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, 81 (1), 206-18

Role for Golgi Reassembly and Stacking Protein (GRASP) in Polysaccharide Secretion and Fungal Virulence


Role for Golgi Reassembly and Stacking Protein (GRASP) in Polysaccharide Secretion and Fungal Virulence

Lívia Kmetzsch et al. Mol Microbiol.


Secretion of virulence factors is a critical mechanism for the establishment of cryptococcosis, a disease caused by the yeast pathogen Cryptococcus neoformans. One key virulence strategy of C. neoformans is the release of glucuronoxylomannan (GXM), a capsule-associated immune-modulatory polysaccharide that reaches the extracellular space through secretory vesicles. Golgi reassembly and stacking protein (GRASP) is required for unconventional protein secretion mechanisms in different eukaryotic cells, but its role in polysaccharide secretion is unknown. This study demonstrates that a C. neoformans functional mutant of a GRASP orthologue had attenuated virulence in an animal model of cryptococcosis, in comparison with wild-type (WT) and reconstituted cells. Mutant cells manifested altered Golgi morphology, failed to produce typical polysaccharide capsules and showed a reduced ability to secrete GXM both in vitro and during animal infection. Isolation of GXM from cultures of WT, reconstituted or mutant strains revealed that the GRASP orthologue mutant produced polysaccharides with reduced dimensions. The mutant was also more efficiently associated to and killed by macrophages than WT and reconstituted cells. These results demonstrate that GRASP, a protein involved in unconventional protein secretion, is also required for polysaccharide secretion and virulence in C. neoformans.


Figure 1
Figure 1. Deletion and complementation of the C. neoformans GRASP ortholog
A. Scheme for the construction of the mutant strain. GRASP gene was replaced with the hygromicin resistant marker (HygR) cassette (gray box). GRASP 5’ and 3’ flanks (5 GRASP and 3 GRASP, respectively) were fused with HygR cassette by Delsgate methodology (Garcia-Pedrajas et al., 2008). The resulting targeting vector (TV) was used for C. neoformans transformation. The wild type locus of GRASP (WT) and the position of primers used for GRASP gene disruption are also indicated. The black bar scale corresponds to 500 base pairs (bp). The cleavage sites of EcoRI restriction enzyme are indicated in the deletion scheme. B. Southern blot analysis. Genomic DNA (10 µg) from WT (lane 1), grasp mutant (lane 2) and grasp::GRASP reconstituted (lane 3) strains were digested with EcoRI restriction enzyme. The 5’ gene flank was used as probe in Southern hybridization. Numbers at left indicate the hybridization signal sizes based upon the position of molecular size marker. C. Semi-quantitative RT-PCR with cDNA from WT (lane 1), grasp mutant (lane 2) and grasp::GRASP reconstituted (lane 3) strains as template. Numbers at rigth indicate the length of the transcript amplification for GRASP (upper panel) and ACT1 (lower panel) genes. Lane 4: positive control with genomic DNA as template. NC: negative control of the PCR reaction.
Figure 2
Figure 2. Phylogenetic analysis of the C. neoformans GRASP ortholog
A. Alignment of the GRASP PDZ domains (PDZ-1 and PDZ-2) from R. norvegicus (AAB81355.2), C. elegans (NP_501354), D. melanogaster (AAF49092), S. cerevisiae (NP_010805), D. discoideum (EAL60823), S. pombe (NP_593015.1), P. falciparum (AAN35366), A. nidulans (Broad Institute accession number ANID_11248), U. maydis (Broad Institute accession number UM01076), C. albicans (Broad Institute accession number CAWG_01021) and C. neoformans (Broad Institute accession number CNAG_03291) using ClustalX2. B. Phylogenetic analysis applying the Neighbor-Joining method including GRASP sequences from distinct eukaryotic organisms listed above. The phylogeny tree splits into two major clades. C-I and C-II represents clades I and II, respectively. The bar marker indicates the genetic distance, which is proportional to the number of amino acid substitutions.
Figure 3
Figure 3
Modified morphology of the Golgi apparatus in C. neoformans GRASP ortholog mutant cells. WT cells (A) and the GRASP ortholog mutant (B) were sequentially incubated with C6-NBD-ceramide for Golgi visualization (green) and Uvitex 2B for staining of the cell wall (blue). Scale bar, 3 µm.
Figure 4
Figure 4. Virulence phenotype of the GRASP ortholog mutant
A. Growth rates of WT, mutant and complemented cells. B. The GRASP ortholog mutant exhibited attenuated virulence in an animal model of cryptococcosis. Mice were lethally infected with C. neoformans for daily monitoring of survival. Animals infected with the mutant strain survived significantly longer (P<0.01). C. Association of FITC-labeled C. neoformans cells with murine phagocytes. The similarity in the fluorescence levels of macrophages after infection with non-opsonized WT and reconstituted cells is indicative of similar indices of association between fungal and host cells. Higher fluorescence levels were observed for the mutant, suggesting increased phagocytosis. D. Survival of cryptococci after interaction with the phagocytes. The GRASP ortholog mutant was significantly more susceptible to killing by macrophages than WT (*) and complemented (**) cells (P<0.01).
Figure 5
Figure 5. Absence of GRASP does not affect pigmentation (A–B) or urease activity (C) in cryptococcal cultures
A. Pigmentation of C. neoformans cells after growth in the presence of L-DOPA. Pigmented pellets of WT (a), mutant (b) and reconstituted cells (c) are shown. B. Release of pigment-like molecules into C. neoformans cultures. C. Urease activity was detected (pink color) in cultures of WT, mutant and reconstituted cells, but not in cultures of a urease deletion mutant of C. neoformans (ure1Δ, yellow).
Figure 6
Figure 6. GRASP is required for normal GXM secretion and capsule assembly
A. India ink counterstaining of C. neoformans cells. Yeast strains are indicated on the top of each panel. B. Reactivity of C. neoformans cells with calcofluor white (blue fluorescence) and a monoclonal antibody raised against GXM (green fluorescence). Scale bars in A and B represent 20 and 10 µm, respectively. C. Determination of capsule size of the C. neoformans cells illustrated in A and B. GXM determination in culture supernatants (D) and infected brains (E) are shown, indicating that C. neoformans GRASP ortholog mutant shows a reduced content of extracellular GXM. Statistical analysis of the results shown in C, D and E indicates that values obtained for the GRASP ortholog mutant are significantly smaller than those found for WT and complemented systems (P<0.05 in all cases).
Figure 7
Figure 7. Effective diameter determination of GXM obtained from cultures of WT, mutant, and reconstituted cells
The upper panel shows diameter determination under regular conditions of GXM analysis. The lower panel illustrates diameter determination after incubation of polysaccharide fractions with 1 mM CaCl2.

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