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BACKGROUND: The American cutaneous leishmaniasis (ACL) is expanding in peri-urban environments. METHODS: An entomological survey was conducted in the area of the occurrence of an autochthonous urban case of ACL. Sandflies and a parasitological slide of the human case were submitted for molecular diagnosis. RESULTS: Nyssomyia whitmani and Ny. antunesi were the most frequently collected species. Ny. whitmani and Bichromomyia flaviscutellata were positive for Leishmania guyanensis and L. lainsoni, respectively. The human case tested positive for L. lainsoni. CONCLUSIONS: Sandflies and Leishmania parasites present in urban forest may occur frequently in nearby domiciliary environments; thus, these areas must be monitored.

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Protozoan parasites of the genus Leishmania are responsible for leishmaniases, one of the most important anthropozoonotic diseases affecting millions of people worldwide. To date, there are no approved vaccines against leishmaniases for humans. At present, available treatment options lack specificity, which may lead to drug resistance and often cause adverse effects. Genomic analysis of Leishmania spp. revealed that most of the annotated genes encode hypothetical proteins, yet the functions of those proteins are still unknown. Characterization of these proteins is, hence, of utmost importance for the discovery of new therapeutic targets against leishmaniases. Reporter gene systems, or reporters, are powerful tools that enable the detection and measurement of targeted gene expression when introduced to a biological system. Over the years, numerous expression systems containing various reporters have been employed in characterizing several novel genes essential for parasite development. Such systems can be used to predict the subcellular localization of targeted proteins, screen antileishmanial drugs, and monitor the progression of infection within the vector and vertebrate hosts, among other uses. Therefore, it is critical to comprehend the available reporter gene expression systems to choose the most suitable for each study.

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Multiple genes and proteins have been identified as differentially expressed in the stages of the Leishmania life cycle. The differentiation processes are implicated in specific transcriptional and proteomic adjustments driven by gene expression regulation mechanisms. Leishmania parasites lack gene-specific transcriptional control, and gene expression regulation mostly depends on posttranscriptional mechanisms. Due to the lack of transcriptional regulation, criticism regarding the relevance of transcript quantification as a possible and efficient prediction of protein levels is recurrent in studies that use transcriptomic information. The advent of high-throughput technologies has improved the analysis of genomes, transcriptomes and proteomes for different organisms under several conditions. Nevertheless, defining the correlation between transcriptional and proteomic profiles requires arduous and expensive work and remains a challenge in Leishmania. In this review, we analyze transcriptomic and proteomic data for several Leishmania species in two different stages of the parasite life cycle: metacyclogenesis and amastigogenesis (amastigote differentiation). We found a correlation between mRNA and protein levels of 60.9% and 69.8% for metacyclogenesis and amastigogenesis, respectively; showing that majority mRNA and protein levels increase or decrease concomitantly. Among the analyzed genes that did not present correlation indicate that transcriptomic data should be carefully interpreted as protein expression. We also discuss possible explanations and mechanisms involved for this lack of correlation.

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[This corrects the article DOI: 10.3389/fcimb.2022.826039.].

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Visceral leishmaniasis caused by Leishmania (Leishmania) infantum in Latin America progress with hepatosplenomegaly, pancytopenia, hypergammaglobulinemia, and weight loss and maybe lethal mainly in untreated cases. miRNAs are important regulators of immune and inflammatory gene expression, but their mechanisms of action and their relationship to pathogenesis in leishmaniasis are not well understood. In the present study, we sought to quantify changes in miRNAs associated with immune and inflammatory pathways using the L. (L.) infantum promastigote infected- human monocytic THP-1 cell model and plasma from patients with visceral leishmaniasis. We identified differentially expressed miRNAs in infected THP-1 cells compared with non-infected cells using qPCR arrays. These miRNAs were submitted to in silico analysis, revealing targets within functional pathways associated with TGF-ß, chemokines, glucose metabolism, inflammation, apoptosis, and cell signaling. In parallel, we identified differentially expressed miRNAs in active visceral leishmaniasis patient plasma compared with endemic healthy controls. In silico analysis of these data indicated different predicted targets within the TGF-ß, TLR4, IGF-I, chemokine, and HIF1α pathways. Only a small number of miRNAs were commonly identified in these two datasets, notably with miR-548d-3p being up-regulated in both conditions. To evaluate the potential biological role of miR-548d-3p, we transiently transfected a miR-548d-3p inhibitor into L. (L.) infantum infected-THP-1 cells, finding that inhibition of miR-548d-3p enhanced parasite growth, likely mediated through reduced levels of MCP-1/CCL2 and nitric oxide production. Further work will be required to determine how miR-548d-3p plays a role in vivo and whether it serves as a potential biomarker of progressive leishmaniasis.

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MicroRNAs are small non-coding RNAs that regulate cellular processes by the post-transcriptional regulation of gene expression, including immune responses. The shift in the miRNA profiling of murine macrophages infected with Leishmania amazonensis can change inflammatory response and metabolism. L-arginine availability and its conversion into nitric oxide by nitric oxide synthase 2 (Nos2) or ornithine (a polyamine precursor) by arginase 1/2 regulate macrophage microbicidal activity. This work aimed to evaluate the function of miR-294, miR-301b, and miR-410 during early C57BL/6 bone marrow-derived macrophage infection with L. amazonensis. We observed an upregulation of miR-294 and miR-410 at 4 h of infection, but the levels of miR-301b were not modified. This profile was not observed in LPS-stimulated macrophages. We also observed decreased levels of those miRNAs target genes during infection, such as Cationic amino acid transporters 1 (Cat1/Slc7a1), Cat2/Slc7a22 and Nos2; genes were upregulated in LPS stimuli. The functional inhibition of miR-294 led to the upregulation of Cat2 and Tnfa and the dysregulation of Nos2, while miR-410 increased Cat1 levels. miR-294 inhibition reduced the number of amastigotes per infected macrophage, showing a reduction in the parasite growth inside the macrophage. These data identified miR-294 and miR-410 biomarkers for a potential regulator in the inflammatory profiles of microphages mediated by L. amazonensis infection. This research provides novel insights into immune dysfunction contributing to infection outcomes and suggests the use of the antagomiRs/inhibitors of miR-294 and miR-410 as new therapeutic strategies to modulate inflammation and to decrease parasitism.

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ABSTRACT Background: The American cutaneous leishmaniasis (ACL) is expanding in peri-urban environments. Methods: An entomological survey was conducted in the area of the occurrence of an autochthonous urban case of ACL. Sandflies and a parasitological slide of the human case were submitted for molecular diagnosis. Results: Nyssomyia whitmani and Ny. antunesi were the most frequently collected species. Ny. whitmani and Bichromomyia flaviscutellata were positive for Leishmania guyanensis and L. lainsoni, respectively. The human case tested positive for L. lainsoni. Conclusions: Sandflies and Leishmania parasites present in urban forest may occur frequently in nearby domiciliary environments; thus, these areas must be monitored.

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American Tegumentary Leishmaniasis (ATL) is an endemic disease in Latin America, mainly caused in Brazil by Leishmania (Viannia) braziliensis. Clinical manifestations vary from mild, localized cutaneous leishmaniasis (CL) to aggressive mucosal disease. The host immune response strongly determines the outcome of infection and pattern of disease. However, the pathogenesis of ATL is not well understood, and host microRNAs (miRNAs) may have a role in this context. In the present study, miRNAs were quantified using qPCR arrays in human monocytic THP-1 cells infected in vitro with L. (V.) braziliensis promastigotes and in plasma from patients with ATL, focusing on inflammatory response-specific miRNAs. Patients with active or self-healed cutaneous leishmaniasis patients, with confirmed parasitological or immunological diagnosis, were compared with healthy controls. Computational target prediction of significantly-altered miRNAs from in vitro L. (V.) braziliensis-infected THP-1 cells revealed predicted targets involved in diverse pathways, including chemokine signaling, inflammatory, cellular proliferation, and tissue repair processes. In plasma, we observed distinct miRNA expression in patients with self-healed and active lesions compared with healthy controls. Some miRNAs dysregulated during THP-1 in vitro infection were also found in plasma from self-healed patients, including miR-548d-3p, which was upregulated in infected THP-1 cells and in plasma from self-healed patients. As miR-548d-3p was predicted to target the chemokine pathway and inflammation is a central to the pathogenesis of ATL, we evaluated the effect of transient transfection of a miR-548d-3p inhibitor on L. (V.) braziliensis infected-THP-1 cells. Inhibition of miR-548d-3p reduced parasite growth early after infection and increased production of MCP1/CCL2, RANTES/CCL5, and IP10/CXCL10. In plasma of self-healed patients, MCP1/CCL2, RANTES/CCL5, and IL-8/CXCL8 concentrations were significantly decreased and MIG/CXCL9 and IP-10/CXCL10 increased compared to patients with active disease. These data suggest that by modulating miRNAs, L. (V.) braziliensis may interfere with chemokine production and hence the inflammatory processes underpinning lesion resolution. Our data suggest miR-548d-3p could be further evaluated as a prognostic marker for ATL and/or as a host-directed therapeutic target.

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Seven isolates from patients with American cutaneous leishmaniasis in the Amazon region of Brazil were phenotypically suggestive of Leishmania (Viannia) guyanensis/L. (V.) shawi hybrids. In this work, two molecular targets were employed to check the hybrid identity of the putative hybrids. Heat shock protein 70 (hsp70) gene sequences were analyzed by three different polymerase chain reaction (PCR) approaches, and two different patterns of inherited hsp70 alleles were found. Three isolates presented heterozygous L. (V.) guyanensis/L. (V.) shawi patterns, and four presented homozygous hsp70 patterns involving only L. (V.) shawi alleles. The amplicon sequences confirmed the RFLP patterns. The high-resolution melting method detected variant heterozygous and homozygous profiles. Single-nucleotide polymorphism genotyping/cleaved amplified polymorphic site analysis suggested a higher contribution from L. (V.) guyanensis in hsp70 heterozygous hybrids. Additionally, PCR-RFLP analysis targeting the enzyme mannose phosphate isomerase (mpi) gene indicated heterozygous and homozygous cleavage patterns for L. (V.) shawi and L. (V.) guyanensis, corroborating the hsp70 findings. In this communication, we present molecular findings based on partial informative regions of the coding sequences of hsp70 and mpi as markers confirming that some of the parasite strains from the Brazilian Amazon region are indeed hybrids between L. (V.) guyanensis and L. (V.) shawi.

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In this study, we measured aluminum (Al) bioconcentration in the brain, ovaries, and liver of Oreochromis niloticus females, and analyzed the effects of exposure to Al and acidic pH on the gene expression of follicle-stimulating hormone (ßfsh) and luteinizing hormone (ßlh) in these animals. Mature females were divided into 4 groups, thus being maintained for 96 h in one of the following conditions: control at neutral pH (Ctr); Al at neutral pH (Al); acidic pH (Ac), and Al at acidic pH (Al-Ac). pH alone did not influence Al bioconcentration in the brain. The animals from the Al-Ac group bioconcentrated more Al in the ovaries than those from the Al group, while no differences were observed in the liver. Aluminum bioconcentration was higher in the brain than in the liver and ovaries in Al-exposed animals (Al and Al-Ac), and higher in the brain than in the ovaries in the Ctr and Ac groups. The liver bioconcentrates more Al than the ovaries in the females from the Ctr and Ac groups. Aluminum and/or acidic pH did not alter ßfsh gene expression, while ßlh gene expression decreased in females from the Al group. Aluminum acted as an endocrine disruptor, suggesting deleterious effects in reproduction that could result in ovulation failure. Aluminum can act directly and/or indirectly in the pituitary, affecting ovarian steroidogenesis and altering the reproductive endocrine axis of mature O. niloticus females in an acute period of exposure.

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