Transcriptomic profiling revealed immune-related signaling pathways in response to experimental infection of Leishmania donovani in two desert lizards from Northwest China.

Little is known about the immune response of lizards to Leishmania parasties. In this study, we conducted the first liver transcriptome analysis of two lizards (Phrynocephalus przewalskii and Eremias multiocellata) challenged with L. donovani, endemic to the steppe desert region of northwestern China. Our results revealed that multiple biological processes and immune-related signaling pathways are closely associated with the immune response to experimental L. donovani infection in the two lizards, and that both lizards show similar changes to mammals in terms of immunity to Leishmania. However, the interspecific divergence of the two lizards leads to different transcriptomic changes. In particular, in contrast to P. przewalskii, the challenged E. mutltiocellata was characterized by the induction of down-regulation of most DEGs. These findings will contribute to the scarce resources on lizard immunity and provide a reference for further research on immune mechanisms in reptiles.

Opportunistic pathogens increased and probiotics or short-chain fatty acid-producing bacteria decreased in the intestinal microbiota of pneumonia inpatients during SARS-CoV-2 Omicron variant epidemic.

The global pandemic of COVID-19 has been over four years, and the role of intestinal microbiota in the occurrence and development of COVID-19 needs to be further clarified. During the outbreak of SARS-CoV-2 Omicron variant in China, we analyzed the intestinal microbiome in fecal samples from inpatients with pneumonia and normal individuals in January 2023. The microbiota composition, alpha diversity, beta diversity, differential microbial community, co-occurrence networks, and functional abundance were analyzed. The results showed significant differences in microbiota composition between the two groups. In pneumonia group, the abundance of Bifidobacterium, Blautia, Clostridium, and Coprococcus decreased, while the abundance of Enterococcus, Lactobacillus, and Megamonas increased. Through LEfSe analysis, 37 marker microbiota were identified in pneumonia group. Co-occurrence network analysis found that Lachnospiraceae was critical for the interaction of intestinal microbiota, and the anti-inflammatory bacteria Blautia was negatively correlated with the pro-inflammatory bacteria Ruminococcus. Functional prediction found the up-regulation of steroid biosynthesis, geraniol degradation, and mRNA surveillance pathway in pneumonia group. In conclusion, opportunistic pathogens increased and probiotics, or short-chain fatty acid-producing bacteria, decreased in the intestinal microbiota of pneumonia inpatients during the Omicron epidemic. Blautia could be used as a probiotic in the treatment of pneumonia patients in the future.

Upregulation of PD-1/PD-L1 and downregulation of immune signaling pathways lead to more severe visceral leishmaniasis in undernutrition mice.

BACKGROUND: Leishmaniasis is mainly prevalent in tropical and subtropical developing countries, where chronic undernutrition often co-exists. Undernutrition is reported to promote the progression of leishmaniasis, but its immune mechanisms have not been fully elucidated. METHODS: To simulate chronic undernutrition of patients in epidemic areas and explore the immune mechanism of undernutrition promoting leishmaniasis, BALB/c mouse models with different nutritional imbalances were established, including undernutrition 75%, undernutrition 65% and obesity mouse models. After infection with Leishmania donovani in these model mice, we focused on evaluating the progress of leishmaniasis in the spleen and liver, the expression of important immunosuppressive and immunoactivation molecules, and changes of spleen transcriptome. The immune signaling pathways enriched by differentially expressed genes and hub genes were analyzed. RESULTS: The results showed that among the mouse infection models, undernutrition 75% + infection group had the highest parasite load in the spleen and liver at the 8th week post-infection, possibly due to the continuous increase of PD-1, PD-L1 and TCR. Spleen RNA-seq results suggested that some immune signaling pathways were downregulated in undernutrition 75% + infection group, including neutrophil extracellular trap formation, IL-17 signaling pathway, natural killer cell-mediated cytotoxicity, etc. Among them, neutrophil extracellular trap formation pathway had the largest number of downregulated genes. This also explained why undernutrition 75% + infection group had the highest parasite load. Through PPI network analysis, hub genes such as Lcn2, Ltf, Mpo, Dnaja1, Hspa1a, Hspa1b and Hsph1 were screened out and might play important roles in the process of undernutrition promoting leishmaniasis. CONCLUSIONS: Undernutrition upregulated PD-1 and PD-L1 expression and downregulated immune signaling pathways in mice with visceral leishmaniasis. The signaling pathways and hub genes may serve as drug targets or intervention targets for the treatment of leishmaniasis patients with undernutrition.

LdCyPA attenuates MAPK pathway to assist Leishmania donovani immune escape in host cells.

BACKGROUND: Visceral leishmaniasis is a neglected tropical disease affecting millions of people worldwide. Macrophages serve as the primary host cells for L. donovani, the immune response capability of these host cells is crucial for parasites' intracellular survival. L. donovani peptidyl-prolyl cis/trans isomerase Cyclophilin A (LdCypA) is a key protein for L. donovani intracellular proliferation, while the molecular mechanism conducive to intracellular survival of parasites remains elusive. METHODS: In this study, we generated a macrophage cell line overexpressing LdCyPA to investigate its role in controlling host immunity and promoting intracellular immune escape of L. donovani. RESULTS: It was discovered that the overexpression of the LdCyPA cell line regulated the host immune response following infection by downregulating the proportion of M1-type macrophages, promoting the secretion of the anti-inflammatory factor IL-4, and inhibiting the secretion of pro-inflammatory factors like IL-12, IFN-γ, TNF-α, and INOS. Transcriptome sequencing and mechanistic validation, meanwhile, demonstrated that cells overexpressing LdCyPA controlled the immune responses that followed infection by blocking the phosphorylation of P38 and JNK1/2 proteins in the MAPK signaling pathway and simultaneously increasing the phosphorylation of ERK proteins, which helped the L. donovani escape immune recognition. CONCLUSION: Our findings thus pave the way for the development of host-directed antiparasitic drugs by illuminating the pro-Leishmania survival mechanism of L. donovani cyclophilin A and exposing a novel immune escape strategy for L. donovani that targets host cellular immune regulation.

TLR-2 agonist Pam3CSK4 has no therapeutic effect on visceral leishmaniasis in BALB/c mice and may enhance the pathogenesis of the disease.

Most of the existing Leishmania-related research about TLR-2 agonists was focusing on their role as adjuvants in the vaccine, few studied its therapeutic effect. This paper aims to explore the therapeutic effect of TLR-2 agonist Pam3CSK4 on Leishmania-infected mice and the underlying immune molecular mechanisms. In L. donovani-infected BALB/c mice, one group was treated with Pam3CSK4 after infection and the other group was not treated. Normal uninfected mice treated with Pam3CSK4 or untreated were used as controls. Parasite load, hepatic pathology and serum antibodies were detected to assess the severity of the infection. The expression of immune-related genes, spleen lymphocyte subsets and liver RNA-seq were employed to reveal possible molecular mechanisms. The results showed that the liver and spleen parasite load of infected mice in Pam3CSK4 treated and untreated groups had no statistical difference, indicating Pam3CSK4 might have no therapeutic effect on visceral leishmaniasis. Infected mice treated with Pam3CSK4 possessed more hepatic inflammation focus, lower IgG and IgG2a antibody titers, and a lower proportion of spleen CD3+CD4+ T cells. Transcriptome analysis revealed that Th1/Th2 differentiation, NK cells, Th17 cell, complement system and calcium signaling pathways were down-regulated post-treatment of Pam3CSK4. In this study, TLR-2 agonist Pam3CSK4 showed no therapeutic effect on visceral leishmaniasis in BALB/c mice and might enhance the pathogenesis of the disease possibly due to the down-regulation of several immune-related pathways, which can improve our understanding of the role of TLR-2 in both treatment and vaccine development.

TLR9 agonist CpG ODN 2395 promotes the immune response against Leishmania donovani in obesity and undernutrition mice.

As important immunomodulators, CpG ODNs have broad application prospects in the treatment and prevention of leishmaniasis. In order to explore the immunomodulatory effect of CpG ODNs on mice infected with Leishmania parasites in different nutritional status, TLR9 agonist CpG ODN 2395 or TLR9 antagonist CpG ODN 2088 was injected into normal, obesity and undernutrition BALB/c mice infected with Leishmania donovani, respectively. Subsequently, spleen and liver parasite loads, spleen and liver immune gene expression, spleen T cell subsets proportion and PD-1 expression, serum lipids, serum cytokines, and anti-Leishmania antibodies were measured to assess the immune response of mice with different nutritional status. The results displayed that at the 8th week after infection, the spleen parasite load of obesity and undernutrition mice was significantly higher than that of normal mice, but the liver parasite load showed no statistical difference among the three groups. The treatment of CpG ODN 2395 or CpG ODN 2088 significantly reduced the spleen parasite load of obesity and undernutrition infected mice, but did not reduce that of normal infected mice. In obesity infected mice, CpG ODN 2395 promoted the up-regulation of TCR, ICOS and TLR4 in spleen, promoted the secretion of IFN-γ and anti-Leishmania total IgG and IgG1 antibodies, and increased the content of serum HDL-C. In undernutrition infected mice, CpG ODN 2395 promoted the up-regulation of spleen CD28 and TLR9, increased the proportion of spleen CD3+ T cells, and decreased the content of serum IL-10. Our results demonstrated that CpG ODN 2395 enhanced the immune response and clearance of Leishmania parasites in obesity and undernutrition mice, which might be used as a therapeutic agent for obesity and undernutrition leishmaniasis patients in the future.

Screening Novel Vaccine Candidates for Leishmania Donovani by Combining Differential Proteomics and Immunoinformatics Analysis.

Visceral leishmaniasis (VL), also known as kala-azar, is the most dangerous form of leishmaniasis. Currently no effective vaccine is available for clinical use. Since the pathogenicity of different Leishmania strains is inconsistent, the differentially expressed proteins in Leishmania strains may play an important role as virulence factors in pathogenesis. Therefore, effective vaccine candidate targets may exist in the differentially expressed proteins. In this study, we used differential proteomics analysis to find the differentially expressed proteins in two Leishmania donovani strains, and combined with immunoinformatics analysis to find new vaccine candidates. The differentially expressed proteins from L. DD8 (low virulent) and L. 9044 (virulent) strains were analyzed by LC-MS/MS, and preliminarily screened by antigenicity, allergenicity and homology evaluation. The binding peptides of MHC II, IFN-γ and MHC I from differentially expressed proteins were then predicted and calculated for the second screening. IFN-γ/IL-10 ratios and conserved domain prediction were performed to choose more desirable differentially expressed proteins. Finally, the 3D structures of three vaccine candidate proteins were produced and submitted for molecular dynamics simulation and molecular docking interaction with TLR4/MD2. The results showed that 396 differentially expressed proteins were identified by LC-MS/MS, and 155 differentially expressed proteins were selected through antigenicity, allergenicity and homology evaluation. Finally, 16 proteins whose percentages of MHC II, IFN-γ and MHC I binding peptides were greater than those of control groups (TSA, LmSTI1, LeIF, Leish-111f) were considered to be suitable vaccine candidates. Among the 16 candidates, amino acid permease, amastin-like protein and the hypothetical protein (XP_003865405.1) simultaneously had the large ratios of IFN-γ/IL-10 and high percentages of MHC II, IFN-γ and MHC I, which should be focused on. In conclusion, our comprehensive work provided a methodological basis to screen new vaccine candidates for a better intervention against VL and associated diseases.

Mutation Characteristics and Phylogenetic Analysis of Five Leishmania Clinical Isolates.

Leishmaniasis is a neglected tropical disease threatening millions of people worldwide. The emergence of antimony-resistant Leishmania strains have brought difficulties to the treatment and elimination of leishmaniasis. This study performed genome sequencing, phylogenetic analysis and mutation analysis of five Leishmania clinical isolates, especially the Leishmania strain L_HCZ isolated in 2016, which shows strong virulence and antimony resistance. By phylogenetic analysis, four isolates (L_DD8, L_801, L_Liu and L_9044) were identified as Leishmania donovani, the isolate L_HCZ was identified as Leishmania infantum and the isolate L_DD8 as a standard strain of L.donovani. Genome-wide mutation analysis was applied to identify mutations related to the drug resistance and virulence of the newly isolated L_HCZ. Compared with the other four Leishmania isolates, L_HCZ had the most mutations in genes associated with antimony resistance, including the ABC transporter, ascorbate-dependent peroxidase, gamma-glutamylcysteine synthetase, glucose-6-phosphate 1-dehydrogenase, ATP-binding cassette protein subfamily A and multi-drug resistance protein-like genes. Among the genes associated with virulence, L_HCZ had the most mutations in cysteine peptidase A, cysteine peptidase B, cysteine peptidase C, heat-shock protein 70, gp63, acid phosphatase, kinesin k39, kinesin, phosphoglycan beta 1, amastin-like surface protein and amastin-like proteins. The mutations in L_HCZ might possibly contribute to its antimony resistance and strong virulence in clinical patients. Whole-genome resequencing has exhibited broad application prospects and may be put into clinical use in the future for parasite identifying and epidemiological investigations.

Sandfly Fever Sicilian Virus-Leishmania major co-infection modulates innate inflammatory response favoring myeloid cell infections and skin hyperinflammation.

BACKGROUND: The leishmaniases are a group of sandfly-transmitted diseases caused by species of the protozoan parasite, Leishmania. With an annual incidence of 1 million cases, 1 billion people living in Leishmania-endemic regions, and nearly 30,000 deaths each year, leishmaniasis is a major global public health concern. While phlebotomine sandflies are well-known as vectors of Leishmania, they are also the vectors of various phleboviruses, including Sandfly Fever Sicilian Virus (SFSV). Cutaneous leishmaniasis (CL), caused by Leishmania major (L. major), among other species, results in development of skin lesions on the infected host. Importantly, there exists much variation in the clinical manifestation between individuals. We propose that phleboviruses, vectored by and found in the same sandfly guts as Leishmania, may be a factor in determining CL severity. It was reported by our group that Leishmania exosomes are released into the gut of the sandfly vector and co-inoculated during blood meals, where they exacerbate CL skin lesions. We hypothesized that, when taking a blood meal, the sandfly vector infects the host with Leishmania parasites and exosomes as well as phleboviruses, and that this viral co-infection results in a modulation of leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: In vitro, we observed modulation by SFSV in MAP kinase signaling as well as in the IRF3 pathway that resulted in a pro-inflammatory phenotype. Additionally, we found that SFSV and L. major co-infection resulted in an exacerbation of leishmaniasis in vivo, and by using endosomal (Toll-like receptor) TLR3, and MAVS knock-out mice, deduced that SFSV's hyperinflammatory effect was TLR3- and MAVS-dependent. Critically, we observed that L. major and SFSV co-infected C57BL/6 mice demonstrated significantly higher parasite burden than mice solely infected with L. major. Furthermore, viral presence increased leukocyte influx in vivo. This influx was accompanied by elevated total extracellular vesicle numbers. Interestingly, L. major displayed higher infectiveness with coincident phleboviral infection compared to L. major infection alone. CONCLUSION/SIGNIFICANCE: Overall our work represents novel findings that contribute towards understanding the causal mechanisms governing cutaneous leishmaniasis pathology. Better comprehension of the potential role of viral co-infection could lead to treatment regimens with enhanced effectiveness.

Development of dominant epitope-based vaccines encoding Gp63, Kmp-11 and Amastin against visceral leishmaniasis.

The most dangerous form of leishmaniasis is Visceral leishmaniasis (VL). The elimination of VL depends not only on agent treatments but also on effective vaccines against Leishmania parasites. Epitope-based vaccines composed of alternative short antigenic epitopes have the advantages of MHC epitope easy designing, which has broad application prospects. In a previous study, we analyzed Leishmania Gp63, Kmp-11 and Amastin protein sequence in silico, and found that the amino acid fragments of Gp63 (138-360aa), Kmp-11 (1-91aa) and Amastin (1-72aa) were rich in dominant epitopes. In this study, we used the three amino acid fragments as multi-epitope vaccine candidates to construct DNA and protein vaccines. BALB/c mice were vaccinated with the DNA and protein vaccines by DNA prime-protein boost strategy and challenged with Leishmania promastigotes. To evaluate vaccine immunogenicity and immunoprotection, serum specific antibody titers and cytokines were detected using ELISA, splenic CD3+, CD4+ and CD8+ cells were analyzed by flow cytometry, livers were made into pathological sections to observe pathological changes, and splenic parasitic loads were quantified using qPCR. The results showed that the increased specific IgG titers from vaccinated mice supported the vaccine immunogenicity. The increased cytokines (IFN-γ, IL-12 and TNF-α), splenic CD3+, CD4+ and CD8+ T cells and hepatic granulomas, and the decreased splenic parasitic loads (parasite reduction rates of Gp63, Kmp-11 and Amatin groups were 89%, 86% and 79%, respectively) from immunized mice post-infection were suggested the good immunoprotection of the vaccines. Our study demonstrated that vaccines based on the dominant epitopes of Gp63, Kmp-11 and Amastin with DNA prime-protein boost vaccination strategy showed significant immune effects against Leishmania, especially the Gp63 group showed a nearly 90% parasites reduction rate. This study will provide references for visceral leishmaniasis epitope vaccine design and immune strategy selection.

[Construction and Expression of Eukaryotic and Prokaryotic Recombinant Vectors of Pepck and Gp63 Dominant Epitopes of Leishmania infantum].

OBJECTIVE: To construct eukaryotic and prokaryotic recombinant vectors containing Pepck- Gp63 and to achieve protein expression by selecting the dominant epitope genes of Pepck and Gp63 of Leishmania infantum. METHODS: The secondary structure and HLA epitopes of phosphoenolpyruvate carboxylase (PEPCK) were predicted by in silico analysis, and the dominant epitopes were picked out. According to the analysis results of glycoprotein of 63×10 3(GP63) epitopes identified by the same method in our laboratory, the dominant epitope genes of Pepck and Gp63 were used to construct pET32a- Pepck- Gp63 and pVAX1- Pepck- Gp63 by overlapping PCR and enzyme reaction. Then, for protein expression, the prokaryotic vectors were transfected into E.coil while the eukaryotic vectors were transfected into NIH3T3 cells by liposome transfection. RESULTS: There were multiple dominant epitopes in Pepckand there were Pepck-Gp63 sequences in the polyclonal site of expression vector. The expression of Pepck-Gp63 in E.coil appeared in inclusion form and led to 74 kDa band in SDS-PAGE. The immunofluorescence results of NIH3T3 cells transfected by pVAX1- Pepck-Gp63 were positive. CONCLUSION: The recombinant prokaryotic expression plasmids pET32a- Pepck-Gp63 and eukaryotic expression plasmids pVAX1- P epck -Gp63 were successfully constructed, and it was shown that the recombinant plasmids were able to express the corresponding target proteins in E. coli and NIH3T3 cells, respectively, providing a preliminary experimental basis for the subsequent study of immunization strategies.

Records of three mammal tick species parasitizing an atypical host, the multi-ocellated racerunner lizard, in arid regions of Xinjiang, China.

BACKGROUND: Ticks are ectoparasites that feed on blood of a broad taxonomical range of terrestrial and flying vertebrates and are distributed across a wide range of environmental settings. To date, the species identity, diversity, and relationships among the ticks on lizards in China have been poorly understood. METHODS: In this study, 30 ticks, collected from the multi-ocellated racerunner (Eremias multiocellata) lizard in the Tarim Basin and adjacent Yanqi Basin of the Xinjiang Uygur Autonomous Region in China, were identified by morphological observation and confirmed by DNA-based techniques. The mitochondrially encoded 12S rRNA, 16S rRNA, and COI gene fragments of ticks were amplified and sequenced. To understand the genetic polymorphisms, 47 ticks collected from hedgehogs and 1 from brushwood in the Tarim Basin were also included. Species identification was based on both morphological and molecular characters. The median-joining network approach was used to evaluate the intraspecific genealogies of the ticks and their relatedness with the geographical origin or hosts. RESULTS: The sequence similarity analysis confirmed that the 30 ticks belong to three genera and three species including 11 individuals of Hyalomma asiaticum, 3 of Rhipicephalus turanicus, and 16 of Haemaphysalis sulcata. A network approach revealed paraphyletic populations of R. turanicus and Hy. asiaticum at the intraspecies level regarding geographical origin and low host specificity. For R. turanicus and Hy. asiaticum, common ancestry was observed between COI sequences from lizards and other sequence types from different hosts and countries. CONCLUSIONS: To our knowledge, our study is the first to conduct a molecular survey of ticks from lizards in the arid regions of Xinjiang, China. Eremias multiocellata is an atypical host of the three tick species. Notably, two species of ticks, Hy. asiaticum and R. turanicus, have been collected and identified from lizards in China for the first time. Star-like networks suggest both of them might have experienced recent population expansion. The discoveries are closely related to the geographical environments in Xinjiang and will provide information for the control of ticks and tick-borne pathogens in Northwest China.

Vaccine design based on 16 epitopes of SARS-CoV-2 spike protein.

The global outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) urgently requires an effective vaccine for prevention. In this study, 66 epitopes containing pentapeptides of SARS-CoV-2 spike protein in the IEDB database were compared with the amino acid sequence of SARS-CoV-2 spike protein, and 66 potentially immune-related peptides of SARS-CoV-2 spike protein were obtained. Based on the single-nucleotide polymorphisms analysis of spike protein of 1218 SARS-CoV-2 isolates, 52 easily mutated sites were identified and used for vaccine epitope screening. The best vaccine candidate epitopes in the 66 peptides of SARS-CoV-2 spike protein were screened out through mutation and immunoinformatics analysis. The best candidate epitopes were connected by different linkers in silico to obtain vaccine candidate sequences. The results showed that 16 epitopes were relatively conservative, immunological, nontoxic, and nonallergenic, could induce the secretion of cytokines, and were more likely to be exposed on the surface of the spike protein. They were both B- and T-cell epitopes, and could recognize a certain number of HLA molecules and had high coverage rates in different populations. Moreover, epitopes 897-913 were predicted to have possible cross-immunoprotection for SARS-CoV and SARS-CoV-2. The results of vaccine candidate sequences screening suggested that sequences (without linker, with linker GGGSGGG, EAAAK, GPGPG, and KK, respectively) were the best. The proteins translated by these sequences were relatively stable, with a high antigenic index and good biological activity. Our study provided vaccine candidate epitopes and sequences for the research of the SARS-CoV-2 vaccine.

The immunogenicity and protective immunity of multi-epitopes DNA prime-protein boost vaccines encoding Amastin-Kmp-11, Kmp11-Gp63 and Amastin-Gp63 against visceral leishmaniasis.

Visceral leishmaniasis (VL) is the most fatal form of leishmaniasis if left untreated and 50,000 to 90,000 new cases of VL occur worldwide each year. Although various vaccines had been studied in animal models, none of them was eligible to prevent human from infections. In this study, according to the silico analysis of Leishmania Amastin, Kmp-11 and Gp63 protein, dominant epitope sequences of these proteins were selected and linked to construct dominant multi-epitopes DNA and protein vaccines (Amastin-Kmp-11, Amastin-Gp63 and Kmp-11-Gp63) against VL. BALB/c mice were immunized with a DNA prime-protein boost immunization strategy and challenged with a new Leishmania parasite strain isolated from a VL patient. After immunization, the results including specific antibody titers, IL-4 and TNF-α levels, and CD4 and CD8 T cell proportion suggested the potent immunogenicity of the three vaccines. After infection, the results of spleen parasite burdens in the three vaccine groups were significantly lower than those of control groups, and the parasite reduction rates of Amastin-Kmp-11, Amastin-Gp63 and Kmp-11-Gp63 groups were 89.38%, 91.01% and 88.42%, respectively. Spleen smear observation and liver histopathological changes showed that all vaccine groups could produce significant immunoprotection against VL and Amastin-Gp63 vaccine was the best. In conclusion, our work demonstrated that the three dominant multi-epitopes Amastin-Kmp-11, Amastin-Gp63 and Kmp-11-Gp63 DNA prime-protein boost vaccines might be new vaccine candidates for VL, and the Amastin-Gp63 vaccine have best efficacy.

Evaluation of in vitro antileishmanial efficacy of cyclosporin A and its non-immunosuppressive derivative, dihydrocyclosporin A.

BACKGROUND: New therapeutic drugs are urgently needed against visceral leishmaniasis because current drugs, such as pentavalent antimonials and miltefosine, produce severe side effects and development of resistance. Whether cyclosporine A (CsA) and its derivatives can be used as therapeutic drugs for visceral leishmaniasis has been controversial for many years. METHODS: In this study, we evaluated the efficacy of CsA and its derivative, dihydrocyclosporin A (DHCsA-d), against promastigotes and intracellular amastigotes of Leishmania donovani. Sodium stibogluconate (SSG) was used as a positive control. RESULTS: Our results showed that DHCsA-d was able to inhibit the proliferation of L. donovani promastigotes (IC50: 21.24 µM and 12.14 µM at 24 h and 48 h, respectively) and intracellular amastigotes (IC50: 5.23 µM and 4.84 µM at 24 and 48 h, respectively) in vitro, but CsA treatment increased the number of amastigotes in host cells. Both DHCsA-d and CsA caused several alterations in the morphology and ultrastructure of L. donovani, especially in the mitochondria. However, DHCsA-d showed high cytotoxicity towards cells of the mouse macrophage cell line RAW264.7, with CC50 values of 7.98 µM (24 h) and 6.65 µM (48 h). Moreover, DHCsA-d could increase IL-12, TNF-α and IFN-γ production and decrease the levels of IL-10, IL-4, NO and H2O2 in infected macrophages. On the contrary, CsA decreased IL-12, TNF-α, and IFN-γ production and increased the levels of IL-10, IL-4, NO and H2O2 in infected macrophages. The expression of L. donovani cyclophilin A (LdCyPA) in promastigotes and intracellular amastigotes and the expression of cyclophilin A (CyPA) in RAW 264.7 cells were found to be significantly downregulated in the CsA-treated group compared to those in the untreated group. However, no significant changes in LdCyPA and CyPA levels were found after DHCsA-d or SSG treatment. CONCLUSIONS: Our findings initially resolved the dispute regarding the efficacy of CsA and DHCsA-d for visceral leishmaniasis treatment. CsA showed no significant inhibitory effect on intracellular amastigotes. DHCsA-d significantly inhibited promastigotes and intracellular amastigotes, but it was highly cytotoxic. Therefore, CsA and DHCsA-d are not recommended as antileishmanial drugs.

Multi-locus characterization and phylogenetic inference of Leishmania spp. in snakes from Northwest China.

BACKGROUND: Leishmaniasis caused by protozoan parasite Leishmania is a neglected disease which is endemic in the northwest of China. Reptiles were considered to be the potential reservoir hosts for mammalian Leishmaniasis, and Leishmania had been detected in lizards from the epidemic area in the northwest of China. To date, few studies are focused on the natural infection of snakes with Leishmania. METHODS: In this study, 15 snakes captured from 10 endemic foci in the northwest of China were detected Leishmania spp. on the base of mitochondrial cytochrome b, heat shock protein 70 gene and ribosomal internal transcribed spacer 1 regions, and identified with phylogenetic and network analyses. RESULT: In total, Leishmania gene was found in 7 snakes. The phylogenetic inference trees and network analysis suggests that the species identification was confirmed as Leishmania donovani, L. turanica and L. (Sauroleishmania) sp. CONCLUSION: Our work is the first time to investigate the natural Leishmania spp. infection of snakes in the northwest of China. Mammalian Leishmania (L. donovani and L. turanica) was discovered in snakes and the reptilian Leishmania (Sauroleishmania sp.) was closely related to the clinical strains both prompt the importance of snakes in the disease cycle. To indicate the epidemiological involvement of snakes, a wide sample size in epidemic area and the pathogenic features of reptilian Leishmania promastigotes are recommended in the future research.

Bioinformatics analysis of four proteins of Leishmania donovani to guide epitopes vaccine design and drug targets selection.

Visceral leishmaniasis (VL) is a serious and widespread parasitic disease caused by Leishmania donovani complex. The threat of this fatal disease continues due to the lack of ideal drugs or vaccines. In this study, we selected Amastin, CaNA2, Kmp-11 and PDI proteins of Leishmania donovani for study, which are VL vaccine candidates or possible drug targets. Eleven bioinformatics tools were used to analyze different aspects of these proteins, including amino acid composition, topology, signal peptide, secondary structure, surface properties, phosphorylation sites and kinases, protein binding sites, 3D homology modeling, B cell epitopes, MHC class Ⅰ and Ⅱ epitopes and protein-protein interactions. Finally, the functionally related amino acid sites and dominant epitopes of these proteins were founded. Some possible relationships between protein structure, phosphorylation sites, protein binding sites and epitopes were also discovered. High flexibility and random coils regions of protein have a tendency to be phosphorylated, bind proteins and present epitopes. Since some phosphorylation sites and their kinases are involved in Leishmania invasion and survival in host cells, they may be potential drug targets. Bioinformatics analysis helps us better understand protein function and find dominant epitopes to guide drug design and vaccine development.

DNA prime-protein boost vaccine encoding HLA-A2, HLA-A24 and HLA-DR1 restricted epitopes of CaNA2 against visceral leishmaniasis.

Visceral leishmaniasis is a tropical and neglected disease with an estimated 200 000-400 000 cases and 60 000 deaths worldwide each year. Currently, no clinically valid vaccine is available for this disease. In this study, we formulated DNA and protein vaccines encoding HLA-A2, HLA-A24 and HLA-DR1 restricted epitopes of CaNA2 against visceral leishmaniasis. We predicted the secondary and tertiary structures, surface properties, subcellular localization, potential binding sites and HLA-A2, HLA-A24 and HLA-DR1 restricted epitopes of CaNA2. The best candidate CpG ODN (2395, M362, D-SL03 or 685) was screened out as a DNA vaccine adjuvant. We also prepared Kmp-11 and Kmp-11/CaNA2 DNA and protein vaccines, respectively, for comparison. BALB/c mice were immunized with a DNA prime-protein boost immunization strategy and challenged with a newly isolated Leishmania strain from an individual with visceral leishmaniasis. The IgG antibody titers showed that our vaccine had strong immunogenicity with a long duration, especially cellular immunity. The spleen parasite burden of each group demonstrated that the CaNA2 vaccine had a certain immune protective effect on visceral leishmaniasis in BALB/c mice, and the amastigote reduction rate reached 76%. Preliminary safety tests confirmed the safety of the vaccine. Our work demonstrates that the HLA-A2, HLA-A24 and HLA-DR1 restricted epitope CaNA2 DNA prime-protein boost vaccine may be a safe and effective epitope vaccine candidate against visceral leishmaniasis.

Direct and Indirect Inhibition Effects of Resveratrol against Toxoplasma gondii Tachyzoites In Vitro.

Toxoplasma gondii is one of the most widespread obligatory parasitic protozoa and infects nearly all warm-blooded animals, leading to toxoplasmosis. The therapeutic drugs currently administered, like the combination of pyrimethamine and sulfadiazine, show high rates of toxic side effects, and drug resistance is encountered in some cases. Resveratrol is a natural plant extract with multiple functions, such as antibacterial, anticancer, and antiparasite activities. In this study, we evaluated the inhibitory effects of resveratrol on tachyzoites of the Toxoplasma gondii RH strain extracellularly and intracellularly. We demonstrate that resveratrol possesses direct antitoxoplasma activity by reducing the population of extracellularly grown tachyzoites, probably by disturbing the redox homeostasis of the parasites. Moreover, resveratrol was also able to release the burden of cellular stress, promote apoptosis, and maintain the autophagic status of macrophages, which turned out to be regulated by intracellular parasites, thereby functioning indirectly in eliminating T. gondii In conclusion, resveratrol has both direct and indirect antitoxoplasma effects against RH tachyzoites and may possess the potential to be further evaluated and employed for toxoplasmosis treatment.

Recombinant Mip-PilE-FlaA dominant epitopes vaccine candidate against Legionella pneumophila.

Legionella pneumophila is the main causative agent of Legionnaires' disease, which is a severe multi-system disease with pneumonia as the primary manifestation. We designed a recombinant Mip-PilE-FlaA dominant epitopes vaccine against Legionella pneumophila to prevent the disease and evaluated its immunogenicity and protective immunity. The protein structures of Mip, PilE and FlaA were analyzed using a computer, and the gene sequences of the dominant epitopes of the three proteins were selected to construct and optimize the vaccine. The optimized mip, pilE, flaA and recombinant mip-pilE-flaA gene sequences were cloned, expressed and purified. The purified proteins were used as dominant epitopes vaccines to immunize BALB/c mice and determine the protective immunity and immunogenicity of these purified proteins. The identification confirmed that the recombinant mip-pilE-flaA was successfully cloned and expressed. ELISA revealed that the Mip-PilE-FlaA group produced the highest IgG response, and this protein may considerably improve the production of some cytokines in BALB/c mice. Histopathology analyses of lungs from mice immunized with Mip-PilE-FlaA revealed a certain protective effect. Our work demonstrated that the recombinant dominant epitopes of Mip-PilE-FlaA exhibited strong immunogenicity and immune protection, and this protein may be an efficient epitopes vaccine candidate against Legionella pneumophila.