The mRNA-bound Proteome of Leishmania mexicana: Novel Genetic Insight into an Ancient Parasite

Abstract

Leishmania parasite infections, termed the leishmaniases, cause significant global infectious disease burden. The lifecycle of the parasite embodies three main stages that require precise coordination of gene regulation to survive environmental shifts between sandfly and mammalian hosts. Constitutive transcription in kinetoplastid parasites means that gene regulation is overwhelmingly reliant on post-transcriptional mechanisms, yet strikingly few Leishmania trans-regulators are known. Using optimized crosslinking and deep, quantified mass spectrometry, we present a comprehensive analysis of 1400 mRNA binding proteins (mRBPs) and whole cell proteomes from the three main Leishmania lifecycle stages. Supporting the validity, although the crosslinked RBPome is magnitudes more enriched, the protein identities of the crosslinked and non-crosslinked RBPomes were nearly identical. Moreover, multiple candidate RBPs were endogenously tagged and found to associate with discrete mRNA target pools in a stage-specific manner. Results indicate that in L. mexicana parasites, mRNA levels are not a strong predictor of the whole cell expression or RNA binding potential of encoded proteins. Evidence includes a low correlation between transcript and corresponding protein expression and stage-specific variation in protein expression versus RNA binding potential. Unsurprisingly, RNA binding protein enrichment correlates strongly with relative replication efficiency of the specific lifecycle stage. Our study is the first to quantitatively define and compare the mRBPome of multiple stages in kinetoplastid parasites. It provides novel, in-depth insight into the trans-regulatory mRNA:Protein (mRNP) complexes that drive Leishmania parasite lifecycle progression.

Keywords: Label-free quantification; Leishmania; Microbes; Microbiology; Molecular biology*; Parasite; Pathogens; Protein Cross-linking*; Protein Identification*; Proteome; RBPome/mRNA binding proteome; Ribonucleoproteins; kinetoplastid; mRNP/trans-regulator.

Graphical abstract

Fig. 1.

Isolation and validation of L. mexicana lifecycle stages. A, Cumulative growth curve of L. mexicana in M199 (purple) and Grace's media (green). Harvested procyclic (PCF, purple circle) and metacyclic-enriched promastigote cultures (META, green circle) were tested for stage-specific traits. Metacyclic-enriched cultures were used to infect immortalized macrophages (J774.2 MØs) and mice to generate AMA (MØ) and AMA (LD) amastigote forms, respectively. Amastigotes forms (24 h post-infection (pi) for AMA (MØ) and 4 months pi for AMA(LD)) were gradient purified. B, FACS analyses compare DNA profiles of PCF, META, AMA (MØ) and AMA (LD) cells. Cell cycle profiles demonstrate PCF are highly proliferative, META are relatively quiescent and distinct AMA populations display a near-identical intermediate replicative capacity. C, Human serum resistance expressed as percentage parasite viability (y axis) of PCF (purple) and META (green) parasites treated with Human Serum (H.S) or Heat Inactivated human serum (H.I). D, Validation of each isolated L. mexicana stage by heightened mRNA expression of histone h4, sherp and amastin relative to nmt transcript levels presented as mean ± S.E. from 3 experimental replicates. One-way ANOVA and Tukey's post-test were conducted; **p < 0.01, ***p < 0.001.

Fig. 2.

Overview of the mRNA-interactome capture workflow. A, Leishmania cells from verified lifecycle stages are UV-irradiated in vivo creating covalent bonds between mRNA and bound proteins in crosslinked (XL) versus non-crosslinked cells (nonXL). Cells are then purified, lysed and mRNP complexes are isolated using oligo(dT)-labeled magnetic beads. Isolated RBPs were precipitated and processed for label-free, quantified mass spectrometry. Numbers indicate identified proteins filtered for triplicate-consistent, high quality reads with at least 2 unique peptides. B, Growth curve and cell morphology after 120” or 180” UV-irradiation with optimized equipment. C, Relative RNA recovery of 18S and nmt transcripts after oligo(dT) magnetic bead mRNA purification in XL, nonXL and WC lysates in PCF (purple), META (green) and AMA (MØ; red) samples.

Fig. 3.

Variability and GO term enrichment among proteomes. A, Principal component analysis (PCA) using relative protein quantification derived from unique peptide intensities was used to examine relatedness between the different biological samples and the variability among replicates. Notably, the triplicate proteomic samples cluster within discrete lifecycle stages for XL, nonXL and WC proteomes displaying low variance among replicates and reproducibility of the results. Colors indicate lifecycle stage of samples: PCF (red), META (green), AMA(MØ) (blue) and AMA(LD) (black). XL Samples (X) are Px = PCF XL, Mx = META XL, Ax = AMA(MØ) XL and Lx = AMA (LD) XL. NonXL Samples (O) are Pn = PCF nonXL, Mn = META nonXL and An = AMA(MØ) nonXL. WC Samples (Δ) are p = PCF WC, M = META WC and A = AMA(MØ) WC. B, Within the specific 234 RBPs Gene Ontology (GO) term enrichment analyses again indicated that this specific L. mexicana subset was enriched in the following terms by the following order: structural constituent of the ribosome (p = 4.67e⁻¹⁰), RNA-binding (p = 1.25e⁻⁶), nucleic acid binding (p = 7.2e⁻⁴) indicating that the Molecular Function subfraction specific to the L. mexicana RBPome is overwhelmingly enriched in RNA-related terms. Red line indicates p ≤ 0.05.

Fig. 4.

L. mexicana lifecycle whole cell and RBPome comparisons. A, The number of proteins containing common RNA-binding domains (RBDs) in the predicted L. mexicana proteome (Tritrypdb.org) and XL-RBPome are presented. 494 out of 8144 genes in the total proteome and 155 out of 1407 proteins in the XL-RBPome contain known RBDs. Domain classes labeled as “Other” represent proteins with either additional, less abundant RBDs not listed separately in the diagram or weak conservation of known RBDs and “Basal” proteins have homology to the translational and splicing machinery. The RBPome RBD total is higher than the number of RBPs identified because of the presence of multiple domain classes in single proteins. B, Volcano plots were generated in Python using p values derived by conducting independent t-tests (3 replicates in stage 1 versus 3 replicates in stage 2). No multiple testing was conducted as the plots are for data visualization. The log₂(FC) is log₂((mean exp. condition 2 + 1)/(mean expression condition 1 + 1)). Red line indicates p ≤ 0.05; protein identities above are significantly enriched in one lifecycle stage. Blue circles indicate identities below this threshold, green above. Red circles indicate proteins in the range of −5 < log₂(FC) < 5 and orange circles indicate highly expressed factors with intensities ≥ 10⁶.

Fig. 5.

Comparing L. mexicana WC and XL Proteomes to Transcriptome data. A, GLM (Generalized Linear Model) of loess algorithm fit displaying a sliding window approach to visualize relative correlation between the WC (Purple) and XL (Green) proteomic data relative to published L. mexicana transcriptomic data (30) in AMA(MØ) versus PCF lifecycle stages. Gray shading = S.E. Relevant statistics provided in Table I. B, A linear regression fit against all proteins which meet XL/WC ratio, Lm [WC∼XL]. Expressed proteins are more likely to bind mRNA in PCF than META with AMA intermediary. Likelihood for expressed proteins to bind RNA correlates well to both replicative and translational efficiency of each lifecycle stage.

Fig. 6.

Validation of novel L. mexicana RBPs. A, Western blots (anti-HA) of whole cell lysate (Input) and immunoprecipitations (Elution) of endogenously-tagged RBP candidates show specific isolation in both PCF and META stages. B, Whole cell expression of candidate transcript targets are shown in both PCF and META stages, normalized to nmt transcript expression. C, HA-RBP RIP-selected mRNA levels are shown relative to WC levels (B) for each target RNA. All qRTPCR data are presented as mean ± S.E. from 3 biological replicates. Two-way ANOVA test and Bonferroni between log and stationary phase samples indicate a stage-regulated association with target transcripts for some RBPs; *p < 0.05, **p < 0.01, ***p < 0.001. ND = Not Detected.