Ethics statement

All the experimental animal work was performed in strict compliance to the guidelines of our institution’s animal ethics committee, who also approved this study (under authorization AWB_2015_09_MP_Malaria), and in accordance with the Federation of European Laboratory Animal Science Associations (FELASA) guidelines.

Experimental animals

Male C57BL/6J mice (6–8 weeks old) were purchased from Charles River Laboratories (Lyon, France). RAG2^-/-γc^-/- mice, kindly provided by the Instituto de Medicina Molecular João Lobo Antunes (iMM JLA, Lisbon, Portugal)’s Silva-Santos laboratory, were bred in specific-pathogen-free (SPF) facilities at iMM JLA. All animals were housed and kept in the SPF rodent facility of the iMM JLA, a licensed establishment that complies with the European Directive 2010/63/EU on the protection of animals used for scientific purposes.

Parasites and infections

The transgenic 90–13 cell-line of the Trypanosoma brucei brucei AnTaT 1.1 pleomorphic strain [31], as well as the monomorphic Lister 427 strain [32] (antigenic type MiTat 1.2, clone 221a, that co-expresses the T7 RNA polymerase and the tetracyclin repressor genes), were employed throughout this study. Bloodstream forms (BSF) were grown in HMI-11 medium at 37°C and 5% CO₂ [33]. Procyclic forms (PCF), obtained by differentiation with cis-aconitate [34] of the monomorphic Lister 427 strain, were grown in Differentiating Trypanosome Medium (DTM) at 27°C. BSF were employed in all the experiments, unless stated otherwise. Mice were infected by intraperitoneal (i.p.) injection of 2 x 10³ motile bloodstream parasites, obtained from cryostabilates of the pleomorphic strain, as previously described [12, 35].

GFP-[36] and Luciferase-[37]expressing Plasmodium berghei ANKA and Plasmodium yoelii yoelii 17XNL rodent malaria parasites were used throughout this study. For intravenous (i.v.) sporozoite injections, P. berghei and P. yoelii sporozoites were obtained by dissection of salivary glands from infected female Anopheles stephensi mosquitoes, reared at iMM JLA. Mice were infected by retro-orbital i.v. injection of 500 or 3 x 10⁴ sporozoites, under isoflurane anaesthesia.

Assessment of Experimental Cerebral Malaria (ECM) symptoms

ECM development was monitored daily using the rapid murine coma and behaviour scale (RMCBS) score, as described in [38]. Mice with RMCBS score equal or below 5/20 were classified as displaying ECM symptoms and euthanized immediately.

Assessment of Plasmodium parasitaemia

P. berghei parasitaemia was assessed using a modified version of the protocol described in Zuzarte-Luis V. et al [39]. Briefly, 5 μl of blood was collected from the tail vein into 45 μl of lysis buffer and preserved at -20°C. Parasite bioluminescence was measured by adding 50 μl of D-Luciferin dissolved in luciferase assay buffer to 15 μl of the blood lysate. Luminescence was immediately measured on a microplate reader (Tecan M200, Switzerland).

Quantification of Plasmodium hepatic infection in vivo

Mouse liver infection by Plasmodium was assessed 46 h after sporozoite inoculation and quantified by quantitative real-time reverse transcriptase-PCR (qRT-PCR), as previously described[40]. For qRT-PCR analyses, 0.7–0.9 mg of livers collected upon euthanasia of infected mice were mechanically homogenized in TRIzol (BioRad, Hercules, CA, USA), RNA was extracted following the manufacturer’s instructions, and converted into complementary DNA (cDNA) as described below. Liver Plasmodium load was quantified by qRT-PCR, as previously described[41], using primers specific for Plasmodium 18S rRNA (Table 1). Expression of the endogenous mouse housekeeping gene hypoxanthine-guanine phosphoribosyltransferase (Hprt) was used for normalization.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 1. List of primer sequences used for qRT-PCR.

https://doi.org/10.1371/journal.pntd.0009912.t001

RNA extraction, complementary DNA synthesis and qRT-PCR

RNA was extracted following the manufacturer’s instructions (BioRad, Hercules, CA, USA) and quantified using a NanoDrop DR 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA USA). cDNA was synthesized from 1 μg of RNA, using the NZYTech cDNA synthesis kit (NZYTech, Lisbon, Portugal), according to the manufacturer’s recommendations, and employing the following thermocycling parameters: 25°C for 10 min, 55°C for 30 min, and 85°C for 5 min. The qRT-PCR reaction was performed in a total volume of 10 μl employing an QuantStudio 5 equipment (Applied Biosystems, Foster City, CA, USA), using the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) and the following thermocycling parameters: 50°C for 2 min, 95°C for 10 min, 40 cycles at 95°C for 15 s and 60°C for 1 min; melting stage employed 95°C for 15 s, 60°C for 1 min, and 95°C for 30 s. Primer pairs used to detect target gene transcripts are listed in Table 1. Gene expression was analysed by the comparative CT (ΔΔCT) method and the expression level of all target genes was normalized to that of Hprt.

Serum transfer

Blood was collected by heart puncture of either naïve mice or mice infected 5 days earlier with 2 x 10³ T. brucei parasites. After clotting, samples were centrifuged at 1,000 x g for 10 min at room temperature (RT). Serum was transferred to a fresh tube and pooled together. 200 or 450 μl were administered into naïve mice by i.v. injection in the tail vein, 30 min prior to P. berghei sporozoite inoculation.

T. brucei lysate preparation

T. brucei lysates were obtained from the monomorphic Lister 427 strain. Trypanosomes were harvested by centrifugation at 3,000 x g for 15 min at 4°C, washed in phosphate-buffered saline (PBS), followed by centrifugation at 3,000 x g for 15 min at 4°C and resuspension in PBS. Trypanosomes were kept on ice and mechanically disrupted, using the Soniprep 150 (MSE, London, UK), following a sequence of 6 cycles of 1 min 30 s sonication at maximum intensity and 30 s pauses. Parasite lysis was confirmed by microscopy, flash frozen in liquid nitrogen and stored at -80°C. T. brucei lysates were inoculated into mice through i.v. injection in the tail vein. Livers were collected from mice 46 h after sporozoite injection and processed as described above for the quantification of Plasmodium liver load by qRT-PCR.

Immunofluorescence microscopy analysis of in vivo P. berghei hepatic infection

Livers were collected at 46 h after P. berghei sporozoite inoculation and fixed in 4% (v/v) paraformaldehyde (PFA) (SantaCruz Biotechnology, Dallas, TX, USA) for at least 12 h at RT. Liver sections of 50 μm thickness were stained and analyzed as previously described [40, 42]. Briefly, slices were incubated in permeabilization/blocking solution containing 1% (w/v) bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) and 0.5% (v/v) Triton-X100 in PBS (Sigma-Aldrich) at RT for 1 h, followed by a 2 h incubation at RT with an anti-UIS4 antibody (goat, homemade; dilution 1:500). Liver sections were further incubated for 1 hour in a 1:500 dilution of anti-GFP-Alexa 488 antibody (Invitrogen, Carlsbad, CA, USA) and anti-goat Alexa-Fluor 568 (Invitrogen, Carlsbad, CA, USA) in the presence of a 1:1,000 dilution of Hoechst 33342 (Invitrogen, Carlsbad, CA, USA). After washing, liver sections were mounted on microscope slides with Fluoromount (SouthernBiotech, Birmingham, AL, USA). Widefield images for the determination of the size of P. berghei intrahepatic forms were acquired employing a Zeiss Axiovert 200M microscope (Carl Zeiss, Oberkochen, Germany). Confocal images were acquired using a Zeiss LSM 510 confocal microscope (Carl Zeiss, Oberkochen, Germany). Images were processed with the ImageJ software (version 1.47, NIH, Bethesda, MD, USA).

Liver histopathology

Livers collected 46 h after P. berghei sporozoite inoculation were formalin-fixed in neutral buffered formalin, paraffin-embedded, cut in 4 μm sections, and stained with hematoxylin (Bio-Optica, Milan, Italy) and eosin (Thermo Fisher Scientific, Waltham, MA USA). Tissue sections were analyzed by a pathologist blinded to experimental groups, using a Leica DM2000 microscope coupled to a Leica MC170 HD microscope camera (Leica Microsystems, Wetzlar, Germany).

Biochemical assessment of liver pathology

Blood was collected from mice either naïve, infected 5 days earlier with T. brucei, or injected 30 min earlier with T. brucei lysates. Blood was collected at the time of P. berghei sporozoite injection, 12 h and 24 h later. After clotting, samples were centrifuged at 1,000 x g for 10 min at 4°C, and serum was transferred to a fresh tube. Liver pathology was assessed by quantification of serum levels of alanine transaminase (ALT), aspartate transaminase (AST), bilirubin (total) and glucose (Idexx Bioresearch, Stuttgart, Germany).

In vivo depletion of macrophages

Macrophages were depleted by i.v. injection of 1 mg of liposome-encapsulated clodronate (Liposoma B.V., Amsterdam, The Netherlands), 48 h prior to P. berghei sporozoite inoculation. An equivalent volume of liposome-encapsulated PBS was injected i.v. into control mice. Livers were collected 46 h after sporozoite injection and processed as described above for the quantification of P. berghei liver load by qRT-PCR.

In vivo inhibition of monocyte migration

Monocyte recruitment was inhibited by i.p. injection of 20 μg anti-CCR2 antibody, kindly provided by Matthias Mack (University Hospital Regensburg), 48 and 24 h prior to inoculation of P. berghei sporozoites, at the time of infection and 24 h later. An equivalent volume of PBS was injected i.p. into control mice. Livers were collected 46 h after sporozoite injection and processed as described above for the quantification of the P. berghei liver load by qRT-PCR.

Isolation and quantification of liver monocytes by flow cytometry

Livers were collected by dissection of mice at 46 hours after P. berghei sporozoite inoculation, mechanically homogenized in 5 ml of PBS containing 2 U/ml DNAse (Sigma-Aldrich, St. Louis, MO, USA), using a 100 μm cell strainer, and centrifuged at 410 x g for 8 min at RT. The cell pellet was fractionated using 10 ml of 35% (v/v) of Percoll gradient medium diluted in non-supplemented RPMI (Gibco-Thermo Fisher Scientific, Waltham, MA USA), followed by centrifugation at 1,360 x g for 20 min without brake at 20°C. The cells deposited in the pellet were washed with PBS and centrifuged at 410 x g for 8 min at RT. The cell pellet was depleted of red blood cells (RBCs) by incubation in 3 ml of ammonium-chloride-potassium solution, following incubation for 3 min at RT and subsequent inactivation with 7 ml of PBS 2% FBS (FACS buffer). The leukocyte suspension was centrifuged at 410 x g for 8 min at RT and resuspended in 500 μl of PBS for subsequent staining. One million leukocytes from each mouse were then plated in 96-well plates, centrifuged at 845 x g for 3 min at 4°C and incubated with α-CD16/CD32 (eBioscience/Thermo Fisher Scientific, Waltham, MA, USA) for 20 min at 4°C. For the staining of lymphoid surface antigens, cells were incubated for 20 min at 4°C with FACS buffer containing Fixable Viability Dye (eFluor 780) and the following conjugated flow cytometry monoclonal antibodies: FITC anti-CD11b (clone M1/70), BV605-Ly6C (clone HK1.4), BV785-MHC II (clone M5/114.15.2), PE-Cy7-Ly6G (clone 1A8) and PE-CD11c (clone N418). All antibodies were from eBioscience (Thermo Fisher Scientific, St. Louis, MO, USA) or Biolegend. Cells were acquired on a FACS LSRFortessa X-20 (BD Biosciences Franklin Lakes, New Jersey, U.S.) and data acquisition and analysis were carried out using the FACSDiva (version 6.2) and FlowJo (version 10.7, FlowJo) software packages, respectively.

In vivo hematopoietic cell ablation

Lymphoid and myeloid cell ablation was obtained by submitting mice to whole-body γ-radiation, using the Gammacell 3000 ELAN irradiator (Elan Drug Technologies, Dublin, Ireland), at a dose of 9 Gy for 2 min at room temperature, 24 h prior to P. berghei sporozoite inoculation. Livers were collected 46 h after sporozoite injection and processed as described above for the quantification of the P. berghei liver load by qRT-PCR.

T. brucei Lysate digestion with DNAse

DNA present in the lysates was digested in PBS containing 20 U/ml of DNAse (Sigma-Aldrich, St. Louis, MO, USA) and 10% (v/v) of enzyme buffer, following incubation for 2 h at 37°C and subsequent DNAse inactivation at 75°C for 10 min. Untreated lysates were submitted to the same incubation periods and temperatures. Treated or untreated lysates of 1 x 10⁸ trypanosomes (200 μl) were injected i.v. into mice 30 min prior to P. berghei sporozoite inoculation. An equivalent volume of PBS containing inactivated DNAse was injected i.v. into control mice.

T. brucei Lysate digestion with proteinase K

Proteins present in the lysates were digested in PBS containing 250 μg/ml of proteinase K (Promega, Wisconsin, USA) and 30% of digestion buffer (0.01 M Tris, 5 mM ethylenediamine tetraacetic acid [EDTA], 0.5% sodium dodecyl sulphate [SDS]), following overnight incubation at 50°C and subsequent proteinase K inactivation at 95°C for 10 min. Untreated lysates were submitted to the same incubation periods and temperature. Treated or untreated lysates of 10⁸ trypanosomes (200 μl) were injected i.v. into mice 30 min prior to P. berghei sporozoite inoculation. An equivalent volume of PBS containing inactivated proteinase K was injected i.v. into control mice.

Trypanosome digestion with phospholipase C (PLC)

Trypanosomes were harvested and pelleted as described above and suspended in PBS containing 0.5 U/ml of PLC from C. perfringens (Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 1 x 10⁸ trypanosomes/100 μl. The parasite suspension was incubated for 30 min at 37°C and then centrifuged at 10,000 x g for 30 s, cleaving and separating the glycosylphosphatidylinositol (GPI)-anchored proteins (supernatant) from the residual intact parasites (pellet). The parasite pellet was suspended in PBS. The PLC present in the soluble GPI-anchored proteins suspension was inactivated at 95°C for 5 min. The equivalent to 10⁸ trypanosomes (200 μl) was injected i.v. into mice 30 min prior to P. berghei sporozoite inoculation. An equivalent volume of PBS containing inactivated PLC was injected i.v. into control mice.

sVSG purification T. brucei lysates

Soluble VSG (sVSG) was obtained through cleavage of membrane form VSG by the endogenous GPI-PLC. T. brucei were grown as described above, transferred to Falcon tubes and incubated on ice for 10 min. The parasites were then centrifuged at 2,500 x g, 0°C, 10 min, washed with trypanosome dilution buffer (20 mM Na₂HPO₄, 2 mM NaH₂PO₄, 80 mM NaCl, 5 mM KCl, 1 mM MgSO₄, 20 mM glucose; pH adjusted to 7.7) and resuspended in a, pre-warmed at 37°C, 10 mM sodium phosphate buffer (pH 8.0) containing 2 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich) and 1:200 Protease Inhibitor Cocktail (PIC) (Sigma-Aldrich), followed by incubation at 37°C for 5 min. The parasite suspension was then centrifuged at maximum speed for 5 min, the supernatant was collected and passed through a column of DE52, pre-equilibrated in 10 mM sodium phosphate (pH 8.0). The eluate containing the sVSG was washed with Milli-Q H₂O (Millipore, Massachusetts, USA) and concentrated using an Amicon Ultra-15 3 kDa (Millipore, Massachusetts, USA). The purity of sVSG was analysed by SDS-PAGE stained with Coomassie Blue. 100 μg of sVSG, equivalent of 1 x 10⁸ trypanosomes, was injected i.v. into mice 30 min prior to P. berghei sporozoite inoculation. An equivalent amount of albumin (Sigma-Aldrich, St. Louis, MO, USA) in PBS was injected i.v. into control mice. Livers were collected 46 h after sporozoite injection and processed as described above for the quantification of P. berghei liver load by qRT-PCR.

Excretion/Secretion of T. brucei proteins

Trypanosomes were harvested and pelleted as described above and incubated in PBS for 1 h at RT at a concentration of 10⁸ trypanosomes/200 μl. The parasite suspension was then centrifuged at 10,000 x g for 30 s, separating excreted/secreted proteins (supernatant) from the parasites (pellet). The equivalent to 10⁸ trypanosomes (200 μl) was injected i.v. in the tail vein of mice 30 min prior to inoculation of P. berghei sporozoites.

Extracellular vesicle purification

Trypanosomes were harvested and pelleted as described above. The supernatant was filtered (0.2 μm) to remove remaining parasites and cell debris. Extracellular vesicles (EVs) were collected by ultracentrifugation at 100,000 x g for 70 min, as described in [43]. EVs were stored in PBS at 4°C. Each mouse was injected with EVs excreted by 10⁸ trypanosomes.

Transmission electron microscopy

EVs were incubated on a glow-discharged, carbon-formvar 100-mesh TEM grid and fixed in 4% formaldehyde in 0.2M Phosphate buffer for 5 min. Samples were washed with dH2O and incubated for 5 min with 2% aqueous uranyl acetate. TEM micrographs were acquired on a Tecnai G2 Spirit BioTWIN TEM (FEI) operated at 120 kV using a side mount 2k x 2k Olympus-SIS Veleta CCD camera.

Mass spectrometry analysis

Protein mass spectrometry was carried out by the inhouse facility at the University of St. Andrews. Samples were reduced with DTT, alkylated with iodoacetamide and digested at 37°C with trypsin. Samples in gels were extracted from diced gel pieces in 5% formic acid to an extraction volume of 20 μl. A proportion of the sample was injected onto an eksigent nano-LC set up in ‘trap elute’ configuration using a Pepmap column and trap (ThermoScientific). Peptides were eluted in a linear gradient over 180 min and flowed directly into a Sciex 5600+ Q-Tof mass spectrometer. Survey scans were carried out between 400–1200 m/z and the strongest 15 peptides from each scan were fragmented to give MS-MS spectra from 100–2000 m/z. Spectra were extracted using the mgf generator script from Sciex and the data searched using the mascot search algorithm against T. brucei strain 427 genome and NCBI databases.

Gene ontology analysis

Gene ontology (GO) categories related to biological processes, molecular function and cell component were identified using TopGo, based on the annotated GO term list for T. brucei TREU927 genome version 2.1 available in TritrypDB [44]. Enriched GO terms among detected proteins were established using the weight01 algorithm and Fisher’s exact test at a false discovery rate (FDR) p-value ≤ 0.05.

Statistical analyses

Data are expressed as mean ± standard error of the mean (SEM). Statistical analyses were performed using the GraphPad Prism 6 software (La Jolla, CA, USA). Statistically significant differences were determined using One-way analysis of variance (ANOVA), Log- Rank (Mantel-Cox) test, Two-tailed Mann-Whitney test or Unpaired t test. Statistical significances are represented as indicated in each figure: ns–not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Total lysates of T. brucei protect mice against P. berghei liver infection

Having previously shown that an ongoing infection by T. brucei impairs a secondary P. berghei hepatic infection [27], we initially assessed whether the observed protection could be transferred to a naïve mouse through passive transfer of the serum of a T. brucei-infected animal. To this end, 200 and 450 μl of serum from donor mice, either naïve or infected 5 days earlier with T. brucei, were collected and injected i.v. into naïve recipient mice. Thirty min later, the recipient mice were inoculated with P. berghei sporozoites and its hepatic infection was assessed by qRT-PCR 46 h later. We found that passive transfer of serum from T. brucei-infected animals into naïve mice led to a dose-dependent impairment of P. berghei hepatic infection relative to those that received serum from healthy animals (Fig 1A).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 1. Total lysates of T. brucei protect mice against P. berghei liver infection.

(A) P. berghei liver infection load quantified by qRT-PCR 46 h after injection of P. berghei sporozoites into mice that received 200 or 450 μl of serum from naïve mice (black symbols), or from mice infected 5 days earlier with T. brucei (green symbols). Symbols represent the individual values of each mouse in one independent experiment and error bars indicate the SEM. (B) Schematic illustration of the experimental design: (i) P. berghei single infection consists of an i.v. injection of sporozoites (Pb—black) and collection of livers 46 h later, (ii) Co-infection consists of infection with T. brucei parasites (Tb/Pb—green) by i.p. injection 5 days prior to sporozoite inoculation, and liver collection 46 h later (iii) T. brucei (TbL/Pb—blue) and P. berghei (PbL—grey) total lysates injection consists of an i.v. injection of the respective lysates 30 min prior to sporozoite inoculation and subsequent liver collection 46 h later. (C) Liver load was determined by qRT-PCR 46 h post-sporozoite inoculation in mice infected with P. berghei (Pb—black symbols), mice infected with T. brucei 5 days prior to sporozoite inoculation (Tb/Pb—green symbols; 2 independent experiments), mice previously injected with three different amounts of T. brucei total lysates (TbL/Pb—blue symbols; 2 independent experiments) or mice injected with three different amounts of blood stage P. berghei total lysates (grey symbols; 1 independent experiment). Symbols represent the Plasmodium parasite load of individual mice. Error bars indicate the SEM. (D) Number of P. berghei-infected hepatocytes per square millimetre of liver section quantified by immunofluorescence microscopy 46 h after injection of P. berghei sporozoites into naïve mice (Pb—black symbols), mice infected 5 days earlier with T. brucei (Tb/Pb—green symbols), or that received T. brucei total lysates prior to Plasmodium sporozoites (TbL/Pb—blue symbols). Symbols represent the values of individual mice from one independent experiment and error bars indicate the SEM. (E) Representative confocal microscopy images of EEFs 46 h after injection of P. berghei sporozoites into naïve mice, mice infected 5 days earlier with T. brucei or mice that received T. brucei total lysates. Blue: Hoechst—nuclear; staining; green: P. berghei GFP labelling showing the parasite UIS4 protein. Scale bars, 10 μm (F) EEF area assessed by immunofluorescence microscopy, at 46 h after injection of P. berghei sporozoites into naïve mice (Pb -black symbols), mice infected 5 days earlier with T. brucei (Tb/Pb—green symbols), or mice that received T. brucei total lysates (TbL/Pb—blue symbols). Results are expressed as mean values of one experiment and error bars indicate the SEM. (G) Assessment of P. berghei prepatency period following inoculation of P. berghei sporozoites into naïve mice (Pb—black line), mice infected 5 days earlier with T. brucei (Tb/Pb—green line), or mice that previously received T. brucei total lysates (TbL/Pb—blue line). Percentage of mice displaying P. berghei parasitaemia, as measured by bioluminescence. The pooled data of 10 mice from two independent experiments is shown. (H) Mouse survival following inoculation of P. berghei sporozoites into naïve mice (Pb—black line), mice infected 5 days earlier with T. brucei (Tb/Pb—green line), or mice that received T. brucei total lysates (TbL/Pb—blue line). Percentage of live mice from a pool of 10 mice employed in two independent experiments. For (C), (D) and (E) the one-way ANOVA with post-test Dunnett was employed to assess the statistical significance of differences between experimental groups. ns, not significant, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. For (G) and (H) the Mantel-Cox (log rank) test was employed to compare the onset of P. berghei parasitaemia.

https://doi.org/10.1371/journal.pntd.0009912.g001

Knowing that the serum contains components from both the host and the parasites, we sought to assess whether the inhibitory component of the serum was trypanosome-derived. To this end, total lysate suspensions of 10⁶, 10⁷ and 10⁸ mechanically lysed trypanosomes were injected intravenously into mice 30 min prior to inoculation of 3 x 10⁴ P. berghei sporozoites (Fig 1B). In parallel, equivalent suspensions of total lysates of P. berghei blood stages were injected into mice (Fig 1B), to test the specificity of the effect of the trypanosome lysates, and to control for the injection of high doses of parasite-derived molecules. Our results show that T. brucei total lysates are able to impair a P. berghei liver infection in a dose-dependent manner, with the suspension containing 10⁸ trypanosomes showing the highest level of protection (~80%) (Fig 1C). This effect appears to be trypanosome-specific, since the control mice that received the P. berghei blood stage lysates displayed a hepatic infection similar to that of animals infected with Plasmodium only. Additionally, we assessed the capacity of trypanosome total lysates to impair hepatic infection by another Plasmodium species, P. yoelii. Our results show that T. brucei infection and T. brucei lysates had a similar ~76% inhibitory effect on P. yoelii liver infection (S1 Fig). Altogether, our results show that T. brucei total lysates administered to mice prior to sporozoite inoculation significantly reduce a subsequent Plasmodium hepatic infection. Moreover, our data suggest that the inhibitory effect is trypanosome-specific and that it is not limited to a single Plasmodium species.

Having observed that the administration of T. brucei total lysates leads to a significant decrease in a subsequent P. berghei hepatic infection, we next investigated whether this effect was due to a reduction in the number of P. berghei-infected hepatocytes or to a defect in the parasite’s ability to replicate inside the hepatocytes. To this end, we analysed sections of livers collected from mice infected only with P. berghei, co-infected with both parasites, or injected with the T. brucei total lysates prior to P. berghei sporozoite inoculation, by immunofluorescence microscopy. We observed that both the co-infected mice and those that received the T. brucei total lysates show a ~75% reduction in the number of infected hepatocytes, when compared to the control mice infected only with P. berghei (Fig 1D). Conversely, no significant differences were observed in the size of the P. berghei EEFs in the three groups of mice (Fig 1E and 1F). Collectively, these data indicate that the impairment of P. berghei liver infection by T. brucei total lysates results from a decrease in the number of infected hepatocytes rather than from an impairment of the intrahepatic replication of Plasmodium parasites.

Next, we investigated whether the reduction in liver infection impacted the ensuing Plasmodium blood stage of infection [27]. To this end, animals were infected by i.v. injection of 500 luciferase-expressing P. berghei sporozoites in order to mimic the inoculum delivered by the bites of five infected mosquitoes [5]. As expected based on previous observations, 100% of the co-infected mice used as controls in this experiment were protected against Plasmodium erythrocytic infection (Fig 1G) [27]. Conversely, 90% of the mice that received the T. brucei total lysates developed an erythrocytic infection. Crucially, however, the prepatent period in these mice was one to two days longer than in mice infected only with P. berghei, indicating that, as expected, the reduction in the P. berghei liver burden in the mice receiving T. brucei total lysates leads to a delay in the onset of Plasmodium parasitaemia [39] (Fig 1G). Also of note, whereas 60% of P. berghei single infected mice died within 8 to 10 days after sporozoite inoculation with signs of experimental cerebral malaria (ECM), only 20% of the mice that received the T. brucei total lysates displayed ECM symptoms or succumbed within that time period (Fig 1H).

Overall, our data show that injection of T. brucei total lysates prior to sporozoite inoculation enhances resistance to P. berghei infection. T. brucei total lysates lead not only to a reduction in P. berghei liver burden, but also to a delay in the appearance of Plasmodium parasitaemia and protection against the development of severe malaria pathology.

The protective effect of T. brucei lysates is time-dependent

Knowing that P. berghei hepatic infection can be impaired by T. brucei total lysates in the absence of an active infection, we next investigated the duration of this inhibitory effect following injection of trypanosome lysates into mice. To achieve this, T. brucei total lysates were injected into mice 48 h, 24 h, 12 h, 6 h and 30 min prior to sporozoite inoculation, and P. berghei liver infection was assessed 46 h after sporozoite injection (Fig 2A). The strongest reduction of P. berghei hepatic infection was observed when the T. brucei total lysates were injected 30 min prior to sporozoite inoculation, and the approximate half maximal inhibitory effect was observed when sporozoites were injected 12 h after lysate administration (Fig 2B). These results show that the protective effect of T. brucei total lysates is time-dependent, since this protection decreases as the period between the injection of the trypanosome lysates and the sporozoites increases.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 2. The protective effect of T. brucei lysates is time dependent.

(A) Schematic illustration of the experimental design: (i) P. berghei single infection group consists of an i.v. injection of sporozoites (Pb—black) and collection of livers 46 h later, (ii) Co-infection consists of infection with T. brucei parasites (Tb/Pb—green) by i.p. injection 5 days prior to sporozoite inoculation, and liver collection 46 h later (iii) T. brucei total lysates group consists of an i.v. injection 30 min, 6 h, 12 h, 24 h, and 48 h prior to sporozoite inoculation (TbL/Pb—blue) and 6 h post sporozoite inoculation (light green) and subsequent liver collection 46 h later. (B) P. berghei liver infection quantified by qRT-PCR, 46 h post-sporozoite inoculation into naïve mice (Pb—black symbols), mice infected with T. brucei 5 days prior to sporozoite inoculation (Tb/Pb—green symbols), or mice that received T. brucei total lysates 30 min, 6 h, 12 h, 24 h and 48 h prior to P. berghei sporozoites inoculation (TbL/Pb—blue symbols). Symbols represent the individual values of each mouse in two independent experiments for T. brucei total lysates and one experiment for P. berghei total lysates, and error bars indicate the SEM. (C) P. berghei liver infection determined by qRT-PCR, 46 h post-sporozoite inoculation into naïve mice (Pb—black symbols), mice infected with T. brucei 5 days prior to sporozoite inoculation (Tb/Pb—green symbols), mice that received T. brucei total lysates 30 min prior to P. berghei sporozoites inoculation (TbL/Pb—blue symbols), or mice that received T. brucei total lysates 6 h post-P. berghei sporozoite inoculation (Pb/TbL—green symbols). Symbols represent the individual values of each mouse in two independent experiments and error bars indicate the SEM. For (B) and (C) one-way ANOVA with post-test Dunnett was employed to assess the statistical significance of differences between experimental groups. ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

https://doi.org/10.1371/journal.pntd.0009912.g002

Having shown the impact of the injection of T. brucei total lysates prior to P. berghei sporozoite inoculation, we then wondered whether the trypanosome lysates would impact a pre-established P. berghei liver infection. To evaluate this, T. brucei total lysates were injected into mice 6 h after the injection of P. berghei sporozoites (Fig 2A). Our results showed that trypanosome lysates inhibit an already established P. berghei liver infection, albeit to an extent lower than that observed when the injection of T. brucei total lysates is performed prior to sporozoite inoculation (Fig 2C).

Altogether, our results indicate not only that the protection elicited by T. brucei total lysates against P. berghei liver infection is time-dependent, but also that the trypanosome lysates are able to inhibit a recently established P. berghei liver infection.

T. brucei lysates do not cause major liver damage

It has been previously described that an infection by T. brucei results in liver damage and impaired hepatic function [27, 45, 46]. Thus, we hypothesized that these hepatic alterations could be the cause of the decreased number of P. berghei-infected hepatocytes following the injection of T. brucei total lysates. To evaluate the hepatocellular damage resulting from the administration of T. brucei lysates, histopathological analyses of livers collected from both groups of animals at 46 h of P. berghei hepatic infection were performed. As expected, the livers of co-infected mice displayed hepatocyte damage and immune cell infiltrates, namely mononuclear inflammatory cells. Conversely, the livers of mice that received trypanosome lysates, similarly to those of mice infected only with Plasmodium, displayed minimal tissue damage and no infiltration of immune inflammatory cells (Fig 3A).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 3. T. brucei lysates do not severely compromise liver function.

(A) Representative microphotographs of liver 46 h after injection of P. berghei sporozoites into naïve mice, mice infected 5 days earlier with T. brucei, or mice that received T. brucei total lysates; depicted are the inflammatory cell infiltrates (“i”), hepatocellular damage/apoptosis (“n”) and P. berghei EEFs (“p”). Hematoxylin and Eosin. Original magnification 10x. (B) Quantification of levels of biochemical parameters in serum. The graphics show the ALT, AST, bilirubin and glucose quantification in serum of naïve mice (Naïve—red dots), mice infected 5 days earlier with T. brucei (Tb—green lines) or mice that received lysates of trypanosomes (TbL—blue lines). The time points indicated on the XX-axis correspond to the hours of P. berghei infection, if sporozoites had been injected. Dots represent the mean values of three to six mice from one independent experiment, with error bars indicating the SEM.

https://doi.org/10.1371/journal.pntd.0009912.g003

Although T. brucei lysates did not promote microscopically detectable hepatocyte apoptosis, liver function could still be compromised. Thus, we measured several biochemical parameters in naïve mice, mice infected with T. brucei or previously injected with T. brucei total lysates, at a time-point corresponding to the 46 h of P. berghei hepatic infection. In agreement with previous reports, we observed that mice infected by T. brucei display increased alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin and decreased glucose levels compared to naïve mice, suggestive of severe liver injury [47–49]. Conversely, mice that received trypanosome lysates did not display altered serum levels for these biochemical markers, relative to naïve mice (Fig 3B).

Collectively, our data indicate that trypanosome lysates do not induce hepatocyte damage, nor do they severely compromise liver function, hence suggesting that the reduction in P. berghei hepatic load by T. brucei infection does not result from lysate-induced liver injury.

The impairment of P. berghei liver infection by T. brucei lysates is independent of lymphoid and myeloid immune cells

To assess the mechanism of impairment of P. berghei liver infection by T. brucei total lysates, we next assessed the possible involvement of the immune response in this process. To test a possible role of lymphocytes, RAG2^-/-γc^-/- mice, which are genetically deficient for both T, B, natural killer and innate lymphoid cell lymphocytes, were injected with T. brucei total lysates prior to sporozoite inoculation. The quantification of P. berghei hepatic infection at 46 hpi showed that co-infected and trypanosome lysate-injected RAG2^-/-γc^-/- mice displayed a reduction of P. berghei hepatic infection (~80% and 65%, respectively) similar to that observed in wild-type mice (Fig 4A). This result suggests that lymphocytes do not mediate the impairment of P. berghei liver infection by T. brucei lysates.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 4. Impairment of P. berghei liver infection by T. brucei lysates is independent of lymphoid and myeloid immune cells.

(A) P. berghei liver infection quantified by qRT-PCR 46 h after injection of P. berghei sporozoites into wild-type and RAG2^-/-γc^-/- mice, either naïve (Pb—black symbols), infected 5 days earlier with T. brucei (Tb/Pb—green symbols), or mice that received T. brucei total lysates (TbL/Pb—blue symbols) 30 min prior to sporozoite inoculation. Symbols represent the individual values of each mouse of one independent experiment and error bars indicate the SEM. (B) P. berghei liver infection quantified by qRT-PCR 46 h after injection of P. berghei sporozoites into naïve mice (Pb—black symbols), infected 5 days earlier with T. brucei (Tb/Pb—green symbols), or mice that received T. brucei total lysates (TbL/Pb—blue symbols) 30 min prior to sporozoite inoculation, non- or clodronate-treated 48 h prior to P. berghei infection. Symbols represent the individual values of each mouse of one independent experiment and error bars indicate the SEM. (C) P. berghei liver infection quantified by qRT-PCR 46 h after injection of P. berghei sporozoites into naïve mice (Pb—black symbols), infected 5 days earlier with T. brucei (Tb/Pb—green symbols), or mice that received T. brucei total lysates (TbL/Pb—blue symbols) 30 min prior to sporozoite inoculation, administered or not with anti-CCR2. Symbols represent the individual values of each mouse of one independent experiment and error bars indicate the SEM. (D) P. berghei liver infection quantified by qRT-PCR 46 h after injection of P. berghei sporozoites into non- and γ-irradiated mice, either naïve (Pb—black symbols), infected 5 days earlier with T. brucei (Tb/Pb—green symbols), or mice that received T. brucei total lysates (TbL/Pb—blue symbols) 30 min prior to sporozoite inoculation. Symbols represent the individual values of each mouse of one independent experiment and error bars indicate the SEM. The one-way ANOVA with post-test Dunnett was employed to assess the statistical significance of differences between groups. * p<0.05, ** p<0.01 and ***p<0.001.

https://doi.org/10.1371/journal.pntd.0009912.g004

It has been previously shown that macrophages are not involved in the impairment of P. berghei liver infection by a primary T. brucei infection [27]. To investigate whether this is also the case following injection of T. brucei total lysates, phagocytic cells from either naïve mice or from mice that received T. brucei total lysates were depleted by injection of clodronate-filled liposomes 48 h prior to sporozoite inoculation [50]. The efficiency of depletion was confirmed by the quantification of liver mRNA levels of specific macrophage marker genes (Clec4f, F4/80 and CD68) [51] (S2A Fig). We found that phagocytic cell-depleted mice that received T. brucei total lysates displayed a reduced P. berghei liver infection, similar to that observed in their non-treated counterparts (Fig 4B), indicating that the impairment of P. berghei hepatic infection by T. brucei total lysates is independent of macrophages.

Even though the observed transcript levels of Clec4f indicated that very few liver-resident macrophages remained in the liver after clodronate treatment, the fact that the mRNA expression of the other marker genes employed was not negligible may suggest monocyte recruitment to the liver. Thus, in order to evaluate whether liver-infiltrated monocytes were responsible for the inhibition of P. berghei liver infection following injection of T. brucei lysates, monocyte recruitment was blocked by administration of anti-CCR2 antibody 48 and 24 h prior to sporozoite inoculation to either naïve mice, T. brucei-infected mice or mice that received T. brucei total lysates. The ability of the antibody to prevent monocyte recruitment to the liver was confirmed by flow cytometry analysis, which showed that the number of monocytes in the liver was decreased in all groups of mice, relative to their non-treated counterparts (S2B Fig). The quantification of P. berghei hepatic load revealed that mice that received the anti-CCR2 antibody and either lysed or live trypanosomes presented a marked impairment of Plasmodium liver infection, relative to the respective control mice (Fig 4C), indicating that monocytes recruited to the liver are not involved in the phenotype under study.

Redundant or compensatory mechanisms are likely to occur when a subset of cells is depleted [52, 53]. In order to address this issue, mice were immunosuppressed by whole-body lethal γ-irradiation, which markedly decreases the number of circulating leukocytes and mitotically active cells, including bone-marrow hematopoietic cells [54, 55], 24 h prior to sporozoite injection. γ-irradiated mice infected only with P. berghei showed a higher Plasmodium parasite load than their non-γ-irradiated counterparts (S2C Fig), confirming the success of the immunosuppression. We found that γ-irradiated mice that received T. brucei total lysates prior to sporozoite inoculation displayed a reduction in P. berghei infection load relative to the respective control mice (Fig 4D), suggesting that neither lymphoid nor myeloid cells are involved in the trypanosome-mediated inhibitory effect.

We conclude that the impairment of P. berghei liver infection caused by T. brucei total lysates is independent of lymphoid and myeloid immune cells.

The impairment of P. berghei liver infection is mediated by a protein component of T. brucei lysates

A cell lysate is a complex mixture that includes proteins, nucleic acids and lipids, among other molecules. To assess whether trypanosome-derived DNA or proteins were responsible for the reduction of P. berghei hepatic infection, lysates treated with either DNAse or proteinase K to degrade their DNA or proteins, respectively, were administered to mice 30 min prior to P. berghei sporozoite injection, and the Plasmodium liver load was quantified (Fig 5A). We found that mice that received DNAse-treated lysates displayed a ~70% reduction in parasite load relative to mice infected only with P. berghei, similar to that observed in mice that received whole lysates. In contrast, T. brucei lysates treated with proteinase K did not impair P. berghei infection, indicating that the trypanosome lysate component responsible for inhibiting Plasmodium infection is a protein.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 5. The impairment of P. berghei liver infection by total lysates is mediated by their T. brucei protein component.

(A) P. berghei liver infection load quantified by qRT-PCR 46 h after injection of sporozoites into naïve mice (Pb—black symbols), mice infected 5 days earlier with T. brucei (Tb/Pb—green symbols), or mice that received either the T. brucei total lysates (TbL/Pb—blue symbols), treated or not with DNAse or proteinase K, or the respective vehicles 30 min prior to sporozoite inoculation. Symbols represent the individual values of each mouse of two independent experiments and error bars indicate the SEM. (B) P. berghei liver infection load quantified by qRT-PCR 46 h after injection of sporozoites into naïve mice (Pb—black symbols), mice that received either T. brucei total lysates (TbL/Pb—blue symbols), mice that received sVSG (sVSG/Pb—red symbols), or mice that received albumin (Albumin/Pb—dark blue symbols) 30 min prior to sporozoite inoculation. Symbols represent the individual values of each mouse of two independent experiments and error bars indicate the SEM. (C) P. berghei liver infection load quantified by qRT-PCR 46 h after injection of sporozoites into naïve mice (Pb—black symbols), mice that received T. brucei total lysates (TbL/Pb—blue symbols), trypanosome GPI-surface proteins (TbGPI-anchored surface proteins/Pb—dark purple symbols), T. brucei lysates without GPI-surface proteins (TbL without GPI-anchored surface proteins/Pb—light purple symbols) or the respective vehicle (inactivated PLC/Pb—pink symbols) 30 min prior to sporozoite inoculation. Symbols represent the individual values of each mouse of three independent experiments and error bars indicate the SEM. (D) P. berghei liver infection load quantified by qRT-PCR 46 h after injection of sporozoites into naïve mice (Pb—black symbols), mice that received T. brucei total lysates (TbL/Pb—blue symbols), trypanosome excreted proteins (TbExcreted Proteins/Pb—yellow symbols) 30 min prior to sporozoite inoculation. Symbols represent the individual values of each mouse of five independent experiments and error bars indicate the SEM. (E) Enriched GO terms among excreted T. brucei proteins. Biological process category included. (F) Venn diagram of the comparative analysis between T. brucei published secretome (30) (purple) and the excreted proteins identified in this study (yellow). (G) Venn diagram of the comparative analysis between T. brucei proteins identified in plasma form T. b. rhodesiense infected patients (29) (green) and excreted proteins identified in this study (yellow). For (A) to (D) The one-way ANOVA with post-test Dunnett was employed to assess the statistical significance of differences between groups. ns, not significant, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

https://doi.org/10.1371/journal.pntd.0009912.g005

VSG is the most abundant protein in the bloodstream forms of T. brucei, comprising ~10% of total protein content, and it is anchored to the plasma membrane through a glycosylphosphatidylinositol (GPI) [56–59]. Since VSG has been previously found in the parasite‘s excretome [30], we investigated whether this protein was responsible for the observed inhibitory effect on P. berghei hepatic infection. To this end, the soluble form of VSG (sVSG), which is also found in the serum of infected animals, and which consists of a polypeptide and a glycosyl-inositol-phosphate (GIP) moiety [60], was purified (S3 Fig), and an amount of this protein equivalent to that present in 1 x 10⁸ trypanosomes was injected into mice prior to sporozoite inoculation. Quantification of the Plasmodium liver load showed that the administration of sVSG did not reduce P. berghei liver infection relative to that of control mice (Fig 5B), suggesting that VSG is not involved in the phenotype under investigation. As expected, injection of a similar amount of albumin also did not cause an inhibitory effect on P. berghei hepatic infection.

Next, we investigated whether other cell-surface glycoproteins could be responsible for the observed inhibitory effect. Trypanosomes were treated with an exogenous phospholipase C (PLC), which cleaves the GPI-anchors immediately before the phosphate group, releasing the polypeptides and glycan moiety into a soluble fraction, while the dimyristoylglycerol remains attached to the parasite surface [60]. The soluble fraction was then injected into mice, followed by injection of sporozoites. As expected, we observed that the parasite liver load of mice that received the GPI-anchored surface proteins fraction was not different from that of control mice. Additionally, we observed that the T. brucei lysates without the GPI-anchored surface proteins retained their inhibitory effect, which was not impacted by PLC’s enzymatic activity (Fig 5C). Altogether, these results indicate that the polypeptides of GPI-anchored proteins are not involved in the inhibitory phenotype.

We subsequently tested whether the T. brucei protein(s) responsible for the impairment of Plasmodium liver infection is/were present in more than one stage of the trypanosome life cycle. To investigate this, total lysates of either T. brucei bloodstream forms (BSF) or procyclic forms (PCF, found in the insect vector) were administered to mice prior to sporozoite injection, and Plasmodium hepatic load was assessed. Interestingly, we found that the lysates of both trypanosome life cycle stages reduced P. berghei hepatic infection, relative to that of mice infected only with Plasmodium (S4 Fig). This result suggests that the protein(s) that impair liver infection by P. berghei is/are either expressed in more than one stage of the trypanosome life cycle, or that a different set of proteins has a similar Plasmodium inhibitory activity in both stages of the trypanosome life cycle.

To identify the inhibitory trypanosome protein(s), we started by assessing whether they were excreted. To assess this, trypanosomes were incubated in PBS for 1 h at room temperature and the proteins excreted during this period were collected. The fraction containing the excreted proteins was subsequently injected into mice prior to sporozoite inoculation and P. berghei liver infection was quantified (Fig 5D). We found that the excreted protein fraction reduces P. berghei liver infection to ~40% of the liver load observed mice infected only with P. berghei, indicating that the impairment of P. berghei liver infection is mediated by one or more excreted T. brucei proteins.

Extracellular vesicles are used by several microorganisms as vehicles for delivery of proteins, lipids and nucleic acids [61]. It has been shown that T. brucei also releases EVs that fuse with the host erythrocyte membrane, altering the physical properties of the membrane, and causing the RBC to be cleared from circulation [43]. Since the excreted protein fraction of T. brucei reduces P. berghei liver infection, we hypothesized that T. brucei EVs might be involved in this process. To assess this, we purified T. brucei EVs and used transmission electron microscopy (TEM) to assess their shape and size (≈150 nm) and confirm their identity (S5A Fig). EVs were injected into mice prior to sporozoite inoculation and Plasmodium liver infection was assessed as above. Mice that received T. brucei EVs did not present an impairment of Plasmodium liver infection, relative to the respective control mice, which indicates that T. brucei EVs are not involved in the phenotype under study (S5B Fig).Mass spectrometry analysis of the active excreted fraction (which we call here, excretome) revealed it to be composed by 376 proteins (S1 Table). Gene ontology (GO) analysis showed that most of the proteins belong to biological processes of interaction with the host, protein folding and translation, cell cycle and cellular organization (Figs 5E and S6). Having previously excluded proteins with a GPI-anchor, we used a GPI-anchor predictor [62] to disregard 13 predicted GPI-anchored proteins (S1 Table), from the list of candidate proteins responsible for the impairment of P. berghei liver infection. A comparative analysis between our 376 excreted proteins and the published T. brucei secretome [30] indicated that 127 proteins are common between the two data sets (Fig 5F). This difference was not surprising since the secretion/excretion treatments were different between the two studies. Interestingly, 60 proteins (S2 Table) found in the excretome have been previously identified in the plasma of T. b. rhodesiense infected patients [29] (Fig 5G). Thus, it is possible that one of these 60 proteins could be responsible for the observed inhibitory activity.

Collectively, our data show that trypanosome BSF excreted protein(s) impair(s) P. berghei hepatic infection. Importantly, VSG, the most abundant surface BSF trypanosome protein, does not mediate the impairment of P. berghei liver infection.