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Systematic identification of genes encoding cell surface and secreted proteins that are essential for in vitro growth and infection in Leishmania donovani

['Adam J. Roberts', 'Cell Surface Signalling Laboratory', 'Wellcome Sanger Institute', 'Hinxton', 'Cambridge', 'United Kingdom', 'Han B. Ong', 'Simon Clare', 'Pathogen Support Team', 'Cordelia Brandt']

Date: 2022-04

Prolonged in vitro culture of Leishmania spp. has previously been attributed to a loss of virulence in experimental settings [ 25 , 26 ], and so to ensure the virulence of the line, mice were inoculated and parasites recovered from the spleen of an infected mouse after 37 days. Parasites were cloned by limiting dilution and 12 selected clones were again tested for their ability to target the PF16 locus as above; the clone that resulted in the most rapid generation of resistance to both drug selection markers was selected. Infection parameters of this cloned line were determined using bioluminescence in mice and were shown to infect both the liver and spleen of BALB/c mice at similar levels and kinetics to those observed with the parental strain [ 19 ] ( Fig 1C and 1D ) [ 19 ]. This parasite line was named Ld-LUC-T7-Cas9 and used in all subsequent experiments.

( A ) Stable expression of a FLAG-tagged Cas9 nuclease in a bioluminescent L. donovani parasite line (LUC). Total cell lysates from 5x10 6 L. donovani promastigotes were resolved by SDS-PAGE under reducing conditions, blotted, and detected with an anti-FLAG antibody. ( B ) Schematic of diagnostic PCR to demonstrate expected targeting, gDNA from wild type (WT) or doubly-drug resistant (mutant) parasites is amplified using a shared sense primer (F) and specific antisense primers targeting the gene of interest (G), PAC (P) or BLA (B) (left panel). Products from a diagnostic PCR demonstrating replacement of the endogenous PF16 gene in L. donovani using Cas9 genome editing. Primers specific for the endogenous PF16 locus (primers F and G) amplified a product of the expected size from genomic DNA from the parental (lane 1) but not the drug-resistant parasites (lane 2); targeted integration into the PF16 locus was demonstrated with shared and drug-specific primers (P and B) for the puromycin (lane 3) and blasticidin resistance genes (lane 4) respectively (right panel). ( C ) Representative images of mice infected with Ld-LUC-T7-Cas9 at weeks 2 and 11 post infection including the gating strategy to quantify parasitemia in the liver and spleen. ( D ) Virulence of the Ld-LUC-T7-Cas9 line in an experimental murine infection model. Groups of 3 to 5 female BALB/c mice were infected with stationary phase promastigotes of the Ld-LUC-T7-Cas9 line and parasitemia in both liver and spleen quantified using bioluminescent imaging. Data points represent means ± s.d. Data are from a representative experiment. Each unit of bioluminescence represents 1 x 10 5 photons per second.

To identify L. donovani genes that are predicted to encode cell surface and secreted proteins that are required for either in vitro growth or host infection, we first needed to generate a transgenic bioluminescent parasite that expressed both the Cas9 nuclease and T7 RNA polymerase [ 17 , 24 ]. We selected an L. donovani LV9 parasite strain stably expressing both the fluorescent protein mCherry and firefly luciferase as a parental cell line to facilitate in vitro and in vivo characterisation [ 19 ]. Parasites were electroporated with a plasmid encoding both SpCas9 and T7 RNA polymerase, and we confirmed that the resulting transgenic parasites expressed the SpCas9 protein ( Fig 1A ). To demonstrate functional expression of the T7 RNA polymerase and usability of the parasite line for genetic targeting, we transfected the line with four PCR products: two containing templates for sgRNAs driven by a T7 promoter matching the 5’ and 3’ untranslated regions of the gene encoding the flagellum protein PF16, and two encoding genes that confer resistance to the drugs puromycin and blasticidin, each flanked by 30 nucleotides of homology 5’ and 3’ of the cut sites [ 17 , 24 ]. A parasite population resistant to both puromycin and blasticidin was only observed in parasites electroporated in the presence of the four PCR products, and diagnostic PCR analysis of the genomic DNA confirmed targeted disruption of the PF16 locus ( Fig 1B ).

A library of gene-targeted L. donovani parasites encoding cell surface and secreted proteins identified four genes required for in vitro culture

To identify genes encoding cell surface or secreted proteins that are required for L. donovani cell viability in vitro and in vivo, we used the Ld-LUC-T7-Cas9 line to systematically select gene-deficient parasites from a curated list of 92 putative cell surface and secreted Leishmania proteins for which there was evidence of protein expression from published proteomics data; for convenience, each candidate was given a systematic “LD” number (S1 Table). To increase the chances of correct gene targeting, we sequenced the genome of the Ld-LUC-T7 parental strain and compared it to a reference genome sequence [14] to identify polymorphisms in the predesigned targeting and repair primers, incorporating any differences as appropriate. Of the 92 candidates targeted, doubly-drug resistant parasite populations with confirmed locus-specific biallelic knockout with each of the drug selection markers was confirmed for 68 genes (74%) by PCR (S1 Fig). Typically, parasite strains successfully targeted at both alleles required just one or two attempts, demonstrating that these genes were readily dispensable for in vitro promastigote proliferation (S1 Table). Just under a fifth (17/92) of genes demonstrated evidence of genomic rearrangements in at least one targeting attempt because despite the correct incorporation of both drug resistance markers, a PCR product showing the retention of the native locus was also observed, most likely due to selection-induced aneuploidy [27]. There was not an exclusive relationship, however, between the retention of the native locus after targeting, and the chromosomes on which the targeted genes were located, and this was true for chromosomes where multiple genes had been targeted. Furthermore, analysis of the normalised read depth of the sequencing data from the parental strain suggested the presence of trisomy and tetrasomy on chromosomes 26 and 31 respectively in Ld-LUC-T7-Cas9, and both these chromosomes contained genes that we were able to target successfully using only the two selectable markers as evidenced by the disruption of the native loci of LD40, which is present on chromosome 31, and LD30, LD31 and LD73 that are all located on chromosome 26 (Fig 2A and S1 Table). A possible mechanism for this observation is the integration of a selectable marker into multiple alleles. The results of PCR genotyping for three genes (LdBPK_090870.1 (LD17), LdBPK_141150.1 (LD21) and LdBPK_140570.1 (LD55)) were consistently ambiguous, because we were unable to demonstrate the presence of the endogenous locus and so these genes were not further investigated in this study.

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TIFF original image Download: Fig 2. The failure to obtain some null mutants is unlikely to be due to aneuploidy or inability to correctly target the gene. (A) Graph showing the success of gene targeting on individual chromosomes. The number of successfully targeted genes is shown in teal, failure to recover viable promastigotes in pink, and dual drug resistant parasites retaining a copy of the native loci in purple. Chromosomes 26 and 31 are present in multiple copies per haploid genome. (B) Schematic of the diagnostic PCRs used to genotype parasites. (C) Diagnostic PCRs demonstrating the expected monoallelic targeting of genes with just the puromycin-selection cassette for genes that had failed to recover doubly-drug resistant parasites. The indicated genomic DNAs are: wild-type control, C or puromycin-resistant mutant parasites, M, which were used as PCR templates using a shared sense primer (F) and the indicated antisense primer as shown in the schematic. (D) Table showing the frequency of being able to recover doubly drug resistant parasites in the presence (Rescue) or absence (Parent) of a non-targetable version of the named genes. (E) Confirmation of correct integration of drug resistance markers into the LdBPK_292250.1 (LD10) and LdBPK_211610.1 (LD11) loci only in the presence of a non-targetable copy of the gene under constitutive expression. Genotyping for LD10 and LD11 is shown for two independently selected populations. https://doi.org/10.1371/journal.ppat.1010364.g002

The targeting of four genes: LdBPK_211610.1 (LD11), LdBPK_100590.1 (LD18), LdBPK_111030.1 (LD67) and LdBPK_010280.1 (LD81) failed to recover any viable parasites despite a minimum of five independent attempts (Table 1). To confirm that the targeting and repair constructs for these genes were functioning as expected, we attempted single allele replacement for each gene using just the puromycin selection cassette. For all four of the genes (LD11, LD18, LD67 and LD81), we were able to recover viable puromycin-resistant parasites, validating the specificity of the designed targeting sequences (Fig 2B and 2C).

To demonstrate that these genes were required for in vitro parasite viability, we attempted to replace both endogenous alleles in either the presence or absence of a constitutively expressed version of the same gene that lacked the CRISPR targeting sequences. Three independent transfections were performed and doubly drug-resistant parasite populations were recovered for both LD11 and LD81 only in the presence of a non-targetable copy with a 100% success rate demonstrating their essentiality (Fig 2D). PCR genotyping confirmed the correct targeting of both endogenous alleles of LD11 in the presence of the ectopic rescue (Fig 2E); however, we were unable to obtain PCR genotyping for the LD81 parasite populations despite multiple attempts. Viable drug resistant parasites were not recovered from electroporations targeting LD18 or LD67 in either the absence or presence of their appropriate overexpressed open reading frame. This suggested that our failure to replace both endogenous alleles of LD18 and LD67 might be because the genetic rescue was insufficient to support the loss of both endogenous copies, or allelic variation in the targeting regions acquired by the parasite post genome sequencing prevented the replacement of the second allele with the drug resistance marker [28,29].

Leishmania parasites exhibit remarkable genomic plasticity, with the parasites being able to tolerate aneuploidy of different chromosomes both in vitro and in vivo [14,27]. The ability to modulate gene copy number may be used by the parasite as a mechanism to regulate gene expression, and it is possible to observe this plasticity experimentally in classical gene knockout studies of genes that have been chemically validated to be essential in this parasite [28,29]. In our experiments, we observed that targeting attempts for 17 genes on at least one occasion resulted in doubly-drug resistant parasites that nevertheless showed evidence of retention of the endogenous gene (S1 Table). This may indicate that these genes are essential for promastigote growth and so we investigated the gene LdBPK_292250.1 (LD10) which encodes a putative component of the ergosterol biosynthesis pathway in more detail. In three independent attempts to generate a null mutant for LD10 in the parental strain, doubly-drug resistant parasites were recovered, but at a lower frequency (one out of three attempts) compared to knockouts attempted in the presence of the “rescue” construct (two out of three attempts, Fig 2D). Indeed, it was only in the presence of the rescue construct that correct targeting of both LD10 alleles was observed in both populations (Fig 2E), confirming that C8 sterol isomerase is an essential gene for the viability of L. donovani promastigotes in vitro. We conclude that a further 17 genes may be required for L. donovani promastigote growth in vitro but the plasticity of the parasite genome makes further detailed investigation of this class of genes challenging.

[END]

[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010364

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