Bruno Souza Bonifácio

Lysine acetylation plays a crucial role in cellular processes and is found across various evolutionary organisms. Recent advancements in proteomic techniques revealed the presence of acetylation in thousands of non‐histone proteins. Here, we conducted extensive meta‐analysis of 48 acetylomes spanning diverse organisms, including archaea, bacteria, fungi, protozoa, worms, plants, insects, crustacea, fish, and mammals. Our analyzes revealed a predominance of a single acetylation site in a protein detected in all studied organisms, and proteins heavily acetylated, with > 5–10 acetylated‐sites, were represented by Hsp70, histone, or transcription GTP‐biding domain. Moreover, using gene enrichment approaches we found that ATP metabolic processes, glycolysis, aminoacyl‐tRNA synthetase pathways and oxidative stress response are among the most acetylated cellular processes. Finally, to better explore the regulatory function of acetylation in glycolysis and oxidative stress we used aldolase and superoxide dismutase A (SODA) enzymes as model. For aldolase, we found that K147 acetylation, responsible to regulate human enzyme, conserved in all phylogenic clade, suggesting that this acetylation might play the same role in other species; while for SODA, we identified many lysine residues in different species present in the tunnel region, which was demonstrated for human and Trypanosoma cruzi, as negative regulator, also suggesting a conserved regulatory mechanism. In conclusion, this study provides insights into the conservation and functional significance of lysine acetylation in different organisms emphasizing its roles in cellular processes, metabolic pathways, and molecular regulation, shedding light in the extensive function of non‐histone lysine acetylation.

Leishmania presents a complex life cycle that involves both invertebrate and vertebrate hosts. By regulating gene expression, protein synthesis, and metabolism, the parasite can adapt to various environmental conditions. This regulation occurs mainly at the post‐transcriptional level and may involve epitranscriptomic modifications of RNAs. Recent studies have shown that mRNAs in humans undergo a modification known as N4‐acetylcytidine (ac4C) catalyzed by the enzyme N‐acetyltransferase (NAT10), impacting mRNAs stability and translation. Here, we characterized the NAT10 homologue of L. mexicana, finding that the enzyme exhibits all the conserved acetyltransferase domains although failed to functionally complement the Kre33 mutant in Saccharomyces cerevisiae. We also discovered that LmexNAT10 is nuclear, and seems essential, as evidenced by unsuccessful attempts to obtain null mutant parasites. Phenotypic characterization of single‐knockout parasites revealed that LmexNAT10 affects the multiplication of procyclic forms and the promastigote‐amastigote differentiation. Additionally, in vivo infection studies using the invertebrate vector Lutzomyia longipalpis showed a delay in the parasite differentiation into metacyclics. Finally, we observed changes in the cell cycle progression and protein synthesis in the mutant parasites. Together, these results suggest that LmexNAT10 might be important for parasite differentiation, potentially by regulating ac4C levels.

Leishmania presents a complex life cycle that involves both invertebrate and vertebrate hosts. By regulating gene expression, protein synthesis, and metabolism, the parasite can adapt to various environmental conditions. This regulation occurs mainly at the post-transcriptional level and may involve epitranscriptomic modifications of RNAs. Recent studies have shown that mRNAs in humans undergo a modification known as N4-acetylcytidine (ac4C) catalyzed by the enzyme N-acetyltransferase (NAT10), impacting mRNAs stability and translation. Here, we characterized the NAT10 homologue of L. mexicana , finding that the enzyme exhibits all the conserved acetyltransferase domains and although failed to functionally complement the Kre33 mutant in Saccharomyces cerevisia e, has in vitro acetyltransferase activity. We also discovered that LmexNAT10 is nuclear, and seems essential, as evidenced by unsuccessful attempts to obtain null mutant parasites. Phenotypic characterization of single-knockout parasites revealed that LmexNAT10 affects the multiplication of procyclic forms and the promastigote-amastigote differentiation. Additionally, in vivo infection studies using the invertebrate vector Lutzomyia longipalpis showed a delay in the parasite differentiation into metacyclics. Finally, we observed changes in the cell cycle progression and protein synthesis in the mutant parasites. Together, these results suggest that LmexNAT10 might be important for parasite differentiation, potentially by regulating ac4C levels.

Cellular invasion by Trypanosoma cruzi metacyclic trypomastigotes (MTs) or tissue culture trypomastigotes (TCTs) is a complex process involving host–parasite cellular and molecular interactions. Particularly, the involvement of host cell actin cytoskeleton during trypomastigote invasion is poorly investigated, and still, the results are controversial. In the present work, we compare side by side both trypomastigote forms and employ state‐of‐the‐art live‐cell imaging showing for the first time the dynamic mobilization of host cell actin cytoskeleton to MT and TCT invasion sites. Moreover, cytochalasin D, latrunculin B, and jasplakinolide‐pretreated cells inhibited MT and TCT invasion. Furthermore, our results demonstrated that TCT invasion decreased in RhoA, Rac1, and Cdc‐42 GTPase‐depleted cells, whereas MT invasion decreased only in Cdc42‐and RhoA‐depleted cells. Interestingly, depletion of the three studied GTPases induced a scattered lysosomal distribution throughout the cytosol. These observations indicate that GTPase depletion is sufficient to impair parasite invasion despite the importance of lysosome spread in trypomastigote invasion. Together, our results demonstrate that the host cell actin cytoskeleton plays a direct role during TCT and MT invasion.