Glucuronoxylomannan, galactoxylomannan, and mannoprotein occupy spatially separate and discrete regions in the capsule of Cryptococcus neoformans

Figures & data

Figure 1 Construction of MP98(CDA2) deletion in strain JEC 34 (serotype D). PC R was used to fuse the promoter region of MP98 (P_MP98) and its terminator (T_MP98) with the ORF of URA5 to generate a cassette for gene replacement, as shown in (A). Primers are designated in lower case (a–h) and their sequences are given in Table 1. Domains of the MP98(CDA2) protein (signal peptide, catalytic and Serine/Threonine-rich) are boxed with “Y” used to identify putative N-glycosylation sites.28 As shown in (B), the strategy to confirm gene replacement for Ura⁺ transformants was by PC R. Genomic DNAs were used as templates with five sets of primers (g–n, Table 1) to generate products (A–E). Shown below the maps is an electrophoretogram comparing PC R products from wild-type control DNA (1) with a JEC34 Ura⁺ transformant (2). Each of the five PC R products of the transformant were indicative of gene replacement and a new genotype (mp98::URA5).

Figure 2 Strain deleted of MP98(CDA2) lacks MP98 protein. Supernatant of a cell homogenate of untransformed strain JEC34 was compared to that of the Ura⁺ transformant with genotype mp98(cda2)::URA5 ascribed by PCR analysis in Figure 1. Unstimulated sample had no protein added. Values are the average of four measurements. This bioassay incorporated the specificity of the T-cell hybridoma P1D6 in recognizing an epitope on the MP98(CDA2) protein; recognition activates IL-2 secretion.

Figure 3 mAb 18F2 is limited to strain JEC21. Immunofluorescence using anti-MP98 MAb 18F2 with serotype D and serotype A, C. neoformans strains. MP98 is located in a punctate pattern in B3501 and near the cell wall in cap67 (top parts). The cda1::URA5 mutant shows an accumulation of MP98 protein in the emerging bud. The negative control, mp98(cda2)::URA5 mutant shows only background fluorescence. Parental strains KN99a and KN99α, mutant cda2Δ and the cda1Δcda2Δcda3Δfpd1Δ quadruple mutant each display only background fluorescence (lower parts). Scale bar is 5 µm.

Figure 4 Capsules from B3501 and KN99α had similar permeability to dextran. A dextran penetration assay was performed using tetramethylrhodamine-labeled dextran of molecular weights 10, 40 and 70 kDa. The penetration of dextrans into the capsule was similar for both strains.

Figure 5 Alignment of MP98 protein sequences encoded by H99, JEC 21 and WM 276 (Serotype B) C. neoformans strains. Sequence highlighted in yellow is for the MP98 protein encoded by the JEC21 allele that was expressed in E. coli and used for generating mAbs. The green areas are putative N-linked glycosylation sites. The red areas are not N-glycosylated in protein encoded by other alleles. The gray areas are possible sites of O-glycosylation differences among protein sequences.

Figure 6 Reconstruction of capsular components. 3D confocal reconstructions from Z-series stacks of B3501 and cap67 cells. Green: GalXM, red: GXM, blue: MP98. Scale bar is 5 µm.

Figure 7 Colocalization studies of MP98 and other mannoproteins using mAb 18F2 and Con A lectin respectively. C. neoformans strain B3501 parts (A–F) and MP98 mutant strain parts (G–I). Left parts are C. neoformans treated with mAb 18F2 (green), middle parts Con A (red) and right parts are the merged images. In B3501 (parts A–F) Con A stains the mannoproteins of cell wall and mannoproteins found on the capsular edge, the mAb 18F2 specifically recognizes and binds to MP98. Parts (A–C) also show that MP98 is mostly localized next to the cell wall during budding. Parts (D–F) shows that 18F2 localizes to bud scars. Parts (G–I) shows that 18F2 is specific for MP98 but that other MPs are available to bind to Con A. Scale bar is 5 µm.

Table 1 Primers to make MP98(CDA2) deletion cassette and confirm gene replacement

	Primer	Sequence1
a2	NFJEC21	tga tgc cga gga ggt tat
b	MP98R	cat tcg taa tcc cgt gcc
c	MP98URA5-F	cgc ctt tta atc aca cag gca ctt gat cAT GTC CTC CC
d	MP98URA5-R	GGG AGG ACA Tga tca agt gcc tgt gtg att aaa agg cg
e	URA5MP98-F	CGG TGT TCA GAG GTC TTA Agt gat gtg ctt tga cga agg
f	URA5MP98-R	cct tcg tca aag cac atc acT TAA GAC CTC TGA ACA CC G
g	NF-1	tta atc aca cag gcg aa
h	NR-5′	tgc ttg agc tgc ttc cat ct
i	NR-5	tcg ttg agc tgc ttc cat ct
j	NF-5	tgg acc ggt gtc aac tac aa
k	MP98-F	tgg cat ctg acc ttg ttg
l	URA-R	CC T CGA TGA AGG CAA CTT
m	URA-F	AGC TGC AGG AGG TCA GGA A
n	MP98-R2	cag tta cca cca gtc aga

1 Lower case designates MP98(CDA2) sequence and upper case URA5 sequence.

2 Primer designations in Figure 1B.

Table 1 Primers to make CDA1 deletion cassette and confirm gene replacement

	Primer	Sequence1
a2	CDA1-F	cac gtt tga ttg cga acg ct
b	CDA1-R	ccg acc taa gac gat cat
c	CDA1URA5-F	cca gaa gcg gaa ttt cgc cct tga tcA TGT CCT CCC
d	CDA1URA5-R	GGG AGG ACA Tga tca agg gcg aaa ttc cgc ttc tgg
e	URA5CDA1-F	CGG TGT TCA GAG GTC TTA Atc taa cga tcg atg ggg t
f	URA5CDA1-R	Acc cca tcg atc gtt aga TTA AGA CC T CTG AAC ACC G
g	CDA1-F4	atc ggt cga cag tag cat
h	CDA1-R5	gga aga aga gtc aga ggt
i	CDA1-TAA	tta ggc gac ata tac cat acc gac cgc a
j	CDA1-F5	ggt gac att gat gac cgt
k	CDA1-F3	Cca ggc ata cgt tca agt
l	URA-R	CC T CGA TGA AGG CAA CTT
m	URA-F	AGC TGC AGG AGG TCA GGA A
n	CDA1-R3	ctg tcc aca gtg cat caa

1 Lower case designates CDA1 sequence and upper case URA5 sequence.

2 Primer locations are the same or similar to those designated in Figure 1B if the CDA1 gene was substituted for MP98(CDA2).

Levitz SM, Nong S, Mansour MK, Huang C, Specht CA. Molecular characterization of a mannoprotein with homology to chitin deacetylases that stimulates T cell responses to Cryptococcus neoformans. Proc Natl Acad Sci USA 2001; 98:10422 - 10427

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