Role of Protein Glycosylation in Candida parapsilosis Cell Wall Integrity and Host Interaction

Abstract

Candida parapsilosis is an important, emerging opportunistic fungal pathogen. Highly mannosylated fungal cell wall proteins are initial contact points with host immune systems. In Candida albicans, Och1 is a Golgi α1,6-mannosyltransferase that plays a key role in the elaboration of the N-linked mannan outer chain. Here, we disrupted C. parapsilosis OCH1 to gain insights into the contribution of N-linked mannosylation to cell fitness and to interactions with immune cells. Loss of Och1 in C. parapsilosis resulted in cellular aggregation, failure of morphogenesis, enhanced susceptibility to cell wall perturbing agents and defects in wall composition. We removed the cell wall O-linked mannans by β-elimination, and assessed the relevance of mannans during interaction with human monocytes. Results indicated that O-linked mannans are important for IL-1β stimulation in a dectin-1 and TLR4-dependent pathway; whereas both, N- and O-linked mannans are equally important ligands for TNFα and IL-6 stimulation, but neither is involved in IL-10 production. Furthermore, mice infected with C. parapsilosis och1Δ null mutant cells had significantly lower fungal burdens compared to wild-type (WT)-challenged counterparts. Therefore, our data are the first to demonstrate that C. parapsilosis N- and O-linked mannans have different roles in host interactions than those reported for C. albicans.

Keywords: Candida parapsilosis; cell wall; host-fungus interplay; mannosylation pathway; mannosyltransferase; virulence.

Figure 1

CpOCH1 is the functional ortholog of CaOCH1. The CpOCh1 open reading frame was expressed in the Caoch1Δ null mutant, as described in materials and methods, and the ability of cells to bind Alcian blue, an indirect measurement of the cell wall phosphomannan content, and therefore of mannan length was analyzed (A). In addition, cells were grown in minimal medium with GlcNAc added to induce the N-acetylhexosaminidase activity, broken to obtain a cell homogenate and protein samples were used to perform electrophoretic mobility shift assays of Hex1, a highly N-linked glycosylated N-acetylhexosaminidase (B). ^*P < 0.05. Strains used are: NGY152 (WT), Caoch1Δ (NGY357); Caoch1Δ + CaOCH1 (NGY328), Caoch1Δ + CpOCH1 (HMY163).

Figure 2

Loss of OCH1 affects C. parapsilosis morphological transition. Cells from the Cpoch1Δ null mutant grown on dextrose-Sabouraud plates at 28°C did not show differences in colonial morphology (A), but tended to form aggregates in dextrose-Sabouraud broth (B). Upon growth in RPMI 1640 plates supplemented with 10% fetal bovine serum at 37°C, the null mutant displayed rounded and well-defined colony edges, whereas the control strains generated filamentous projections outside the colony (C). When filamentation was induced using liquid medium (RPMI 1640 added with 10% fetal bovine serum and 37°C), control strains underwent morphological transition, but Cpoch1Δ null mutant was unable to form pseudohyphae (D). The strains used are: CLIB-214 (WT), CPRI (WT^(R)), AP (OCH1/och1Δ), AP-1 (och1Δ/och1Δ), and AP-2 (och1Δ/och1Δ + OCH1).

Figure 3

C. parapsilosis och1Δ null mutant displays enhanced susceptibility to specific cell wall perturbing agents. The wild type (open diamonds), wild-type plus CmLEU2 and CdHIS1 (open squares), OCH1/och1Δ (open circles), och1Δ/och1Δ (closed triangles), and och1Δ + OCH1 (X symbol) cells were incubated, using a micro-dilution method, with different concentrations of either Calcofluor White, Congo Red, Tunicamycin, or SDS, and growth was determined after incubation for 16 h at 30°C. Growth data were normalized as percentage of those generated with the same strains without treatment. The Cpoch1Δ null mutant (closed triangles) exhibited a higher susceptibility to all the perturbing agents tested. Data are means ± SD of three independent experiments performed in duplicates.

Figure 4

Chitin and β1,3-glucan are significantly exposed at the cell surface of the C. parapsilosis och1Δ null mutant. Live or heat-killed (HK) yeast cells were incubated with either fluorescein isothiocyanate-wheat germ agglutinin conjugate (closed bars) or IgG Fc-Dectin-1 chimera (open bars) as described in Materials and Methods, inspected under fluorescence microscopy, and the fluorescence associated to 50 individual cells recorded. The strains used are: CLIB-214 (WT), CPRI (WT^(R)), AP (OCH1/och1Δ), AP-1 (och1Δ/och1Δ), and AP-2 (och1Δ + OCH1). ^*P < 0.05, when compared with live cells from CLIB-214, CPR1, AP, and AP-2 strains.

Figure 5

Loss of proper cell wall mannosylation affects the ability of C. parapsilosis to stimulate cytokine production by human PBMCs. Yeast cells where co-incubated with human PBMCs, the supernatant saved and used to quantify pro- and anti-inflammatory cytokines. Results (means ± SD) where obtained using samples from six donors, each assayed in duplicate wells. The strains used are: CLIB-214 (WT), CPRI (WT^(R)), AP (OCH1/och1Δ), AP-1 (och1Δ/och1Δ), and AP-2 (och1Δ + OCH1). ^*P < 0.05, when compared with live cells; ^†P < 0.05, when compared with untreated cells; ^‡P < 0.05, when compared with cells subjected to the same treatment.

Figure 6

Blocking of dectin-1 and TLR4 affects IL-1β stimulation by C. parapsilosis cells. Human PBMCs were pre-incubated with either laminarin, antibody to TLR4, or irrelevant IgG₁ for 1 h at 37°C, before incubation with yeast cells. After 24 h incubation at 37°C the supernatant were saved and used to quantify IL-1β. Results (means ± SD) where obtained using samples from six donors, each assayed in duplicate wells. The strains used are: CLIB-214 (WT), AP-1 (och1Δ/och1Δ), and AP-2 (och1Δ + OCH1). ^*P < 0.05, when compared to same cell type without treatment. NT, non-treated cells.

Figure 7

The C. parapsilosis och1Δ null mutant has decreased virulence in the mouse model of systemic candidiasis. Wild-type BALB/c mice were infected i.v. with 2 × 10⁷ cells from either C. parapsilosis wild type (CLIB-214), och1Δ (AP-1) or och1Δ + OCH1 (AP-2), and fungal burdens in kidneys, liver, and spleen were determined at 1, 2, or 7 days post-infection and expressed as CFU/g tissue (mean ± SEM). Results are pooled data from 2 separate experiments with a total of 5–8 mice per group. ^**P < 0.01, ^***P < 0.001.

Figure 8

Conceptualization of the stimulation of IL-1β production by C. parapsilosis. (A) In the wild-type cells, N-linked mannans mask both β1,3-glucan and the ligand of TLR4, most likely O-linked mannans, resulting in a relatively limited production of IL-1β. (B) Once the N-linked mannan outer chain is removed, such as in the och1Δ null mutant, the ligands for dectin-1 and TLR4 are exposed enabling the induction of a strong stimulation for IL-1β production.