Candida albicans Iff11, a secreted protein required for cell wall structure and virulence

Abstract

The Candida albicans cell wall is the immediate point of contact with the host and is implicated in the host-fungal interaction and virulence. To date, a number of cell wall proteins have been identified and associated with virulence. Analysis of the C. albicans genome has identified the IFF gene family as encoding the largest family of cell wall-related proteins. This family is also conserved in a range of other Candida species. Iff11 differs from other family members in lacking a GPI anchor, and we have demonstrated it to be O glycosylated and secreted in C. albicans. A null mutant lacking IFF11 was hypersensitive to cell wall-damaging agents, suggesting a role in cell wall organization. In a murine model of systemic infection the null mutant was highly attenuated in virulence, and survival-standardized infections suggest it is required to establish an infection. This work provides the first evidence of the importance of this gene family in the host-fungal interaction and virulence.

FIG. 1.

Localization and modification of Iff11. (A) Secreted, cell wall, and cytoplasmic protein extracts were analyzed by Western blotting with an anti-V5 antibody. Fractions were secreted proteins (lanes 1 and 2), cell wall extracts from treatments with β-mercaptoethanol (lanes 3 and 4), mild alkali (lanes 5 and 6), or HF-pyridine (lanes 7 and 8) and soluble proteins (lanes 9 and 10). The extracts were prepared from wild-type NGY152 (lanes 1, 3, 5, 7, and 9) and SBC121 (overexpressing V5-tagged Iff11; lanes 2, 4, 6, 8, and 10). All secreted and cell wall extracts were prepared sequentially from the same set of cells, and extracts were resuspended at a volume to represent the proportion of original culture volume. For soluble protein extracts, 50 μg of protein was analyzed. The faint low-molecular-weight bands in lanes 9 and 10 are nonspecific. (B) Glycosylation status of Iff11. The secreted protein extracts from SBC121 (overexpressing Iff11-V5) were untreated (lanes 1 and 3) or treated with endo H (lane 2) or jack bean mannosidase (lane 4); soluble cytoplasmic extracts were included as a control (lane 5). Note that the low-molecular-weight band in each lane is nonspecific.

FIG. 2.

Sensitivity of the iff11Δ null mutant to cell wall perturbing agents. Sensitivity was tested quantitatively by serial dilution of agents in multiwell plates. Agents to which iff11Δ displayed hypersensitivity (Calcofluor White, Congo red, and SDS) are shown. Strains tested were wild type (▪), iff11Δ (□), reintegrant (▵), iff11Δ-overexpressing Iff11 (⧫) and iff11Δ-overexpressing Iff11-V5 (⋄). Error bars indicate the means ± the standard deviation.

FIG. 3.

Attenuation of virulence in the iff11Δ null mutant. The wild-type (▪), iff11Δ (□), and reintegrant (▵) strains were tested for virulence in a mouse model of systemic infection. Six mice per strain were intravenously infected with 2 × 10⁴ CFU/g (body weight).