<i>Leishmania amazonensis</i> Amastigotes Highly Express a Tryparedoxin Peroxidase Isoform That Increases Parasite Resistance to Macrophage Antimicrobial Defenses and Fosters Parasite Virulence

<div><p>Professional phagocytes generate a myriad of antimicrobial molecules to kill invading microorganisms, of which nitrogen oxides are integral in controlling the obligate intracellular pathogen <i>Leishmania</i>. Although reactive nitrogen species produced by the inducible nitric oxide synthase (iNOS) can promote the clearance of intracellular parasites, some <i>Leishmania</i> species/stages are relatively resistant to iNOS-mediated antimicrobial activity. The underlying mechanism for this resistance remains largely uncharacterized. Here, we show that the amastigote form of <i>L. amazonensis</i> is hyper-resistant to the antimicrobial actions of cytokine-activated murine and human macrophages as compared to its promastigote counterpart. Amastigotes exhibit a marked ability to directly counter the cytotoxicity of peroxynitrite (ONOO⁻), a leishmanicidal oxidant that is generated during infection through the combined enzymatic activities of NADPH oxidase and iNOS. The enhanced antinitrosative defense of amastigotes correlates with the increased expression of a tryparedoxin peroxidase (TXNPx) isoform that is also upregulated in response to iNOS enzymatic activity within infected macrophages. Accordingly, ectopic over-expression of the TXNPx isoform by <i>L. amazonensis</i> promastigotes significantly enhances parasite resistance against ONOO⁻ cytotoxicity. Moreover, TXNPx-overexpressing parasites exhibit greater intra-macrophage survival, and increased parasite growth and lesion development in a murine model of leishmaniasis. Our investigations indicate that TXNPx isoforms contribute to <i>Leishmania's</i> ability to adapt to and antagonize the hostile microenvironment of cytokine-activated macrophages, and provide a mechanistic explanation for persistent infection in experimental and human leishmaniasis.</p></div

<i>L. amazonensis</i> amastigotes antagonize macrophage antimicrobial defenses.

<p>Survival of promastigotes (Pm, A) and amastigotes (Am, B) in control MΦs (white symbols/bars) and IFN-γ/LPS-activated MΦs (black symbols/bars). MΦs were activated with LPS (100 ng/mL) and IFN-γ (100 U/mL) for 16 h prior to infection. C) Pm survival in wild-type (WT) and iNOS-deficient (iNOS^−/−) control and IFN-γ/LPS-activated MΦs at 2 days post-infection. A–C) Parasite loads determined by qRT-PCR normalizing the constitutively expressed parasite ubiquitin to MΦ Gapdh transcript levels. % survival was calculated comparing the parasite burden at the indicated time relative to the parasite burden after 1 h of internalization (t = 0). All infections were performed with an MOI of 2. The data represent the mean % survival ± SD of 4–8 independent observations from at least 2 separate experiments. ** <i>p</i><0.01, *** <i>p</i><0.001 compared to infected controls.</p

Overexpressing the 190-amino acid TXNPx1 isoform boosts promastigote resistance to ONOO⁻- and macrophage-mediated parasite killing.

<p>A) ONOO⁻ susceptibility and B) intracellular survival of promastigotes stably transformed with pXG (open symbols, bars) or pXG::TXNPx1 (black symbols, bars) in control and cytokine-activated macrophages. MΦs were activated with LPS (100 ng/mL) and IFN-γ (100 U/mL) for 16 h prior to infection. % survival was calculated by comparing the number of parasites in treated vs. untreated controls, or the parasite burden 3 days post-infection relative to the parasite burden after 1 h of internalization (t = 0). Infections were performed with an MOI of 2. The data represent the mean % survival ± SD of 3–5 independent observations from 2 separate experiments. ** <i>p</i><0.01, *** <i>p</i><0.001 compared to empty vector transformed controls.</p

TXNPx1 expression fosters parasite growth and disease progression in a murine model of cutaneous leishmaniasis.

<p>C57BL/6 mice were inoculated s.c. in the right hind foot with 5×10⁶ stationary-phase promastigotes stably transformed with pXG (open symbols) or pXG::TXNPx1 (closed symbols). A) Lesion development was measured by biweekly caliper measurements. B) Parasite loads were determined by qRT-PCR based on the mRNA level of the constitutively transcribed <i>Ubiq</i> gene. C) <i>TXNPx1</i> expression levels in lesions were determined by qRT-PCR. Relative <i>TXNPx</i> mRNA was calculated by using <i>Ubiq</i> mRNA levels to normalize for parasite load, setting control levels to 1. D) Linear regression analysis of relative <i>TXNPx</i> mRNA and parasite load. The data correspond to the mean ± S.E.M. of data from 5 mice. * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001 compared to empty vector controls.</p

<i>L. amazonensis</i> amastigotes are hyper-resistant to oxyradicals and nitrogen oxides.

<p>Susceptibility of luciferase-expressing promastigotes (Pm) or amastigotes (Am) to increasing concentrations of A) hydrogen peroxide (H₂O₂), B) an NO releaser spermine NONOate (sNO), or C) peroxynitrite (ONOO⁻) in phosphate-buffered saline for 4 h. Percent survival was calculated by comparing luciferase activity in treated vs. untreated parasites.</p

A unique tryparedoxin peroxidase (TXNPx1) isoform is highly expressed by mammalian stage amastigotes.

<p>TXNPx1 isoform protein levels in promastigotes (Pm), amastigotes (Am), and lesion-derived amastigotes (L-Am) determined by immunoblot. Relative density represents the mean band intensity from two independent immunoblots. Ponceau S staining of the membrane was used as a loading control.</p

<i>L. amazonensis</i> TXNPx1 induction in infected MΦs is iNOS-dependent.

<p>A) Expression of TXNPx1 in control and IFN-γ/LPS-activated wild-type (WT) and iNOS^−/− MΦs at 4 days after infection with luciferase-expressing promastigotes (MOI 2). Host β-actin was used as a loading control and luciferase (luc) as an indicator of parasite loads. <b>B</b>) TXNPx1 expression in control and IFN-γ/LPS-activated WT and iNOS^−/− MΦs at 48 h post-infection with luciferase-expressing axenic amastigotes (MOI 2). Host β-actin was used as a loading control and luciferase (luc) as an indicator of parasite loads. C) Amastigotes were harvested 2 days post-infection from control MΦs (light grey bar) or cytokine-activated MΦs with (dark grey bar) or without (black bar) the iNOS inhibitor aminoguanidine, and the virulence of MΦ-derived amastigotes was compared to axenic amastigotes (Ax, white bar) in IFN-γ/LPS-activated MΦs. Parasite loads were determined at 48 h post-infection by using qPCR and calculated by the ΔΔC(t) method. * <i>p</i><0.05, ** <i>p</i><0.01.</p

IL-10 and WNV RNA levels in the skin and draining lymph nodes following intradermal inoculation with 10⁴ pfu of WNV in 10 µl of PBS.

<p>RNA levels were assessed by real-time RT-PCR at 24 and 72 h post-infection and normalized to those of the GAPDH gene. The means ± standard deviation are shown, and an asterisk signifies a statistically significant difference (p<0.05) as compared to the group infected with WNV alone as determined by Mann-Whitney test. Control groups were mock infected with PBS alone. n = 5/group; displayed results are representative of two experimental replicates.</p

<i>In vitro</i> expression of cytokines in SINV-infected resident peritoneal macrophages.

<p>IFN-β, IL-10, and iNOS levels were assessed by real-time RT-PCR at 24 and 48 h post-infection (MOI  = 1). Their mRNA expression levels were normalized to those of the GAPDH gene. The means ± standard deviation are shown, and an asterisk signifies a statistically significant difference (p<0.05) as compared to the group infected with SINV alone as determined by Mann-Whitney test. IFN-γ, IL-12, and IL-1β mRNA expression, as well as SINV titers (assessed by titration on cell culture) were found not to vary among groups (data not shown). Control groups were mock infected with PBS alone. These results are representative of two independent replicates.</p

Cytokine expression and WNV titers in resident peritoneal macrophages following <i>in vitro</i> infection in the presence mosquito salivary gland extract (SGE).

<p>IFN-β, IL-10, IL-12, and iNOS levels were assessed by real-time RT-PCR at 24 and 48 h post-infection (MOI = 5). Their mRNA expression levels were normalized to those of the GAPDH gene. WNV production was quantified by titration on vero cells. The means ± standard deviation are shown, and an asterisk signifies a statistically significant difference (p<0.05) as compared to the group infected with WNV alone as determined by Mann-Whitney test. IFN-γ and IL-1β mRNA expression were found not to vary among groups (data not shown). Control groups were mock infected with PBS alone. Results were similar in three independent replicates.</p