Proteomic analysis of a mosquito host cell response to persistent Wolbachia infection

Abstract

Wolbachia pipientis, an obligate intracellular bacterium associated with arthropods and filarial worms, is a target for filarial disease treatment and provides a gene drive agent for insect vector population suppression/replacement. We compared proteomes of Aedes albopictus mosquito C/wStr1 cells persistently infected with Wolbachia strain wStr, relative to uninfected C7-10 control cells. Among approximately 2500 proteins, iTRAQ data identified 815 differentially abundant proteins. As functional classes, energy and central intermediary metabolism proteins were elevated in infected cells, while suppressed proteins with roles in host DNA replication, transcription and translation suggested that Wolbachia suppresses pathways that support host cell growth and proliferation. Vacuolar ATPase subunits were strongly elevated, consistent with high densities of Wolbachia contained individually within vacuoles. Other differential level proteins had roles in ROS neutralization, protein modification/degradation and signaling, including hypothetical proteins whose functions in Wolbachia infection can potentially be manipulated by RNAi interference or transfection. Detection of flavivirus proteins supports further analysis of poorly understood, insect-specific flaviviruses and their potential interactions with Wolbachia, particularly in mosquitoes transinfected with Wolbachia. This study provides a framework for future attempts to manipulate pathways in insect cell lines that favor production of Wolbachia for eventual genetic manipulation, transformation and transinfection of vector species.

Keywords: Aedes albopictus; Flavivirus; Intracellular bacterium; Mosquito cell lines; Transinfection; Wolbachia pipientis.

Fig. 1

Sample preparation for MS/MS analysis. Samples were prepared according to the flow chart at left as detailed earlier by Baldridge et al. [27]. Underlined, bold text designates fractions used in the analyses at right. The shaded rectangle shows samples D (p16), E (p9), F (p16) and G (p16), from which host proteins were identified in a standard analysis, and the shaded oval represents samples H (p35), I, (p16 and p35) and J (p16 and p35) used for iTRAQ analysis; note that “p” designates passage number. Samples for MS data sets D and E were separated by SDS-PAGE and recovered from gel slices that were trypsin-digested in situ for protein identification by LTQ MS/MS. Data sets F and G were derived from the Wolbachia-enriched gradient fraction GF-50/60 digested in-solution with trypsin for HPLC separation of peptides and protein identification by Orbitrap MS/MS [27]. For the iTRAQ analysis, aliquots of total, cytoplasmic or mitochondrial fractions (pooled GF-30/40 and GF-40/50) from C7–10 and C/wStr1 cells were labeled with isobaric tags in three 4-plex reactions for protein identification by Orbitrap MS/MS.

Fig. 2

Representative transmission electron micrographs of C/wStr1 cells at near confluency. A) Host cell containing coccoid and elongate (arrowheads) wStr whose profiles range from ≈ 0.4–1.1 microns. Some inclusions appear to contain Wolbachia undergoing degradation (arrow). N, nucleus. B) Wolbachia in vertical and horizontal cross-section. Note the separation between the host-derived vacuolar membrane (arrowheads) and the bacterial cell wall, electron-lucent periplasmic space and inner periplasmic membrane (arrow).

Fig. 3

Distribution of identified Aedes host proteins by functional class. Individual protein identifications (8,313) in standard data sets D and E (see Table 1 and Fig. 1) and iTRAQ data sets H, I and J (3,177 identifications) were grouped into functional classes and normalized to numbers in the largest class, Protein modification/chaperones, defined as 100%. Functional classes were sorted left to right based on decreasing percentages in iTRAQ (gray bars). Diamond symbols indicate functional classes in which the ratio of C/wStr1 (black bars) relative to C7–10 (white bars) ranged from 0.5–0.9 (white diamonds) or 1.2–1.7 (black diamonds). Note that over 30% of proteins in the small Inorganic ion metabolism class (Table S1, sheet 1) have roles in ion transport and have been merged with the larger transporters class.

Fig. 4

Distribution of Aedes host proteins with differential iTRAQ ratios by functional class. Average ratios by functional class were calculated from 1,307 observations (representing 815 consensus proteins listed in Table S4). Ratios are depicted in decreasing order on a linear scale, which was converted to log scale for statistical analyses described in the text. Diamonds indicate mean ratios (listed in Table 4), filled boxes represent first through third quartiles, and bars indicate minimum and maximum values. A small number of proteins with differential ratios in the inorganic ion and extracellular matrix class were included in the transporter and cytoskeleton/cell membrane classes, respectively.