The Cif proteins from Wolbachia prophage WO modify sperm genome integrity to establish cytoplasmic incompatibility

R. Kaur, B. A. Leigh, I. T. Ritchie and S. R. Bordenstein,  PLOS Biology,  20:e3001584. 2022.

Inherited microorganisms can selfishly manipulate host reproduction to drive through populations. In Drosophila melanogaster, germline expression of the native Wolbachia prophage WO proteins CifA and CifB cause cytoplasmic incompatibility (CI) in which embryos from infected males and uninfected females suffer catastrophic mitotic defects and lethality; however, in infected females, CifA expression rescues the embryonic lethality and thus imparts a fitness advantage to the maternally transmitted Wolbachia. Despite widespread relevance to sex determination, evolution, and vector control, the mechanisms underlying when and how CI impairs male reproduction remain unknown and a topic of debate. Here, we use cytochemical, microscopic, and transgenic assays in D. melanogasterto demonstrate that CifA and CifB proteins of wMel localize to nuclear DNA throughout the process of spermatogenesis.

Fig 5. CifA, CifB, and the protamine deficiency are transferred with the mature sperm to the female reproductive tract. (A) Schematic representation of Drosophila melanogaster female reproductive system. Mature oocytes leave the OV and reach the UT, where they can be fertilized prior to being laid. Sperms from males are stored in specialized organs—SP and SR shown in the box, which open into the UT for fertilization to occur. Schematic is created with (B) Transgenic cifABexpressing and wMel− males were crossed to wMel− females. Four hours postfertilization, sperms isolated from females were decondensed and immunostained for localizing CifA (green) and CifB (red). DAPI stain (blue) was used to label nuclei. CifA is absent in sperm heads (empty arrowheads) and puctae are seen along the sperm tails (arrows). CifB is present in apical acrosomal tip of all of the sperm heads (solid arrowheads), with more distant signal in the more decondensed sperm nuclei. No Cifs are present in the sperms transferred from wMel− negative control males. (C) Individual sperm intensity quantification shows that protamine deficiency of sperms from wMel+ and transgenic cifAB males persists after transfer in the females compared to wMel− males. Sperm protamine deficiency from transgenic cifAB males also persists in the reproductive tract of wMel+ females. Vertical bars represent mean, and error bars represent standard deviation. Letters indicate statistically significant (p < 0.05) differences as determined by multiple comparisons based on a Kruskal–Wallis test and Dunn multiple test correction. All of the p-values are reported in S1 Table, and rawdata underlying this panel can be found in S1 Data file. (D) Representative images of CMA3-stained mature sperms (arrows) transferred from wMel−, wMel+ and transgenic cifAB males in wMel− and wMel+ female reproductive systems are shown. CMA3, chromomycin A3; OV, ovary; SP, spermathecae; SR, seminal receptacle; UT,uterus.
Image from Kaur et al 2022 10.1038/s41587-022-01325-y

Cif proteins cause abnormal histone retention in elongating spermatids and protamine deficiency in mature sperms that travel to the female reproductive tract with Cif proteins. Notably, protamine gene knockouts enhance wild-type CI. In ovaries, CifA localizes to germ cell nuclei and cytoplasm of early-stage egg chambers; however, Cifs are absent in late-stage oocytes and subsequently in fertilized embryos. Finally, CI and rescue are contingent upon a newly annotated CifA bipartite nuclear localization sequence. Together, our results strongly support the Host modification model of CI in which Cifs initially modify the paternal and maternal gametes to bestow CI-defining embryonic lethality and rescue

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