Leishmania (Leishmania)amazonensisis an intracellular protozoan parasite responsible for the cutaneous leishmaniasis. The parasite replicates inside mammalian macrophage to establish infection. Host-pathogen interactions result in microRNA-mediated post-transcriptional regulation of host genes involved in inflammatory immune response. We analyzed macrophage miRNA profiles duringL. (L.)amazonensisinfection. The regulation of macrophage miRNA expression by the parasite correlates with/depends on parasite arginase activity during infection.L. (L.)amazonensis (La-WT) presented significant miRNA profile alteration (27%) compared toL. (L.)amazonensisarginase knockout (La-arg⁻) (~40%) in relation to uninfected-macrophages. We observed that 78% of the altered miRNAs were up-regulated inLa-WT infection, while only 32% were up-regulated inLa-arg⁻-infected macrophages. In contrast toLa-WT, the lack ofL. (L.)amazonensisarginase led to the inhibition of miR-294 and miR-721 expression. The expression of miR-294 and miR-721 was recovered to levels similar toLa-WT inLa-arg⁻addback mutant. The inhibition of miR-294/Nos2 and miR721/Nos2interactions increased NOS2 expression and NO production, and reducedL. (L.)amazonensisinfectivity, confirmingNos2as target of these miRNAs. The role of miR-294 and miR-721 in the regulation of NOS2 expression duringLeishmaniareplication in infected macrophages pointing these miRNAs as potential new targets for drug development.

In Leishmania, de novo polyamine synthesis is initiated by the cleavage of L-arginine to urea and L-ornithine by the action of arginase (ARG, E.C. 3.5.3.1). Previous studies in L. major and L. mexicana showed that ARG is essential for in vitro growth in the absence of polyamines and needed for full infectivity in animal infections. The ARG protein is normally found within the parasite glycosome, and here we examined whether this localization is required for survival and infectivity. First, the localization of L. amazonensis ARG in the glycosome was confirmed in both the promastigote and amastigote stages. As in other species, arg⁻ L. amazonensis required putrescine for growth and presented an attenuated infectivity. Restoration of a wild type ARG to the arg⁻ mutant restored ARG expression, growth and infectivity. In contrast, restoration of a cytosol-targeted ARG lacking the glycosomal SKL targeting sequence (argΔSKL) restored growth but failed to restore infectivity. Further study showed that the ARGΔSKL protein was found in the cytosol as expected, but at very low levels. Our results indicate that the proper compartmentalization of L. amazonensis arginase in the glycosome is important for enzyme activity and optimal infectivity. Our conjecture is that parasite arginase participates in a complex equilibrium that defines the fate of L-arginine and that its proper subcellular location may be essential for this physiological orchestration.

Leishmaniases are zoonotic vector-borne diseases caused by protozoan parasites of the genus Leishmania that affect millions of people around the globe. There are various clinical manifestations, ranging from self-healing cutaneous lesions to potentially fatal visceral leishmaniasis, all of which are associated with different Leishmania species. Transmission of these parasites is complex due to the varying ecological relationships between human and/or animal reservoir hosts, parasites, and sand fly vectors. Moreover, vector-borne diseases like leishmaniases are intricately linked to environmental changes and socioeconomic risk factors, advocating the importance of the One Health approach to control these diseases. The development of an accurate, fast, and cost-effective diagnostic tool for leishmaniases is a priority, and the implementation of various control measures such as animal sentinel surveillance systems is needed to better detect, prevent, and respond to the (re-)emergence of leishmaniases.

Leishmania is a protozoan parasite that alternates its life cycle between the sand fly and the mammalian host macrophages, involving several environmental changes. The parasite responds to these changes by promoting a rapid metabolic adaptation through cellular signaling modifications that lead to transcriptional and post-transcriptional gene expression regulation and morphological modifications. Molecular approaches such as gene expression regulation, next-generation sequencing (NGS), microRNA (miRNA) expression profiling, in cell Western blot analyses and enzymatic activity profiling, have been used to characterize the infection of murine BALB/c and C57BL/6 macrophages, as well as the human monocytic cell-lineage THP-1, with Leishmania amazonensis wild type (La-WT) or arginase knockout (La-arg^-). These models are being used to elucidate physiological roles of arginine and polyamines pathways and the importance of arginase for the establishment of the infection. In this review, we will describe the main aspects of Leishmania-host interaction, focusing on the arginine and polyamines pathways and pointing to possible targets to be used for prognosis and/or in the control of the infection. The parasite enzymes, arginase and nitric oxide synthase-like, have essential roles in the parasite survival and in the maintenance of infection. On the other hand, in mammalian macrophages, defense mechanisms are activated inducing alterations in the mRNA, miRNA and enzymatic profiles that lead to the control of infection. Furthermore, the genetic background of both parasite and host are also important to define the fate of infection.

Protozoan parasites of the genus Leishmania cause a large spectrum of clinical manifestations known as Leishmaniases. These diseases are increasingly important public health problems in many countries both within and outside endemic regions. Thus, an accurate differential diagnosis is extremely relevant for understanding epidemiological profiles and for the administration of the best therapeutic protocol. Exploring the High Resolution Melting (HRM) dissociation profiles of two amplicons using real time polymerase chain reaction (real-time PCR) targeting heat-shock protein 70 coding gene (hsp70) revealed differences that allowed the discrimination of genomic DNA samples of eight Leishmania species found in the Americas, including Leishmania (Leishmania) infantum chagasi, L. (L.) amazonensis, L. (L.) mexicana, L. (Viannia) lainsoni, L. (V.) braziliensis, L. (V.) guyanensis, L. (V.) naiffi and L. (V.) shawi, and three species found in Eurasia and Africa, including L. (L.) tropica, L. (L.) donovani and L. (L.) major. In addition, we tested DNA samples obtained from standard promastigote culture, naturally infected phlebotomines, experimentally infected mice and clinical human samples to validate the proposed protocol. HRM analysis of hsp70 amplicons is a fast and robust strategy that allowed for the detection and discrimination of all Leishmania species responsible for the Leishmaniases in Brazil and Eurasia/Africa with high sensitivity and accuracy. This method could detect less than one parasite per reaction, even in the presence of host DNA. The different clinical forms of the Leishmaniases range from cutaneous to visceral infections and are caused by organisms belonging to the genus Leishmania. Controversy over the validity of different molecular methods to correctly identify a species hinders the association of a given species with different clinical forms, complicating the prognosis and the development of suitable treatment protocols. A correct identification leads to a better understanding of the action and consequent development of new drugs and immunological reactions. It also provides important information about the relationship of each species with its hosts (humans, animal reservoirs and sandflies) in different geographical areas and ecological situations, helping to design control strategies. Today, PCR is the most commonly used method for Leishmania identification, but even though several targets have been described, no simple and direct protocol has emerged. In this paper, we coupled hsp70 real-time PCR with the determination of amplicon melting profiles in order to explore polymorphic regions by HRM analysis. This methodology yielded discriminatory melting temperature (Tm) values for Brazilian and Eurasian/African Leishmania species. The protocol has proven to be 100% reliable with both clinical and experimental samples. The major advantage of the presently described method is that it is simple, less expensive, highly sensitive and easily automated.

Leishmaniasis is one of the leading globally neglected diseases, affecting millions of people worldwide. Leishmania infection depends on the ability of insect-transmitted metacyclic promastigotes to invade mammalian hosts, differentiate into amastigotes, and replicate inside macrophages. To counter the hostile oxidative environment inside macrophages, these protozoans contain anti-oxidant systems that include iron-dependent superoxide dismutases (SODs) in mitochondria and glycosomes. Increasing evidence suggests that in addition to this protective role, Leishmania mitochondrial SOD may also initiate H₂O₂-mediated redox signaling that regulates gene expression and metabolic changes associated with differentiation into virulent forms. To investigate this hypothesis, we examined the specific role of SODA, the mitochondrial SOD isoform in Leishmania amazonensis. Our inability to generate L. amazonensis SODA null mutants and the lethal phenotype observed following RNAi-mediated silencing of the Trypanosoma brucei SODA ortholog suggests that SODA is essential for trypanosomatid survival. L. amazonensis metacyclic promastigotes lacking one SODA allele failed to replicate in macrophages and were severely attenuated in their ability to generate cutaneous lesions in mice. Reduced expression of SODA also resulted in mitochondrial oxidative damage and failure of SODA/ΔsodA promastigotes to differentiate into axenic amastigotes. SODA expression above a critical threshold was also required for the development of metacyclic promastigotes, as SODA/ΔsodA cultures were strongly depleted in this infective form and more susceptible to reactive oxygen species (ROS)-induced stress. Collectively, our data suggest that SODA promotes Leishmania virulence by protecting the parasites against mitochondrion-generated oxidative stress and by initiating ROS-mediated signaling mechanisms required for the differentiation of infective forms.