A cysteine-rich extracellular protein containing a PA14 domain mediates quorum sensing in Dictyostelium discoideum

Abstract

Much remains to be understood about quorum-sensing factors that allow cells to sense their local density. Dictyostelium discoideum is a simple eukaryote that grows as single-celled amoebae and switches to multicellular development when food becomes limited. As the growing cells reach a high density, they begin expressing discoidin genes. The cells secrete an unknown factor, and at high cell densities the concomitant high levels of the factor induce discoidin expression. We report here the enrichment of discoidin-inducing complex (DIC), an approximately 400-kDa protein complex that induces discoidin expression during growth and development. Two proteins in the DIC preparation, DicA1 and DicB, were identified by sequencing proteolytic digests. DicA1 and DicB were expressed in Escherichia coli and tested for their ability to induce discoidin during growth and development. Recombinant DicB was unable to induce discoidin expression, while recombinant DicA1 was able to induce discoidin expression. This suggests that DicA1 is an active component of DIC and indicates that posttranslational modification is dispensable for activity. DicA1 mRNA is expressed in vegetative and developing cells. The mature secreted form of DicA1 has a molecular mass of 80 kDa and has a 24-amino-acid cysteine-rich repeat that is similar to repeats in Dictyostelium proteins, such as the extracellular matrix protein ecmB/PstA, the prespore cell-inducing factor PSI, and the cyclic AMP phosphodiesterase inhibitor PDI. Together, the data suggest that DicA1 is a component of a secreted quorum-sensing signal regulating discoidin gene expression during Dictyostelium growth and development.

FIG. 1.

Profiles of discoidin-inducing activity in different purification steps. (A) Discoidin-inducing activity in fractions from a gel filtration column. (B) Activity of fractions from the anion-exchange column. (C) Activity of eluates of slices of a native polyacrylamide gel. Discoidin expression was determined with luciferase as a reporter using arbitrary units. Active fractions increase discoidin expression over buffer control. Buffer controls (diamonds on the y axis) are indicated for each column.

FIG. 2.

Protein composition of the active fraction on polyacrylamide gels. (A) The enriched activity migrates as one complex on native polyacrylamide gels. (B) N-terminal sequences of the 49-, 53-, and 120-kDa proteins were identical to that of PDE. The 80-kDa fraction was N-terminally blocked. Peptide sequences were obtained after digestion with LysC, and the obtained sequences are indicated in quotation marks. Proteins in panels A and B were detected by silver staining.

FIG. 3.

Recombinant DicA1 induces the expression of discoidin in cells grown and starved at low cell density. (A) Wild-type cells were grown at a low cell density to prevent induction of discoidin and then starved at a low cell density in the indicated concentration of either crude recombinant thioredoxin-DicA1, crude recombinant thioredoxin-DicB, or a crude preparation of the thioredoxin protein (control). After 3 h, cells were fixed and stained for discoidin I by immunofluorescence and the number of positive cells and the total number of cells were counted in three randomly chosen microscope fields containing a total of approximately 150 to 200 cells. (B) A similar induction was done with Gβ⁻ cells (33). For both panels A and B, results are the means ± SEMs from three (A) or four (B) independent experiments. The absence of error bars indicates the error was smaller than the plot symbol. When cells were grown to 1 × 10⁶ cells/ml to induce discoidin expression by endogenous PSF, 79% ± 11% of Ax4 cells and 43% ± 14% of Gβ⁻ cells expressed discoidin.

FIG. 4.

Expression pattern of the DicA1 mRNA. RNAs from cells growing in shaking culture on bacteria at various cell densities (left panel; numbers are densities [10⁶ cells/ml]; S, early stationary phase; ON, stationary overnight) and cells developing for various times on filter pads (right panel, numbers are developmental time in hours) were hybridized to a probe for DicA1 and a discoidin probe as a control. Equal loading was verified by ethidium bromide staining.