Fatty Acid Profiles of Leishmania major Derived from Human and Rodent Hosts in Endemic Cutaneous Leishmaniasis Areas of Tunisia and Algeria

Leishmaniasis is a protozoal vector-borne disease that affects both humans and animals. In the Mediterranean Basin, the primary reservoir hosts of Leishmania spp. are mainly rodents and canids. Lipidomic approaches have allowed scientists to establish Leishmania spp. lipid profiles for the identification of cell stage specific biomarkers, drug mechanisms of action, and host immune response. Using an in silico approach of global network interaction between genes involved in fatty acid (FA) synthesis followed by the GC-MS approach, we were able to characterize the fatty acid profiles of L. major derived from human and rodent hosts. Our results revealed that the lipid profile of L. major showed similarities and differences with those already reported for other Leishmania species. Phospholipids are the predominant lipid class. FA composition of rodent parasites was characterized by a lower abundance of the precursor C18:2(n-6). One of the rodent clones, which also expressed the lowest lipid abundance in PL and TAG, was the least sensitive clone to the miltefosine drug and has the lowest infection efficiency. Our findings suggest that the lipid composition variation may explain the response of the parasite toward treatment and their ability to infect their host.

High-resolution melting analysis identifies reservoir hosts of zoonotic Leishmania parasites in Tunisia

Background Leishmaniasis is endemic in Tunisia and presents with different clinical forms, caused by the species Leishmania infantum, Leishmania major, and Leishmania tropica. The life cycle of Leishmania is complex and involves several phlebotomine sand fly vectors and mammalian reservoir hosts. The aim of this work is the development and evaluation of a high-resolution melting PCR (PCR-HRM) tool to detect and identify Leishmania parasites in wild and domestic hosts, constituting confirmed (dogs and Meriones rodents) or potential (hedgehogs) reservoirs in Tunisia. Methods Using in vitro-cultured Leishmania isolates, PCR-HRM reactions were developed targeting the 7SL RNA and HSP70 genes. Animals were captured or sampled in El Kef Governorate, North West Tunisia. DNA was extracted from the liver, spleen, kidney, and heart from hedgehogs (Atelerix algirus) (n = 3) and rodents (Meriones shawi) (n = 7) and from whole blood of dogs (n = 12) that did not present any symptoms of canine leishmaniasis. In total, 52 DNA samples were processed by PCR-HRM using both pairs of primers. Results The results showed melting curves enabling discrimination of the three Leishmania species present in Tunisia, and were further confirmed by Sanger sequencing. Application of PCR-HRM assays on reservoir host samples showed that overall among the examined samples, 45 were positive, while seven were negative, with no Leishmania infection. Meriones shawi were found infected with L. major, while dogs were infected with L. infantum. However, co-infections with L. major/L. infantum species were detected in four Meriones specimens and in all tested hedgehogs. In addition, multiple infections with the three Leishmania species were found in one hedgehog specimen. Sequence analyses of PCR-HRM products corroborated the Leishmania species found in analyzed samples. Conclusions The results of PCR-HRM assays applied to field specimens further support the possibility of hedgehogs as reservoir hosts of Leishmania. In addition, we showed their usefulness in the diagnosis of canine leishmaniasis, specifically in asymptomatic dogs, which will ensure a better evaluation of infection extent, thus improving elaboration of control programs. This PCR-HRM method is a robust and reliable tool for molecular detection and identification of Leishmania and can be easily implemented in epidemiological surveys in endemic regions. Graphical Abstract

Specific Human ATR and ATM Inhibitors Modulate Single Strand DNA Formation in Leishmania major Exposed to Oxidative Agent

Leishmania parasites are the causative agents of a group of neglected tropical diseases known as leishmaniasis. The molecular mechanisms employed by these parasites to adapt to the adverse conditions found in their hosts are not yet completely understood. DNA repair pathways can be used by Leishmania to enable survival in the interior of macrophages, where the parasite is constantly exposed to oxygen reactive species. In higher eukaryotes, DNA repair pathways are coordinated by the central protein kinases ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR). The enzyme Exonuclease-1 (EXO1) plays important roles in DNA replication, repair, and recombination, and it can be regulated by ATM- and ATR-mediated signaling pathways. In this study, the DNA damage response pathways in promastigote forms of L. major were investigated using bioinformatics tools, exposure of lineages to oxidizing agents and radiation damage, treatment of cells with ATM and ATR inhibitors, and flow cytometry analysis. We demonstrated high structural and important residue conservation for the catalytic activity of the putative LmjEXO1. The overexpression of putative LmjEXO1 made L. major cells more susceptible to genotoxic damage, most likely due to the nuclease activity of this enzyme and the occurrence of hyper-resection of DNA strands. These cells could be rescued by the addition of caffeine or a selective ATM inhibitor. In contrast, ATR-specific inhibition made the control cells more susceptible to oxidative damage in an LmjEXO1 overexpression-like manner. We demonstrated that ATR-specific inhibition results in the formation of extended single-stranded DNA, most likely due to EXO1 nucleasic activity. Antagonistically, ATM inhibition prevented single-strand DNA formation, which could explain the survival phenotype of lineages overexpressing LmjEXO1. These results suggest that an ATM homolog in Leishmania could act to promote end resection by putative LmjEXO1, and an ATR homologue could prevent hyper-resection, ensuring adequate repair of the parasite DNA.

Antibody Responses to Phlebotomus papatasi Saliva in American Soldiers With Cutaneous Leishmaniasis Versus Controls

Leishmania major, transmitted in Iraq by the bite of a sand fly Phlebotomus papatasi, causes cutaneous leishmaniasis (CL). The sand fly saliva is immunogenic, with both systemic humoral and cellular human immune responses resulting from natural exposure. 248 Americans who developed L. major infection in Iraq were sex, race/ethnicity, year of Iraq deployment-matched to controls without CL. Using a case-control study design, we compared sand fly saliva-specific human IgG levels and recognized antigens between the two groups. Serologic responses to Ph. papatasi salivary gland homogenate were studied with ELISA and Western blot, using serial samples obtained from before travel, during CL treatment (CL) or at time of return to US (controls), as well as (for CL cases) six to 24 months after return to non-endemic US. The mean change in optical density (MCOD), reflecting the change in sand fly saliva-specific IgG before and after exposure in Iraq, was 0.296 (range -0.138 to 2.057) in cases and 0.151 (range -0.454 to1.085) in controls, p<0.001. Low levels of sand fly saliva specific antibody were noted in CL cases by 7-8 months after return to the US. The most frequently recognized Ph. papatasi salivary antigens were MW30 (PpSP32) and MW64, although other salivary proteins recognized were MW12/14, 15, 18, 28, 32, 36, 42, 44, 46, 52. Logistic regression suggested that MW15, 28 and 42 were associated with the largest effect on the MCOD. MW30 was the most frequently recognized antigen suggesting a role as biomarker for sand fly exposure and CL risk. Anti-Ph. papatasi saliva IgG waned within months of return to the US. We also discuss vector antigenic saliva proteins in the context of CL presentation and identify some salivary antigens that may correlate with less lesion area, ulcer versus papule/plaque, race among those with CL.

Effect of Beauveria bassiana and Metarhizium anisopliae on different stages of Phlebotomus papatasi (Diptera: Psychodidae)

The sand fly, Phlebotomus papatasi (Scopoli, 1786) (Diptera: Psychodidae), is the main vector of Leishmania major Yakimoff and Schokhor, 1914, the causative agent of zoonotic cutaneous leishmaniasis North Africa, the Middle East, and North Sinai. The purpose of this study was to determine the effect of fungi on P. papatasi larvae, pupae, and adults using light microscopic analysis, scanning electron microscope (SEM), and histopathological studies. Specifically, larvae, pupae, and adult P. papatasi were infected with the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae. Scanning electron microscope and histopathological methods were used to investigate the destructive impact of the fungi on the external and internal structures of P. papatasi. The results revealed propagation of the conidia on the cuticles of all P. papatasi life stages, including on the compound eyes, leg setae, thorax, wings, and abdomen of the adults. Histological sections of the control and treated larvae, pupae, and adults showed many alterations and malformations in the body and tissues of all life stages after 72 h. These results demonstrated that B. bassiana was more effective than M. anisopliae as a biological control of phlebotomine sand flies. Further studies to determine the best methods for delivery and application in the diverse ecological settings of the various leishmaniasis vectors are recommended.

The role of Leishmania GP63 in the modulation of innate inflammatory response to Leishmania major infection

Leishmaniasis is a disease caused by the protozoan parasite Leishmania and is known to affect millions of individuals worldwide. In recent years, we have established the critical role played by Leishmania zinc-metalloprotease GP63 in the modulation of host macrophage signalling and functions, favouring its survival and progression within its host. Leishmania major lacking GP63 was reported to cause limited infection in mice, however, it is still unclear how GP63 may influence the innate inflammatory response and parasite survival in an in vivo context. Therefore, we were interested in analyzing the early innate inflammatory events upon Leishmania inoculation within mice and establish whether Leishmania GP63 influences this initial inflammatory response. Experimentally, L. major WT (L. majorWT), L. major GP63 knockout (L. majorKO), or L. major GP63 rescue (L. majorR) were intraperitoneally inoculated in mice and the inflammatory cells recruited were characterized microscopically and by flow cytometry (number and cell type), and their infection determined. Pro-inflammatory markers such as cytokines, chemokines, and extracellular vesicles (EVs, e.g. exosomes) were monitored and proteomic analysis was performed on exosome contents. Data obtained from this study suggest that Leishmania GP63 does not significantly influence the pathogen-induced inflammatory cell recruitment, but rather their activation status and effector function. Concordantly, internalization of promastigotes during early infection could be influenced by GP63 as fewer L. majorKO amastigotes were found within host cells and appear to maintain in host cells over time. Collectively this study provides a clear analysis of innate inflammatory events occurring during L. major infection and further establish the prominent role of the virulence factor GP63 to provide favourable conditions for host cell infection.

Synthetic Perturbations in IL6 Biological Circuit Induces Dynamical Cellular Response

Macrophage phenotype plays a crucial role in the pathogenesis of Leishmanial infection. Pro-inflammatory cytokines signals through the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway that functions in parasite killing. Suppression of cytokine signaling (SOCS) is a well-known negative feedback regulator of the JAK/STAT pathway. However, change in the expression levels of SOCSs in correlation with the establishment of infection is not well understood. IL6 is a pleotropic cytokine that induces SOCS1 and SOCS3 expression through JAK-STAT signaling. Mathematical modeling of the TLR2 and IL6 signaling pathway has established the immune axis of SOCS1 and SOCS3 functioning in macrophage polarization during the early stage of Leishmania major infection. The ratio has been quantified both in silico and in vitro as 3:2 which is required to establish infection during the early stage. Furthermore, phosphorylated STAT1 and STAT3 have been established as an immunological cross talk between TLR2 and IL6 signaling pathways. Using synthetic biology approaches, peptide based immuno-regulatory circuits have been designed to target the activity of SOCS1 which can restore pro-inflammatory cytokine expression during infection. In a nutshell, we explored the potential of synthetic biology to address and rewire the immune response from Th2 to Th1 type during the early stage of leishmanial infection governed by SOCS1/SOCS3 immune axis.