Dietary cholesterol modulates pathogen blocking by Wolbachia

Abstract

The bacterial endosymbiont Wolbachia pipientis protects its hosts from a range of pathogens by limiting their ability to form infections inside the insect. This "pathogen blocking" could be explained by innate immune priming by the symbiont, competition for host-derived resources between pathogens and Wolbachia, or the direct modification of the cell or cellular environment by Wolbachia. Recent comparative work in Drosophila and the mosquito Aedes aegypti has shown that an immune response is not required for pathogen blocking, implying that there must be an additional component to the mechanism. Here we have examined the involvement of cholesterol in pathogen blocking using a system of dietary manipulation in Drosophila melanogaster in combination with challenge by Drosophila C virus (DCV), a common fly pathogen. We observed that flies reared on cholesterol-enriched diets infected with the Wolbachia strains wMelPop and wMelCS exhibited reduced pathogen blocking, with viral-induced mortality occurring 2-5 days earlier than flies reared on Standard diet. This shift toward greater virulence in the presence of cholesterol also corresponded to higher viral copy numbers in the host. Interestingly, an increase in dietary cholesterol did not have an effect on Wolbachia density except in one case, but this did not directly affect the strength of pathogen blocking. Our results indicate that host cholesterol levels are involved with the ability of Wolbachia-infected flies to resist DCV infections, suggesting that cholesterol contributes to the underlying mechanism of pathogen blocking.

Figure 1. Survival curves and total cholesterol levels for Wolbachia -infected Drosophila melanogaster fed cholesterol-enriched food.

Survival curves for wMelPop- (A), wMelCS- (B), and wMel-infected flies (C) reared on cholesterol-enriched diets then challenged with DCV by injection into the haemocoel. Each curve represents one of three experiments for that strain. There was a clear effect of diet on survival, where Wolbachia-infected flies (dashed lines with rhomboid markers) reared on the Intermediate (red lines) and High (blue lines) cholesterol-enriched diets had a shorter average survival time than those reared on Standard food (black lines). Each line depicts the mean survival over time (± s.e.m.) for the three vials from each fly line x diet combination. Data were compared statistically using Cox Regression. A delay in virus-induced mortality was observed in Wolbachia-infected flies compared with uninfected (solid lines with square markers). This pathogen blocking effect occurred for all strains with the weakest blocking occurring for the wMel infection. PBS-injected flies (dotted lines) experienced a high rate of survival with (circles) and without (triangles) Wolbachia, indicating that the death observed in DCV-infected flies was not due to trauma or buffer contamination. Mean levels (± s.e.m.) of total cholesterol and cholesteryl esters for wMelPop- (D), wMelCS- (E), and wMel-infected flies (F) reared on Standard (black bars), Intermediate (red) and High (blue) cholesterol diets. Cholesterol quantification was performed on flies from the same bottles used in the survival assays. Data were compared statistically using univariate ANOVA followed by student's t-tests. Flies reared on high cholesterol diets typically had higher cholesterol levels, and this was generally associated with lower mean survival after challenge with DCV.

Figure 2. DCV levels of Wolbachia -infected flies fed cholesterol-enriched food.

Mean normalised DCV:CycK expression ratios (median ± interquartile range) for wMelPop- (A) and wMelCS-infected flies (B), five days-post DCV infection. Total DCV copies were quantified using qPCR and normalised against CycK expression levels. Data were compared statistically using Mann-Whitney U-tests. Both strains had higher DCV levels after rearing on the Intermediate (Int) and High (High) cholesterol-enriched diets than for Standard diet (Ctrl), this suggests that it is likely that rearing on high cholesterol diets increases the rate of viral accumulation.

Figure 3. Wolbachia levels of flies reared on cholesterol-enriched food.

Mean (± s.e.m.) normalized wsp:rps17 expression ratios for wMelPop- (A) and wMelCS-infected flies (B) during the age window that survival assays were performed. Flies were collected simultaneously with survival experiment two for wMelPop and survival experiment one for wMelCS. Total Wolbachia levels were quantified using qPCR. Data were compared statistically using ANOVA and student's t-tests corrected for multiple comparisons. For wMelPop-infected flies, Wolbachia levels were significantly lower after rearing on the High cholesterol diet, than on the Intermediate (Student's t test: t = 3.370, df = 37, P<0.01) or Standard diets (Student's t test: t = 2.586, df = 36, P<0.05). Interestingly, this difference in density did not have a corresponding difference in survival was between flies from the two cholesterol-enriched diets. There was no effect of diet on Wolbachia levels in wMelCS-infected flies.