Intracellular Density of Wolbachia Is Mediated by Host Autophagy and the Bacterial Cytoplasmic Incompatibility Gene cifB in a Cell Type-Dependent Manner in Drosophila melanogaster

Expression of Wolbachia cytoplasmic incompatibility genes CifA and CifB modulate wMel density in the hub. Representative confocal z-stacks of hubs with overexpressed Wolbachia Cif genes. Unpaired (Upd) was used to overexpress Cif constructs specifically in the hub and not male germ line and soma in the testis. DNA is cyan, D cadherin (labeling the hub) is yellow, and a fluorescently labeled DNA probe to detect Wolbachia is in magenta. Grayscale insets display the Wolbachia-only channel. (A) Sibling control hubs displayed low relative wMel density. (B) Overexpression of CifB resulted in higher relative wMel density. (C) Overexpression of CifA in the hub resulted in a trend for lower relative wMel density. (D) Overexpression of both CifA and CifB resulted in wMel hub density similar to that of the control. (E) A Kruskal-Wallis test of significance revealed a significant difference in the data set (P < 0.0001). Quantification of relative wMel hub density showed overexpression of CifA results in a nonsignificant trend for lower wMel densities (N^cont = 29, N^CifA = 11, P < 0.0905). Overexpression of CifB results in a significant increase in wMel density compared to that in the control (N^cont = 29, N^CifB = 33, P < 0.00001) and to those with both CifA and CifB overexpression (N^CifB = 33, N^cifA-B = 36, P < 0.0019). Overexpression of both CifA and CifB resulted in no difference from the control (N^cont = 29, N^CifA-B = 36, P = 1.0). P values reported for individual comparisons were from a Kruskal-Wallis post hoc Dunn’s test with Bonferroni correction. Scale bars, 5 µM.

Epistasis analysis of Wolbachia CifB and Atg1 genes reveal Wolbachia effector CifB acts in the autophagy pathway. Representative confocal z-stacks of hubs. Unpaired (Upd) was used to express siRNAs or Cif constructs specifically in the hub and not male germ line and soma in the testis. DNA was not acquired for this experiment; D cadherin labeling the hub is yellow, and a fluorescently conjugated DNA probe to detect Wolbachia is in magenta. Grayscale insets display the Wolbachia-only channel. (A) Control hubs displayed low relative wMel density. (B) Overexpression of CifB resulted in higher relative wMel density. (C) Expression of Atg1 RNAi in the hub resulted in higher relative wMel density. (D) Overexpression of both CifB and Atg1 RNAi resulted in high wMel hub density, similar to that with Atg1 RNAi. (E) A Kruskal-Wallis test of significance revealed a significant difference in the data set (P < 0.0001). Quantification of relative wMel hub density showed overexpression of CifB results in a statistically significant increase in wMel density (N^cont = 20, N^CifB = 22, P < 0.0001). Expression of Atg1 RNAi result in a significant increase in wMel density (N^cont = 29, N^Atg1RNAi = 35, P < 0.0001). Coexpression of CifB and Atg1 RNAi results in a significant increase in wMel density compared to that in the control (N^Cont = 29, N^{CifB-Atg1RNAi} = 33, P < 0.0001) and with CifB alone (N^{CifB-Atg1RNAi} = 33, N^CifB = 22, P < 0.0075) and a density similar to that with Atg1 RNAi expression alone (N^Atg1RNAi = 35, N^{CifB-Atg1RNAi} = 33, P = 1.0). P values reported for individual comparisons were from a Kruskal-Wallis post hoc Dunn’s test with Bonferroni correction. Scale bars, 5 µM.

Knockdown of autophagy in the female germ line decreases wMel density. Stage-specific confocal analysis reveals a decrease in relative Wolbachia density upon knockdown of autophagy genes during multiple stages of development. NGT;nos was used to knockdown Atg1, and MTD was used to knockdown Atg8a. DNA is colored cyan, D cadherin (labeling the follicle cells) is yellow, and a fluorescently conjugated DNA probe detecting Wolbachia is in magenta. Grayscale images display the Wolbachia-only channel. (A) Representative confocal z-stack images of control and Atg1 knockdown in the germ line of wMel-infected flies. (B) Quantification of wMel-infected stage-specific egg chambers upon Atg1 knockdown. Statistically significant P values are reported only. (C) Representative confocal z-stack images of wMel-infected stage-8 control egg chambers show high levels of germ line Wolbachia. (D) Representative confocal z-stack image of stage-8 egg chambers with Atg8 RNAi expression displays reduced wMel density in the germ line. (E) Quantification of wMel-infected stage-8 egg chambers for the control and Atg8 knockdown. Scale bars, 10 µM for stages 2 and 3, 20 µM for stages 4 to 6, and 40 µM for stages 7 and 8. Student’s t tests were conducted to determine significance.

Knockdown of autophagy in the female germ line decreases wMelCS density. Stage-specific confocal analysis reveals a decrease in relative Wolbachia density upon knockdown of autophagy genes during multiple stages of development. NGT;nos was used to knockdown Atg1, and MTD was used to knockdown Atg8a. DNA is colored cyan, D cadherin (labeling the follicle cells) is yellow, and a fluorescently labeled DNA probe detecting Wolbachia is in magenta. Grayscale images display the Wolbachia-only channel. (A) Representative confocal z-stack images of control and Atg1 knockdown in the germ line of wMelCS-infected flies. (B) Quantification of wMelCS-infected stage-specific egg chambers upon Atg1 knockdown. Statistically significant P values are reported only. (C) Representative confocal z-stack images of wMelCS-infected stage-8 egg chambers for control flies display a high Wolbachia density. (D) Representative confocal z-stack images of wMelCS-infected stage-8 egg chambers with Atg8 RNAi expressed display reduced germ line Wolbachia density. (E) Quantification of wMelCS-infected stage-8 egg chambers for the control and Atg8 knockdown reveal decreased density upon germ line expression of Atg8 RNAi. Scale bars, 10 µM for stages 2 and 3, 20 µM for stages 4 to 6, and 40 µM for stages 7 and 8. Student’s t tests were conducted to determine significance.

Ref(2)p does not regulate Wolbachia density in stage-8 egg chambers. (A) Representative confocal z-stack image of wMel-infected control stage-8 egg chamber shows moderate density of Wolbachia. NGT;nos was used to drive knockdown of Ref(2)p. DNA is colored cyan, D cadherin (labeling the follicle cells) is yellow, and a fluorescently labeled DNA probe to detect Wolbachia is in magenta. Grayscale images display the Wolbachia-only channel. (B) Representative confocal z-stack image of wMel-infected stage-8 egg chamber with Ref(2)p knocked down shows similar moderate density to that of the control. (C) Quantification of relative germ line Wolbachia density reveals no difference in density upon the expression of Ref(2)p RNAi. (D) Representative confocal z-stack image of wMelCS-infected control stage-8 egg chamber shows high germ line density. (E) Representative confocal z-stack image of wMelCS-infected stage-8 egg chamber with Ref(2)p knocked down displays similar germ line density to that of the control. (F) Scale bars, 40 µM. P values determined by Student’s t test.

Differentially regulated metabolic pathways in Atg1 RNAi mutant ovaries of wMel-infected flies in the context of a metabolic model. All significantly differentiated pathways are reported between wMel-infected ovaries with autophagy knocked down (Atg1 RNAi) and the wild type. Downregulated metabolic pathways are highlighted with red, while upregulated pathways are green. Results are reported in the context of a proposed model of how they interact and could affect Wolbachia. NES, normalized enrichment score from fGSEA MetaboAnalyst results.