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Expression profiling of Sudanese visceral leishmaniasis patients pre- and post-treatment with sodium stibogluconate

ABSTRACT: Visceral leishmaniasis (VL) in Sudan caused by Leishmania donovani is fatal in susceptible individuals if untreated. Treatment with Sodium Stibogluconate (SSG) leads to post kala azar dermal leishmaniasis (PKDL) in 58% of patients. Here Affymetrix microarrays were used to identify genes differentially expressed in lymph nodes (N=9 paired samples) pre- and post-treatment with SSG. Using the Bioconductor package limma, 438 genes from 28,869 post quality-control probe-sets were differentially expressed (Pnominal≤0.02) post- versus pre-treatment. Canonical pathway analysis using Ingenuity Pathway Analysis identified “Role of NFAT in Regulation of Immune Response” (Pnominal=1.35x10-5; PBH-adjusted=4.79x10-3), “B Cell Development” (Pnominal=2.04x10-4; PBH-adjusted=0.024), “Fcγ Receptor-mediated Phagocytosis in Macrophages and Monocytes” (Pnominal=2.04x10-4; PBH-adjusted=0.024), and “OX40 Signaling” (Pnominal=2.82x10-4; PBH-adjusted=0.025) as pathways differentially regulated post- versus pre-treatment. Major network hub genes included TP53, FN1, MYC, BCL2, JUN, SYK, RUNX2, MMP1 and ACTA2. Top endogenous upstream regulators included IL-7 (P=2.28x10-6), TNF (P=4.26x10-6), APP (P=4.23x10-5) and SPI1/PI.1 (P=1.17x10-7). Top predicted chemical drug regulators included the flavonoid genistein (P=4.56x10-7) and the quinoline alkaloid camptothecin (P=5.14x10-5). These results contribute to our understanding of immuno-pathology associated with VL and response to SSG treatment. Further replication could identify novel therapeutic strategies that improve on SSG treatment and reduce the likelihood of progression to PKDL.

ORGANISM(S): Homo sapiens

PROVIDER: GSE95625 | GEO | 2017/03/03

SECONDARY ACCESSION(S): PRJNA377721

REPOSITORIES: GEO

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Project description:Among the most central questions in Leishmania research is why some species remain in the skin dermis at the site of infection by the sand fly vector whereas other species migrate to visceral organs where they cause fatal visceral leishmaniasis. Although L. donovani is the species typically responsible for visceral leishmaniasis, an atypical L. donovani strain is the etiologic agent for cutaneous leishmaniasis in Sri Lanka. To identify molecular determinants for visceral disease, we have analysed the phenotype and genotype of two L. donovani clinical isolates from Sri Lanka where one isolate was derived from a cutaneous leishmaniasis patient (CL) and the other from a visceral leishmaniasis patient (VL). These isolates cause dramatically different pathology when introduced into mice; notably the CL isolate has lost the ability to survive in visceral organs while the VL isolate was highly virulent in visceral organs of BALB/c mice. Whole genome sequencing of the CL and VL isolates revealed that these genomes were very similar as there were no gene deletions and few individual gene amplifications. Indels resulting in frame shifts and loss/gain of stop codons resulted in 13 distinct pseudogenes present in each of the CL and VL isolates. There were 154 non-synonymous SNPs specific to the CL isolate and 193 non-synonymous SNPs specific to the VL isolate. Genome wide gene expression analysis revealed several transcript level differences, including the A2 virulence gene resulting in higher expression of A2 proteins in the VL isolate than in the CL isolate. Genotypic variations relevant to pathology and tropism in Leishmania can be interrogated by reverse genetics. Experimentally increasing A2 expression in the CL isolate through gene transfer significantly increased it’s ability to survive in the spleen of BALB/c mice and conversely, down-regulating A2 expression in the VL isolate abrogated attenuated its survival in BALB/c mice. These observations reveal that there are relatively few genetic differences between the CL and VL isolates apart from the A2 genes, but collectively these have profound effects on human disease and experimentally infected mice.

Project description:Among the most central questions in Leishmania research is why some species remain in the skin dermis at the site of infection by the sand fly vector whereas other species migrate to visceral organs where they cause fatal visceral leishmaniasis. Although L. donovani is the species typically responsible for visceral leishmaniasis, an atypical L. donovani strain is the etiologic agent for cutaneous leishmaniasis in Sri Lanka. To identify molecular determinants for visceral disease, we have analysed the phenotype and genotype of two L. donovani clinical isolates from Sri Lanka where one isolate was derived from a cutaneous leishmaniasis patient (CL) and the other from a visceral leishmaniasis patient (VL). These isolates cause dramatically different pathology when introduced into mice; notably the CL isolate has lost the ability to survive in visceral organs while the VL isolate was highly virulent in visceral organs of BALB/c mice. Whole genome sequencing of the CL and VL isolates revealed that these genomes were very similar as there were no gene deletions and few individual gene amplifications. Indels resulting in frame shifts and loss/gain of stop codons resulted in 13 distinct pseudogenes present in each of the CL and VL isolates. There were 154 non-synonymous SNPs specific to the CL isolate and 193 non-synonymous SNPs specific to the VL isolate. Genome wide gene expression analysis revealed several transcript level differences, including the A2 virulence gene resulting in higher expression of A2 proteins in the VL isolate than in the CL isolate. Genotypic variations relevant to pathology and tropism in Leishmania can be interrogated by reverse genetics. Experimentally increasing A2 expression in the CL isolate through gene transfer significantly increased itM-bM-^@M-^Ys ability to survive in the spleen of BALB/c mice and conversely, down-regulating A2 expression in the VL isolate abrogated attenuated its survival in BALB/c mice. These observations reveal that there are relatively few genetic differences between the CL and VL isolates apart from the A2 genes, but collectively these have profound effects on human disease and experimentally infected mice. 6 Samples in total, 3 each from VL and CL causing isolates were analyzed by Splice Leader RNASeq. These three samples from each of the isolates were grown to form one of the following three lifestages, Promastigotes, Macrophage derived Amastigotes, Axenic Amastigotes.

Project description:Post Kala Azar Dermal Leishmaniasis (PKDL) is a non-fatal dermal sequel of Visceral Leishmaniasis (VL), caused by protozoan parasite Leishmania donovani. On the global scenario, PKDL in mainly restricted to distinct zones of South Asia (India, Nepal and Bangladesh) and East Africa, mainly Sudan. In India, the most prominent fatal form of leishmaniasis present is VL, affecting entirely the eastern region of the country. The clinical presentation of VL and PKDL differ substantially; where VL patients suffer from sustained fever, hepatospleenomegaly, weight loss and anemia, PKDL patients manifest dermal lesions in the form of macular, nodular and polymorphic. Although, compared to VL, the mortality rate of PKDL is significantly lower, yet it is observed as a stigmatizing disease that brings a substantial socioeconomic burden, further intensified by a hesitancy of patients, to obtain treatment, or due to noncompliance. Lesions, especially the polymorphic forms, are parasite-rich, concluding that PKDL plays a crucial role in the anthroponotic transmission of VL. PKDL patients with Macular lesions, having comparatively lower parasitic load, and thus lacks detection sensitivity with standard techniques like rK39 (73% sensitivity), Leishmanin skin test (54% sensitivity) and Histopathological studies with patients’ skin biopsy sample (7-33% sensitivity) and is often misdiagnosed as Vitiligo cases. In order to completely eliminate Kala-azar in Southeast Asia, proper identification of various routes of the disease is essential, which could efficiently distinguish between cured PKDL and active PKDL as well as active PKDL from other cross disease like Leprosy and Vitiligo. Till now very less work has been done towards biomarker identification for PKDL from patients’ plasma. Most of the methods for disease identification are based on highly invasive tissue biopsy followed by PCR/qPCR. Recently investigations from our group have demonstrated the presence of novel glycoproteins in the circulating immune complexes (CICs) from serum, as biomarkers for early identification of PKDL patients. An Immune complex (IC), sometimes called an antigen-antibody complex, is a molecule formed from the integral binding of an antibody to a soluble antigen. After an antigen-antibody reaction, the immune complexes can be subject to any number of responses, including complement deposition, opsonisation, phagocytosis, or processing by proteases. Accumulation of ICs leads to a broad spectrum of pro-inflammatory effects, including activation of the complement cascade and induction of cytokine secretion. These complexes may also be deposited in tissues and vessel walls, leading to inflammation, tissue damage and, ultimately, disease manifestations. Immune complexes also affect disease progression and outcome in various disorders through the induction of pro-inflammatory or anti-inflammatory cytokines. In the present study we hypothesize that the CICs’ proteome is altered among PKDL patients with different dermal manifestations (Macular and Polymorphic) as compared to Cured individuals (CR) and Healthy controls (HI). The aim of this current study is to identify differentially expressed CIC proteomic profile among different forms PKDL patients as compared to HI. The differential protein expression of specific unique peptide fragments, representing specific proteins, could be used as diagnostic biomarkers in early detection of both Macular and Polymorphic PKDL cases. In this prospective study, we included PKDL patients with Macular and/or Polymorphic lesion, Cured individuals and HI. Among the enrolled patients, blood samples were collected from 20 primarily confirmed PKDL patients with Macular lesions, 20 primarily confirmed PKDL patients with Polymorphic lesions, 12 Cured individuals previously suffering from PKDL and 12 Healthy individuals; collected during the period of 2015-2016, stored at -80°C. Next the CICs were purified from PKDL patients’ plasma and LC MS/MS analysis was performed using Q-ExactivePlusOrbitrap mass spectrometer. This work for the first time revealed the differential CICs proteome profile among different forms of PKDL patients as compared to their Cured and Healthy counterparts.

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Expression profiling of Sudanese visceral leishmaniasis patients pre- and post-treatment with sodium stibogluconate

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