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Cryptococcus neoformans is resistant to surfactant protein A mediated host defense mechanisms.

Giles SS, Zaas AK, Reidy MF, Perfect JR, Wright JR - PLoS ONE (2007)

Bottom Line: Using in vitro binding assays we confirmed that SP-A does not directly bind to a fully encapsulated strain of C. neoformans (H99).We found that the immune response assessed by cellular counts, TNFalpha cytokine production, and fungal burden in lungs and bronchoalveolar lavage fluids during early stages of infection were equivalent.Our results suggest that unlike a variety of bacteria, viruses, and other fungi, progression of disease with an inhalational challenge of C. neoformans does not appear to be negatively or positively affected by SP-A mediated mechanisms of pulmonary host defense.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Duke University Medical Center, Durham, North Carolina, United States of America.

ABSTRACT
Initiation of a protective immune response to infection by the pathogenic fungus Cryptococcus neoformans is mediated in part by host factors that promote interactions between immune cells and C. neoformans yeast. Surfactant protein A (SP-A) contributes positively to pulmonary host defenses against a variety of bacteria, viruses, and fungi in part by promoting the recognition and phagocytosis of these pathogens by alveolar macrophages. In the present study we investigated the role of SP-A as a mediator of host defense against the pulmonary pathogen, C. neoformans. Previous studies have shown that SP-A binds to acapsular and minimally encapsulated strains of C. neoformans. Using in vitro binding assays we confirmed that SP-A does not directly bind to a fully encapsulated strain of C. neoformans (H99). However, we observed that when C. neoformans was incubated in bronchoalveolar fluid, SP-A binding was detected, suggesting that another alveolar host factor may enable SP-A binding. Indeed, we discovered that SP-A binds encapsulated C. neoformans via a previously unknown IgG dependent mechanism. The consequence of this interaction was the inhibition of IgG-mediated phagocytosis of C. neoformans by alveolar macrophages. Therefore, to assess the contribution of SP-A to the pulmonary host defenses we compared in vivo infections using SP-A mice (SP-A-/-) and wild-type mice in an intranasal infection model. We found that the immune response assessed by cellular counts, TNFalpha cytokine production, and fungal burden in lungs and bronchoalveolar lavage fluids during early stages of infection were equivalent. Furthermore, the survival outcome of C. neoformans infection was equivalent in SP-A-/- and wild-type mice. Our results suggest that unlike a variety of bacteria, viruses, and other fungi, progression of disease with an inhalational challenge of C. neoformans does not appear to be negatively or positively affected by SP-A mediated mechanisms of pulmonary host defense.

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SP-A can bind C. neoformans via an IgG dependent mechanism.(A.) C. neoformans yeast (1×105) were treated with AF488 labeled SP-A (Panel A), PE-Texas Red labeled anti-GXM IgG (Panel B) and PE-Texas Red labeled anti-GXM IgG and AF488 labeled SP-A (Panel C). Calcofluor was added to all samples to visualize the cell wall. SP-A binding to IgG opsonized (Panel C) but not non-opsonized (Panel A) C. neoformans was observed. Scale bar length is equal to 10 µm. (B.) IgG opsonized C. neoformans were treated with SP-A concentrations of 1, 10, 20, 40 µg/ml and binding was quantified by flow cytometric analysis. EDTA (1 mM) was included to determine if SP-A binding required the carbohydrate recognition domain. Histograms represent the mean fluorescence intensity of the AF 488 channel. At least 10,000 particles were interrogated for each sample.
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pone-0001370-g002: SP-A can bind C. neoformans via an IgG dependent mechanism.(A.) C. neoformans yeast (1×105) were treated with AF488 labeled SP-A (Panel A), PE-Texas Red labeled anti-GXM IgG (Panel B) and PE-Texas Red labeled anti-GXM IgG and AF488 labeled SP-A (Panel C). Calcofluor was added to all samples to visualize the cell wall. SP-A binding to IgG opsonized (Panel C) but not non-opsonized (Panel A) C. neoformans was observed. Scale bar length is equal to 10 µm. (B.) IgG opsonized C. neoformans were treated with SP-A concentrations of 1, 10, 20, 40 µg/ml and binding was quantified by flow cytometric analysis. EDTA (1 mM) was included to determine if SP-A binding required the carbohydrate recognition domain. Histograms represent the mean fluorescence intensity of the AF 488 channel. At least 10,000 particles were interrogated for each sample.

Mentions: Several studies have reported that naturally occurring IgG antibodies can non-specifically bind to pathogenic fungi. In addition, antibody binding can alter the physical properties of the C. neoformans capsule and affect the ability of other opsonins, such as complement to bind [16]. Therefore, we investigated whether IgG antibody binding to capsule enabled SP-A binding to C. neoformans. C. neoformans cells were opsonized with anti-GXM IgG and then treated with AF488 labeled SP-A (20 µg/ml) or left untreated as a control. Calcofluor was used to visualize the cell wall. While SP-A did not bind directly to C. neoformans cells (Figure 2A), the addition of anti-GXM IgG to the interaction resulted in robust SP-A binding that appeared to co-localize with anti-GXM IgG binding and was generally uniform throughout the capsular matrix (Figure 2A). Due to the size of the capsule, some areas bound by SP-A and anti-GXM IgG appear larger than the yeast. Further analysis by flow cytometry demonstrated that SP-A binding (1, 10, 20, and 40 µg/ml) to anti-GXM IgG opsonized C. neoformans cells occurred in a dose-dependent manner (Figure 2B). The carbohydrate recognition domain of SP-A is known to bind sugars in a calcium dependent manner. We observed that EDTA (1 mM) did not affect SP-A binding to anti-GXM IgG opsonized C. neoformans, suggesting that the carbohydrate recognition domain was not involved in this mechanism (Figure 2B). Furthermore, this result suggested that SP-A binding to anti-GXM IgG opsonized C. neoformans is not due to antibody mediated exposure of carbohydrate ligands.


Cryptococcus neoformans is resistant to surfactant protein A mediated host defense mechanisms.

Giles SS, Zaas AK, Reidy MF, Perfect JR, Wright JR - PLoS ONE (2007)

SP-A can bind C. neoformans via an IgG dependent mechanism.(A.) C. neoformans yeast (1×105) were treated with AF488 labeled SP-A (Panel A), PE-Texas Red labeled anti-GXM IgG (Panel B) and PE-Texas Red labeled anti-GXM IgG and AF488 labeled SP-A (Panel C). Calcofluor was added to all samples to visualize the cell wall. SP-A binding to IgG opsonized (Panel C) but not non-opsonized (Panel A) C. neoformans was observed. Scale bar length is equal to 10 µm. (B.) IgG opsonized C. neoformans were treated with SP-A concentrations of 1, 10, 20, 40 µg/ml and binding was quantified by flow cytometric analysis. EDTA (1 mM) was included to determine if SP-A binding required the carbohydrate recognition domain. Histograms represent the mean fluorescence intensity of the AF 488 channel. At least 10,000 particles were interrogated for each sample.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2147053&req=5

pone-0001370-g002: SP-A can bind C. neoformans via an IgG dependent mechanism.(A.) C. neoformans yeast (1×105) were treated with AF488 labeled SP-A (Panel A), PE-Texas Red labeled anti-GXM IgG (Panel B) and PE-Texas Red labeled anti-GXM IgG and AF488 labeled SP-A (Panel C). Calcofluor was added to all samples to visualize the cell wall. SP-A binding to IgG opsonized (Panel C) but not non-opsonized (Panel A) C. neoformans was observed. Scale bar length is equal to 10 µm. (B.) IgG opsonized C. neoformans were treated with SP-A concentrations of 1, 10, 20, 40 µg/ml and binding was quantified by flow cytometric analysis. EDTA (1 mM) was included to determine if SP-A binding required the carbohydrate recognition domain. Histograms represent the mean fluorescence intensity of the AF 488 channel. At least 10,000 particles were interrogated for each sample.
Mentions: Several studies have reported that naturally occurring IgG antibodies can non-specifically bind to pathogenic fungi. In addition, antibody binding can alter the physical properties of the C. neoformans capsule and affect the ability of other opsonins, such as complement to bind [16]. Therefore, we investigated whether IgG antibody binding to capsule enabled SP-A binding to C. neoformans. C. neoformans cells were opsonized with anti-GXM IgG and then treated with AF488 labeled SP-A (20 µg/ml) or left untreated as a control. Calcofluor was used to visualize the cell wall. While SP-A did not bind directly to C. neoformans cells (Figure 2A), the addition of anti-GXM IgG to the interaction resulted in robust SP-A binding that appeared to co-localize with anti-GXM IgG binding and was generally uniform throughout the capsular matrix (Figure 2A). Due to the size of the capsule, some areas bound by SP-A and anti-GXM IgG appear larger than the yeast. Further analysis by flow cytometry demonstrated that SP-A binding (1, 10, 20, and 40 µg/ml) to anti-GXM IgG opsonized C. neoformans cells occurred in a dose-dependent manner (Figure 2B). The carbohydrate recognition domain of SP-A is known to bind sugars in a calcium dependent manner. We observed that EDTA (1 mM) did not affect SP-A binding to anti-GXM IgG opsonized C. neoformans, suggesting that the carbohydrate recognition domain was not involved in this mechanism (Figure 2B). Furthermore, this result suggested that SP-A binding to anti-GXM IgG opsonized C. neoformans is not due to antibody mediated exposure of carbohydrate ligands.

Bottom Line: Using in vitro binding assays we confirmed that SP-A does not directly bind to a fully encapsulated strain of C. neoformans (H99).We found that the immune response assessed by cellular counts, TNFalpha cytokine production, and fungal burden in lungs and bronchoalveolar lavage fluids during early stages of infection were equivalent.Our results suggest that unlike a variety of bacteria, viruses, and other fungi, progression of disease with an inhalational challenge of C. neoformans does not appear to be negatively or positively affected by SP-A mediated mechanisms of pulmonary host defense.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Duke University Medical Center, Durham, North Carolina, United States of America.

ABSTRACT
Initiation of a protective immune response to infection by the pathogenic fungus Cryptococcus neoformans is mediated in part by host factors that promote interactions between immune cells and C. neoformans yeast. Surfactant protein A (SP-A) contributes positively to pulmonary host defenses against a variety of bacteria, viruses, and fungi in part by promoting the recognition and phagocytosis of these pathogens by alveolar macrophages. In the present study we investigated the role of SP-A as a mediator of host defense against the pulmonary pathogen, C. neoformans. Previous studies have shown that SP-A binds to acapsular and minimally encapsulated strains of C. neoformans. Using in vitro binding assays we confirmed that SP-A does not directly bind to a fully encapsulated strain of C. neoformans (H99). However, we observed that when C. neoformans was incubated in bronchoalveolar fluid, SP-A binding was detected, suggesting that another alveolar host factor may enable SP-A binding. Indeed, we discovered that SP-A binds encapsulated C. neoformans via a previously unknown IgG dependent mechanism. The consequence of this interaction was the inhibition of IgG-mediated phagocytosis of C. neoformans by alveolar macrophages. Therefore, to assess the contribution of SP-A to the pulmonary host defenses we compared in vivo infections using SP-A mice (SP-A-/-) and wild-type mice in an intranasal infection model. We found that the immune response assessed by cellular counts, TNFalpha cytokine production, and fungal burden in lungs and bronchoalveolar lavage fluids during early stages of infection were equivalent. Furthermore, the survival outcome of C. neoformans infection was equivalent in SP-A-/- and wild-type mice. Our results suggest that unlike a variety of bacteria, viruses, and other fungi, progression of disease with an inhalational challenge of C. neoformans does not appear to be negatively or positively affected by SP-A mediated mechanisms of pulmonary host defense.

Show MeSH
Related in: MedlinePlus