Wolbachia strain wAu differs in cellular perturbation and virus inhibition profiles from previously characterised Wolbachia strains

Some strains of the inherited bacterium Wolbachia have been shown to be effective at reducing the transmission of dengue and other positive-sense RNA viruses by Aedes aegypti in both laboratory and field settings and are being deployed for dengue control. The degree of virus inhibition varies between Wolbachia strains; density and tissue tropism can contribute to these differences but there are also indications that this is not the only factor involved: for example, strains wAu and wAlbA are maintained at similar densities but only wAu produces strong dengue inhibition. We previously reported perturbations in lipid transport dynamics, including sequestration of cholesterol in lipid droplets, with strains wMel / wMelPop in Ae. aegypti. Here we show that strain wAu does not produce the same cholesterol sequestration phenotype despite displaying strong virus inhibition and moreover, in contrast to wMel, wAu antiviral activity was not rescued by cyclodextrin treatment. To further investigate the cellular basis underlying these differences, proteomic analysis of midguts was carried out on Ae. aegypti lines and revealed that wAu-carrying midguts showed a distinct proteome when compared to Wolbachia-free, wMel- or wAlbA-carrying midguts, in particular with respect to lipid transport and metabolism. The data suggest a possible role for perturbed RNA processing pathways in wAu virus inhibition. Together these results indicate that wAu shows unique features in its inhibition of arboviruses compared to previously characterized Wolbachia strains. Author Summary Wolbachia endosymbionts can block transmission of dengue virus by Aedes aegypti mosquitoes, and Wolbachia release programs for dengue control are now being undertaken in several countries. Understanding the mechanisms of Wolbachia-mediated antiviral activity is important for maximizing the efficacy of this control approach. Using functional and proteomic analyses, this study indicates that different strains of Wolbachia perturb cellular functions in diverse ways and display different antiviral profiles. These differences raise the possibility that Wolbachia strain switching could be used to counteract viral escape mutations, should they arise and threaten the efficacy of dengue control programmes.


51
The maternally inherited intracellular symbiotic bacteria Wolbachia are common in insects and can 52 spread through insect populations by inducing cytoplasmic incompatibility (CI), a sperm modification 53 that results in a pattern of crossing sterility that gives Wolbachia-carrying females a relative fitness   An intervention trial using wAlbB in Malaysia showed 40-80% reduction in dengue incidence over 78 multiple release sites (18). With the continued field deployment of Wolbachia it is increasingly 79 important to understand the molecular mechanisms underlying Wolbachia-mediated antiviral activity.

80
Knowledge of the viral inhibition mechanisms will allow more informed monitoring and mitigation of 81 potential operational problems, such as the possibility of viral resistance mutations that confer 82 resistance to or the instability of particular strains of the symbiont in given environments. When Ae. 83 aegypti larvae are reared at temperatures above ~35°C the density and maternal transmission of wMel 84 is lowered -potentially compromising its capacity to inhibit dengue in hot conditions and elevating the 85 risk of selection of escape mutations (8,20-24). If Wolbachia strains can be identified for use in release 86 programs that have mechanistic differences to wMel / wAlbB in their viral inhibition, this would be 87 highly valuable for long-term success of the strategy, in providing a means to either reduce the risk 88 of selection of viral escape mutations, and / or allow a means of mitigation against viral escape should 89 it occur.

90
In light of the unusually efficient viral inhibition conferred by strain wAu, which does not seem to be a 91 consequence solely of its relatively high intracellular density (8,16), we sought to examine whether any 92 differences could be identified relative to other Wolbachia strains in terms of the viruses inhibited or

111
The density of Wolbachia strains such as wAu and wMel has been shown to correlate with the ability 112 to block viruses (15,16). However, the strain wAlbA is found in similar densities and with similar tissue 113 tropism to wAu, but shows relatively low anti-viral activity (8,16). Therefore, these strains can be 114 compared to wildtype (wt) mosquitoes to determine mechanisms related to antiviral activity 115 independent of density. In order to gain further insight into the mechanisms of wAu antiviral activity, a 116 proteomic analysis was carried out on age-matched female midguts of wAu, wAlbA and wt Ae. aegypti.

117
Midguts were chosen as previous proteomic analysis had shown results obtained from these tissues 118 were robust for the study of Wolbachia/viral interactions (17). In total, 3821 proteins were detected, of 119 which 27 were identified as Wolbachia proteins, which were subsequently excluded from the KEGG 120 pathway analysis. From the total proteins identified, 3379 were quantified in all sample groups and 121 were therefore used for differential expression analysis.     (Table S1), where wAu is consistently different to wMel.

142
Upregulation of proteins involved in the protein unfolding response and increases in ER stress proteins 143 were observed in the presence of wMel; however, the opposite was observed for wAu (Table S1)

148
Since there is a large dynamic response to Wolbachia, a global analysis was undertaken using

187
Of key interest, given the differing levels of inhibition between the wAu and wAlbA lines, were KEGG

286
Interestingly wMel also appears to have an effect on gene splicing (47) ; therefore as wAu perturbs the 287 spliceosome it would be interesting to look at transcript profiles in wAu and wMel-carrying mosquitoes.

288
The clear evidence for differences between strains of Wolbachia in proteomic changes that are likely to

307
hand-held microinjector, with a pulled glass capillary. 48hr after injection the mosquitoes were blood 308 fed using a Hemotek artificial blood-feeding system (Hemotek, UK) using defibrinated sheep blood 309 (TCS Biosciences, UK). Mosquitoes were allowed to recover for 72hr before midguts were dissected 310 and stained as described below.

311
In Aa23 (Aedes albopictus) cells which had been cleared of Wolbachia, wMel and wAu strains were 312 introduced from Drosophila simulans STCP lines (50) as follows: Aa23 cells were plated the day 313 before in a 96-well plate. For each Wolbachia strain to be transferred, around 200 mated Drosophila 314 flies were placed in a BugDorm rearing cage (W17.5 x D17.5 x H17.5 cm) with a Petri dish containing 315 grape agar (3% agar, 1% sucrose, 25% grape juice, water) and a spot of yeast paste in the centre to 316 stimulate egg-laying. After one hour, around 500 Drosophila eggs were collected from the agar plate 317 with a brush and rinsed in sterile water. Eggs were further dechorionated and surface-sterilized in 2.5% bleach for 2 min, 70% ethanol for 5 min twice and were rinsed in sterile water three times. Sterilized 319 eggs were transferred to a 1.5 mL Eppendorf tube, resuspended in PBS and homogenized with a sterile 320 pestle. The egg homogenate was centrifuged at 2,500 g for 10 min at 4°C to remove cellular debris and 321 the supernatant was filtered through a 5 μm and a 2.7 μm Millex syringe filters. The filtered homogenate 322 was finally centrifuged at 18,500 g for 5 min at 4°C to pellet the bacteria. The bacterial pellet was 323 resuspended in 100 µl Schneider's media with 10% FBS and overlaid onto the aa23 cells. Finally, the 324 cell plate was centrifuged at 2,500g for 1h at 15°C. In the following days, fully confluent cells were 325 serially passaged from the 96-well plate, to 48, 24 and 12-well plates. Cells were later maintained in 25 326 cm 3 flasks with Schneider's media with 10% FBS at 28℃. Cells were checked regularly for Wolbachia 327 density using quantitative PCR as described previously (8).

405
Acquisitions were arranged by Xcalibur to inject ions for all available parallelisable time.

407
TMT data peak lists were converted from centroided .raw to .mgf format using Mascot Distiller (version 408 2.6.1, Matrix Science) and MS3 spectra were concatenated into their parent MS2 spectra for database

415
Proteome Software) and a second search run against the same database using X!Tandem was run.

416
Protein identifications were filtered to require a maximum protein and peptide FDR of 1% with a

596
(c) Cellprofiler was used to calculate the number of Topfluor spots per cell in 3 independent replicates for each 597 treatment (d) Cells were treated for 48hr with either PBS or 2HPCD and then infected with Zika at an MOI of 1.

598
72hr post infection total RNA was isolated. Data is normalised to the mosquito gene RPS17. Each bar 599 corresponds to 3 independent experiments with 2 biological replicates per experiment (* P< 0.05 show 600 significant differences in comparisons between the PBS control and each 2HPCD concentration for a given