiments considered and error bar represents SEM. p values *< 0.05, **<0.01 and ****< 0.0001 were significant as determine by one-way ANOVA.

Transcriptomics analyses C. albicans cells upon EDTA treatment.

(A) A Volcano plot depicting differentially expressed genes (DEGs) in CAET cells. The red dots indicate upregulated genes (411) and blue dots indicates the downregulated genes (388). (B) Heat map showing relative abundance of top 25 significantly upregulated and downregulated genes in Ca and CAET. RNA sequencing was carried out in triplicates. Red colour indicates upregulation and blue indicates downregulation as denoted by the Z score. (C) Double donut chart indicates top 100 significantly upregulated genes categorized into four main groups i.e., metal transporters (light green), cell wall- and membrane-associated genes other than metal transporters (blue), others (includes drugs resistance-associated, morphology-associated, biosynthetic and catalytic processes; represented in grey), and uncharacterized genes (cream). Inner donut depicts the number of DEGs from the above categories associated with virulence and pathogenesis in C. albicans as reported by published literatures. (D) Double donut chart depicting top 100 significant downregulated gene categorized as metabolic pathways (green), cell-wall associated (blue), ribosomal (grey), others (includes morphology-associated, resistance-associated; represented in yellow), and uncharacterized (cream). Outer circle represents the total number of genes in each category and inner circle indicates the virulence-related genes out of the total number of genes in that specific category. (E) Gene ontology enrichment analysis for 411 upregulated DEGs. (F) Gene ontology enrichment analysis for 388 downregulated DEGs were plotted. Both in (E) and (F), x-axis represents the number of DEGs and y-axis represents various processes like BP (biological processes), MF (molecular functions), and CC (cellular components) indicated in maroon, blue, and grey colors, respectively. (G) STRING analyses showing relationship between DEGs. Interaction amongst 15 connected out of 31 upregulated DEGs involved in metal transport and homeostasis is shown. (H) STRING analysis shows 9 connected out of 33 upregulated DEGs involved in pathogenesis. (I) Among the down-regulated DEGs, 70 connected out of 74 DEGs belonging to cytosolic ribosomal genes as determined by STRING. (J) 38 connected out of 56 DEGs belonging to mitochondrial ribosomal components were connected by as determined STRING.

A brief summary of RNA sequencing reads obtained from Illumina Novaseq

List of top 100 upregulated genes in EDTA treated C. albicans cell (CAET)

List of top 100 downregulated genes in EDTA treated C. albicans cell (CAET)

Effect of EDTA on the cell wall and polysome of C. albicans.

(A) Showing TEM images of ultrathin sections of untreated (Ca) and EDTA-treated (CAET) C. albicans at different resolution. Samples visualized at 14000 X magnification, arrow indicates the marked cell, and box indicates the focus area where thickness was measured. Scale bar = 2 µm for intact cells (i); scale bar = 1 µm for a single cell (ii); scale bar=500 nm for zoomed in image of an individual cell wall (iii). (B) C. albicans cells were stained with Con A and analyzed by flow cytometry (i) and mean fluorescence intensity was measured to estimate the mannan level (ii). (C) C. albicans cells were stained with aniline blue and analyzed by flow cytometry (i) and mean fluorescence intensity was measured to estimate the β-glucan level (ii). (D) C. albicans cells were stained with calcofluor white and analyzed by flow cytometry (i) and mean fluorescence intensity was measured to estimate the chitin level (ii). Mean average of three independent biological replicates with error bars is shown. p values *< 0.05 and ***< 0.001 as determined by unpaired t-test. (E) Cell-free total ribosomes were isolated by taking two different concentrations (pellet size of 200 µL and 400 µL) of C. albicans cells (Ca and CAET) and fractionated using sucrose gradient centrifugation. Fractions were analysed and plotted. The position and transition of ribosome subunits, monosome and polysome peaks were as shown.

C. albicans-Macrophage interaction.

(A) Deep red-stained RAW 264.7 murine macrophage cells were co-cultured with C. albicans (Ca and CAET) in 1:1 ratio. After each time point (1 hr, 2 hrs and 3 hrs) cells were pooled down and double positive cells (CFSE-FITC channel and deep red-APC channel; Q2, P3) were analysed by flow cytometry (i). Mean florescence intensity of three independent biological replicates were plotted using the GraphPad Prism software (ii). (B) From a similar co-culture experiment, C. albicans cells (Ca and CAET) were retrieved from macrophage cells and plated on YPD/Chloramphenicol plate by taking appropriate dilutions. The plates were incubated at 30 °C for 48 hrs. The plates were imaged (i) and colony-forming unit (CFU) was determined (ii). (C) A similar co-culture of macrophage and C. albicans (Ca and CAET) were carried out for 2.5 hrs and the dying macrophages were observed by propidium iodide staining (Red dots). Images were captured with fluorescence microscope (EVOS imaging system; Thermo Fisher Scientific) at 20X and 40X magnifications (i) and the population of dead macrophages were determined (ii). p values *< 0.05, **< 0.01, and ***< 0.01 as determined by two-way ANOVA.

Systemic candidiasis development and progression in C. albicans challenged mice.

(A) Mice (n=6/category) were injected intravenously with 5 × 105 C. albicans cells (Ca: cyan blue and CAET: purple) and saline as control (grey) and their survivability was monitored for 30 days. In a similar set of experiment, mice (n=6/category) were first immunized with CAET (represented in brown) or sham vaccinated with saline (represented in orange), and after 30 days, they were further re-challenged with C. albicans. Their survivability was monitored for another 30 days and a survival curve was plotted. The number of sacrificed mice were mentioned on the particular days in the survival plot. 1° and 2° suggest primary and secondary challenges, respectively. Statistical significance of the survival curve was determined using Mantel-Cox test. p values were as mentioned, otherwise they were non-significant. Y-axis was represented in Log rank scale. (B) Fungal load in the kidney, liver, and spleen of sacrificed mice in Log10 scale was determined by CFU analyses. No statistical significant difference was observed between these groups. (C) PAS-staining for one of the kidneys carried out to visualize the fungal burden in the cortex and medulla regions under 40X magnification. (D) To check the disease progression, a kinetic experiment was planned as indicated in the schematic diagram. A group of mice were injected intravenously with 5 × 105 C. albicans cells (Ca: cyan blue, CAET: purple, and 1° CAET 2°Ca: brown) and saline controls (grey and green) at 0 day, and five mice from each group were sacrificed on each day mentioned (3rd, 7th, 10th, 20th, and 30th day). Approximately 20 µl blood was drawn from the lateral tail vein of the mice prior to sacrifice. (E) Fungal load in the brain, liver, kidney, and spleen in Log10 scale was determined by CFU analyses. (F) Blood parameters such as WBC, Granulocytes, Platelets, and Monocytes levels were analyzed on the above-mentioned days and graphs were plotted using the GraphPad Prism software. The statistical analyses between saline and fungal infected mice groups of same day sacrificed were carried out. p values **<0.01, **<0.01, ***<0.00, and ****< 0.0001 as determined by two-way ANOVA.

Cytokine and chemokine estimation in infected mice.

The mice (n=3/category) were inoculated with Ca or CAET or saline and sacrificed on various days as indicated and cytokine and chemokine levels in the blood serum were quantified by using Bio-plex ProTM cytokine multiplex kit. (A) A panel of Th-1 cytokines, i.e., IL-1α, IL-1β, IL2, IL-10, IL-12(p40), IL-12(p70), IFN-γ, TNF-α, and GM-CSF, (B) Th-2/Th-17 cytokines, i.e., IL-3, IL-4, IL-5, IL-6, IL-9, IL-13, and IL-17, and (C) Chemokines i.e., RANTES, MCP-1, MIP-1α, MIP-1β, KC, and EOTAXIN. The statistical analyses between saline and fungal infected mice groups of same day sacrificed were carried out. p values **<0.01, **<0.01, ***<0.00, and ****< 0.0001 as determined by two-way ANOVA.

Model depicting the attributes of CAET cells.

EDTA alters the cell wall thickness by altering its composition. Metal transporters and several cell wall and membrane associated genes get upregulated. To mitigate the essential metal scarcity, genes involved in ribosome biogenesis and one-carbon metabolism were down-regulated. CAET cells get phagocytosed efficiently and eliminated faster by macrophages. CAET infected mice survived and induced robust host immune responses to protect the lethal challenge. Thus, CAET is a potential live whole-cell vaccine candidate.