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. 2017 Jul 14;11(7):e0005774.
doi: 10.1371/journal.pntd.0005774. eCollection 2017 Jul.

Arginase activity in pathogenic and non-pathogenic species of Leishmania parasites

Alireza Badirzadeh  1   2 , Tahereh Taheri  1 , Yasaman Taslimi  1 , Zahra Abdossamadi  1 , Maryam Heidari-Kharaji  1 , Elham Gholami  1 , Baharehsadat Sedaghat  1 , Maryam Niyyati  2 , Sima Rafati  1
Affiliations

Arginase activity in pathogenic and non-pathogenic species of Leishmania parasites

Alireza Badirzadeh et al. PLoS Negl Trop Dis. .

Abstract

Proliferation of Leishmania (L.) parasites depends on polyamine availability, which can be generated by the L-arginine catabolism and the enzymatic activity of arginase (ARG) of the parasites and of the mammalian hosts. In the present study, we characterized and compared the arginase (arg) genes from pathogenic L. major and L. tropica and from non-pathogenic L. tarentolae. We quantified the level of the ARG activity in promastigotes and macrophages infected with pathogenic L. major and L. tropica and non-pathogenic L. tarentolae amastigotes. The ARG's amino acid sequences of the pathogenic and non-pathogenic Leishmania demonstrated virtually 98.6% and 88% identities with the reference L. major Friedlin ARG. Higher ARG activity was observed in all pathogenic promastigotes as compared to non-pathogenic L. tarentolae. In vitro infection of human macrophage cell line (THP1) with pathogenic and non-pathogenic Leishmania spp. resulted in increased ARG activities in the infected macrophages. The ARG activities present in vivo were assessed in susceptible BALB/c and resistant C57BL/6 mice infected with L. major, L. tropica and L. tarentolae. We demonstrated that during the development of the infection, ARG is induced in both strains of mice infected with pathogenic Leishmania. However, in L. major infected BALB/c mice, the induction of ARG and parasite load increased simultaneously according to the time course of infection, whereas in C57BL/6 mice, the enzyme is upregulated solely during the period of footpad swelling. In L. tropica infected mice, the footpads' swellings were slow to develop and demonstrated minimal cutaneous pathology and ARG activity. In contrast, ARG activity was undetectable in mice inoculated with the non-pathogenic L. tarentolae. Our data suggest that infection by Leishmania parasites can increase ARG activity of the host and provides essential polyamines for parasite salvage and its replication. Moreover, the ARG of Leishmania is vital for parasite proliferation and required for infection in mice. ARG activity can be used as one of the main marker of the disease severity.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Real-time qPCR analysis of total RNA from cultured species of Leishmania promastigotes.
Arginase transcript levels were quantified in extracted total RNAs of (A) logarithmic and (B) stationary growth phase of promastigotes (2×108 parasites/ml) for each parasite separately. Data reported are those of duplicate samples, and the experimental procedure was repeated at least three times with identical outcomes. Error bars are SD (**p ≤ 0.01, ****p ≤ 0.001 and *****p ≤ 0.0001).
Fig 2
Fig 2. Arginase activities in the cultured species of pathogenic and non-pathogenic Leishmania promastigotes.
Promastigotes from (A) logarithmic and (B) stationary growth phases were obtained from cultures (2×108 parasites/ml). Data reported are those of duplicate samples, and the experimental procedure was repeated at least three times with identical outcomes. Error bars are SD (**p ≤ 0.01, ****p ≤ 0.001 and *****p ≤ 0.0001).
Fig 3
Fig 3. Comparative levels of ARG activity and NO production in infected THP1 cells.
Cells from the human macrophages cell line (THP1) were infected with stationary growth phase of pathogenic and non-pathogenic Leishmania promastigotes (2×108 parasites/ml) with a MOI of 1:10 and after 48 hours post-infection the amount of (A) ARG and (B) NO released into culture supernatants was measured. Data reported are those of duplicate samples, and the experimental procedure was repeated at least three times with identical outcomes. Error bars are SD (**p≤ 0.01, ****p≤ 0.001 and *****p≤ 0.0001).
Fig 4
Fig 4. Lesion development, ARG activity and parasite burden in L. major infected mice in footpads and draining lymph nodes.
Groups of susceptible BALB/c and resistant C57BL/6 mice were infected with 2×106 metacyclic L. major promastigotes in the left hind footpad. (A) The lesion size was monitored by measuring the increase in footpad thickness and width weekly by using a caliper. At various times after infection (ending at 10 week) mice were sacrificed and ARG activity was determined in (B) footpads and (D) lymph nodes. (C) Parasite number was quantified in draining lymph nodes at 1, 5 and 10 weeks after infection. Data reported are those of duplicate samples, and the experimental procedure was repeated at least two times with similar outcomes. Error bars are SD (**p≤ 0.01, ****p≤ 0.001 and *****p≤ 0.0001).
Fig 5
Fig 5. Lesion development, ARG activity and parasite burden in L. tropica infected mice in footpads and draining lymph nodes.
Groups of susceptible BALB/c and resistant C57BL/6 mice were infected with 2×107 metacyclic L. tropica promastigotes in the left hind footpad. (A) The lesion size was monitored by measuring the increase in footpad thickness and width weekly using a caliper. At various times after infection (ending at 10 week), mice were sacrificed and ARG activity was determined in (B) footpads and (D) lymph nodes. (C) Parasite number was quantified in draining lymph nodes at 1, 5 and 10 weeks after infection. Data reported are those of duplicate samples, and the experimental procedure was repeated at least two times with similar outcomes. Error bars are SD (**p≤ 0.01, ****p≤ 0.001 and *****p≤ 0.0001).”
Fig 6
Fig 6. Lesion development, ARG activity and parasite burden in mice injected with non-pathogenic L. tarentolae.
Groups of susceptible BALB/c and resistant C57BL/6 mice were infected with 2×107 metacyclic L. tarentolae promastigotes in the left hind footpad. (A) The lesion size was monitored by measuring the footpad thickness and width weekly using a caliper. At various times after infection (ending at 10 week) mice were sacrificed and ARG activity was determined in (B) footpads and (D) lymph nodes. (C) Parasite number was quantified in draining lymph nodes at 1, 5 and 10 weeks after infection. Data reported are those of duplicate samples, and the experimental procedure was repeated at least two times with similar outcomes. Error bars are SD (**p≤ 0.01, ****p≤ 0.001 and *****p≤ 0.0001).
Fig 7
Fig 7. Correlation analysis between parasite numbers and ARG activity in infected draining lymph nodes (LN).
At various times after infection (A) BALB/c and (B) C57BL/6 mice infected with pathogenic (L. major: 2×106 parasites/ml and L. tropica: 2×107 parasites/ml) and non-pathogenic Leishmania (L. tarentolae: 2×107 parasites/ml) promastigotes were sacrificed, and parasite numbers as well as ARG activity in the draining LNs was determined. Data reported are those of duplicate samples, and the experimental procedure was repeated at least two times with similar outcomes. Error bars are SD (**p≤ 0.01, ****p≤ 0.001 and *****p≤ 0.0001) (W: Week).
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Grants and funding

AB is a PhD student who was supported by the Shahid Beheshti University of Medical Sciences, Tehran, Iran. This article has been extracted from the Ph.D. thesis written by AB in the Department of Medical Parasitology and Mycology, School of Medicine at Shahid Beheshti University of Medical Sciences (Registration No.: 26). This work was financially supported by the Pasteur Institute of Iran Grant number 730 to TT and by the Iran National Science Foundation Grant ID 94013422 and 940007 to SR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.