Sialic Acid Expression in the Mosquito Aedes aegypti and Its Possible Role in Dengue Virus-Vector Interactions

Figure 1

Ae.CSAS functional expression evaluation. (a) RT-PCR analysis of Ae.CSAS and Ae.ST. The figure shows the bands obtained with the internal and external primers of each enzyme using a whole extract of Ae. aegypti mosquito. Lanes 1-2: Ae.CSAS (147 bp) and Ae.ST (125 bp) sequences obtained using the internal primers. Lanes 3-4: Ae.CSAS (786 bp) and Ae.ST (1396 bp) complete sequences obtained with the external primers. Lane 5: Ae. actin (298 bp) was used as a housekeeping gene control. (b) RT-PCR analysis of Ae.CSAS and ST using total RNA from five pairs of Ae. aegypti SGs (lane 1) and five midguts (lane 2): Ae.CSAS (147 bp); Ae.ST (125 bp); and actin control (298 bp). (c) cDNA and aa sequences of Ae.CSAS. Identical residues in yellow show multiple alignments with different sequences from other organisms (Figure S1), whereas conserved residues are indicated in blue. (d) Flow cytometry analysis using LEC29.Lec32 untransfected and transfected cells with Ae.CSAS, which were incubated with MAA lectin to evaluate Sia expression. Red: isotype control; black: LEC29.Lec32 cells transfected with empty p3XFlag-CMV vector (negative control); green: untransfected cells in the presence of secondary antibody only; blue: LEC29.Lec32 transfected with Ae.CSS cDNA; and magenta: wild-type CHO cells (positive control for the expression of α-2,3Sia). The bars show the percentage of fluorescence intensity. Approximately 30% of LEC32.Lec29-transfected cells expressed Sia (blue bar) compared with 100% Sia expression in the positive control CHO cells (magenta bar). (e) Affinocytochemistry and confocal microscopy assays using MAA lectin staining to assess Sia expression. Left: LEC29.Lec32-transfected cells with an empty pFlag vector. Center: LEC29.Lec32-transfected cells with the Ae.CSAS pFlag vector. Right: wild-type CHO positive control transfected with an empty pFlag vector.

(a)

(b)

(c)

(d)

(e)