Mechanistic and structural insights into vitamin B2 metabolizing enzyme riboflavin kinase from Leishmania donovani.

Leishmania donovani relies on specific vitamins and cofactors crucial for its survival and pathogenesis. Tailoring therapies to disrupt these pathways offers a promising strategy for the treatment of Visceral Leishmaniasis. Current treatment regimens are limited due to drug resistance and high costs. The dependency of Leishmania parasites on Vitamin B2 and its metabolic products is not known. In this study, we have biochemically and biophysically characterized a Vitamin B2 metabolism enzyme, riboflavin kinase from L. donovani (LdRFK) which converts riboflavin (vitamin B2) into flavin mononucleotide (FMN). Sequence comparison with human counterpart reflects 31.58 % identity only, thus opening up the possibility of exploring it as drug target. The rfk gene was cloned, expressed and the recombinant protein was purified. Kinetic parameters of LdRFK were evaluated with riboflavin and ATP as substrates which showed differential binding affinity when compared with the human RFK enzyme. Thermal and denaturant stability of the enzyme was evaluated. The rfk gene was overexpressed in the parasites and its role in growth and cell cycle was evaluated. In the absence of crystal structure, homology modelling and molecular dynamic simulation studies were performed to predict LdRFK structure. The data shows differences in substrate binding between human and parasite enzyme. This raises the possibility of exploring LdRFK for specific designing of antileishmanial molecules. Gene disruption studies can further validate its candidature as antileishmanial target.

Mechanistic study of inhibitory peptides with SHP-1 in hypertonic environment for infection model.

Cutaneous Leishmaniasis, an infectious disease is globally the most prevalent form of leishmaniasis accounting for approximately 1 million cases every year as per world health organization. Infected individuals develop skin lesion which has been reported to be infiltrated by immune cells and parasite with high sodium accumulation creating hypertonic environment. In our work, we tried to mimic the hypertonic environment in virtual environment to study dynamicity of SHP-1 and NFAT5 along with their interactions through molecular dynamics simulation. We validated the SHP-1 and NFAT5 dynamics in infection and HSD conditions to study the impact of hypertonicity derived NFAT5 mediated response to L.major infection. We also evaluated our therapeutic peptides for their binding to SHP-1 and to form stable complex. Membrane stability with the peptides was analyzed to understand their ability to sustain mammalian membrane. We identified PepA to be a potential candidate to interact with SHP-1. Inhibition of SHP-1 through PepA to discern IL-10 and IL-12 reciprocity may be assessed in future and furnish us with a potential therapeutic molecule. HSD mice exhibited high pro-inflammatory response to L.major infection which resulted in reduced lesion size. Contrary to observations in HSD mice, infection model exhibited low pro-inflammatory response and increased lesion size with high parasite load. Thus, increase in NFAT5 expression and reduced SHP-1 expression may result in disease resolving effect which can be further studied through incorporation of synthetic circuit using PepA to modulate IL-10 and IL-12 reciprocity.

Single-cell ATAC sequencing identifies sleepy macrophages during reciprocity of cytokines in L. major infection.

The hallmark characteristic of macrophages lies in their inherent plasticity, allowing them to adapt to dynamic microenvironments. Leishmania strategically modulates the phenotypic plasticity of macrophages, creating a favorable environment for intracellular survival and persistent infection through regulatory cytokine such as interleukin (IL)-10. Nevertheless, these effector cells can counteract infection by modulating crucial cytokines like IL-12 and key components involved in its production. Using sophisticated tool of single-cell assay for transposase accessible chromatin (ATAC) sequencing, we systematically examined the regulatory axis of IL-10 and IL-12 in a time-dependent manner during Leishmania major infection in macrophages Our analysis revealed the cellular heterogeneity post-infection with the regulators of IL-10 and IL-12, unveiling a reciprocal relationship between these cytokines. Notably, our significant findings highlighted the presence of sleepy macrophages and their pivotal role in mediating reciprocity between IL-10 and IL-12. To summarize, the roles of cytokine expression, transcription factors, cell cycle, and epigenetics of host cell machinery were vital in identification of sleepy macrophages, which is a transient state where transcription factors controlled the epigenetic remodeling and expression of genes involved in pro-inflammatory cytokine expression and recruitment of immune cells.IMPORTANCELeishmaniasis is an endemic affecting 99 countries and territories globally, as outlined in the 2022 World Health Organization report. The disease's severity is compounded by compromised host immune systems, emphasizing the pivotal role of the interplay between parasite and host immune factors in disease regulation. In instances of cutaneous leishmaniasis induced by L. major, macrophages function as sentinel cells. Our findings indicate that the plasticity and phenotype of macrophages can be modulated to express a cytokine profile involving IL-10 and IL-12, mediated by the regulation of transcription factors and their target genes post-L. major infection in macrophages. Employing sophisticated methodologies such as single-cell ATAC sequencing and computational genomics, we have identified a distinctive subset of macrophages termed "sleepy macrophages." These macrophages exhibit downregulated housekeeping genes while expressing a unique set of variable features. This data set constitutes a valuable resource for comprehending the intricate host-parasite interplay during L. major infection.

Synthetic biology for combating leishmaniasis.

Leishmaniasis is a neglected tropical disease caused by protozoan parasites of the Leishmania genus. Despite the efforts to control and treat the disease, it still remains a major public health problem in many countries. Synthetic biology is a rapidly evolving interdisciplinary field that combines biology, engineering, and computer science to design and construct novel biological systems. In recent years, synthetic biology approaches have shown great promise for developing new and effective strategies to combat leishmaniasis. In this perspective, we summarize the recent advances in the use of synthetic biology for the development of vaccines, diagnostic tools, and novel therapeutics for leishmaniasis.

Immuno-metabolic signaling in leishmaniasis: insights gained from mathematical modeling.

Motivation: Leishmaniasis is a global concern especially in underdeveloped and developing subtropical and tropical regions. The extent of infectivity in host is majorly dependent on functional polarization of macrophages. Classically activated M1 macrophage can eliminate parasite through production of iNOS and alternatively activated M2 macrophages can promote parasite growth through by providing shelter and nutrients to parasite. The biological processes involved in immune signaling and metabolism of host and parasite might be responsible for deciding fate of parasite. Results: Using systems biology approach, we constructed two mathematical models and inter-regulatory immune-metabolic networks of M1 and M2 state, through which we identified crucial components that are associated with these phenotypes. We also demonstrated how parasite may modulate M1 phenotype for its growth and proliferation and transition to M2 state. Through our previous findings as well as from recent findings we could identify SHP-1 as a key component in regulating the immune-metabolic characterization of M2 macrophage. By targeting SHP-1 at cellular level, it might be possible to modulate immuno-metabolic mechanism and thereby control parasite survival. Availability and implementation: Mathematical modeling is implemented as a workflow and the models are deposited in BioModel database. FactoMineR is available at: https://github.com/cran/FactoMineR/tree/master.

Decoding systems immunological model of sphingolipids with IL-6/IL-17/IL-23 axes in L. major infection.

IL-6, IL-17, IL-23 and IL-1ß are the crucial cytokines controlling inflammatory and immune response during L. major infection. During cutaneous leishmaniasis, an important T helper cell type CD4+ Th17 subset plays a deterministic role in lesion formation through channelling infected macrophages and production of IL-1ß, IL-6, IL-23 and IFN-γ. Ceramide derived sphingosine precursors may assist in pro-inflammatory cytokine response. However, the role of these metabolites in inflammation with pleiotropic pro-inflammatory cytokines in L. major infection is unknown. The present study indicates IL-6/IL-17/IL-23 and SPHK1-S1P-S1PRs signaling axes with the overexpression of SATB1 aiding in disease progression. Targeting SATB1 might modulate the secretion of pro-inflammatory cytokines and abnormal immune functioning, thereby killing the intracellular parasite. Systems immunological methods assisted in a step towards identifying the key to the mystery of crucial components and serving as an approach for therapeutic intervention in L. major infection.

Mechanobiology of immune cells: Messengers, receivers and followers in leishmaniasis aiding synthetic devices.

Cytokines are influential molecules which can direct cells behavior. In this review, cytokines are referred as messengers, immune cells which respond to cytokine stimulus are referred as receivers and the immune cells which gets modulated due to their plasticity induced by infectious pathogen leishmania, are referred as followers. The advantage of plasticity of cells is taken by the parasite to switch them from parasite eliminating form to parasite survival favoring form through a process called as reciprocity which is undergone by cytokines, wherein pro-inflammatory to anti-inflammatory switch occur rendering immune cell population to switch their phenotype. Detailed study of this switch can help in identification of important targets which can help in restoring the phenotype to parasite eliminating form and this can be done through synthetic circuit, finding its wider applicability in therapeutics.

Artificial intelligence channelizing protein-peptide interactions pipeline for host-parasite paradigm in IL-10 and IL-12 reciprocity by SHP-1.

Identification of molecular targets in any cellular phenomena is a challenge and a path that one endeavors upon independently. We have identified a phosphatase SHP-1 as a point of intervention of IL-10 and IL-12 reciprocity in leishmaniasis. The therapeutic model that we have developed uniquely targets this protein but the pipeline in general can be used by the researchers for their unique targets. Naturally occurring peptides are well known for their biochemical participation in cellular functions hence we were motivated to use this uniqueness of physico-chemical properties of peptides conferred by amino acids through machine learning to channelize a mode of therapeutic exploration in infectious disease. Using computational approaches, we identified high order sequence conservation and similarity in SHP-1 sequence which was also evolutionarily conserved, complete structure of Mouse SHP-1 was predicted and validated, a unique motif of the same was identified against which library of synthetic peptides were designed and validated followed by screening the library by docking them with MuSHP-1 protein structure. Our findings showed 3 peptides had high binding affinity and in future can be validated using cell based and in vivo assays.

Computational System Level Approaches for Discerning Reciprocal Regulation of IL10 and IL12 in Leishmaniasis.

IL12 and IL10 are two of the major cytokines which control the fate of Leishmaniasis. This paper presents two models healthy state and diseased state which shows how secretion of IL12 is responsible for parasite elimination and IL10 can jeopardize the parasite elimination and promote its survival. Epigenetic modification in the host IL12 and IL10 promoter can decide the fate of parasites. It was observed that reciprocal relationship exists between IL12 and IL10 and that is majorly controlled by a transcription factor NFAT5 from Rel family of transcription factors. By targeting this transcription factor at the cellular level, it might be possible to modulate the release of powerful pro-inflammatory cytokines, thereby reducing parasite survival. The mathematical models developed here serves as a step towards finding a key component that can pave a way for therapeutic investigation.