Table 1.
GXM levels in tissues of C57BL/6 mice (n = 5) 24 h post-intranasal administration.
Fig 1.
Exogenous glucuronoxylomannan (GXM) administration and Cryptococcus neoformans H99 strain pulmonary infection reduces survival in C57BL/6 mice.
(A) Experimental timeline for the intranasal (i.n.) GXM challenge and fungal intratracheal (i.t.) infection model. Mice were sensitized with 125 μg/mL of GXM 24 h pre-inoculation with 105 C. neoformans cells. Then, survival studies and colony forming units (CFU) determinations, histopathology, and microscopy were performed 3- and 7-days post-infection (dpi). The C57BL/6 mouse cartoon in the timeline is a creative commons clip art inserted using Microsoft Office Power Point 2019. This diagram was drawn by Luis R. Martinez. (B) Survival differences of untreated (e.g., sterile phosphate-buffered saline (PBS)-sensitization) and GXM-treated C57BL/6 mice infected with 105 fungi (n = 10 per group). P-value significance (* P < 0.05) was calculated by log rank (Mantel-Cox) analysis. Fungal burden in (C) lungs (numbers of CFU/gram (g) of tissue) and (D) blood (numbers of CFU/0.1 mL of blood) collected from untreated and GXM-treated mice i.t. infected with 105 cryptococci (n = 8 per group) 3- and 7-dpi. For panels C and D, bars and error bars denote the means and standard deviations (SDs), respectively. Asterisks indicate P-value significance (** P < 0.01 and **** P < 0.0001) calculated using multiple student’s t-test analyses. ns represents not statistically significant comparisons. For panels A-D, these experiments were performed twice, similar results were obtained each time, and all the results combined are presented. (E) Histological analysis of lungs removed 7-dpi from untreated (left panels) and GXM-treated (right panels) C57BL/6 mice infected with 105 C. neoformans H99 strain cells. Representative hematoxylin and eosin (H&E, host tissue morphology; upper panels; Scale bar: 100 μm)- and mucin carmine (fungi; red staining; lower panels)-stained sections of the lungs sequentially examined are shown. Lower panel images are magnifications of the smaller boxes in the H&E-stained sections to better show the cryptococcal cells stained with mucin carmine (indicated with arrows; Scale bar: 20 μm).
Fig 2.
Exogenous GXM challenge enhances C. neoformans migration into the central nervous system (CNS) of C57BL/6 mice.
(A) Fungal burden (numbers of CFU/gram of tissue) in brain collected from untreated and GXM-treated mice i.t. infected with 105 cryptococci (n = 8 per group) 3- and 7-dpi. (B) Confocal microscopy of cryptococcal cells (yellow arrowheads) in the brain parenchyma of GXM-treated mice 7-dpi. Scale bar: 20 μm. (C) Immunofluorescent (IF) image of a 7-dpi hippocampal tissue section displaying a large cryptococcoma (red arrows) filled with yeasts cells (yellow arrowhead) and abundant amounts of capsular polysaccharide released (white arrowheads). Scale bar: 150 μm. (D) GXM accumulation (white arrowheads) in the cortex and (E) blood vessel of GXM-treated and C. neoformans infected mice 7-dpi. Scale bar: 200 μm. IF staining of brain lesions (cryptococcomas; white arrows) caused by C. neoformans 7-dpi in (F) untreated or (G) GXM-treated animals. Scale bar: 150 μm. For B to G, FITC-labeled (green) GXM-specific monoclonal antibody (mAb) 18B7 was used to label cryptococci. MAP-2 (red) and DAPI (blue) staining were used to label the cell bodies and nuclei of neurons, respectively. Each tissue section was sequentially examined using multiple slides. (H) Brain lesions per mouse (n = 7 per group) and (I) lesion area (n = 10 per group) analyses in tissue sections of untreated and GXM-treated mice infected with C. neoformans. The areas of 10 brain lesions per condition were measured using NIH ImageJ software. Each symbol represents a single lesion. For A, H and I, bars and error bars denote the mean value and SDs, respectively. Asterisks denote P-value significance (* P < 0.05, *** P < 0.001, and **** P < 0.0001) calculated using single or multiple student’s t-test analyses.
Fig 3.
GXM challenge alters tight junction protein (TJ) expressions in vivo.
(A) Confocal microscopy of cryptococcal GXM [FITC-labeled (green; arrowheads) GXM-specific mAb 18B7] distribution in the brain parenchyma (blue; nuclei of neurons) of GXM (125 μg/mL)-treated mice 24 h after i.n. administration. Scale bar: 20 μm. (B) Western blot (WB) analyses of brain tissue from untreated (e.g., sterile PBS-sensitization) or GXM (7.8, 15.6, 31.25, 62.5, and 125 μg/mL)-challenged C57BL/6 mice were performed to compare the expression of the TJ proteins claudin-5 (23 kDa), ZO-1 (200 kDa), and occludin (59 kDa) and the adhesion protein JAM-A (32 kDa). Actin (42 kDa) was used as a housekeeping protein control. Quantitative measurements of individual band intensity in WB analyses described in panel B for (C) claudin-5, (D) ZO-1, (E) occludin, and (F) JAM-A, using NIH ImageJ software. Actin was used as a control to determine the relative intensity ratio (A.U. denotes arbitrary units). Bars represent the means of 5 independent gel results (black circles) and error bars indicate SDs. Asterisks denote P-value significance calculated using one-way analysis of variance (ANOVA) and adjusted using the Tukey’s post-hoc analysis. *P < 0.05, for the reduction in the intensity of the band of TJ and adhesion molecules as compared to actin.
Fig 4.
GXM alters the distribution of TJs on human brain endothelial cells (HBECs).
(A) IF images of claudin-5 and occludin distribution on the surface of HBECs after incubation in absence (untreated) and presence of 10 μg/mL of ethylenediaminetetraacetic acid (EDTA; positive control) or GXM for 4 h at 37°C and 5% CO2. After co-incubation with EDTA or GXM, the cells were washed and incubated with claudin-5-FITC (green) and occludin-rhodamine (red)-specific antibodies. DAPI (blue) was used to stain the cell nuclei. Scale bar, 100 μm. (B) Quantification of (B) claudin-5 and (C) occludin intensity on the surface of HBECs was performed using NIH ImageJ software. Bars and error bars denote the means and SDs, respectively. Each symbol denotes a single cell measurement (n = 10 cells per group). Asterisks indicate P-value significance (* P < 0.05, ** P < 0.01 and **** P < 0.0001) calculated using one-way ANOVA and adjusted using the Tukey’s post-hoc analysis. ns represents not statistically significant comparisons.
Fig 5.
GXM augments the expression of activated RhoA and modulates the cytoskeleton in HBECs in a time-dependent manner.
(A) WB analyses of HBEC lysates stimulated with 10 μg/mL of GXM were performed to assess the expression of activated RhoA protein (GTP-bound) and phosphorylated myosin light chain (pMLC) over the course of 180 min. Quantitative measurements of individual band intensity in WB analyses described in panel A for (B) activated RhoA and (C) phosphor-MLC using LiCOR Image Studio software. Bars represent the means of 3 independent gel results (black circles) and error bars indicate SDs. P-value significance (* P < 0.05, ** P < 0.01 and *** P < 0.001) was calculated using one-way ANOVA and adjusted using the Tukey’s post-hoc analysis. (D) IF images of actin remodeling in HBECs after incubation in the absence (0 min) and the presence of 10 μg/mL GXM for the indicated times (15–180 min). Cells were stained with Phalloidin-594 (red) and DAPI (blue). Arrows indicate actin stress fiber formation; asterisks represent perinuclear relocation of actin. Scale bar, 20 μM.
Fig 6.
GXM decreases the trans-endothelial electrical resistance (TEER) and transmigration of C. neoformans through HBECs in a blood brain barrier (BBB) model.
Graphic representation of the HBECs and pericytes BBB model used for the (A) TEER measurements, (C) HBEC barrier permeability assay, and HBEC model used for the (E) fungal transmigration assay. For A and C, HBECs and pericytes were grown separately (HBECs, 0.4 μm pore insert; pericytes, bottom of a well) and co-incubated using a microtiter transwell system that permits chemotactic exchange through the supernatant. (B) Relative TEER of HBECs incubated with GXM (10, 50, or 100 μg/mL) for 5 h at 37°C and 5% CO2. HBECs incubated alone (negative), with EDTA (10 μg/mL; positive), or with mock extract from acapsular strain cap59 (negative) were used as controls. Time points are the averages of the results for three different TEER measurements, and error bars denote SDs. Symbols (*, #, ϕ, and x) indicate P value significance (P < 0.05) calculated using one-way ANOVA and adjusted using the Tukey’s post-hoc analysis. *, #, ϕ, and x indicate significantly higher TEER than in the 10 μg/mL of EDTA-, 10 μg/mL of GXM-, 50 μg/mL of GXM-, and 100 μg/mL of GXM-treated groups, respectively. (D) Relative HBEC barrier permeability to streptavidin-horseradish peroxidase (HRP) after incubation with GXM (10, 50, or 100 μg/mL) for 30 min at 37°C and 5% CO2. HBECs incubated alone (negative), with EDTA (10 μg/mL; positive), or with mock extract (negative) were used as controls. (E) HBECs were cultured for 2 h with 10 μg/mL of GXM in a microtiter transwell system (5 μm pore insert). Then, 105 cryptococci were added to the well and fungal transmigration through the HBECs was determined for 4 h using CFU. (F) CFU determinations after fungal transmigration are shown. HBECs incubated alone or with EDTA (10 μg/mL) were used as negative and positive controls, respectively. For B, D, and F, bars and error bars denote the means and SDs, respectively. Each symbol denotes a single well measurement (n = 3 wells per group for B and D) or CFU determinations from a single well (n = 5 wells per group for F). Asterisks indicate P-value significance (* P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001) calculated using one-way ANOVA and adjusted using the Tukey’s post-hoc analysis. These experiments were performed thrice, similar results were obtained each time, and all the results combined are presented.
Fig 7.
GXM causes vasodilation of mouse blood vessels after ex vivo treatment.
(A) Murine superior mesenteric arteries (SMA) incubated in absence (untreated) or presence of 25 μg/mL GXM were evaluated in response to phenylephrine (PE; test for vasoconstriction) or acetylcholine (ACh; test for endothelium-dependent vasodilation). Cumulative concentration-response curves are shown. Each point represents the average of 5 individual SMA measurements. The concentration–response curve was log-transformed, normalized to percent maximal response, and fitted using a nonlinear regression. P values of <0.05 were considered significant. (B) pD2 for ACh curve. Each symbol (n = 5, untreated; n = 3, GXM-treated SMA rings) represents a single SMA ring. Bars and error bars denote the mean value and SDs, respectively. Asterisk denotes P-value significance (* P < 0.05) calculated using student’s t-test analysis.
Fig 8.
Model of GXM-mediated disruption of the BBB during cryptococcal infection.
GXM stimulates activation of RhoA, which activates Rho-associated protein kinase (ROCK). ROCK inhibits the myosin light chain phosphatase (MLCP), leading to actin stress fiber formation through enhancement of MLC phosphorylation. Actin stress fibers contribute to the internalization and lysosomal degradation of claudin and occludin, disrupting TJs between brain microvascular endothelial cells (BMVECs).