Cryptococcus neoformans serotype A virulence and pathogenicity are capsular glucuronoxylomannan (GXM) motif composition dependent

Abstract

The Cryptococcus neoformans capsular extracellular polysaccharide (EPS) plays an essential role in virulence and host immunomodulation. In this study, we analyzed the virulence of four serotype A C. neoformans strains with different polysaccharide structures: two multi-motif (H99 and KN99α) and two single-motif (Mu-1 and ATCC-24064 (24064)). Mice were infected by the intranasal (IN) or intravenous (IV) route. Survival, fungal load (colony-forming unit), cell lung population, cytokines, and histology were analyzed. No differences in cell size or capsule size were found between the four strains. However, polysaccharide sizes were highly variable, and no significant differences were observed for all other virulence factors. Nitrite production in cell culture dendritic cells was reduced for the H99 or KN99α strains of EPS. The reactive oxygen species in cell culture M1 macrophages and dendritic cells were increased for the H99 or KN99α strain's EPS. Cytokines differed from the IN and IV infections. Lung histology showed increased inflammatory cell presence after infection with Mu-1 or 24064 strains, with no cryptococcal cells observed in the lung parenchyma or alveoli, whereas the opposite pattern was seen after challenges with H99 or KN99α. There were differences in lung neutrophil infiltration between the analyzed strains, and a higher presence of activated macrophages and CD4+ lymphocytes for the Mu-1 or 24064 strains. Polysaccharides from single and multi-motif strains elicited different effects on macrophage-like cells in vitro. These results suggest that variation in polysaccharide composition and structure can translate into differences in virulence and pathogenicity.

Importance: Cryptococcosis is a systemic fungal infection that causes approximately 1 million cases globally, leading to approximately 625,000 annual deaths. Two species are responsible for the majority of cases, Cryptococcus neoformans and Cryptococcus gattii. C. neoformans usually causes disease in immunosuppressed hosts, whereas C. gattii can infect and cause disease in immunocompetent hosts. The capsule of Cryptococcus spp. is one of its major virulence factors, due to its immunomodulation abilities. The capsule is composed of three major different molecules: glucuronoxylomannan (GXM), galactoxylomannan (GalXM), and mannoproteins. GXM is composed of six different structures called motifs. The exact mechanism and structures associated with its immunomodulation are still not well elucidated. Here, we looked at different immune responses based on the capsule composition. Our results strongly suggest that the capsule motif composition can affect the virulence, pathogenicity, and immunomodulatory capabilities of the Cryptococcus capsule.

Keywords: Cryptococcus neoformans; GXM; capsular polysaccharide; capsule; exopolysaccharide; extracellular polysaccharide; glucuronoxylomannan; motifs; pathogenesis; virulence.

Fig 1

GXM motif prevalence in EPS from the different serotype A strains of C. neoformans determined by proton NMR. EPS was produced after the growth of different serotype A strains of C. neoformans in MM for 1 week at 30°C under agitation (150 rpm). (A) Representative 1D [1H] NMR spectra of the SRG region with peak chemical shift assignments for each GXM motif. (B) Strain GXM motif makeup as determined by the integral percentage of motif assigned chemical shifts. Sequence and linkage of GXM motif repeats using glycobiology illustrative shorthand. NF = not found.

Fig 2

Phenotypic characterization of the EPS from serotype A strains of C. neoformans. After growth of different serotype A strains of C. neoformans in MM for 1 week at 30°C under agitation (150 rpm), the media were filtered with a 0.22 µm size filter. (A) Analysis of H99 EPS size and polydispersity by DLS (n = 5 per group). (B) Analysis of KN99α EPS size and polydispersity by DLS (n = 5 per group). (C) Analysis of Mu-1 EPS size and polydispersity by DLS (n = 5 per group). (D) Analysis of 24064 EPS size and polydispersity by DLS (n = 5 per group). (E) YPD Cell body size (n = 50 per group). (F) SAB cell body size (n = 50 per group). (G) MM cell body size (n = 50 per group). (H) YPD Capsule relative size (n = 50 per group). (I) SAB capsule relative size (n = 50 per group). (J) MM capsule relative size (n = 50 per group). (K) Biofilm formation at 30°C (n = 8 per group). (L) Biofilm formation at 37°C, respectively (n = 8 per group). (M) Percentage of cell surface hydrophobicity (n = 3 per group). No significant statistics were observed (t-test and/or ANOVA). Multiple comparisons were corrected by Šídák’s multiple comparisons test.

Fig 3

Different virulence factors of C. neoformans. Serotype A strains of C. neoformans (cap59∆, H99, KN99α, Mu-1, or 24064) were grown in different culture media (YPD, SAB, MM, or UB) and at different temperatures (30°C or 37°C), and their main virulence mechanisms were evaluated. (A) YPD 30°C growth rate (n = 4 per group). (B) SAB 30°C Growth rate (n = 4 per group). (C) MM 30°C growth rate (n = 4 per group). (D) YPD 37°C growth rate (n = 4 per group). (E) SAB 37°C growth rate (n = 4 per group). (F) MM 37°C growth rate (n = 4 per group). The unusual shape of growth curves for cap59Δ reflects the fact that under certain conditions, it aggregates. (G) Laccase activity (n = 6 per group). H99 and KN99α laccase knockout (lac1Δ) were used as a negative control. (H) Urease activity (n = 9 per group), H99 and KN99α urease knockout (ure1Δ) were used as negative control. (I) Phospholipase activity (n = 3 per group). (J) Mean gray value of the melanin production (n = 6 per group). * = P < 0.05, ** = P < 0.01, **** = P < 0.0001 (t-test and or ANOVA). Multiple comparisons were corrected by Šídák’s multiple comparisons test.

Fig 4

Capsular defective (acapsular) strain cap59∆ re-encapsulation. The re-encapsulation was performed by incubating the cap59Δ strains with the EPS from the H99, KN99α, Mu-1, or 24064 serotype A strains of C. neoformans at room temperature (24°C) for 1 h under rotation. (A) Phagocytosis assay showing that the re-encapsulation is preventing the acapsular strain (cap59∆) from being phagocytized by J774 macrophages after 2 h of interaction when compared with their EPS donor counterpart (H99, KN99α, Mu-1, or 24064). (B) The FOV (mathematical representation of the phagocytosis) area occupied by the fluorescent (Uvitex 2B) C. neoformans strains. The images represent the sum of nine squares (3 × 3) with 0.1 mm² area each (0.9 mm² total area), 40× magnification. Non-phagocytosed yeast was removed by the washing steps before fixation.

Fig 5

Mitochondrial activity of J774 macrophages after incubation with EPS from different serotype A strains of C. neoformans (cap59∆, H99, KN99α, Mu-1, or 24064) or EPS + β-glucans (Zymosan A). Mitochondrial activity was measured by the formation of formazan after the addition and incubation of MTT [3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide] for 2 h. (A) Mitochondrial activity of J774 cells incubated with EPS from cap59∆, H99, KN99α, Mu-1, or 24064, showing that the EPS from KN99α, Mu-1, or 24064 compromised the function of the macrophage mitochondria. Statistical analyses also showed that the influence of the EPS on the mitochondria varied depending on the strain. Control cells (c) incubated with cell culture media only (n = 8 per group). (B) Mitochondrial activity of J774 cells incubated with EPS from cap59∆, H99, KN99α, Mu-1, or 24064 + β-glucans, showing that the EPS from all analyzed strains + β-glucans compromised the function of the macrophage mitochondria. Statistical analyses also showed that the influence of the EPS + β-glucans in the mitochondria varied, depending on the strain. Control cells (c) incubated with cell culture media + β-glucans (n = 8 per group). (C) Grouped analysis comparing the difference in the mitochondrial activity between the cells incubated with only EPS from cap59∆, H99, KN99α, Mu-1, or 24064 and EPS from cap59∆, H99, KN99α, Mu-1, or 24064 + β-glucans (n = 8 per group). * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001 (t-test and or ANOVA). Multiple comparisons were corrected by Šídák’s multiple comparisons test.

Fig 6

Nitrite production by bone marrow-derived cells, macrophages (BMDM, GM-CSF, and M1), or dendritic cells (DCs), after incubation with EPS from different serotype A strains of C. neoformans (cap59∆, H99, KN99α, Mu-1, or 24064) or EPS + β-glucans (Zymosan A). Nitrite was measured after 12 h of incubation. BMDM = M0 macrophages (no activation or differentiation), GM-CSF = M0 macrophages stimulated with 20 ng/mL of GM-CSF overnight, M1 = M1 macrophages activated and differentiated overnight with 100 U/mL of IFN-γ and 500 ng/mL of LPS. (A) Nitrite production by BMDM cells after incubation with EPS from cap59∆, H99, KN99α, Mu-1, or 24064 shows no nitrite production. Control cells (c) were incubated with cell culture media only in all panels (n = 4 per group). (B) Nitrite production by BMDM cells after incubation with EPS from cap59∆, H99, KN99α, Mu-1, or 24064 + β-glucans. The EPS from the KN99α strain elicited nitrite production compared with the H99 EPS. Control cells (c) incubated with cell culture media + β-glucans (n = 4 per group). (C) Grouped analysis comparing the difference in nitrite production of the BMDM cells incubated with only EPS from cap59∆, H99, KN99α, Mu-1, or 24064 and EPS from cap59∆, H99, KN99α, Mu-1 + β-glucans. The incubation of the cells with EPS from cap59∆, H99, KN99α, Mu-1, or 24064 + β-glucans increased the nitrite production when compared with the cells incubated with only EPS from cap59∆, H99, KN99α, Mu-1, or 24064 (n = 4 per group). (D) Nitrite production by GM-CSF-stimulated cells after incubation with EPS from cap59∆, H99, KN99α, Mu-1, or 24064 showed no nitrite production. Control cells (c) were incubated with cell culture media only (n = 4 per group). (E) Nitrite production by GM-CSF-stimulated cells after incubation with EPS from cap59∆, H99, KN99α, Mu-1, or 24064 + β-glucans. Statistical analyses showed that the influence of the EPS + β-glucans on the nitrite production also varied depending on the strain. Control cells (c) were incubated with cell culture media + β-glucans (n = 4 per group). (F) Grouped analysis comparing the difference in nitrite production of the GM-CSF stimulated cells incubated with only EPS from cap59∆, H99, KN99α, Mu-1, or 24064 and EPS from cap59∆, H99, KN99α, Mu-1, or 24064 + β-glucans. The incubation of the cells with EPS from cap59∆, H99, KN99α, Mu-1, or 24064 + β-glucans increased the nitrite production when compared with the cells incubated with only EPS from cap59∆, H99, KN99α, Mu-1, or 24064 (n = 4 per group). (G) Nitrite production by M1 cells after incubation with EPS from cap59∆, H99, KN99α, Mu-1, or 24064. Cells treated with cap59∆ and 24064 EPS had lower nitrite production levels than those treated with H99 EPS. Control cells (c) incubated with cell culture media only (n = 4 per group). (H) Nitrite production by M1 cells after incubation with EPS from cap59∆, H99, KN99α, Mu-1, or 24064 + β-glucans. Statistical analyses showed that the Mu-1 EPS + β-glucans had lower nitrite production levels when compared with the control cells. Control cells (c) incubated with cell culture media + β-glucans (n = 4 per group). (I) Grouped analysis comparing the difference in nitrite production of the M1 cells incubated with only EPS from cap59∆, H99, KN99α, Mu-1, or 24064 and EPS from cap59∆, H99, KN99α, Mu-1 + β-glucans (n = 4 per group). (J) Nitrite production by DC cells after incubation with EPS from cap59∆, H99, KN99α, Mu-1, or 24064 showing no nitrite production. Control cells (c) incubated with cell culture media only (n = 4 per group). (K) Nitrite production by DC cells after incubation with EPS from cap59∆, H99, KN99α, Mu-1, or 24064 + β-glucans. Statistical analyses showed that the incubation of the DC cells with EPS from H99, KN99α + β-glucans had lower nitrite production compared with the control cells. Control cells (c) incubated with cell culture media + β-glucans (n = 4 per group). (L) Grouped analysis comparing the difference in nitrite production of the DC cells incubated with only EPS from cap59∆, H99, KN99α, Mu-1, or 24064 and EPS from cap59∆, H99, KN99α, Mu-1 + β-glucans. The incubation of the cells with EPS from cap59∆, H99, KN99α, Mu-1, or 24064 + β-glucans increased the nitrite production when compared with the cells incubated with only EPS from cap59∆, H99, KN99α, Mu-1, or 24064 (n = 4 per group). * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001 (t-test and or ANOVA). Multiple comparisons were corrected by Šídák’s multiple comparisons test.

Fig 7

ROS production by bone marrow-derived cells, macrophages (BMDM, GM-CSF, and M1), or dendritic cells (DCs), after incubation with EPS from different serotype A strains of C. neoformans (cap59∆, H99, KN99α, Mu-1, or 24064) or EPS + β-glucans (Zymosan A). ROS was measured after 12 h of incubation. BMDM = M0 macrophages (no activation or differentiation), GM-CSF = M0 macrophages stimulated with 20 ng/mL of GM-CSF overnight, M1 = M1 macrophages activated and differentiated overnight with 100 U/mL of IFN-γ and 500 ng/mL of LPS. (A) ROS production by BMDM cells after incubation with EPS from cap59∆, H99, KN99α, Mu-1, or 24064. Statistical analyses showed that the control cells had the lowest levels of ROS production. Control cells (c) incubated with cell culture media only (n = 4 per group). (B) ROS production by BMDM cells after incubation with EPS from cap59∆, H99, KN99α, Mu-1, or 24064 + β-glucans. Statistical analyses showed that the control cells had the lowest levels of ROS production. Control cells (c) incubated with cell culture media + β-glucans (n = 4 per group). (C) Grouped analysis comparing the difference in ROS production of the BMDM cells incubated with only EPS from cap59∆, H99, KN99α, Mu-1, or 24064 and EPS from cap59∆, H99, KN99α, Mu-1 + β-glucans (n = 4 per group). (D) ROS production by GM-CSF-stimulated cells after incubation with EPS from cap59∆, H99, KN99α, Mu-1, or 24064. Control cells (c) incubated with cell culture media only (n = 4 per group). (E) ROS production by GM-CSF-stimulated cells after incubation with EPS from cap59∆, H99, KN99α, Mu-1, or 24064 + β-glucans. Control cells (c) incubated with cell culture media + β-glucans (n = 4 per group). (F) Grouped analysis comparing the difference in ROS production of the GM-CSF stimulated cells incubated with only EPS from cap59∆, H99, KN99α, Mu-1, or 24064 and EPS from cap59∆, H99, KN99α, Mu-1, or 24064 + β-glucans. Statistical analyses showed that Mu-1 and 24064 EPS had an increase in ROS production with the addition of + β-glucans (n = 4 per group). (G) ROS production by M1 cells after incubation with EPS from cap59∆, H99, KN99α, Mu-1, or 24064. Control cells (c) incubated with cell culture media only (n = 4 per group). (H) ROS production by M1 cells after incubation with EPS from cap59∆, H99, KN99α, Mu-1, or 24064 + β-glucans. Statistical analyses showed that the cap59∆, H99, and KN99α EPS + β-glucans had higher ROS production levels when compared with the control cells. Control cells (c) incubated with cell culture media + β-glucans (n = 4 per group). (I) Grouped analysis comparing the difference in ROS production of the M1 cells incubated with only EPS from cap59∆, H99, KN99α, Mu-1, or 24064 and EPS from cap59∆, H99, KN99α, Mu-1 + β-glucans (n = 4 per group). (J) ROS production by DC cells after incubation with EPS from cap59∆, H99, KN99α, Mu-1, or 24064. Statistical analyses showed that cap59Δ, H99, or KN99α had higher ROS production levels when compared with the control cells. Control cells (c) incubated with cell culture media only (n = 4 per group). (K) ROS production by DC cells after incubation with EPS from cap59∆, H99, KN99α, Mu-1, or 24064 + β-glucans. Statistical analyses showed that the incubation of the DC cells with EPS from cap59∆, H99, KN99α, or 24064 + β-glucans had higher ROS production levels when compared with the control cells. Control cells (c) incubated with cell culture media + β-glucans (n = 4 per group). (L) Grouped analysis comparing the difference in ROS production of the DC cells incubated with only EPS from cap59∆, H99, KN99α, Mu-1, or 24064 and EPS from cap59∆, H99, KN99α, or Mu-1 + β-glucans. The incubation of the cells with EPS from cap59∆ + β-glucans increased the ROS production when compared with the cells incubated with only EPS from cap59∆ (n = 4 per group). * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001 (t-test and/or ANOVA). Multiple comparisons were corrected by Šídák’s multiple comparisons test.

Fig 8

Survival and fungal burden studies (CFU). Mice were infected with C. neoformans strains H99, KN99α, Mu-1, or 24064, either by intranasal (IN) route or intravenous (IV) route, with 5 × 10⁵ yeast suspended in 20 µL of PBS (10 µL per nostril) or 5 × 10⁵ yeast in 100 µL of PBS, respectively. Mice were euthanized 5 or 20 days after infection. (A) Survival of IN-infected mice (n = 5 per group). (B) Survival of IV-infected mice (n = 5 per group). (C) Lung CFU of IN-infected mice (n = 5 per group). (D) Brain CFU of IN-infected mice (n = 5 per group). (E) Lung CFU in IV-infected mice after 20 days of infection (n = 4 for the H99 group, n = 2 for the KN99α group, n = 2 for the Mu-1 and 24064 groups, and n = 3 for the Mu-1 [20 days] and 24064 [20 days]). (F) Brain CFU of IV-infected mice after 20 days of infection (n = 4 for the H99 group, n = 2 for the KN99α group, n = 2 for the Mu-1 and 24064 groups, and n = 3 for the Mu-1 [20 days] and 24064 [20 days]). The red line represents the original inoculum (5 × 10⁵). For the IV infection, dead animals from the H99 (1) or KN99α (3) group had similar CFU counts but were removed from the final analysis because they died 12 h before euthanasia for CFU determination. Data represent the second independent replicate.

Fig 9

IN-infected mice lungs, cytokines after 20 days of infection. (A) IFN-γ (n = 5 per group). (B) IL-1β (n = 5 per group). (C) IL-4 (n = 5 per group). (D) IL-6 (n = 5 per group). (E) IL-10 (n = 5 per group). All infected lungs had higher levels of IFN-γ, IL-1β, and IL-4 compared with the lungs from non-infected mice (SHAM). * = P < 0.05 (t-test and or ANOVA). Multiple comparisons were corrected by Šídák’s multiple comparisons test.

Fig 10

Histological findings after 20 days of IN infection. After euthanasia, randomly selected lungs had a small tissue sample piece aseptically excised and preserved in formalin until analysis. (A) H99-infected mice 1× magnification. (B) H99-infected mice 6× magnification; insert box 20× magnification and black arrow showing a cryptococcal cell. (C) KN99α 1× magnification. (D) KN99α 6× magnification; insert box 20× magnification and black arrow showing a cryptococcal cell. (E) Mu-1 infected mice 1× magnification. (F) Mu-1 infected mice 6× magnification. (G) 24064 infected mice 1× magnification. (H) 24064 infected mice 6× magnification. HE stains.

Fig 11

Flow cytometry of the lung of IN-infected mice after 20 days of infection. (A) Neutrophils (n = 2 for the sham group, n = 2 for the H99 group, n = 4 for the KN99α group, n = 5 for the Mu-1 and 24064). (B) Dendritic cells (n = 2 for the sham group, n = 2 for the H99 group, n = 4 for the KN99α group, n = 5 for the Mu-1 and 24064). (C) Activated macrophages (n = 2 for the sham group, n = 2 for the H99 group, n = 4 for the KN99α group, n = 5 for the Mu-1 and 24064). (D) M2 macrophages (n = 2 for the sham group, n = 2 for the H99 group, n = 4 for the KN99α group, n = 5 for the Mu-1 and 24064). (E) CD4+ T lymphocytes (n = 2 for the sham group, n = 2 for the H99 group, n = 4 for the KN99α group, n = 5 for the Mu-1 and 24064). (F) CD8+ T lymphocytes (n = 2 for the sham group, n = 2 for the H99 group, n = 4 for the KN99α group, n = 5 for the Mu-1 and 24064). * = P < 0.05, ** = P < 0.01, **** = P < 0.0001 (t-test and or ANOVA). Multiple comparisons were corrected by Šídák’s multiple comparisons test.

Fig 12

Lungs cytokines after 5 and/or 20 days of IV infection. (A) IFN-γ (n = 5 for the sham group, n = 4 for the H99 group, n = 2 for the KN99α group, n = 5 for the Mu-1 and 24064). (B) IL1-β (A) IFN-γ (n = 5 for the sham group, n = 4 for the H99 group, n = 2 for the KN99α group, n = 5 for the Mu-1 and 24064). (C) IL-4 (n = 5 for the sham group, n = 4 for the H99 group, n = 2 for the KN99α group, n = 5 for the Mu-1 and 24064). (D) IL-6 (n = 5 for the sham group, n = 4 for the H99 group, n = 2 for the KN99α group, n = 5 for the Mu-1 and 24064). (E) IL-10 (n = 5 for the sham group, n = 4 for the H99 group, n = 2 for the KN99α group, n = 5 for the Mu-1 and 24064). All infected lungs had higher levels of IFN-γ, IL-1β, and IL-4 when compared with the lungs from non-infected mice (SHAM). ** = P < 0.01 (t-test and or ANOVA). Multiple comparisons were corrected by Šídák’s multiple comparisons test.

Fig 13

Histological findings after 5 days of IV infection. After euthanasia, randomly selected lungs had a small tissue sample piece aseptically excised and preserved in formalin until analysis. (A) H99-infected mice 1× magnification. (B) H99-infected mice 6× magnification; insert box 20× magnification and black arrow showing a cryptococcal cell. (C) KN99α 1× magnification. (D) KN99α 6× magnification; insert box 20× magnification and black arrow showing a cryptococcal cell. (E) Mu-1 infected mice 1× magnification. (F) Mu-1 infected mice 6× magnification. (G) 24064 infected mice 1× magnification. (H) 24064 infected mice 6× magnification. HE stains.

Fig 14

Lung fungal load (CFU) after 12 h of infection. Mice were infected with C. neoformans strains (H99, KN99α, Mu-1, or 24064) by intratracheal (IT) route with 5 × 10⁵ yeast suspended in 50 µL of PBS (n = 4 for the H99 group, n = 5 for the KN99α group, n = 4 for the Mu-1, and n = 4 for the 24064). The euthanasia occurred 12 h after infection. Data represent the one experiment * = P < 0.05. (t-test and or ANOVA). Multiple comparisons were corrected by Šídák’s multiple comparisons test.