The impact of episporic modification of Lichtheimia corymbifera on virulence and interaction with phagocytes

Abstract

Fungal infections caused by the ancient lineage Mucorales are emerging and increasingly reported in humans. Comprehensive surveys on promising attributes from a multitude of possible virulence factors are limited and so far, focused on Mucor and Rhizopus. This study addresses a systematic approach to monitor phagocytosis after physical and enzymatic modification of the outer spore wall of Lichtheimia corymbifera, one of the major causative agents of mucormycosis. Episporic modifications were performed and their consequences on phagocytosis, intracellular survival and virulence by murine alveolar macrophages and in an invertebrate infection model were elucidated. While depletion of lipids did not affect the phagocytosis of both strains, delipidation led to attenuation of LCA strain but appears to be dispensable for infection with LCV strain in the settings used in this study. Combined glucano-proteolytic treatment was necessary to achieve a significant decrease of virulence of the LCV strain in Galleria mellonella during maintenance of the full potential for spore germination as shown by a novel automated germination assay. Proteolytic and glucanolytic treatments largely increased phagocytosis compared to alive resting and swollen spores. Whilst resting spores barely (1-2%) fuse to lysosomes after invagination in to phagosomes, spore trypsinization led to a 10-fold increase of phagolysosomal fusion as measured by intracellular acidification. This is the first report of a polyphasic measurement of the consequences of episporic modification of a mucormycotic pathogen in spore germination, spore surface ultrastructure, phagocytosis, stimulation of Toll-like receptors (TLRs), phagolysosomal fusion and intracellular acidification, apoptosis, generation of reactive oxygen species (ROS) and virulence.

Keywords: AFM, Atomic Force Microscopy; Atomic Force Microscopy (AFM); CD14, Cluster of differentiation 14; CFW, Calcofluor white; Galleria mellonella; HEK, human embryonic kidney; HSI, Hyperspectral imaging; Hyperspectral imaging (HIS); IPS, Insect physiological saline; Intracellular survival; LCA, Lichtheimia corymbifera attenuated; LCV, Lichtheimia corymbifera virulent; MD-2, Myeloid Differentiation factor 2; MH-S, Murine alveolar macrophages; MM6, Acute monocytic leukemia derived human monocyte Mono-Mac-6; Monocytes; NF-κB, Nuclear factor 'kappa-light-chain-enhancer' of activated B-cells; PBS, Phosphate buffer saline solution; PI, Phagocytosis index; ROS, Reactive oxygen species; TEM, Transmission Electron Microscopy; TLRs, Toll like receptors; Transmission Electron Microscopy (TEM).

Graphical abstract

Fig. 1

phagocytosis the spores of L. corymbifera by alveolar macrophages (MH-S). A) Comparative phagocytosis assay between two L. corymbifera strains: L. corymbifera virulent strain (LCV) and L. corymbifera attenuated strain (LCA) in a time-dependent interaction with murine alveolar macrophages at MOI of 5 as indicated by fold change phagocytosis index normalized to the minimal time point of 1 h. The statistical analysis using t-test applies the 1 h time point as reference for comparison with 3 h and 5 h time points. B) Phagocytosis after swelling and opsonization with normal human serum in comparison to resting spores of two L. corymbifera strains: L. corymbifera virulent strain LCV and L. corymbifera attenuated strain LCA confronted with murine alveolar macrophages for 3 h as indicated by fold change phagocytosis index normalized to the resting spore condition. The statistical analysis using t-test applies the resting spore condition as reference for comparison with swollen and opsonized spores. ns…non-significant, * P < 0.05, ** P < 0.01, *** P < 0.001.

Fig. 2

Phagocytosis after physical and enzymatic treatments of spores of two L. corymbifera strains: L. corymbifera virulent strain (LCV) and L. corymbifera attenuated strain (LCA) in interaction with murine alveolar macrophages for 3 h (MOI = 5) as indicated by phagocytic indices normalized to resting spores. The table below displays the spore wall components structurally and qualitatively affected during the treatment. + indicates, affected‘, - indicates, not affected‘. The statistical analysis using t-test applies the resting spore condition as reference for comparison with the other spore treatments: ns non-significant, * P < 0.05, ** P < 0.01, and *** P < 0.001.

Fig. 3

Time lapse analysis of spore germination of L. corymbifera strains (LCA and LCV) after different cell wall treatments. Germination was documented by video microscopy and analyzed with the ImageJ plugin HyphaTracker. The area of each spore/germling was determined at each time points. A,B. Mean values and standard deviation of spore/germling area vs time of resting spores or spores treated with kitalase, pronase, trypsin or vinotaste as indicated. C. Parameters obtained by fitting the experimental data with a lag-exponential model showing offset.. Significance values are given in Table 1.

Fig. 4

Phagolysosomal acidification of spores of L. corymbifera strains (LCV and LCA) during interaction with murine alveolar macrophages (MOI = 5). (A) Phagolysosomal acidification during the phagocytosis of kitalase, vinotaste, and pronase E-treated spores at time point 3 h of confrontation with MH-S cells compared to untreated (resting) and heat-killed spores for negative and positive control, respectively, (B) Temporal kinetics of phagolysosomal acidification after tryptic treatment, (C) Influence of chloroquine on phagolysosomal acidification triggered by resting versus heat-killed spores for LCV spores. Relative acidification was determined by counting the number of acidified phagocytosed spores in relation to 100 phagocytosed spores. Significance values were conducted with t-test: ns non-significant, * P < 0.05, ** P < 0.01, and *** P < 0.001.

Fig. 5

Induction of apoptosis by Lichtehimia corymbifera after physical treatments of spores in murine alveolar macrophages (MH-S). Resting spores, heat-killed spores, and UV-treated spores of LCV and LCA strains of L. corymbifera were compared to MH-S cells treated with staurosporine for positive control for the induction of apoptosis in MH-S. The measures represent additive values of the apoptotic stages (early and late apoptosis, and necrosis). (A) The whole apoptotic rate of MH-S cells (early and late apoptosis process), (B) the rate of late apoptotic of MH-S cells, (C) the rate of early apoptosis process of MH-S cells, (D) The rate of necrotic MH-S cells. Three independent biological measurements were performed by Flow Cytometry. Charts of flow cytometry of 1st replicate are shown in Supplementary Fig. 5 as an example. The statistical analysis was based on the percentages of apoptotic and necrotic MH-S cells co-incubated with resting, heat-killed, and UV-treated spores to MH-S cells and compared to MH-S cells treated with staurosporine as positive control. The statistical analysis using t-test applies this positive control as reference for comparison with swollen and opsonized spores: ns… non-significant, * P < 0.05, ** P < 0.01, and *** P < 0.001.

Fig. 6

Generation of reactive oxygen species (ROS) after 1 h (A), 3 h (B), and 5 h (C) of incubation of MH-S macrophages with L. corymbifera. Resting spores, heat-killed spores, and UV-treated spores of LCV and LCA strains of L. corymbifera were compared to MH-S cells treated with PMA as positive control. The statistical analysis was based on the percentages of ROS production by MH-S cells co-incubated with resting, heat-killed, and UV-treated spores to MH-S cells and compared to MH-S cells treated with PMA as positive control. The statistical analysis using t-test applies this positive control as reference for comparison with swollen and opsonized spores: ns…non-significant, * P < 0.05, ** P < 0.01, *** P < 0.001.

Fig. 7

Virulence of L. corymbifera strains LCV (A and C) and LCA (B and D) after physical and enzymatic treatments of spores in Galleria mellonella. Larvae were infected with 10⁷ spores/larva and incubated at 30 °C in the dark. Survival was monitored every 24 h for six days. (LCV1/LCA1) indicate resting spores of two strains of L. corymbifera, (LCV2/LCA2) refer to swollen spores of LCA and LCA strains, (LCV3/LCA3) show the heat-killed spores of LCV and LCA strains, (LCV4/LCA4) refer to the vinotaste-treated spores of LCV and LCA strains, (LCV5/LCA5) show tween 20 treated spores of LCV and LCA strains, (LCV6/LCA6) refer to pronase E-treated spores of LCV and LCA strains, (LCV7/LCA7) show trypsinized spores of LCV and LCA strains, (LCV8/LCA8) show UV-treated spores of LCV and LCA strains, and (LCV9/LCA9) show kitalase-treated spores of LCV and LCA strains. Kaplan-Meier curves represent average survival data of all three independent survival assays and were analyzed for significance by Log-Rank (Mantel Cox) test utilizing graph pad prism software.

Fig. 8

Topography AFM images show the morphology and ultrastructure of spores of L. corymbifera strains (LCV and LCA). A – Resting spores; B – kitalase-treated spores; C – Vinotaste-treated spores; D – trypsin-treated spores, E – Pronase E-treated spores, F – heat-killed spores.

Fig. 9

Ultrastructure of cross-sections from modified epispores of L. corymbifera strain LCV performed by Transmission Electron Microscopy (TEM). The cross-sections were taken from sub-sections of the spore surfaces from (i) resting spores and compared to resting spores treated with: (ii) trypsin, (iii) pronase E, (iv) heat, (v) Vinotaste and (vi) Tween 20. The episporic ultrastructures display differences in cell wall integrity as given by (i) cell wall continuity, (ii) cell wall thickness and (iii) electron density. The scale bar indicates 200 nm.

Fig. 10

Luciferase reporter activation after stimulation of Toll-like receptors (TLRs) -expressing Flp-In™-293 NF-κB cells with spores of Lichtheimia corymbifera after different treatments for 5 h. Flp-In™-293 NF-κB cells stably expressing different Toll like receptors (TLR1,2,4,5,6) and cofactors (CD14, MD2) and additional knock out of TLR5 (TLR5-ko) were generated for TLR activation assays. All cell lines have a stably integrated promotor that is responding to the transcription factor NF-κB (nuclear factor 'kappa-light-chain-enhancer' of activated B-cells) that translocates from cell plasma to the nucleus upon TLR activation. The responsive promotor regulates the expression of the luciferase reporter gene. The selected TLR combinations were: TLR2-CD14 with TLR5-ko, TLR2-TLR6-CD14, TLR2-TLR1 -CD14, CD14- MD2- TLR4 and TLR5. The basic cell line with luciferase reporter gene was used as comparison and has an additional TLR5-ko of the natural gene. Cell lines were seeded into 96-well-plates. The activity of the luciferase after 5 h of stimulation was normalized to the mean value of the pre run (2 h before stimulation). Data was analyzed by two-way ANOVA and Bonferroni posttest (*** indicates P < 0.001, * indicates P < 0.05).

Fig. 11

Luciferase reporter activation after stimulation of TLR 2-CD14 expressing Flp-In™-293 NF-κB cells with spores of Lichtheimia corymbifera after different treatments for 10 h. The capability of kitalase-treated spores, heat-killed first then kitalase –treated spores, vinotaste-treated spores, heat-killed spores, pronase E, heat-killed spores then pronase E, UV-treated spores, and resting spores of LCV and LCA strains to stimulate expression of TLR2-CD14 was evaluated. Treating the spores of LCV and LCA strains with kitalase and heat-killed spores following kitalase treatment stimulated the expression of TLR2-CD14. Other treatments were comparable to resting spores of both strains. Data were analyzed by two-way ANOVA and Bonferroni posttest (*** indicates P < 0.001, * indicates P < 0.05).

Supplementary figure 1

Influence of the origin of the sera used for opsonization of resting spores of two strains of L. corymbifera (LCV and LCA) on the phagocytosis rate by MH-S cells as indicated by fold change of phagocytic index normalized to resting spores after 3 hours confrontation with murine alveolar macrophages (MOI = 5). The statistical analysis using t-test applies the resting spore condition as reference for comparison with opsonization using murine and human normal sera. The sera were heat-inactivated for negative control: ns… non-significant, *P < 0.05, **P < 0.01, ***P  < 0.001.

Supplementary figure 2

Phagocytosis of resting spores of L. corymbifera strains: LCV (A) and LCA (B) after swelling and opsonization with normal human serum in a time-dependent interaction with murine alveolar macrophages (MOI = 5) as indicated by fold change phagocytosis index normalized to resting spores confronted to the minimal time point of 1 hour. The statistical analysis using t-test applies the 1 h time point of the resting spore condition as reference for comparison with 3 h and 5 h time points: ns… non-significant, **P < 0.05, ** P < 0.01, *** P  < 0.001.

Supplementary figure 3

Phagocytosis of resting spores of L. corymbifera strains: LCV (A) and LCA (B) after swelling and opsonization with normal human serum in a time-dependent interaction with murine alveolar macrophages (MOI = 5) as indicated by fold change phagocytosis index normalized to resting spores confronted to the minimal time point of 1 hour. The statistical analysis using t-test applies the 1 h time point of the resting spore condition as reference for comparison with 3 h and 5 h time points: ns non-significant, * * P < 0.05, ** P < 0.01, *** P  < 0.001.

Supplementary figure 4a

Influence of physical and enzymatic treatments of heat-killed spores of L. corymbifera strains (LCV and LCA) on phagolysosomal acidification during interaction with apoptotic and non-apoptotic human monocytes (MOI = 3) in a single cell analysis as conducted by Hyperspectral Imaging (HSI)-assisted measurements. (A) During phagocytosis of resting spores of LCV and LCA strains, the monocytes recover from apoptosis as indicated by blue and red graphs, respectively. (B) The blue graph refers to non-apoptotic cells indicating cell recovery from acidic pH for positive control whereas the red graph refers to the fate of apoptotic cells lacking cell recovery for negative control. (C) pH series measurement of pH rodo red dye using different buffers ranging from pH 4 to 7. The spectral raw data of pH rodo red labeled particles in the pH buffer solutions of 4, 5, 6, and 7. X-axis indicates the position that can be converted to the wavelength and y-axis represents signal intensity emitted by the fluorescent dye. The scaling of the plots is not similar as the pH value reduces the maximum intensity of signal increases.

Supplementary figure 4b

Supplementary figure 5

Hyperspectral Image microscopy of human monocytes infected with resting spores of L. corymbifera strains LCV (A) versus LCA (B) after physical and enzymatic treatments in a multiple cell analysis. The signal intensity was measured up to 48 hours in live versus apoptotic under infection versus uninfected cells.

Supplementary figure 6a

Induction of apoptosis by L. corymbifera after physical treatments of the spores in murine alveolar macrophages as revealed by Flow Cytometry. A – inhibition of apoptosis with resting, heat-killed and UV-treated spores of LCV and LCA; B – negative and positive controls of unstained and stained macrophages. Q1 indicates the percent of necrotic MH-S cells, Q2 represent the percent of late apoptosis MH-S, Q3 refers the percent of MH-S live cells and Q4 is the percent of early apoptosis of MH-S cells. Values in the quadrants indicate percentages of cells related to the population as a whole. The percentages of the living cells are indicated in yellow, whilst the highly significant, differential values are indicated in red versus black (starting values).

Supplementary figure 6b

Supplementary figure 7

Virulence of LCV after physical and enzymatic treatments of spores in Galleria mellonella which were infected with a wide range of physically and enzymatically treated spores (107/larva) in a time-dependent manner as indicated by Kaplan-Meier curves. IPS and resting spore stages were used as negative and positive control, respectively.

Supplementary figure 8

Virulence of LCA after physical and enzymatic treatments of spores in Galleria mellonella which were infected with a wide range of physically and enzymatically treated spores (107/larva) in a time-dependent manner as indicated by Kaplan-Meier curves. IPS and resting spore stages were used as negative and positive control, respectively.