Animals

Three-week-old female BALB/c mice were purchased from Jackson Laboratories. Four to 6-week-old male Golden Syrian hamsters were purchased from Harlan Laboratories. Animals were housed under pathogen-free conditions at the National Institute of Allergy and Infectious Diseases (NIAID) Twinbrook animal facility in Rockville, Maryland. All animal experiments were approved by NIAID Animal Care and Use Committee under the LMV23E and LMVR4E animal protocols. The NIAID DIR Animal Care and Use Program complies with the Guide for the Care and Use of Laboratory Animals and with the NIH Office of Animal Care and Use and Animal Research Advisory Committee guidelines. We have complied with all relevant ethical regulation for animal use.

Mice diet

Post-weaning, 3-week-old BALB/c mice were assigned randomly to a diet group. Animals assigned to the malnourished (MN) group were fed for a minimum of 4 weeks with a polynutrient deficient diet, containing 3% protein, and low iron (10 ppm), and zinc (1 ppm) (TD.99075, Envigo) until stunted weight was observed as described¹¹ prior to initiation of experiments. In parallel, animals assigned to the well-nourished (WN) group were fed with a nutrient-rich diet containing 16.9% protein, normal zinc (30 ppm), and iron (100 ppm) (TD.99103, Envigo), containing similar nutritional components (19.5% protein, 81 ppm zinc, and 360 ppm iron) as a standard diet commonly used in laboratories⁵⁵. MN mice received 90% volume by weight of food compared to the WN mice. Mice diet was supplied every day at the same time frame. Animal weight was recorded every week during the experiment. According to the LMV23E animal protocol, infected mice were euthanized by the investigator at a loss of 20% or more of pre-infection weight, the study endpoint.

Sand fly infections

Four to 6-day-old Lutzomyia longipalpis (Jacobina colony) female sand flies were artificially infected as described previously¹⁴. Rabbit defibrinated blood (Noble Life Science) was spiked with 5 × 10⁶ Leishmania donovani (MHOM/SD/62/1S) amastigotes per mL and allowed the sand flies to feed for 2 h in the dark. Amastigotes were isolated from a sick L. donovani infected hamster. After infection, fed flies were sorted out and were maintained with 30% sucrose at 26 °C with 75% humidity with 12 h light/dark cycle until transmission. Prior to transmission, the maturity of the infection was assessed by counting the total number of parasites per midgut and the percent of metacyclic promastigotes from infected sand fly midguts (n = 5–10) at day 8–12 post-infection.

Mice anesthesia

Ketamine (100 mg/kg) with xylazine (10 mg/kg) were used to anesthetize mice by intra-peritoneal administration. Eyes were kept moist by the application of LubriFresh ophthalmic ointment (Major Pharmaceuticals) to prevent dryness.

Transmission by vector bites

Briefly, 20 infected L. longipalpis sand flies were applied to a mouse ear and allowed to feed for 1–2 h in the dark. The number of fed sand flies per ear were determined post-transmission.

Needle challenge by intradermal injection

Parasites were maintained in Schneider’s media (Gibco) supplemented with 20% fetal bovine serum (Thermo Fisher Scientific) and kept at 26 °C until metacyclic promastigotes developed at day 4–5. Culture metacyclic promastigotes were purified using a Ficoll gradient and 10⁵ parasites in a volume of 10 μl were injected intradermally in the ear pinnae of mice¹⁴.

Flow cytometry

At 24 h and 72 h post-infection, mice ears were collected and processed as previously described¹⁶. Briefly, single cell suspensions were stained with the LIVE/DEAD™ fixable Aqua dead cell marker (Thermo Fisher Scientific, L34957, 1:2000 dilution), anti-mouse CD16/32 (TruStain FcX, clone 93; Biolegend, Cat No. 101320, 1:50 dilution), PE-Cy7 CD11b (clone: M1/70; Biolegend, Cat No. 101216), Alexa Fluor 700 Ly6C (clone HK1.4; Biolegend, Cat No. 128024), and PerCP-eFluor 710-Ly6G (clone: 1A; eBioscience, Cat No. 46966882) for 30 min. Cells were fixed (Biolegend, Cat No. 420801) and permeabilized (Biolegend, Cat No. 421002), followed by intracellular staining with APC IL1-β pro-form (clone: NJTEN3; eBioscience, Cat No. 17-7114-80) overnight. All antibodies were used at 1:100 dilution. All cells were acquired using MACSQuant 16 (Miltenyi Biotec) and data analyzed using FlowJo 10 software. Gates were established using Fluorescence Minus One (FMO) and unstained controls.

HO-1 Western blot—skin lysate

Western blot from heme oxygenase (HO-1) was performed as described previously¹⁶. Briefly, mouse ear lysate was collected as previously described¹⁴ 72 h after transmission, and 30 μg of protein lysate was loaded in NuPAGE 4%–12% Bis-Tris protein precast gels (Thermo Fisher Scientific) and transferred to an iBlot 2 Transfer Stack 0.45 mm nitrocellulose membrane using the 7 min standard protocol in the iBlot 2 Gel Transfer Device (Thermo-Fisher Scientific). After transfer, the membrane was incubated with Rabbit anti-HO-1 antibody at 1:100 dilution (abcam, Cat No. ab13243), Rabbit anti-HSP90 (Cell Signaling Technology, Cat No. 4877) at 1:200 dilution, and Goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (HRP) (Cell Signaling Technology, Cat No. 70745) at 1:1000 dilution, following the iBIND apparatus instructions (Invitrogen). After 3 h of incubation, the membrane was developed using the Flash Western Chemiluminescence Kit (Azure Biosystems), and the signal was captured after 1–2 min with Azure c600 chemiluminescence imager (Azure Biosystems).

DNA extraction and Leishmania detection by qPCR

The whole organ was collected and macerated in 1 mL of DNA/RNA shield (Zymo). After maceration, DNA was extracted from 100–200 μL following the DNA/RNA Plus kit tissue protocol (Zymo). A total of 40 ng of DNA was used as template for all tissues. Amplification of kinetoplast minicircle DNA of Leishmania was achieved using primers JW11 (5’-CCTATT TTACACCAA CCCCCAGT-3’), JW12 (5’-GGGTAGGG GCGTTCTGCGAAA-3’), and TaqMan probe (5’-[Aminoc6+TxRed] RAAARKKVRTRCAGAAAYCCCGT [BHQ2]-3’) as previously described¹⁴. A standard curve was produced by spiking naive mice tissue with a known number of L. donovani parasites from culture. The number of parasites from experimental samples was determined by fitting the cycle threshold (Ct) values in the equation of the line. All samples were run in duplicate or triplicate in CFX96 Touch Real-Time PCR Detection System, Bio-Rad. The parasite burden per tissue was calculated per mL for all tissues with exception of the spleen and liver. For the spleen and the liver, the parasites per mL were calculated and then divided by the mean weight of the spleen or liver of unchallenged (MN or WN) animals (Fig. S3c).

Sysmex blood-cell analysis

A 100 μL of blood was collected in EDTA treated tubes (BD) and diluted 1:6 in PBS (Lonza). Diluted blood (60 μl) was run in the Sysmex XN 1000 hematology analyzer for the percent of neutrophils, monocytes, and lymphocytes in the blood.

Serum collection

Blood was collected in serum collection tubes (BD) by cardiac puncture at the experimental endpoint and centrifuged at 10,000 × g for 10 min. Serum was separated and aliquots were kept at −20 °C until analysis.

Chemical metabolic panel

A comprehensive chemical metabolic pathway was performed on the collected serum and analyzed using the Abaxis comprehensive diagnostic profile mouse panel (Cat No, 500-1038). Metabolic panel included the following analytes: total protein, albumin, globulin, glucose, amylase, alkaline phosphatase (AKP), alanine aminotransferase (ALT), and urea nitrogen.

HO-1 serum ELISA

Mouse serum was diluted using the kit’s diluent buffer at 1:2 ratio and the ELISA was performed following the recommendations of the mouse Heme Oxygenase 1 Simple Step ELISA Kit (Abcam, Cat No. ab204524).

Cytokine Luminex multiplex

A panel of 11 serum cytokines was analyzed using the ProcartaPlex Mouse Th1/Th2 Cytokine Panel, 11 plex kit (Invitrogen, Cat No. EPX110-20820-901). Briefly, serum was diluted at a 1:1 ratio and incubated with the magnetic beads overnight at 4 °C using an orbital shaker. The next day, the assay was performed following the manufacturer’s instructions and data were acquired in the Luminex 100 analyzer. The limit of quantification of each cytokine reported in pg/ml is as follows: IL-10 (7.67), IL-4 (1.42), IL-5 (2.26), IL-6 (4.88), IL17-A (1.45), IL-18 (4.6), IL-2 (1.81), IL-22 (11), IFN-γ (0.93), IL-12p70 (1.89), TNF-α (3.17).

Metagenomic 16S sequencing

Genomic DNA extraction

Genomic DNA was extracted from fecal samples using the Cetyltrimethylammonium bromide (CTAB) method. DNA concentration and purity was monitored by running a 1% agarose gel and DNA was diluted to 1 ng/µL in molecular graded water.

Amplicon generation

16S rRNA/18SrRNA/ITS genes of distinct regions (16SV4/16SV3/16SV3- V4/16SV4-V5, 18SV4/18SV9, ITS1/ITS2, ArcV4) were amplified using specific barcoded primers (e.g. 16SV4:515F- 806R, 18SV4: 528F-706R, 18SV9: 1380F-1510R). All PCR reactions were carried out with 15 μL of Phusion® High-Fidelity PCR Master Mix (New England Biolabs); 0.2 μM of forward and reverse primers, and 10 ng DNA. PCR conditions, initial denaturation at 98 °C for 1 min, followed by 30 cycles of denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s, and elongation at 72 °C for 30 s and 72 °C for 5 min. PCR products were purified.

Library preparation and sequencing

Sequencing libraries were generated, and indexes were added. Quality control of the DNA was checked by Qubit (Invitrogen), qPCR for quantification, and bioanalyzer. Quantified libraries were pooled and sequenced on Illumina platforms, according to effective library concentration and data amount required.

Bioinformatics analysis pipeline

Following sequencing, trimmed reads were imported into QIIME2 (v. 2021.4) for processing using the import command⁵⁶. Quality control, denoising, and ASV picking were performed using the DADA2 plugin⁵⁷. Trimming parameters were set to 0 as sequences had already been trimmed by the sequencing facility. To create phylogenetic trees, sequences were aligned with mafft⁵⁸ and were then used to construct a phylogeny with fasttree2⁵⁹ using the command align-to-tree-mafft-fasttree. Taxonomic classification was performed using the QIIME2 naive Bayes classifier, pre-trained on the Silva 138-99 database, containing trimmed sequences representing the region between the 341F/806R primers. The final ASV table, tree file, and taxonomy files were imported into R (v. 4.3.2) for downstream processing and analysis.

Files were combined into one R object using the phyloseq package (v 1.46.0)⁶⁰. We performed additional read processing, including removing reads that were not classified past the Kingdom level, removing reads assigned to Eukaryota and Archaea, and removing singletons. Rarefaction curves were also generated to determine whether sufficient read coverage had been achieved, following which samples were rarefied using the lowest sample read count (104,715). Lastly, ASVs with no taxonomic information were grouped together under the next taxonomic level containing information: i.e., if an ASV was classified at the Family level but not at the Genus level, the genus and species level information would be filled in as Family_NameOfASVFamily.

Alpha diversity plots showing both observed richness and Shannon Index values were created using the phyloseq and ggplot2 (v 3.4.4) packages⁶¹. Distance matrices were generated using phyloseq, and PCoA coordinates were calculated using the ape package (v 5.7-1)⁶². Additional PERMANOVA and PERMDISP tests were performed using the adonis2 and betadisper commands from the vegan package (v 2.6-4)⁶³. To create bar plots, the phyloseq object was transformed to a microtable object using the package file2meco (v 0.7), and plots were then generated using the microeco package (v 1.4.0)⁶⁴. The differential abundance analysis was performed using ANCOM-BC (v 2.4.0) and the corresponding raw abundance boxplots were generated using ggplot2^65,66.

Statistics and reproducibility

All experiments were conducted blinded, and the blinded code was broken post-data analysis. Statistical analysis was performed using R software. +p ≤ 0.9, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. Refer to Supplementary Data file 2 for the full statistical report.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.