**1. Introduction**

*Leishmania donovani* is a protozoan parasite that causes the lethal visceral leishmaniasis, also known as *Kala azar*. It is a vector-borne pathogen, transmitted by female sandflies of the genus *Phlebotomus*, in particular *P. argentipes*. *Leishmania* exists in two main developmental stages. Promastigotes, elongated flagellates, proliferate rapidly in the sandfly gut. When transmitted to humans, the parasites are phagocytized by antigen-presenting cells and once inside the phagosomes, convert into ovoid, aflagellated amastigotes as which they may persist in the host for months or years.

The leishmaniae differ from their human host and from most other eukaryotes by their lack of gene-specific transcription regulation [1–3], relying on modulated RNA stability [4], inducible translation [5] and reversible gene amplification [6,7] instead.

In addition, *Leishmania* spp. have a full complement of protein kinases [8] and phosphatases [9] to modulate protein activity via phosphorylation and dephosphorylation. Heat shock proteins are important substrates for life cycle stage-dependent phosphorylation [10], but protein kinases also affect parasite morphology, infectivity and viability [8,11–13]. Methylation, acetylation and glycosylation of proteins, i.e., modifications of amino acid side chains, have also been described for *Leishmania* [14,15].

Another type of post-translational protein modifications (PTMs), the conjugation of modifying polypeptides to target proteins is not as well researched in *Leishmania*, but known to exist, e.g., the conjugation of a mitochondrial associated ubiquitin fold modifier (UFM) [16,17]. Conjugation of

another modifier, small ubiquitin-like modifier (SUMO) was studied in *Trypanosoma* spp: SUMOylation of proteins was described for *Trypanosoma cruzi* [18] and *T. brucei* [19,20], where this PTM is involved in surface antigen expression and nuclear organization. A putative ortholog of *SUMO* is present in the *L. donovani* genome and expressed [5,21].

For SUMOylation to happen, the SUMO precursor must first undergo a proteolytic cleavage by a sentrin-specific protease (SENP), which removes the C-terminal amino acids and leaves an exposed, reactive, C-terminal di-glycine group [22,23]. A putative SENP ortholog is also encoded in the *L. donovani* genome and expressed [5,24]. In humans, the di-glycine is further activated by the E1 protein, transesterified to the E2 SUMO-conjugating enzyme and finally transferred to the substrate protein by the E3 SUMO ligase. DeSUMOylation is also facilitated by SENP [25], establishing SENP as a pivotal enzyme to control the SUMOylation state of substrate proteins.

SUMOylation of proteins may have different consequences and result in (i) interference with binding of partner proteins, (ii) additional interaction sites for other proteins, or (iii) SUMO-induced conformational changes of the modified protein [23]. SUMOylation may interfere or promote other PTMs, such as phosphorylation [26] or ubiquitination [27,28]. The SUMOylation status of proteins is highly dynamic, dependent on cell cycle phases, differentiation and stress exposure [23]. Incorrect or excessive SUMOylation is also associated with severe disease, such as cardiovascular or neurological dysfunctions, but also cancer [28]. It is therefore conceivable that in an organism such as *Leishmania*, which is highly dependent on post-transcriptional gene expression regulation, SUMOylation of proteins may play an important role in its adaption to vectors and hosts.

Here, we describe a reverse genetic analysis of SUMO and SENP in *L. donovani*. We test the SUMO-specific proteolytic activity of SENP *in vivo* and examine its impact on vitality and intracellular survival.