Proteomic profiling of a robust Wolbachia infection in an Aedes albopictus mosquito cell line

Abstract

SummaryWolbachia pipientis, a widespread vertically transmitted intracellular bacterium, provides a tool for insect control through manipulation of host-microbe interactions. We report proteomic characterization of wStr, a Wolbachia strain associated with a strong cytoplasmic incompatibility phenotype in its native host, Laodelphax striatellus. In the Aedes albopictus C/wStr1 mosquito cell line, wStr maintains a robust, persistent infection. MS/MS analyses of gel bands revealed a protein 'footprint' dominated by Wolbachia-encoded chaperones, stress response and cell membrane proteins, including the surface antigen WspA, a peptidoglycan-associated lipoprotein and a 73 kDa outer membrane protein. Functional classifications and estimated abundance levels of 790 identified proteins suggested that expression, stabilization and secretion of proteins predominate over bacterial genome replication and cell division. High relative abundances of cysteine desulphurase, serine/glycine hydroxymethyl transferase, and components of the α-ketoglutarate dehydrogenase complex in conjunction with above average abundances of glutamate dehydrogenase and proline utilization protein A support Wolbachia genome-based predictions for amino acid metabolism as a primary energy source. wStr expresses 15 Vir proteins of a Type IV secretion system and its transcriptional regulator. Proteomic characterization of a robust insect-associated Wolbachia strain provides baseline information that will inform further development of in vitro protocols for Wolbachia manipulation.

Reliance of Wolbachia on High Rates of Host Proteolysis Revealed by a Genome-Wide RNAi Screen of Drosophila Cells

“…Our results are in accord with proteomic and genomic studies indicating that Wolbachia in Aedes and B. malayi must rely on host-derived amino acids (Foster et al 2005;Baldridge et al 2014). In addition, studies demonstrate that amino acids may be limiting because Wolbachia competes with its host for amino acids (Caragata et al, 2014).…”

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

“…Aedes albopictus C7–10 (control) and C/ w Str1 (Wolbachia-infected) cells were maintained in Eagle’s minimal medium supplemented with glucose, non-essential amino acids, glutamine, vitamins, penicillin/streptomycin and 5% fetal bovine serum at 28°C in a 5% CO 2 atmosphere as described previously [25, 27, 30]. For proteomic analysis, unsynchronized populations of exponentially growing cells from six 25-cm 2 flasks were recovered by gentle pipetting, followed by centrifugation of pooled cells to remove medium.…”

“…Biological replicates included cells from passages (p) 9, 16 and 35; data from all samples were combined for the final analysis. Cells were washed with serum-free medium, and subcellular fractions were prepared by sonication, filtration, centrifugation and sucrose gradient fractionation as detailed previously [27] and summarized at left in Fig. 1.…”

“…The C/ w Str1 cell line maintains a robust infection with the CI-inducing Wolbachia strain w Str [24, 25], which replicates in individual vacuoles surrounded by a host-derived membrane [26]. Among 790 Wolbachia proteins, we established a molecular ‘footprint’ of w Str infection dominated by chaperones, stress response and Wolbachia surface proteins [27]. …”

Effects of mimosine on Wolbachia in mosquito cells: cell cycle suppression reduces bacterial abundance

“…Aside from potential effects on tyrosine pools, mimosine interferes with folate metabolism by inhibiting serine hydroxymethyltransferase (Oppenheim et al . 2000), a Wolbachia enzyme with relatively high expression in C/ w Str1 cells (Baldridge et al . 2014).…”