Genes that encodes NAGT , MIF 1 and MIF 2 are not virulence factors for kala-azar caused by Leishmania infantum

Introduction: Kala-azar is a disease resulting from infection by Leishmania donovani and Leishmania infantum. Most patients with the disease exhibit prolonged fever, wasting, anemia and hepatosplenomegaly without complications. However, some patients develop severe disease with hemorrhagic manifestations, bacterial infections, jaundice, and edema dyspnea, among other symptoms, followed by death. Among the parasite molecules that might infl uence the disease severity are the macrophage migration inhibitory factor-like proteins (MIF1 and MIF2) and N-acetylglucosamine-1-phosphotransferase (NAGT), which act in the fi rst step of protein N-glycosylation. This study aimed to determine whether MIF1, MIF2 and NAGT are virulence factors for severe kala-azar. Methods: To determine the parasite genotype in kala-azar patients from Northeastern Brazil, we sequenced the NAGT genes of L. infantum from 68 patients as well as the MIF1 and MIF2 genes from 76 different subjects with diverse clinical manifestations. After polymerase chain reaction (PCR), the fragments were sequenced, followed by polymorphism identifi cation. Results: The nucleotide sequencing of the 144 amplicons revealed the absence of genetic variability of the NAGT, MIF1 and MIF2 genes between the isolates. The conservation of these genes suggests that the clinical variability of kala-azar does not depend upon these genes. Additionally, this conservation suggests that these genes may be critical for parasite survival. Conclusions: NAGT, MIF1 and MIF2 do not alter the severity of kala-azar. NAGT, MIF1 and MIF2 are highly conserved among different isolates of identical species and exhibit potential for use in phylogenetic inferences or molecular diagnosis.

Leishmaniasis is a group of diseases that compromise skin, mucous membranes and visceral organs.Leishmaniasis diseases are zoonotic, widely distributed globally and are closely associated with poverty 2 .As such, leishmaniasis is an important public health problem 1 .The annual incidence is estimated to be approximately two million people, and approximately 350 million people are at risk of contracting the disease 2,3 .Among the types of leishmaniasis, kala-azar is well known and is considered by the World Health Organization (WHO) to be one of the six most signifi cant endemic diseases in the World.
The majority of infected individuals are asymptomatic [4][5][6] ; however, visceral leishmaniasis (VL) exhibits clinical and laboratory presentations that include a prolonged course of fever, pallor, weight loss, hepatosplenomegaly and pancytopenia 7 .With these classic manifestations, clinical presentations including bleeding and bacterial infections are present in the most severe forms of the disease and are associated with mortality 8 .
The identifi cation of genetic variants is critical for the detection of a predisposition to various diseases 9 .High conservation of genes suggests that a small number of speciesspecific genes are relevant to pathogenicity or virulence.Leishmania, the etiological agent of kala-azar, has 34-36 chromosomes.From a total of approximately   genes, followed by L. infantum, with 27 genes specifi c to the species; the least divergent is L. major, with fi ve genes 10 .
The existence of two distinct patient profi les (classic and severe) might be a consequence of several factors such as the infective strength level 11 , host immunosuppression 8,12,13 , and the genetic background of the host 14,15 , or it might be genetically determined by parasitic virulence factors 16 .The identifi cation of genetic changes in the pathogenic target genes is a starting point in the search for virulence factors 17 .The term 'virulence factors' is used to describe proteins that are closely related to the severity of the disease or to genes encoding such peptides 18,19 .Genes such as A2, which causes a virulent phenotype when introduced in L. major 20 , or proteins such as the zinc metalloprotease glycoprotein (GP63) (leishmanolysin) which confer protection against lysis by the host 10 .
The NAGT gene of Leishmania is a single-copy gene with 1,401 base pairs per haploid genome 21 .The translated protein is a transmembrane protein of the endoplasmic reticulum (ER), identifi ed as N-acetylglucosamine-1-phosphotransferase (NAGT), whose enzymatic action is the first step of N-glycosylation 22 .Proteins that are extensively modifi ed posttranslationally, including GP63, can become unstable because of mutations affecting N-glycosylation, probably due to the decreased structural stability of leishmanolysin.This instability decreases the response against proteolytic degradation and causes the strain to be less virulent 23 .
Two genes that encode orthologues of human macrophage migration inhibitory factor (MIF) in the Leishmania genus have been described [24][25][26] .Human MIF is a major mediator of infl ammation.This gene encodes a cytokine involved in cellmediated immunity, immunoregulation, and infl ammation.MIF plays a role in the regulation of macrophage function in host defense through the suppression of the anti-infl ammatory effects of glucocorticoids.The presence of MIF orthologues in other parasites (Eimeria sp., Trichinella sp., Plasmodium sp. and Brugia malay) is related to immune modulation [27][28][29] .Macrophage migration inhibitory factor gene-defi cient mice (MIF -/-) are susceptible to L. major infection and develop signifi cantly larger lesions and greater parasite burdens than do wild type resistant mice (MIF +/+) 30 .Other studies report that recombinant MIF activates murine macrophages to kill L. major by increasing the levels of tumor necrosis factor alpha (TNF-α) and nitric oxide production 31,32 .
The existence of two genes that exhibit signifi cant sequence identity with mammalian MIF in the genome of Leishmania genus suggests a physiological role for macrophage migration inhibitory factor-like protein (MIF1 and MIF2) in the parasite life cycle and the establishment of these orthologues as virulence factors [24][25][26] .To date, the biological activities described for the L. major MIF orthologues include the induction of macrophage migration and an anti-apoptotic activity that could be responsible for the intracellular persistence of Leishmania in macrophages; thus, these MIF orthologues may be related to the clinical outcome 25 .
We investigated these three genes to determine whether the eventual presence of genetic heterogeneity such as single nucleotide polymorphism (SNPs) or indels in N-acetylglucosamine-1-phosphotransferase (NAGT) and in macrophage migration inhibitory factor-like protein (MIF1 and MIF2) of L. infantum could explain the severity of New World kala-azar.

Study design
Two sets of patients participated in the study.For the NAGT study, 35 complication-free kala-azar patients and 33 kala-azar patients with complications (bleeding, opportunistic infections, sepsis or death) were included as shown in Table 1.For the study of the MIF orthologues, 43 L. infantum isolates from other patients with uncomplicated kala-azar and 33 isolates from other patients with complications were analyzed for the presence of genetic polymorphisms (Table 1).The isolates were obtained from patients recruited from a reference hospital in Teresina, Brazil.The patients were referred from endemic areas of L. infantum infection in the neighboring States of Piauí and Maranhão.The clinical and laboratory diagnoses were confi rmed by typical clinical presentations, such as fever, wasting, anemia and hepatosplenomegaly, in addition to reactive serology and the presence of the parasites in the bone marrow.The species identifi cation was performed using monoclonal antibodies.The parasites were cultured in Novy-MacNeal-Nicolle (NNN) media with Schneider's insect medium (Sigma, St. Louis, United States of America) supplemented with fetal calf serum (Cultilab, Campinas, Brazil).The cultures were incubated at 28ºC for fi ve days before DNA extraction.

Phylogenetic analyses
Multiple sequence alignments were performed using Clustal W 34 in Molec ular Evolutionary Genetics Analysis -Mega 5 35 .To calculate the evolutionary distances, phylogenetic trees were constructed by the maximum-likelihood estimation method, and the tree topologies were evaluated using Mega 5 35 .All 68 NAGT sequences were selected, with Leishmania infantum NAGT [GenBank: AF205934], L. infantum NAGT var.For the MIF orthologues, neighbor-joining phylogenetic trees were constructed using the nucleotide and amino acid sequences of MIF1 (HLc1), MIF2 (HLc2), Lin33.2090,Lin33.2100,Lmj1740, Lmj1750, MIF-Lbra and MIF human; the nucleotide and amino acid percentages of identity among the Leishmania MIF orthologues were calculated using LALIGN software 36 .should be investigated 10 .Because of a likely polygenic infl uence on the phenotype of the disease, as suggested by the multiple molecular mechanisms associated with Leishmania survival in vertebrate hosts 38,39 , and the crucial involvement of the host response, additional kala-azar patients and other genes should be observed before a defi nitive conclusion is reached on the role of virulence factors in the pathogenesis of severe kala-azar.This type of information could be critical for drug and vaccine development because the virulence factors might be targeted by specifi c drugs and may be related to the quality and magnitude of the host immune response 40 .
The data support the hypothesis that these genes are conserved in the genome of Leishmania, particularly those belonging to the L. donovani complex; all of the species that we used from this complex were arranged in a single cluster (Figure 2).We hypothesize the importance of NAGT for the visceralization of Leishmania as a predisposing factor for VL.After the genome sequencing of L. major, L. infantum and L. braziliensis, it was observed that only a minority of genes are species-specifi c genes 10,41 .Thus, the difference in the clinical presentation of visceral, cutaneous and mucocutaneous leishmaniasis could not be the result of signifi cant genetic differences throughout the genome.The clinical presentations are likely a consequence of critical point mutations in a few genes or of post-transcriptional or post-translational mechanisms 41 .We could not exclude the possibility that the studied isolates were differed with respect to gene or protein expression levels, which might be related to the different clinical outcomes of kala-azar.
The high degree of conservation of these genes between the isolates and with the published sequence of L. infantum suggests that these genes might be critical for the survival of the parasites in the vertebrate host.This conclusion is drawn from the lack of genetic variability that could impair the MIF orthologue functions involved with parasite survival inside macrophages or the structural stability of leishmanolysin from N-glycosylation, in the case of NAGT.
The NAGT, MIF1 and MIF2 genes have a high sequence similarity to the L. infantum JPCM5 (NCBI) genes used for comparison and are not involved with the severity of kala-azar at the coding level, which suggests a critical role of these genes in the biological cycle of the parasite.Considering their conservation level in identical species, the products of these genes might be used in phylogenetic inferences or, eventually, for diagnostic purposes or second-generation vaccine development.
8,300 genes in each Leishmania species, slightly more than 200 are differentially distributed between the genomes of Leishmania infantum, Leishmania major and Leishmania braziliensis.The most divergent species is L. braziliensis, containing 49 different