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Background: In the Mediterranean basin, three Leishmania species have been identified: L. infantum, L. major and L. tropica, causing zoonotic visceral leishmaniasis (VL), zoonotic cutaneous leishmaniasis (CL) and anthroponotic CL, respectively. Despite animal models and genomic/transcriptomic studies provided important insights, the pathogenic determinants modulating the development of VL and CL are still poorly understood. This work aimed to identify host transcriptional signatures shared by cells infected with L. infantum, L. major, and L. tropica, as well as specific transcriptional signatures elicited by parasites causing VL (i.e., L. infantum) and parasites involved in CL (i.e., L. major, L. tropica). Methodology/Principal findings: U937 cells differentiated into macrophage-like cells were infected with L. infantum, L. major and L. tropica for 24h and 48h, and total RNA was extracted. RNA sequencing, performed on an Illumina NovaSeq 6000 platform, was used to evaluate the transcriptional signatures of infected cells with respect to non-infected cells at both time points. The EdgeR package was used to identify differentially expressed genes (fold change > 2 and FDR-adjusted p-values < 0.05). Then, functional enrichment analysis was employed to identify the enriched ontology terms in which these genes are involved. At 24h post-infection, a common signature of 463 dysregulated genes shared among all infection conditions was recognized, while at 48h post-infection the common signature was reduced to 120 genes. Aside from a common transcriptional response, we evidenced different upregulated functional pathways characterizing L. infantum-infected cells, such as VEGFA-VEGFR2 and NFE2L2-related pathways, indicating vascular remodeling and reduction of oxidative stress as potentially important factors for visceralization. Conclusions: The identification of pathways elicited by parasites causing VL or CL could lead to new therapeutic strategies for leishmaniasis, combining the canonical anti-leishmania compounds with host-directed therapy. Author summary: Leishmaniasis is a zoonosis caused by the intracellular parasite of the genus Leishmania. In the Mediterranean region, the human disease can be present mainly in two forms: cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL), which can be fatal if untreated. The clinical presentation of the disease depends on the parasite species and host characteristics. However, the molecular mechanisms underlying the development of the visceral form of leishmaniasis are not yet fully understood. In this study, we have determined the gene expression profile in cells infected by three Leishmania species (L. infantum, L. major, L. tropica) responsible for different forms of the disease. The results have allowed us to identify an expression profile (and the related pathways) shared by cells infected by all Leishmania species. Aside this common response, we also identified a transcriptional response distinguishing cells infected by L. infantum, L. major or L. tropica, as well as the relevant pathways in which these genes participate. Therefore, our results contribute to the knowledge of the host cell mechanisms that are activated along infection with different Leishmania species causing different forms of the disease and, therefore, may help develop new host-directed therapy for Leishmaniasis. [ABSTRACT FROM AUTHOR]

Background: Leishmaniasis is one of the most important vector-borne diseases and it represents a serious world health problem affecting millions of people. High levels of Leishmania infections, affecting both humans and animals, are recognized among Italian regions. Among these, Sicily has one of the highest prevalence of Leishmania infection. Methodology/Principal Findings: Seventy-eight Leishmania strains isolated from human and animal samples across Sicily, were analyzed for the polymorphic k26-gene and genotypes were assigned according to the size of the PCR products. A multilocus microsatellite typing (MLMT) approach based on the analysis of 11 independent loci was used to investigate populations structure and genetic diversity of the isolated strains. Six L. infantum reference strains were included in the analysis for comparison. Bayesian clustering analysis of microsatellite data showed that all the isolated strains clustered in two genetically distinct populations, corresponding to human and canine isolates respectively. A further subdivision was observed between the two main groups, giving a good correlation between human strains and their geographic origin, conversely canine population showed a great genetic variability diffused in the territory. Conclusions/Significance: Among the 78 Leishmania isolates, K26 analysis detected 71 samples (91%) as MON-1 zymodeme, confirming it as the predominant strain in Mediterranean area and 7 human samples (9%) as non-MON-1. MLMT gives important insights into the epidemiology of leishmaniases and allows characterization of different strains to a higher resolution than possible with zymodeme typing. Two main populations presented a strong correlation respect to the different hosts, exhibiting a co-circulation of two distinct populations of L. infantum. The population found in infected humans exhibited a correlation with geographic origin. These clusters could represent a geographically restricted population of strains with the same or related genotypes. This study can contribute to an understanding of Leishmania epidemiology, including the spread of reservoirs and sand fly vectors in the different foci of infection, characterizing parasites within the different hosts. Author summary: High levels of Leishmania spp. infections affecting both humans and animals are recognized among Italian regions; in particular, Sicily is an endemic area for Leishmania infantum. In this study 78 Sicilian L. infantum strains isolated from humans and dogs were assessed to investigate their biodiversity by genetic tools. Results were compared with 6 L. infantum reference strains included in the analysis. The evaluation of K26 genetic markers identified 91% of samples as belonging to the MON-1 zymodeme, confirming it as the predominant strain in the Mediterranean area and 9% of the samples–all isolated from humans–as non-MON-1. Multilocus microsatellite typing has proven to be a powerful tool to discriminate strains showing all the isolated strains clustered in two genetically distinct populations, corresponding to human and canine isolates, respectively. A further subdivision was observed between the two main groups, giving in the human population a correlation between microsatellite profile and geographical origin. Our results demonstrate that genetic tools are able to discriminate Leishmania strains and to give useful insights into the epidemiology of leishmaniasis, raising questions on the role of dogs as main reservoirs for human leishmaniasis in the Sicily region. [ABSTRACT FROM AUTHOR]