Antimony resistance mechanism in genetically different clinical isolates of Indian Kala-azar patients

The central theme of this enterprise is to find common features, if any, displayed by genetically different antimony (Sb)-resistant viscerotropic Leishmania parasites to impart Sb resistance. In a limited number of clinical isolates (n = 3), we studied the breadth of variation in the following dimensions: (a) intracellular thiol content, (b) cell surface expression of glycan having N-acetyl-D-galactosaminyl residue as the terminal sugar, and (c) gene expression of thiol-synthesizing enzymes (CBS, MST, gamma-GCS, ODC, and TR), antimony-reducing enzymes (TDR and ACR2), and antimonial transporter genes (AQP1, MRPA, and PRP1). One of the isolates, T5, that was genotypically characterized as Leishmania tropica, caused Indian Kala-azar and was phenotypically Sb resistant (T5-LT-SSG-R), while the other two were Leishmania donovani, out of which one isolate, AG83, is antimony sensitive (AG83-LD-SSG-S) and the other isolate, T8, is Sb resistant (T8-LD-SSG-R). Our study showed that the Sb-resistant parasites, regardless of their genotype, showed significantly higher intracellular thiol compared with Sb-sensitive AG83-LD-SSG-S. Seemingly, T5-LT-SSG-R showed about 1.9-fold higher thiol content compared with T8-LD-SSG-R which essentially mirrored cell surface N-acetyl-D-galactosaminyl expression. Except TR, the expression of the remaining thiol-synthesizing genes was significantly higher in T8-LD-SSG-R and T5-LT-SSG-R than the sensitive one, and between the Sb-resistant parasites, the latter showed a significantly higher expression. Furthermore, the genes for Sb-reducing enzymes increased significantly in resistant parasites regardless of genotype compared with the sensitive one, and between two resistant parasites, there was hardly any difference in expression. Out of three antimony transporters, AQP1 was decreased with the concurrent increase in MRPA and PRP1 in resistant isolates when compared with the sensitive counterpart. Interestingly, no difference in expression of the above-mentioned transporters was noted between two Sb-resistant isolates. The enduring image that resonated from our study is that the genetically diverse Sb-resistant parasites showed enhanced thiol-synthesizing and antimony transporter gene expression than the sensitive counterpart to confer a resistant phenotype

Genetic markers for antimony resistant clinical isolates differentiation from Indian Kala-azar

Visceral Leishmaniasis or Kala-azar is caused by the protozoan parasites belonging to the Genus Leishmania. Once thought eradicated from the Indian subcontinent, the disease came back with drug resistance to almost all prevalent drugs. Molecular epidemiological studies revealed the polymorphic nature of the population of the main player of the disease, Leishmania donovani and involvement of other species (L. tropica) and other genus (Leptomonas) with the disease. This makes control measures almost futile. It also strongly demands the characterization of each and every isolate mandatory which is not done. In this background, the present study has been carried out to assess the genetic attributes of each clinical isolates (n = 26) of KA and PKDL patients from India and Bangladesh. All the isolates were characterized through Restriction Fragment Length Polymorphism (RFLP) analysis to ascertain their species identity. 46.2% of the isolates were found to be Sodium Stibogluconate (SSG) resistant by amastigote-macrophage model. When the clinical isolates were subjected to Single Stranded Conformation Polymorphism (SSCP) of Internal Transcribed Spacer 1 (ITS1), Internal Transcribed Spacer 2 (ITS2) and some anonymous markers, the drug resistant Leishmania isolates of SSG can be distinguished from the sensitive isolates distinctly. This study showed for the first time, the genetic markers for SSG drug resistance of Indian Kala-azar clinical isolates

NAD(P)H Cytochrome b5 Oxidoreductase Deficiency in Leishmania major Results in Impaired Linoleate Synthesis Followed by Increased Oxidative Stress and Cell Death

NAD(P)H cytochrome b5 oxidoreductase (Ncb5or) participates in a variety of metabolic conversions. Results: A new Ncb5or acts as a redox partner of �12 fatty acid desaturase. Conclusion: Ncb5or null mutant suffers from impaired linoleate synthesis leading to oxidative stress and cell death. Significance: Our results have exposed a novel fatty acid synthesis pathway in L. major that differs from the mammalian system

Antimony Resistant <i>Leishmania donovani</i> but Not Sensitive Ones Drives Greater Frequency of Potent T-Regulatory Cells upon Interaction with Human PBMCs: Role of IL-10 and TGF-β in Early Immune Response

<div><p>In India the sand fly, <i>Phlebotomus argentipes</i>, transmitted parasitic disease termed kala-azar is caused by <i>Leishmania donovani</i> (LD) in humans. These immune-evading parasites have increasingly developed resistance to the drug sodium antimony gluconate in endemic regions.</p><p>Lack of early diagnosis methods for the disease limits the information available regarding the early interactions of this parasite with either human tissues or cell lineages. We reasoned that peripheral blood mononuclear cells (PBMCs) from healthy human beings could help compare some of their immune signatures once they were exposed for up to 8 days, to either pentavalent antimony sensitive (Sb^S-LD) or resistant (Sb^R-LD) <i>Leishmania donovani</i> isolates.</p><p>At day 2, PBMC cultures exposed to Sb^S-LD and Sb^R-LD stationary phase promastigotes had four and seven fold higher frequency of IL-10 secreting monocyte-macrophage respectively, compared to cultures unexposed to parasites. Contrasting with the CD4⁺CD25⁻CD127⁻ type-1 T-regulatory (Tr1) cell population that displayed similar features whatever the culture conditions, there was a pronounced increase in the IL-10 producing CD4⁺CD25⁺CD127^low/− inducible T-regulatory cells (iTregs) in the PBMC cultures sampled at day 8 post addition of Sb^R-LD.</p><p>Sorted iTregs from different cultures on day 8 were added to anti-CD3/CD28 induced naïve PBMCs to assess their suppressive ability. We observed that iTregs from Sb^R-LD exposed PBMCs had more pronounced suppressive ability compared to Sb^S-LD counterpart on a per cell basis and is dependent on both IL-10 and TGF-β, whereas IL-10 being the major factor contributing to the suppressive ability of iTregs sorted from PBMC cultures exposed to Sb^S–LD. Of note, iTreg population frequency value remained at the basal level after addition of genetically modified Sb^R-LD lacking unique terminal sugar in surface glycan.</p><p>Even with limitations of this artificial <i>in vitro</i> model of <i>L. donovani</i>-human PBMC interactions, the present findings suggest that Sb^R-LD have higher immunomodulatory capacity which may favour aggressive pathology.</p></div

GalT Knock down parasites (KDSb^R-LD) behaves like Sb^S-LD isolates.

<p><b>A</b>. Production of IL-10, <b>B</b>. TGF-β from KDSb^R, Sb^S and Sb^R-Sup. For TGF-β measurement the culture supernatants were acidified followed by neutralization, the level of TGF-β was measured by ELISA. <b>C</b>. Suppression assay performed in presence of neutralizing IL-10 antibody and/or rLAP using iTreg from KDSb^R-LD-PBMC at day eight. Data were analysed by Mann-Whitney test and two-tailed paired Student t test, and levels of significance are indicated by P values.</p

Sb^R-LD parasites induce greater IL-10 production than Sb^S-LD isolates.

<p>Kinetics of IL-10 production during early interaction with different Sb^S and Sb^R-LD isolates. Briefly, freshly isolated PBMCs were incubated with Sb^S and Sb^R-LD isolates and culture supernatants (Sb^S and Sb^R-Sup) were isolated on day- one, two, three and eight, and IL-10 level was measured by ELISA/CBA. The level of IL-10 production at different time points are represented (day one, two, three and eight), n = 10, median values of different time points were indicated. Data were analysed by the Mann-Whitney test, and levels of significance are indicated by P values.</p

Median values of IL-10 production (pg/ml) at different time points.

<p>The median values of IL-10 production at different time points are tabulated. Freshly isolated PBMCs were incubated with different Sb^S and Sb^R-LD isolates and culture supernatants (Sb^S and Sb^R-Sup) were isolated on day- one, two, three and eight, and IL-10 level (pg/ml) was measured by ELISA/CBA.</p

CD14⁺ monocyte/macrophage cells produce IL-10 during initial interaction.

<p>Production of IL-10 from CD14⁺ monocyte/macrophage cells. The frequencies of CD14⁺IL-10⁺ cells from Sb^S-LD-PBMC and Sb^R-LD-PBMC at different time points are represented. <b>A–B</b>. Percentage of IL-10 producing CD14⁺ cells on day two and <b>C</b>–<b>D</b>; on day eight. Pseudo colour plot indicate one representative data set. Data were analysed by the Mann-Whitney test, and levels of significance are indicated by P values.</p

IL-10 producing Tr1 and iTReg cells are induced in response to Sb^S and Sb^R-LD.

<p>Percentages of IL-10 producing Tr1 and iTreg cells from Sb^S-LD-PBMC and Sb^R-LD-PBMC at early and late time point are represented. <b>A, B</b>. Percentage of IL10⁺Tr1 cells on day two, <b>C</b>. Real-Time RTPCR of IL-10 mRNA level in sorted population of Tr1 cells on day two. <b>D, E</b>. Percentage of IL10⁺Tr1 cells on day eight, <b>F</b>. Real-Time RTPCR of IL-10 mRNA level in sorted population of Tr1 cells on day eight. <b>G, H</b>. Percentage of IL10⁺iTreg cells on day two, <b>I</b>. Real-Time RTPCR of IL-10 mRNA level in sorted population of iTeg cells on day two. <b>J, K</b>. Percentage of IL10⁺iTreg cells on day eight, <b>L</b>. Real-Time RTPCR of IL-10 mRNA level in sorted population of iTreg cells on day eight. Dot plots indicate one representative data set. Data were analyzed by the Mann-Whitney test, and levels of significance are indicated by P values.</p