Identification, biochemical characterization, and in-vivo expression of the intracellular invertase BfrA from the pathogenic parasite Leishmania major

Highlights

• A sucrose-metabolizing invertase from the protozoan pathogen Leishmania major was identified.

• RT-PCR experiments demonstrated that this enzyme was expressed in other Leishmania species.

• The expression is dependent on the parasite life stage (overexpressed in promastigotes).

• The cloned and expressed recombinant enzyme has a low affinity for sucrose yet comparable to other invertases.

• These results, coupled to molecular modeling and sequence analysis, suggest that this invertase is an intracellular enzyme.

Abstract

The parasitic life cycle of Leishmania includes an extracellular promastigote stage that occurs in the gut of the insect vector. During that period, the sucrose metabolism and more specifically the first glycosidase of this pathway are essential for growth and survival of the parasite. We investigated the expression of the invertase BfrA in the promastigote and amastigote stages of three parasite species representative of the three various clinical forms and of various geographical areas, namely Leishmania major, L. donovani and L. braziliensis. Thereafter, we cloned, overexpressed and biochemically characterized this invertase BfrA from L. major, heterologously expressed in both Escherichia coli and L. tarentolae. For all species, expression levels of BfrA mRNA were correlated to the time of the culture and the parasitic stage (promastigotes > amastigotes). BfrA exhibited no activity when expressed as a glycoprotein in L. tarentolae but proved to be an invertase when not glycosylated, yet owing low sequence homology with other invertases from the same family. Our data suggest that BfrA is an original invertase that is located inside the parasite. It is expressed in both parasitic stages, though to a higher extent in promastigotes. This work provides new insight into the parasite sucrose metabolism.

Introduction

Leishmaniasis belongs to the group of neglected tropical diseases, as defined by the World Health Organization (WHO), which includes diseases that are endemic in developing countries.¹ The main foci of leishmaniasis are India, Africa, South America, Mediterranean basin and Middle East.2, 3 The microorganism responsible for this widespread zoonosis is a kinetoplastid protozoan parasite of the Leishmania genus, transmitted through inoculation by female sandflies. About twenty Leishmania species can infect humans, and are not only responsible for various clinical forms (cutaneous leishmaniasis, mucocutaneous leishmaniasis or visceral leishmaniasis) and outcomes, depending mainly on the Leishmania species, but also on the immune background of the patient. Whereas cutaneous leishmaniasis is usually a self-healing disease, mucocutaneous leishmaniasis is a more severe non-healing disease, and visceral leishmaniasis is usually fatal in the absence of treatment.⁴

The stages of the parasite life cycle are correlated to the infection stages in the hosts.5, 6, 7 In the vector salivary glands, the parasites are found as extracellular promastigotes. After inoculation by the insect bite, parasites are phagocytosed by mammalian macrophages and differentiate to their amastigote intracellular stage. The adaptation of Leishmania to two different environments is thus highly critical for its survival and growth. In sandflies, the parasite load was shown to be correlated with the feeding with plants containing high levels of sucrose,8, 9, 10, 11 which indicates a critical role for the enzymes involved in sucrose transport and metabolism for Leishmania survival and growth in insect gut. Several pathways have been described for sucrose metabolism,¹² involving either an extracellular13, 14 or an intracellular¹⁵ invertase. Recently, a report from Lyda et al.¹⁶ identified and characterized a secreted invertase from the leishmania species donovani and mexicana, highlighting the potentiality of these enzymes as a therapeutic target to disrupt the parasite metabolism.

Invertases (EC 3.2.1.26), or β-fructofuranosidases, are glycosides hydrolases (GHs) that catalyze the hydrolysis of sucrose into fructose and glucose (Fig. 1) by the recognition of β-d-fructose in the enzyme active site. Invertases belong to the CAZy family GH32 that contains invertases, fructan hydrolases, fructosyltransferases and sucrose-6-phosphate hydrolases.¹⁷ Several X-ray structures of GH32 family from different organisms have been reported in the literature, including bacteria,18, 19, 20, 21 fungi,22, 23 yeast24, 25, 26 and plants.27, 28, 29, 30, 31, 32, 33, 34 Five putative genes encoding invertases have been identified in Leishmania genomes,35, 36, 37, 38 but only one protein was cloned and enzymatically characterized as an extracellular invertase.¹⁶ Unlike Leishmania, no invertase is present in human genome, but another GH is responsible for sucrose hydrolysis, i.e. sucrase or α-d-glucosidase, which interacts with the α-d-glucosyl moiety of sucrose (Fig. 1).

The importance of Leishmania invertases in the survival and growth of the parasite in the insect vector, as well as their absence in humans, make them potential target for the design of anti-Leishmania active compounds.¹⁶ Analyzing their enzymatic behavior, as well as their expression level during the parasite life cycle, is thus necessary to better understand their biological role. Herein, we describe the cloning, purification and biochemical characterization of the first intracellular Leishmania invertase, named BfrA. Noteworthy, this particular enzyme is only highly expressed in the promastigote stage of the parasite, thus is probably essential for the parasite sucrose metabolism in the insect vector.