The Wolbachia cytoplasmic incompatibility enzyme CidB targets nuclear import and protamine-histone exchange factors

  1. John Frederick Beckmann  Is a corresponding author
  2. Gagan Deep Sharma
  3. Luis Mendez
  4. Hongli Chen
  5. Mark Hochstrasser  Is a corresponding author
  1. Auburn University, United States
  2. Yale University, United States
5 figures, 1 table and 2 additional files


Figure 1 with 1 supplement
Cif toxicity in S. cerevisiae.

(a) Five-fold dilutions of yeasts BY4741 and W303-1A carrying galactose-inducible epitope-tagged Wolbachia genes on pRS416GAL1. Three Cif homologs from Wolbachia strains wPip, wHa, and wStr showed strong to mild toxicity. All three showed increased toxicity in W303-1A compared to BY4741 (three replicates). (b) Toxin-antidote behavior was exhibited by the cidABwHa operon. FLAGCidBwHa exhibited toxicity at 36°C when expressed from pRS416GAL1. Co-expression of cognate partner FLAGCidAwHa from the 2-micron plasmid pRS425GAL1 rescues growth. Non-cognate partners did not rescue. Conversely, expression of FLAGCinAwNo from a bidirectionally incompatible Wolbachia strain wNo, enhanced toxicity of FLAGCidBwHa (four replicates). (c) CifA expression alone was nontoxic (three replicates).

Figure 1—figure supplement 1
Expression Analysis of CI Factors in Yeast.

(a) Western blots of total yeast extracts show that some CidB orthologs are not expressed well (three replicates). However, the weakly toxic truncated FLAGCndBwStr(1–1085) is readily detected. Ponceau S staining indicates relative sample loading. (b) CifA orthologs express well and are detected by anti-FLAG Western blots (three replicates). His6CidAwPip serves as negative control. (c) Serial dilutions comparing effects of recombinant protein tags on CidBwPip. FLAG and 3xFLAG tags weaken toxicity of CidB whereas an HA tag enhances toxicity. His6-CidB phenocopies wildtype (untagged). None of these proteins could be detected, except the 3xFLAG-tag which gave sporadic detection and also weakened the phenotypic penetrance (representative of 4 replicates; pictures taken on day 3). All orthologs were expressed from a Gal1 promoter on the CEN vector pRS416GAL1.

Figure 2 with 2 supplements
Yeast Suppressors of CidB.

(a) Seven library plasmids were high-copy suppressors of CidBwPip toxicity. Red genes suppressed when individually sub-cloned. Library plasmid YGPM25o01 includes URA3 and measures screen efficiency since it is an expected suppressor; Backslashes and brackets denote ORF truncations. (b) Five-fold serial dilutions of yeast (W303-1A) with recovered suppressing library plasmids co-transformed with pRS416GAL1-CidB3xFLAG-wPip. Library plasmid suppression varied. Suppression by YGPM25o01 (URA3 control), YGPM26g16, and YGPM32e11 was strong and consistent (three replicates). Plasmids YGPM12h13, YGPM21f02, YGPM32b05, and YGPM11h18, showed weaker and less consistent suppression across four replicates. (c) Individual yeast genes SRP1, RTT103, and HRP1 suppressed CidBwPip toxicity (three replicates). (d) Immunoblot analysis confirmed that suppressor plasmids do not reduce CidB expression. CidB and suppressors were controlled by GAL1 and endogenous promoters, respectively. Asterisk, an unknown cross-reacting yeast protein. Ponceau S staining indicated relative sample loading.

Figure 2—figure supplement 1
Eliminating false positives from the high-copy His6CidBwPip suppression screen.

Plasmids are named YGPMxxxx. (a) Five-fold serial dilution comparing three false positive suppressor plasmids (pulled directly from the screen; red) to the cognate plasmids (pulled directly from the source library; black) showed suppression only after passage through yeast during the screen. Lack of suppression by the original plasmids suggested the screening procedure could produce false positive artifacts (three replicates). Yeast background is BY4741. (b) Restriction digests comparing a false positive with the originating library plasmid. DNAs were simultaneously cleaved with XhoI, ApaI, and XcmI. Differences in banding patterns indicated the pGP564 plasmid backbone had increased the size of the 3.9 and 0.5 kb fragments (one experiment). (c) Sequencing the plasmid revealed that the URA3 marker in pRS416GAL1-His6CidBwPip had recombined into the LEU2 library plasmid (pGP564). This unique recombination event occurred independently in 13 false-positive library plasmids. The recombination event produces a false positive by allowing yeast to lose the CidB toxin plasmid through equivalent growth support on media lacking uracil by the recombinant URA3 marker in the library plasmid. We culled hits with this recombination from our final list.

Figure 2—figure supplement 2
Five-fold serial dilutions of yeast (BY4741) with recovered suppressing library plasmids co-transformed with pRS416GAL1-His6CidB.

These data include all the originally isolated plasmids that were then re-screened in our analysis. Colored plasmids show results that were not reproducible between experiments.

Analysis of high-copy SRP1 suppression of CidBwPip toxicity in yeast.

(a) Differential impact of mutations affecting distinct Srp1 functions. An srp1 mutation impairing NLS binding (S116F) weakened suppression in W303-1A. E145K, which inhibits cotranslational protein degradation, did not impact suppression (three replicates). (b) Immunoblot analysis showed equivalent protein levels in srp1 mutants. Ponceau S staining demonstrated similar loading (three replicates). (c) The srp1-S116F mutation sensitized W303-1A yeast to FLAGCidBwHa-induced toxicity in 6/7 replicates. Wild-type SRP1 complemented the mutation (5th row). Red * indicates an inactive DUB catalytic mutant control (6th row). Blank columns are empty vectors. (d) High-copy SRP1 did not suppress CinBwPip toxicity in BY4741 yeast (three replicates).

Figure 4 with 3 supplements
Drosophila Interactome Analysis.

(a) Experimental pipeline for defining CidA and CidB interactomes. Soluble lysates from Drosophila adults were passed over columns bound to the indicated recombinant proteins and washed. Remaining proteins were eluted and subjected to in-solution LC-MS/MS analysis. (b) Venn diagram of protein identifications from raw biological triplicate measurements. The His6CidB* interactome was dramatically changed when it was bound to FLAGCidA. The interactome of His6CidA itself was modest and showed no overlap with the Drosophila proteins bound to either CidB* or the CidA-CidB* complex.

Figure 4—figure supplement 1
Triplicate Enrichment Interactome for His6CidB*wPip.

(a) Pie chart showing functional categories of protein hits. (b) 45 triplicate-enrichment hits ranked according to peptide frequency F.

Figure 4—figure supplement 2
Triplicate Enrichment Interactome for the FLAGCidAwPip/His6CidB*wPip complex.

(a) Pie chart showing functional categories of protein hits. (b) 67 triplicate-enrichment hits ranked according to peptide frequency F.

Figure 4—figure supplement 3
Triplicate Enrichment Interactome for His6CidAwPip.

(a) Pie chart showing functional categories of protein hits. (B) 15 triplicate-enrichment hits ranked according to peptide frequency F.

Figure 5 with 1 supplement
Suppression of CI in Drosophila.

(a) Transgenic CI was temperature sensitive. (b) Yeast SRP1 and HRP1 did not suppress CI in Drosophila and serve as negative controls. At 22°C, overexpression of D.m.Kap-α1, S.c.Rtt103, GFP, D.m.Nap1, D.m.Kap-α2, D.m.P32 and CidAwMel suppressed transgenic CI relative to the control. Both D.m.P32 and CidAwMel suppression were still highly significant when compared to the GFP control. (c) CI suppressive effects of karyopherin overexpression were countered by its maternal toxicity. (d) D.m. Karyopherins and D.m.P32 significantly suppressed bacterial (wMel) CI; GFP did not. Error bars represent means ± s.d. *p<0.05, **p<0.01, ****p<0.0001 by ANOVA with multiple comparison between all groups and Tukey’s post-hoc analysis; four outliers (x) removed by ROUT analysis.

Figure 5—figure supplement 1
PCR analysis demonstrates that transgenic flies used in this study are not infected with Wolbachia.

VirD4 is a conserved Wolbachia gene. Amplification of the Drosophila melanogaster Histone three gene served as a positive control. NGT is the Nanos-Gal4-Tubulin driver (three replicates).


Table 1
Final refined interactomes of CidB*wPip, CidB*/CidAwPip, and CidAwPip ranked by F-Score.

Peptide spectral matches (PSM) of the top enriched proteins are reported. PSMs are reported as the average of three biological replicates, each a summation of 2 technical replicates; (six total samples, three biological replicates). Mock is an E. coli negative control without plasmid. P-values were calculated by two sample T-test assuming unequal variances of the replicates. Ubiquitin served as an intrinsic positive control.

a His6CidB* Interactome
ProteinkDaUniProtF-ScoreCidB* PSMMock PSMp-value
Moleskin (Kap-β)119Q9VSD6_DROME0.85367.70.042
b His6CidB* + FLAGCidA Interactome
ProteinkDaUniProtF-ScoreCidB*/A PSMMock PSMp-value
AP-3 subunit beta127Q9W4K1_DROME0.7460190.012
AP-3 subunit delta115AP3D_DROME0.7156.716.30.002
c His6CidA Interactome
ProteinkDaUniProtF-ScoreCidA PSMMock PSMp-value

Additional files

Supplementary file 1

Supplementary data within Microsoft excel spreadsheets.

a. Plasmids recovered from iterations of a CidB high copy suppression screen. b. CidB* interactome raw data c. Raw data for biological triplicate CidB* enriched hits d. CidB* + CidA interactome raw data e. Raw data for biological triplicate CidB* + CidA enriched hits f. CidA interactome raw data g. Raw data for biological triplicate CidA enriched hits h. Fly Crossing Hatch-Rate Data i. PCR primer database j. Construct database.
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  1. John Frederick Beckmann
  2. Gagan Deep Sharma
  3. Luis Mendez
  4. Hongli Chen
  5. Mark Hochstrasser
The Wolbachia cytoplasmic incompatibility enzyme CidB targets nuclear import and protamine-histone exchange factors
eLife 8:e50026.