Peptidogalactomannan from Histoplasma capsulatum yeast cell wall: role of the chemical structure in recognition and activation by peritoneal macrophages

Abstract

Histoplasma capsulatum is the causative agent of histoplasmosis, a systemic disease responsible for most reported causes of morbidity and mortality among immunosuppressed individuals. Peptidogalactomannan (pGM) was purified from the yeast cell wall of H. capsulatum isolated from bats, and its structure and involvement in modulating the host immune response were evaluated. Gas chromatography, methylation analysis, and two-dimensional nuclear magnetic resonance (2D-NMR) were used for the structural characterization of pGM. Methylation and 2D-NMR data revealed that pGM comprises a main chain containing α-d-Manp (1 → 6) residues substituted at O-2 by α-d-Manp (1 → 2)–linked side chains, non-reducing end units of α-d-Galf, or β-d-Galp linked (1→ 6) to α-d-Manp side chains. The involvement of H. capsulatum pGM in antigenic reactivity and in interactions with macrophages was demonstrated by ELISA and phagocytosis assay, respectively. The importance of the carbohydrate and protein moieties of pGM in sera reactivity was evaluated. Periodate oxidation abolished much pGM antigenic reactivity, suggesting that the sugar moiety is the most immunogenic part of pGM. Reactivity slightly decreased in pGM treated with proteinase K, suggesting that the peptide moiety plays a minor role in pGM antigenicity. In vitro experiments suggested that pGM is involved in the phagocytosis of H. capsulatum yeast and induction of IL-10 and IFN-γ secretion by peritoneal macrophages from C57BL/6 mice. These findings demonstrated the role of pGM in the H. capsulatum-host interaction.

Introduction

Histoplasma capsulatum is a dimorphic fungus that presents a filamentous form as a saprophytic-geophilic microorganism in the environment. H. capsulatum is distributed worldwide, especially in soils enriched with nitrogen and phosphate as a result of bird and bat guano deposits. When its microconidia are inhaled by human hosts, they convert to the yeast cell phase responsible for respiratory and disseminated disease, even in immunocompetent patients [1]. Histoplasmosis is endemic in the Midwest and Southeast United States and Central and South America, and in micro foci in southern Europe, Africa, and Southeastern Asia [2, 3]. In Brazil, it is highly endemic in Amazonas, Roraima, Pará, Amapá, Ceará, Rio Grande do Norte, Bahia, Minas Gerais, São Paulo, and in the southeastern regions of the country [4].

The cell wall is an essential structure for fungi due to its roles in cell viability, morphology, and protection; moreover, it contains glycosylated molecules that play roles in fungal physiology and host-pathogen interactions [5]. Peptidogalactomannans (pGMs) have been characterized in Fusarium oxysporum and Aspergillus fumigatus, which are recognized by rabbit anti-serum and sera from patients with aspergillosis, respectively [6, 7]. Oliveira and colleagues demonstrated that the carbohydrate moiety was responsible for antigenicity since pGM reactivity against serum decreased after the removal of O-linked oligosaccharide chains or periodate treatment [6]. In addition, F. oxysporum pGM plays a role in fungal recognition and uptake by macrophages and induces TNF-α production [6]. Peptidorhamnomannan (pRM) is another glycoconjugate present in the fungal cell wall of Sporothrix schenckii and Scedosporium/Lomentospora species [8, 9]. In Lomentospora prolificans, pRM is involved in fungal uptake by macrophages, nitrite release, and TNF-α production [10].

Altogether, these data indicate that different peptidopolysaccharides present on the fungal surface play important roles in host-pathogen interactions, thus modulating the host immune response. In this context, this work aimed to characterize a peptidopolysaccharide obtained from H. capsulatum and evaluate its role in the interaction between fungi and peritoneal macrophages.

Material and methods

Microorganisms and growth conditions

H. capsulatum M240/06, isolated from bat liver, was kindly supplied by Dr. Maria Adelaide Dias Galvão, Zoonosis Control Center, São Paulo, Brazil. The yeast phase was obtained by growing the cells at 37 °C in brain-heart infusion (Acumedia, Lansing, MI, USA) supplemented with 0.1% l-cysteine and was inoculated into Erlenmeyer flasks containing liquid medium (200 mL), which were incubated for 7 days with shaking. Cells were centrifuged, washed three times in 150 mM phosphate-buffered saline (PBS; pH 7.2), counted in a Neubauer chamber, and stored at −20 °C.

Extraction and purification of peptidopolysaccharides

Crude peptidopolysaccharides were obtained from H. capsulatum yeast cells by extraction with 0.05 M phosphate buffer (pH 7.2) at 100 °C for 2 h. The mixture was filtered to remove fungal cells, and the filtrate was extensively dialyzed against distilled water and then fractionated by hexadecyltrimethylammonium bromide (Cetavlon, Merck, Darmstadt, Germany), according to Lloyd, 1970 [11].

Colorimetric analysis

Total carbohydrates were determined by the phenol-sulfuric acid method [12]; hexosamine content, by the method of Rondle and Morgan [13]; and protein content, by the Folin phenol reagent method [14].

Monosaccharide composition and methylation analysis of the peptidopolysaccharide

To determine the monosaccharide composition using gas chromatography-mass spectrometry (GC-MS), pGM (2 mg) was hydrolyzed with 5 M trifluoroacetic acid for 4 h at 100 °C and reduced with borohydride, and the alditols were acetylated with acetic anhydride:pyridine (1:1 v/v). The acetylated alditols were dissolved in chloroform and analyzed using a GC-MS instrument (GC-MS-QP2010 Shimadzu, Kyoto, Japan) with a Restek RTX-5MS column, according to the method of Kircher, 1960 [15]. For methylation analysis, the peptidopolysaccharide (5 mg) was subjected to two rounds of methylation as described by Ciucanu and Kerek, 1984 [16]. The methylated peptidopolysaccharide was hydrolyzed, reduced with borohydride, acetylated, and analyzed on a GC-MS instrument, as described above.

The peptidopolysaccharide (5 mg) was dissolved in DMSO (~1 mL), which was diluted with Me₂SO (1 mL) and then with MeI (1 mL). Powdered NaOH (0.3 g) was added, and the mixture was agitated vigorously by vortexing for 30 min and then left for 18 h. After neutralization with acetic acid, water was added, and the per-O-methylated product was extracted with chloroform, which was washed three times with water. After evaporating the chloroform, the resulting per-O-methylated product was converted into partially O-methylated alditol acetates by successive treatments with 3% MeOH-HCl for 2 h at 70 °C, 0.5 M H₂SO₄ for 14 h at 100 °C, reduction with NaBD₄, and acetylation with Ac₂O-pyridine. The alditol acetates of the methylated sugars were dissolved in chloroform and analyzed in a GC-MS unit (GC-MS-QP2010, Shimadzu, Kyoto, Japan) with a Restek RTX-5MS column. The samples were analyzed using a split ratio of 30, helium as the carrier gas, and a temperature gradient of 110–250 °C with 2 °C/min variation. The temperatures of the injector, ion source, and interface were 260 °C, 200 °C, and 230 °C, respectively. The fragments were identified by their retention times and electron impact spectra [17].

Nuclear magnetic resonance spectroscopy

Proton and carbon-13 (¹H and ¹³C) two-dimensional (2D) NMR spectra of pGM were recorded using a 500-MHz nuclear magnetic resonance (NMR) spectrometer (Bruker Biospin, Rheinstetten, Germany) with a triple resonance probe. About 20 mg of peptidopolysaccharides was dissolved in 0.5 mL of 99.9% deuterium oxide (Cambridge Isotope Laboratory, Tewksbury, MA, USA). All spectra were recorded at 50 °C with HOD suppression by presaturation. The 2D ¹H-¹³C multiplicity-edited heteronuclear single quantum coherence (HSQC) spectra were recorded using states-time-proportional phase incrementation for quadrature detection in the indirect dimension. The ¹H/¹³C HSQC spectra were run with 1024×256 points and globally optimized alternating phase rectangular pulses for decoupling. Chemical shifts are displayed relative to external trimethylsilylpropionic acid at 0 ppm for ¹H and relative to methanol for ¹³C [17].

Proteinase K treatment of peptidopolysaccharide

H. capsulatum peptidopolysaccharide (500 ng/well) was treated with proteinase K (500 μg/mL; Sigma Aldrich, St. Louis, MO, USA) in 1% sodium dodecyl sulfate solution for 1 h at 4 °C. After washing, the immunoreactivity of the intact and treated pGM was observed with serum from a histoplasmosis patient by ELISA assay [18].

Partial hydrolysis of peptidopolysaccharide

H. capsulatum peptidopolysaccharide (10 mg) was treated with 0.1 M HCl and heated at 100 °C for 20 min. The degraded molecule was recovered through dialysis against distilled water and freeze-drying of the retained solution [19].

Periodate oxidation of peptidopolysaccharide

H. capsulatum peptidopolysaccharide (4 mg/mL) was dissolved in 1 mL of 50 mM periodic acid and incubated at 4 °C. After 18 h, the residual NaIO₄ was removed with an equimolar amount of glycerol for 15 min, and then the product was reduced with 50 mM NaBH₄ under stirring for 2 h at room temperature. The solution was dialyzed against distilled water for 72 h, and the reduced periodate-oxidized peptidopolysaccharide was lyophilized. The immunoreactivity was measured by ELISA assay [6].

Human sera

Human sera were kindly supplied by Dr. Mauro M. Muniz from the Mycology Laboratory of the Instituto Nacional de Infectologia Evandro Chagas (INI), Fundação Oswaldo Cruz, Brazil. These sera were obtained from histoplasmosis patients (3 donors) and healthy individuals (3 donors).

Rabbit sera

The rabbit immune serum against whole cells of Cladosporium resinae was supplied by Dr. E. Barreto-Bergter, Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Brazil. White male rabbits were inoculated with freeze-dried mycelia of C. resinae (2 mg/mL dry weight) emulsified in an equal volume of complete Freund’s adjuvant, and 1 mL of the emulsion was injected intradermally at weekly intervals for 3 weeks [19]. Then, the same concentration was used in three intravenous injections at 2-day intervals for 1 week. The resulting hyperimmune serum was used in ELISA experiments. Pre-immune serum was collected as a control.

Serological reactivity of glycan

Human sera against H. capsulatum and rabbit immune serum against Cladosporium resinae were used to verify antigenic similarities between the peptidopolysaccharides from H. capsulatum and C. resinae, which were analyzed directly by ELISA assay [20]. The wells of flat-bottomed polyvinyl microtiter plates (Falcon, Becton & Dickinson, Franklin Lakes, NJ, USA) were coated with 100 μL of a 5 μg/mL solution of H. capsulatum yeast pGM or C. resinae pGM, both of which were extracted from cell walls, and maintained for 1 h at 37 °C. After washing with 0.05% PBS-Tween 20, the non-specific sites were blocked using 2% bovine serum albumin in 0.05% PBS-Tween 20. Pooled sera from histoplasmosis patients and rabbit immune serum against whole cells of C. resinae at dilutions of 1:100 to 1:6400 in blocking buffer (100 μL) were added to the wells. Antibody binding was measured using goat anti-human IgG and goat anti-rabbit IgG antibodies conjugated to horseradish peroxidase (Sigma, St. Louis, MO, USA), respectively. Each step was followed by incubation at 37 °C for 1 h and washing with 0.05% PBS-Tween 20. The substrate used was O-phenylenediamine (0.4 mg/mL) with H₂O₂ (0.4 μL/mL) in 0.01 M sodium citrate buffer (pH 5.0). The enzyme reaction was measured at 490 nm using an automated microplate reader (Bio-Rad ELISA Reader, Bio-Rad, Hercules, CA, USA) 20 min after terminating the reaction with 50 μL 1.5 M H₂SO₄ [21]. H. capsulatum pGM treated by periodate oxidation, partial hydrolysis, and proteinase K was tested by ELISA as described above.

Mice

In all experiments, 6–8-week-old female C57BL/6 mice were used.

Macrophage cytotoxicity assay—neutral red technique

A murine macrophage (C57BL/6) viability assay was conducted to test the cytotoxic effect of peptidopolysaccharide using the neutral red dye-uptake method. Peptidopolysaccharide (5–200 μg/mL) was added to a macrophage monolayer in 96-well plates. After 24 h, the cytotoxic effect on macrophages was analyzed by the neutral red technique [10].

Phagocytic assay

Mice received 2 mL of 3% sterile thioglycolate. Three days after injection, macrophages were collected from the peritoneal cavity with ice-cold RPMI-1640 medium. The macrophages were plated in 24-well plates (5 × 10⁵ cells/well). Adherent monolayers were challenged with 300 μL of live yeast suspensions containing 2.5 × 10⁶ cells/well. After incubation at 37 °C in 5% CO₂ for 2 h in RPMI-1640 medium, the cells were rinsed with RPMI medium to remove non-internalized yeast. Samples were fixed in Bouin’s solution and stained with Giemsa [10].

The influence of peptidopolysaccharide on yeast phagocytosis was evaluated. Macrophages were incubated for 30 min at 37 °C with 25 and 50 μg/mL peptidopolysaccharide. Subsequently, the macrophage monolayer was rinsed with RPMI medium, and the live yeast suspensions were added to macrophages and incubated at 37 °C in 5% CO₂ for 2 h. After the interaction, the cultures were rinsed once in RPMI medium to discard free yeast, fixed in Bouin’s solution, and stained with Giemsa. The percentage of infected cells was determined by randomly counting a minimum of 200 cells on each coverslip. The phagocytic index was calculated by multiplying the mean number of ingested fungi per macrophage cell by the percentage of infected cells, as observed by microscopic examination using an immersion objective (Olympus BX50F4, Olympus, Tokyo, Japan) [22].

Macrophage cytokine assay

Macrophages were collected as described above and plated in 96-well plates (2 × 10⁵ cells/well). Different concentrations of H. capsulatum pGM (50, 100, and 200 μg/mL) or 10 ng/well lipopolysaccharide (LPS; O111:B4) were added to the macrophage monolayer and incubated for 18 h. The supernatant was recovered to determine TNF-α, IFN-γ, and IL-10 concentrations using ELISA according to the manufacturer’s instructions (BD OptEIA ELISA Set, BD Biosciences, San Jose, CA, USA). Polymixin B (10 μg/mL) was added 5 min before the addition of the stimulus to rule out the possibility that the stimulating activity was due to lipopolysaccharide contamination [6].

Statistical analysis

Statistical analyses were performed using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA, USA). All data were subjected to analysis of variance (Tukey’s post hoc test) to analyze significant differences between groups. P values of 0.05 or less were considered statistically significant.

Results

Isolation, purification, and monosaccharide composition of H. capsulatum pGM

Crude peptidopolysaccharide from yeast cells of H. capsulatum was obtained by hot extraction and purified by selective precipitation with Cetavlon/sodium tetraborate at pH 8.8, as previously described [7]. It contained 24% protein, 48% total carbohydrates, and 1.5% hexosamine. Acid hydrolysis of the purified material followed by sodium borohydride reduction and acetylation produced alditol acetates of mannitol (Man) and galactitol (Gal) at a molar ratio of 80:20 (GC-MS).

Methylation analysis of pGM

Methylation analysis of pGM extracted from H. capsulatum yeast cells and GC-MS of partially O-methylated alditol acetate derivatives (Table 1) revealed that 2,3,4,6-tetra-O-methylmannose (20%) and 2,3,5,6-tetra-O-methylgalactose (14%) originated from non-reducing end units of mannose and galactose, respectively. Peaks corresponding to 3,4,6-tri-O-methylmannose (22%), 2,3,4-tri-O-methylmannose (23%), and 3,4-di-O-methylmannose (21%) suggest the presence of a main chain of α-(1→6)-mannopyranosyl residues substituted at the 2-positions by mannopyranosyl-containing side chains.

Table 1.

GC-MS analysis of O-methylalditol acetates derived from methylation analysis of peptidogalactomannan extracted from H. capsulatum yeast cells

Derivativea	Rtb (min)	Glycosidic linkage	Mol%
2,3,4,6-Me4Man	31.5	Manp-(1→	20
2,3,5,6-Me4Gal	31.8	Galf-(1→	14
3,4,6-Me3Man	35.9	→2)-Manp-(1→	22
2,3,4-Me3Man	37.5	→6)-Manp-(1→	23
3,4-Me2Man	42.0	→2,6)-Manp-(1→	21

^aPartially methylated alditol acetate derivatives obtained after permethylation with CH₃I hydrolysis, reduction, and acetylation

^bRetention time relative to that of 2,3,4,6-tetra-O-methyl glucitol acetate

NMR spectroscopy

The chemical structure of the pGM purified from yeast cells of H. capsulatum M240/06 was elucidated by NMR. The ¹³C-¹H HSQC spectrum identified more resolved signals (Fig. 1), which were assigned by analogy with published data for similar polysaccharides [23]. The anomeric region of the ¹³C-¹H HSQC spectrum showed six predominant signals: 5.22/108.10, 5.05/103.10, 5.05/101.25, 5.04/103.11, 5.12/99.41, and 4.91/100.52 (units A1, B1, C1, D1, E1 and F1, respectively) (Table 2). HSQC spectroscopy of pGM revealed a typical chemical shift of β-galactofuranose (anomeric of A1 at δ 5.30/108.10), and the signals of B1 and D1 of C¹³ at δ 103.10 ppm typically correspond to α-mannopyranose [21]. According to the analysis of derived partially O-methylated alditol acetates (unit 5) and the report by Ahrazem et al. [23], we can conclude that the signal at 5.12/99.41 (E1) is consistent with the presence of 2,6-di-O-substituted mannopyranose. These substitutions were confirmed due to the C¹³ chemical shift at about ~6 ppm of E2 and E6 (79.61 and 66.71, respectively) when compared to the chemical shift of non-substituted mannose (B2—71.00 and B6—61.90). Based on the chemical shift of the carbon spin system and data previously reported by Ahrazem et al., the signal at 5.05/101.2 ppm (unit C) (Table 2) is consistent with the non-reducing end of α-galactofuranosyl [23].

Fig. 1.

HSQC (¹H-¹³C) spectrum of H. capsulatum pGM showing the signals of CH carbons/protons (phase signals) and those from CH₂ (antiphase). The signals and corresponding units are identified in Table 2

Table 2.

Correlation between the ¹H and ¹³C nucleus found in the HSQC of H. capsulatum pGM

Units	A	B	C	D	E	F
Proposed structure	β-Galf1→	α-Manp1→	α-Galf1→	→6-α-Manp1→	→2,6-α-Manp1→	→2-α-Manp1→
H1/C1	5.22/108.1	5.05/103.1	5.05/101.2	5.05/103.1	5.12/99.4	4.91/100.5
H2/C2	4.15/82.2	4.08/71.0	4.16/77.4	4.08/70.8	4.05/79.6	4.04/79.6
H3/C3	4.10/77.6	3.85/71.5	4.25/75.2	3.83/71.6	3.94/71.7	4.01/71.2
H4/C4	4.08/83.7	3.68/67.7	3.84/82.1	3.82/67.6	3.83/67.7	-
H5/C5	3.84/71.6	3.76/74.18	3.80/72.3	-	3.83/71.7	-
H6′/C6′	3.72/63.6	3.90/61.9	3.63/63.7	3.74/66.9	3.72/66.7	-
H6″/C6″	3.64/63.6	3.78/61.9	3.72/63.7	4.06/66.9	4.02/66.7	-

Complete and definitive interpretation of chemical shifts of the F signal was impaired due to the non-visualization of spin systems on the 2D spectra. However, the F2 signal (4.03/79.6 ppm) had a similar chemical shift to that of E2, and methylation analysis indicated that unit 3 corresponded to a 2-O-substituted mannose (Fig. 1). The glycosidic linkage was defined based on HSQC and NOESY spectra, albeit the latter was not useful due to overlapping NOE signals between inter- and intra-residue signals of the glycosidic linkage. However, the HSQC spectrum (Fig. 1) showed inter-residue contact points between A1^H (5.22 ppm) and E2^C/F2^C (79.61 ppm), E1^H (5.12 ppm) and D6/E6^C (66.9/66.7 ppm), and B1/C1/D1^H (~5.05 ppm) and D6/E6^C (66.9/66.7 ppm). These results suggest a heterogeneous and complex structure consisting of α-d-Manp (1 → 6) in the main chain substituted at O-2 by side chains of α-d-Manp (1 → 2)-linked, the non-reducing end of α-d-Galf ,or β-d-Galf linked to carbon 6 of the α-d-Manp side chain (Fig. 2).

Fig. 2.

Main structures of H. capsulatum pGM. The galactomannan component of H. capsulatum M240/06 pGM consists of a main chain containing (1→6)-linked α-d-Manp residues (D) with branches at C-2 containing α-d-Manp-(1→2) (B), 2,6-α-d-Manp-(1→ (E), α-Galf-(1→6) (A), α-d-Manp-(1→2) (B), and β-Galf-(1→6) (C) as non-reducing end units.

Immunological reactivity of purified H. capsulatum pGM

The antigenicity of H. capsulatum pGM was evaluated by ELISA using sera from histoplasmosis patient. Sera from three patients (serum numbers 25647, 21281, and 26477) reacted with pGM isolated from H. capsulatum yeast (Fig. 3). The highest reactivity was observed in serum number 25647. Healthy human sera were used as a negative control.

Fig. 3.

Reactivity of purified pGM from H. capsulatum M240/06 with sera from histoplasmosis patients (serum numbers 25647, 21281, and 26477). Sera from healthy individuals were used as a negative control. Sera were serially diluted from 1:100 to 1:6400, and reactivity was evaluated by ELISA. The values represent three independent experiments performed in duplicate

Immunological reactivity of the carbohydrate and protein moiety of pGM

To evaluate the role of carbohydrate and protein moieties as important epitopes of H. capsulatum pGM using ELISA, three histoplasmosis serum samples were pooled and tested against pGM subjected to three different treatments: pGM was partially hydrolyzed to remove galactofuranosyl side chain residues, oxidized by periodate to remove all carbohydrate moieties, or treated with proteinase K to remove protein moieties. Our results showed that intact H. capsulatum pGM strongly reacted with sera from histoplasmosis patients, whereas treatment with sodium m-periodate abolished the antigenic activity of this molecule, suggesting that the carbohydrate moiety is the most important epitope in pGM from H. capsulatum yeast cells. Additionally, proteinase K–treated pGM exhibited a weak decrease in reactivity, suggesting a less antigenic property of the peptide moiety when compared to that of the carbohydrate portion. Treatment with 0.1 M HCl, which removed labile galactofuranosyl side chain residues, did not affect the antigenicity of this molecule. No pGM reactivity was seen with the negative control (pooled sera from three healthy individuals) (Fig. 4).

Fig. 4.

Reactivity of sera from histoplasmosis patients against intact and treated H. capsulatum pGM. Three different treatments were performed: partial hydrolysis (0.1 M HCl), which removes labile galactofuranosyl side chain residues; periodate oxidation, which removes all carbohydrate moieties of pGM; and proteinase K treatment, which removes protein moieties. Pooled sera from healthy subjects were used as a negative control. Sera were serially diluted from 1:100 to 1:6400, and reactivity was evaluated by ELISA against all pGM conditions. The values represent three independent experiments performed in duplicate

Antigenic cross-reactivity between H. capsulatum and C. resinae pGMs

Since many fungi, such as Cladosporium resinae, present galactose- and mannose-containing polysaccharides, we tested the antigenic similarity between pGM from H. capsulatum M240/06 and that from C. resinae [19] using pooled sera from histoplasmosis patients and rabbit immune serum against whole cells of C. resinae. In fact, ELISA assay of the pooled sera revealed similar reactivity between H. capsulatum pGM and C. resinae pGM, suggesting the presence of cross-reactive determinants in these fungi. However, much lower reactivity was detected when rabbit immune serum against whole cells of C. resinae was incubated with pGM of H. capsulatum (Fig. 5), suggesting that different epitopes are present in these molecules.

Fig. 5.

Cross-reactivity of pGMs from H. capsulatum and C. resinae were analyzed by ELISA using pooled sera from histoplasmosis patients and rabbit immune serum against whole cells of C. resinae (serum dilution 1:200). The values represent three independent experiments performed in duplicate. Asterisks denote values that are significantly different from that of the control (*P < 0.05, ***P < 0.0001)

Involvement of pGM on H. capsulatum phagocytosis by macrophages

Purified pGM was pre-incubated with macrophages for 30 min before yeast challenge. Treatment with up to 200 μg/mL pGM did not show toxicity toward macrophages. Two different concentrations were chosen, 25 and 50 μg/mL. pGM inhibited yeast uptake by 46% at 25 μg/mL and 53% at 50 μg/mL, suggesting that pGM is an important molecule recognized by macrophage receptors. Although the higher tested pGM concentration showed greater inhibition, there were no significant differences between the tested concentrations (Fig. 6).

Fig. 6.

Involvement of pGM in phagocytosis of H. capsulatum by C57BL/6 peritoneal macrophages. The macrophages were untreated (control) or pre-treated for 30 min with two different pGM concentrations (25 and 50 μg/mL) and then incubated with yeast cells for 1 h. The phagocytic index values represent the mean ± standard deviation of three independent experiments performed in triplicate. Asterisks denote values that are significantly different from those of the control (**P < 0.01)

Cytokine release by H. capsulatum pGM on C57BL/6 macrophages

The role of pGM in the induction of cytokine release by macrophages was evaluated. Macrophages were incubated with various pGM concentrations (50, 100, and 200 μg/mL) for 18 h at 37 °C. At 100 and 200 μg/mL, pGM induced secretion of IL-10, an anti-inflammatory cytokine. Regarding proinflammatory cytokines, 100 and 200 μg/mL pGM induced only IFN-γ release, whereas no effect was observed for TNF-α production (Fig. 7). Treatment with 50 μg/mL pGM was unable to induce any cytokine release.

Fig. 7.

Cytokine release by C57BL/6 peritoneal macrophages was analyzed after pGM exposure for 18 h at 37 °C. Three pGM concentrations (50, 100, and 200 μg/mL) were used, and three cytokines were evaluated: IL-10 (a), IFN-γ (b), and TNF-α (c). Values are the means of three independent experiments. MO, only macrophages are used as a negative control; LPS, 10 ng/well lipopolysaccharide (O111:B4) used as a positive control; LPS + Poly, LPS plus polymixin B (10 μg/mL). Asterisks denote values that are significantly different from those of the control (*P < 0.005, **P < 0.001, ***P < 0.0001). ns, no significance

Discussion

Histoplasmosis is an invasive mycosis caused by the dimorphic fungal pathogen H. capsulatum [24]. Currently, histoplasmosis is one of the most important systemic mycoses in the Americas, with a wide distribution in Brazilian regions [25, 26]. H. capsulatum infection is difficult to diagnose due to similarities in its clinical symptoms and standard diagnostic techniques with those of other systemic infections caused by fungi and bacteria [27–30]. Several studies have been conducted to improve understanding of the biochemistry and cell biology of H. capsulatum in order to elucidate its mechanisms of pathogenicity and to develop better diagnostic and therapeutic approaches [31, 32].

The fungal cell wall comprises polysaccharides and glycoconjugates that are involved in numerous biological activities. The mechanisms of adhesion, colonization, and invasion of host tissues have been studied in several fungal species, including Candida albicans, H. capsulatum, A. fumigatus, Fonsecaea pedrosoi, and Shigella boydii [33–35]. Recognition of glycoconjugates on the fungal surface by host immune cells triggers a response culminating in phagocytosis, antimicrobial compound production, and induction of proinflammatory cytokines that activate and recruit other effector immune cells [36, 37]. However, little is known about glycoproteins from the H. capsulatum yeast cell wall surface.

In this work, the structure of a peptidogalactomannan (pGM) from H. capsulatum M240/06 yeast cells was partially characterized, and its involvement in the recognition and internalization of H. capsulatum by murine peritoneal macrophages were analyzed in addition to the ability of H. capsulatum pGM to induce cytokine secretion by macrophages.

H. capsulatum pGM was found to contain mannose and galactose in an 80:20 molar ratio, which is in agreement with previous results for other H. capsulatum antigens [38–40]. The H. capsulatum pGM structure was partially characterized by the combination of 2D-NMR and methylation techniques. These analyses suggested that pGM consists of a main chain containing (1→6)-linked α-d-Manp residues substituted at O-2 by side chains containing (1→2)-linked α-d-Manp residues. The β-d-Galf and α-d-Galf units are present as non-reducing terminals substituted at O-6 by mannopyranosyl side chains. According to our results, H. capsulatum pGM presents a structure similar to that of Paracoccidioides brasiliensis pGM obtained from mycelium and yeast forms [23]. However, the F. oxysporum pGM structure is completely different, having a main chain containing (1→6)-linked β-d-Galf residues with several branches at C-2 containing α-d-Manp(1→ and short chains containing β-d-Manp(1→2)-Manp [6]. A. fumigatus pGM has a highly branched structure containing α-d-Manp and β-d-Galf as non-reducing end units, a main chain containing (1→6)-linked α-d-Manp substituted at O-2 by side chains containing one to three (1→2)-linked α-d-Manp units, and 5-O-substituted Galf with an average length of 5 units linked to O-6 of the mannan core [7]. Calixto et al. (2010) characterized a pGM from a saprophytic strain of C. (Hormodendrum) resinae, which was very similar to A. fumigatus pGM [19].

Here, the antigenicity of H. capsulatum pGM against sera from histoplasmosis patients was observed, and the degree of reactivity varied between different individuals with histoplasmosis. Moreover, our findings demonstrated the importance of the carbohydrate and protein moieties of H. capsulatum pGM in recognizing the sera from patients with histoplasmosis. Treatment of pGM with sodium metaperiodate and proteinase K revealed the importance of the carbohydrate moiety for the antigenicity of this molecule. Similar findings have been demonstrated previously. A. fumigatus pGM is antigenic and is recognized in ELISA tests of the sera of patients with aspergillosis. It was previously shown that the removal of O-linked oligosaccharides from pGM led to a 50% decrease in serum reactivity with pGM, and treatment with metaperiodate abolished much of the antigenic activity of A. fumigatus pGM [6, 41]. Additionally, pGMs from C. resinae are similar to those found in A. fumigatus [19]. The importance of the carbohydrate moiety of the C. resinae pGM was demonstrated by removal of the β-d-galactofuranose units (1→5)-linked side chains by partial acid hydrolysis. A decrease in the reactivity of this molecule was revealed by ELISA using rabbit serum immunized with C. resinae [19].

The recognition of glycoproteins on the fungal surface initiates an immediate response leading to phagocytosis, production of antimicrobial compounds, and induction of cytokines that activate and recruit other effector immune cells [37]. Here, we showed that pGM isolated from H. capsulatum plays a role in the phagocytosis of yeast cells by murine macrophages and in the induction of inflammatory response. Treatment with 25 and 50 μg/mL pGM inhibited phagocytosis by 46% and 53%, respectively, suggesting that pGM is recognized by macrophage receptors responsible for phagocytosis of H. capsulatum. Popi et al. (2002) and Konno et al. (2009) observed that the glycoprotein Gp43, isolated from P. brasiliensis culture filtrate, also inhibits phagocytosis of both P. brasiliensis and zymosan particles [42, 43]. The authors suggested that this glycoprotein binds to the mannose receptors present in macrophages [42, 43]. The importance of α-glucans for phagocytosis of S. boydii was studied by Bittencourt et al. (2006) [22], who demonstrated that S. boydii α-glucan inhibited conidial phagocytosis in a dose-dependent manner (25, 50, and 100 μg/mL) [22]. In addition, conidia treated with α-amyloglucosidase (which removes α-glucans from the conidial surface) showed less phagocytosis, indicating that α-glucans are accessible on the conidial surface and mediate the interaction of S. boydii conidia with macrophages [22]. Similar results were also observed by Xisto et al. (2015) [10], who found that O-linked oligosaccharides of pRM from L. prolificans were key determinants for phagocytosis by murine peritoneal macrophages and inflammatory response induction [10].

The role of cytokines in modulating the immune response has been studied to better understand their contribution to fungal pathogenesis. Proinflammatory cytokines, such as IL-1 and TNF-α, activate macrophages and induce neutrophil recruitment in response to microorganisms [44]. The proinflammatory cytokine IFN-γ strongly activates phagocytes and leads to decreased intracellular growth of H. capsulatum [45]. IL-10 is known as an anti-inflammatory cytokine initially classified as part of the Th2 cytokine profile, and it is important for decreasing inflammatory damage caused by high proinflammatory cytokine levels [46]. In this work, H. capsulatum pGM induced the release of IL-10 and IFN-γ, but not TNF-α, by peritoneal macrophages. Figueiredo et al. (2010) demonstrated that conidia and N-linked rhamnomannans from S. boydii induced the production of TNF-α and IL-10 [47]. However, L. prolificans pRM induced secretion of TNF-α, but not IL-10, by peritoneal macrophages [10]. Recently, Oliveira et al. (2019) demonstrated that intact F. oxysporum pGM induced the production of a small amount of TNF-α, but removal of the O-linked oligosaccharides increased TNF-α secretion [6]. In histoplasmosis-infected mice, Sahaza et al. (2015) observed high levels of IFN-γ, which can inhibit the intracellular growth of H. capsulatum [48]. They also detected the anti-inflammatory cytokines IL-4 and IL-10, which can be involved in regulating the proinflammatory response [48].

In summary, this study partially characterized pGM from H. capsulatum yeast cells and described its antigenic properties. In addition, pGM was recognized by macrophages, suggesting that it plays a role in the H. capsulatum–macrophage interaction. Cytokines, such as IL-10 and IFN-γ, are induced by pGM, suggesting an immunomodulatory effect of this molecule. Altogether, these data improve the understanding of the role of pGM in host-pathogen interactions, and further studies are needed to clarify the mechanisms by which pGM contributes to fungal pathogenicity.

Author contribution

GMPS, MIDSX, RR-P, and MRP designed the experiments and drafted the manuscript. GMPS, GRCS, MIDSX, and RR-P performed all experiments. GMPS, GRCS, MIDSX, RR-P, ARSB, EMSR, RLDM, EB-B, and MRP analyzed the data. EB-B critically revised the manuscript. All authors read and approved the manuscript.

Funding

This study was funded by Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ) (grant number E-26/200.577/2016). This study was partly funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (Financial Code 001) and Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq).

Data availability

All data generated or analyzed during this study are included in this published article.

Declarations

Ethics approval

This study was approved by the research ethics committee of Instituto Nacional de Infectologia Evandro Chagas, Fiocruz, accession number 19109913.0.0000.5262, and by the Institutional Animal Welfare Committee of the Universidade Federal Fluminense (UFF), accession code 731/2016/CEUA-UFF.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Conflict of interest

The authors declare that there is no conflict of interest.