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Angomonas deanei coevolves in a mutualistic relationship with a symbiotic bacterium that divides in synchronicity with other host cell structures. Trypanosomatid mitochondrial DNA is contained in the kinetoplast and is composed of thousands of interlocked DNA circles (kDNA). The arrangement of kDNA is related to the presence of histone-like proteins, known as KAPs (kinetoplast-associated proteins), that neutralize the negatively charged kDNA, thereby affecting the activity of mitochondrial enzymes involved in replication, transcription and repair. In this study, CRISPR-Cas9 was used to delete both alleles of the A. deanei KAP4 gene. Gene-deficient mutants exhibited high compaction of the kDNA network and displayed atypical phenotypes, such as the appearance of a filamentous symbionts, cells containing two nuclei and one kinetoplast, and division blocks. Treatment with cisplatin and UV showed that Δkap4 null mutants were not more sensitive to DNA damage and repair than wild-type cells. Notably, lesions caused by these genotoxic agents in the mitochondrial DNA could be repaired, suggesting that the kDNA in the kinetoplast of trypanosomatids has unique repair mechanisms. Taken together, our data indicate that although KAP4 is not an essential protein, it plays important roles in kDNA arrangement and replication, as well as in the maintenance of symbiosis.

In trypanosomatids, the kinetoplast is the portion of the single mitochondrion that is connected to the basal body and contains the kDNA, a network composed by circular and interlocked DNA. The kDNA packing is conducted by Kinetoplast Associated Proteins (KAPs), which are similar to eukaryotic histone H1. In symbiont-harboring trypanosomatids (SHTs) such as Angomonas deanei and Strigomonas culicis, a ß-proteobacterium co-evolves with the host in a mutualistic relationship. The prokaryote confers nutritional benefits to the host and affects its cell structure. Atomic force microscopy showed that the topology of isolated kDNA networks is quite similar in the two SHT species. Ultrastructural analysis using high-resolution microscopy techniques revealed that the DNA fibrils are more compact in the kinetoplast region that faces the basal body and that the presence of the symbiotic bacterium does not interfere with kDNA topology. However, RT-PCR data revealed differences in the expression of KAPs in wild-type protozoa as compared to aposymbiotic cells. Immunolocalization showed that different KAPs present distinct distributions that are coincident in symbiont-bearing and in symbiont-free cells. Although KAP4 and KAP7 are shared by all trypanosomatid species, the expanded repertoire of KAPs in SHTs can be used as phylogenetic markers to distinguish different genera.