Ab binding alters gene expression in Cryptococcus neoformans and directly modulates fungal metabolism

Abstract

Abs facilitate humoral immunity via the classical mechanisms of opsonization, complement activation, Ab-dependent cellular cytotoxicity, and toxin/viral neutralization. There is also evidence that some Abs mediate direct antimicrobial effects. For example, Ab binding to the polysaccharide capsule of the human pathogenic fungus Cryptococcus neoformans promotes opsonization but also inhibits polysaccharide release and biofilm formation. To investigate whether Ab binding affects C. neoformans directly, we analyzed fungal gene expression after binding of protective and nonprotective mAbs. The 2 IgM Abs and 1 IgG1 Ab tested each induced different changes in gene expression. The protective IgG1 mAb upregulated genes encoding proteins involved in fatty acid synthesis, the protective IgM mAb downregulated genes encoding proteins required for protein translation, and the nonprotective IgM mAb had modest effects on gene expression. Differences in gene expression correlated with mAb binding to different locations of the capsule. Of the 3 Abs tested, the protective IgG1 mAb bound to C. neoformans closest to the cell wall, produced specific differences in the pattern of phosphorylated proteins, caused changes in lipid metabolism, and resulted in increased susceptibility to the antifungal drug amphotericin B. These results suggest what we believe to be a new mode of action for Ab-mediated immunity and raise the possibility that immunoglobulins mediate cross talk between microbes and hosts through their effects on microbial metabolism.

Figure 1. Categories and numbers of genes changed upon mAb binding to H99.

Pie charts of the categories of genes upregulated or downregulated upon (A) mAb 18B7, (B) 12A1, or (C) 13F1 binding to H99. Numbers indicate the number of genes in each category.

Figure 2. The 3 Abs differ in their location of binding on the C. neoformans capsule.

(A–E) Three-dimensional reconstructions of confocal fluorescence images of mAbs binding H99. (A) mAb 18B7 conjugated to Alexa Fluor 488 (green) binding H99. (B) mAb 12A1 conjugated to Alexa Fluor 488 (green) binding H99. (C) mAb 13F1 conjugated to Alexa Fluor 488 (green) binding H99. (D) mAb 18B7 conjugated to Alexa Fluor 488 (green) and unlabeled mAb 12A1 detected with anti-IgM–TRITC (red) binding H99. (E) mAb 18B7 conjugated to Alexa Fluor 488 (green) and unlabeled mAb 13F1 detected with anti-IgM–TRITC (red) binding H99. In all images, the cell wall is stained with calcofluor white (blue). Scale bar: 5 μm (A–E). (F) Distance in microns between mAb binding and the cell wall (measured from the fluorescence profiles in ImageJ). ANOVA was used to test for differences in the distance to the cell wall.

Figure 3. mAb binding to C. neoformans is associated with changes in protein phosphorylation.

(A) Autoradiogram of phosphorylated proteins in cell lysates with and without mAb. (B) Graph of the total pixels for each band in cell lysates of H99 and mAb 18B7 and H99 and mAb MOPC, normalized to H99 alone. The labeling experiment was done twice. The autoradiogram shown is representative of both experiments. ANOVA was used to test for differences in protein phosphorylation between mAbs 18B7 and MOPC. **P < 0.03.

Figure 4. mAb 12A1 binding can affect the C. neoformans metabolic rate compared with binding of the irrelevant control mAb TEPC, as measured by XTT reduction.

The XTT experiment was done 2 times with 6 wells per concentration per experiment. Data shown is for 1 experiment that is representative of both experiments. ANOVA was used to test for differences in C. neoformans metabolic rate. All mAb/capsule ratios are significantly different except the lowest (P < 0.05).

Figure 5. Growth curves in presence of IgG1 mAb 18B7 and amphotericin B.

C. neoformans cells were grown in the presence of near-saturating concentrations of mAb 18B7, mAb MOPC, or no Ab at all and 0.125 μg/ml amphotericin B (2 times less than in the MIC). The presence of mAb 18B7 led to a delay of approximately 36 hours in the onset of growth.

Figure 6. Neutral lipid composition of C. neoformans in the presence of mAb 18B7, MOPC, or PBS.

(A) Autoradiography of TLC of neutral lipids. H99 cells were grown for 36 hours, and 6 × 10⁷ cells were incubated with 12 mCi ¹⁴C-acetate, together with saturating concentrations of the indicated mAbs for 2 hours at 37°C. Total lipids were extracted and loaded onto a silica TLC plate. Lanes were loaded with equal amounts of total lipids, measured by scintillation counting. The positions of triacylglycerols (TAGS), diacylglycerols (DAGS), fatty acids (FA), sterols esters, and ergosterol are indicated. The left lane shows a standard with 100 nCi of ¹⁴C-palmitic acid (PA). (B) The pixel intensity of the band corresponding to fatty acids was measured and compared with the total pixel intensity of every lane. Mean and SD from 3 replicates are shown. **P < 0.05, ***P < 0.001.