TropicalMed, Vol. 7, Pages 384: Investigating the Leishmania donovani sacp Gene and Its Role in Macrophage Infection and Survival in Mice

Conceptualization, K.P., P.L., W.-W.Z. and G.M.; Methodology, K.P., P.L. and W.-W.Z.; Formal Analysis, K.P.; Investigation, K.P.; Resources, D.J.P.; Writing—Original Draft Preparation, K.P. and G.M.; Writing—Review and Editing, K.P., P.L., W.-W.Z., D.J.P., M.N. and G.M.; Supervision, M.N. and G.M.; and Funding Acquisition, M.N. and G.M. All authors have read and agreed to the published version of the manuscript.

Figure 1. sacp gene copy number in the L. donovani 1S2D used in this study. The read depth coverage by base pair was determined using the module bedtools coverage. An analysis was performed between positions 2,530,900 and 2,548,000 and between positions 2,753,500 and 2,755,800 of chromosome 36 spanning the following genes: hypothetical protein 1 (hp1, LdCL_360075600), the membrane-bound acid phosphatase (macp, LdCL_360075700), the hypothetical protein 2 (hp2, LdCL_360075800), the mitogen-activated protein kinase (mapk, LdCL_360075900), the secreted acid phosphatase (sacp, LdCL_360076000) and hypothetical protein 3 (hp3, LdCL_360080400). The schematic representation (bottom) shows episomal genes in white and non-episomal genes in black. The portion of sacp that is genetically nearly identical to macp is shown in grey. The two genetically different regions with repetitive GC-rich sequences are striped orange and striped blue. The coverage graph (top) shows the sliding window read depth coverage as a function of position in base pairs. The average coverage for diploid genes (2n ≈ 400×) is the solid black line, the average coverage for haploid genes (1n ≈ 200×) is the dotted red line and the average coverage for triploid genes (3n ≈ 600×) is the dotted blue line. Note: the axis was split between 2,548,000 and 2,754,000 to condense the cluster of sacp genes present in the LdSL-CL reference genome to be more representative of the L. donovani 1S2D genome.

Figure 1. sacp gene copy number in the L. donovani 1S2D used in this study. The read depth coverage by base pair was determined using the module bedtools coverage. An analysis was performed between positions 2,530,900 and 2,548,000 and between positions 2,753,500 and 2,755,800 of chromosome 36 spanning the following genes: hypothetical protein 1 (hp1, LdCL_360075600), the membrane-bound acid phosphatase (macp, LdCL_360075700), the hypothetical protein 2 (hp2, LdCL_360075800), the mitogen-activated protein kinase (mapk, LdCL_360075900), the secreted acid phosphatase (sacp, LdCL_360076000) and hypothetical protein 3 (hp3, LdCL_360080400). The schematic representation (bottom) shows episomal genes in white and non-episomal genes in black. The portion of sacp that is genetically nearly identical to macp is shown in grey. The two genetically different regions with repetitive GC-rich sequences are striped orange and striped blue. The coverage graph (top) shows the sliding window read depth coverage as a function of position in base pairs. The average coverage for diploid genes (2n ≈ 400×) is the solid black line, the average coverage for haploid genes (1n ≈ 200×) is the dotted red line and the average coverage for triploid genes (3n ≈ 600×) is the dotted blue line. Note: the axis was split between 2,548,000 and 2,754,000 to condense the cluster of sacp genes present in the LdSL-CL reference genome to be more representative of the L. donovani 1S2D genome.

Tropicalmed 07 00384 g001 Figure 2. Generation of the L. donovani mutant, LdΔSAcP, using CRISPR-Cas9 gene editing. (a) Genetic representation of the sacp loci. The usually diploid chromosome 36 is illustrated from position 2,530,000 to position 2,550,000 containing the macp gene (LdCL_360075700) in pink, the hp gene (LdCL_360075800) in white, the mapk gene (LdCL_360075900) in white and the sacp gene (LdCL_360076000) in blue, with the homologous sequence to the macp gene in pink. The hp gene, the mapk gene and the sacp gene are also encoded on one episome. The black triangles indicate the locations of the Cas9 endonuclease cut sites and the grey arrows represent the primers used in PCR. (b) Gene editing strategy to remove sacp. Promastigotes were transfected with the CRISPR vector pLdCN2 [22] containing two guide RNAs specific to the sacp gene but not to the macp gene. The gRNA sequences are in red, and the PAM sequence is in black with the cut sites indicated by the black arrows. Promastigotes were subsequently transfected with a bleomycin resistance gene (bleoR), and through microhomology end joining, the bleoR gene is integrated into the sacp gene. (c) Gel-electrophoresis of the PCR analysis with the primers LdSAcPF2 and LdSAcPR (Table S1) of the sacp gene from LdWT DNA (1685 bp) and the LdΔSAcP DNA (877 bp). Figure 2. Generation of the L. donovani mutant, LdΔSAcP, using CRISPR-Cas9 gene editing. (a) Genetic representation of the sacp loci. The usually diploid chromosome 36 is illustrated from position 2,530,000 to position 2,550,000 containing the macp gene (LdCL_360075700) in pink, the hp gene (LdCL_360075800) in white, the mapk gene (LdCL_360075900) in white and the sacp gene (LdCL_360076000) in blue, with the homologous sequence to the macp gene in pink. The hp gene, the mapk gene and the sacp gene are also encoded on one episome. The black triangles indicate the locations of the Cas9 endonuclease cut sites and the grey arrows represent the primers used in PCR. (b) Gene editing strategy to remove sacp. Promastigotes were transfected with the CRISPR vector pLdCN2 [22] containing two guide RNAs specific to the sacp gene but not to the macp gene. The gRNA sequences are in red, and the PAM sequence is in black with the cut sites indicated by the black arrows. Promastigotes were subsequently transfected with a bleomycin resistance gene (bleoR), and through microhomology end joining, the bleoR gene is integrated into the sacp gene. (c) Gel-electrophoresis of the PCR analysis with the primers LdSAcPF2 and LdSAcPR (Table S1) of the sacp gene from LdWT DNA (1685 bp) and the LdΔSAcP DNA (877 bp). Tropicalmed 07 00384 g002 Figure 3. Confirmation that the SAcP protein is lost in LdΔSAcP. (a) Western blot analysis of SAcP in L. donovani 1S2D wild type (LdWT) and L. donovani mutant (LdΔSAcP) strains. Cell lysates (p = parasite-contained proteins) and cell-culture supernatants (s = secreted proteins) were Western blotted using the α-SAcP primary rabbit antibody. No remaining SAcP is detected in the LdΔSAcP strain. HSP83 was used as a loading control for the cell lysate. Full, uncropped image available in Figure S2. (b) Acid phosphatase activity was measured in the parasite-contained proteins and in the secreted proteins in the L. donovani 1S2D wild type (shown in orange) and mutant parasites (shown in blue) during logarithmic (n = 6, parasite p-value = 0.4749, secreted p-value = 9.383 × 10−5) and stationary phases of growth (n = 4, parasite p-value = 0.9926, secreted p-value = 2.435 × 10−6), ns > 0.05, **** p Figure 3. Confirmation that the SAcP protein is lost in LdΔSAcP. (a) Western blot analysis of SAcP in L. donovani 1S2D wild type (LdWT) and L. donovani mutant (LdΔSAcP) strains. Cell lysates (p = parasite-contained proteins) and cell-culture supernatants (s = secreted proteins) were Western blotted using the α-SAcP primary rabbit antibody. No remaining SAcP is detected in the LdΔSAcP strain. HSP83 was used as a loading control for the cell lysate. Full, uncropped image available in Figure S2. (b) Acid phosphatase activity was measured in the parasite-contained proteins and in the secreted proteins in the L. donovani 1S2D wild type (shown in orange) and mutant parasites (shown in blue) during logarithmic (n = 6, parasite p-value = 0.4749, secreted p-value = 9.383 × 10−5) and stationary phases of growth (n = 4, parasite p-value = 0.9926, secreted p-value = 2.435 × 10−6), ns > 0.05, **** p Tropicalmed 07 00384 g003

Figure 4. Whole-genome sequencing shows specificity of CRISPR-Cas9 gene knockout. Read depth coverage by base pair was determined using the module bedtools coverage over three separate reference files illustrated at the bottom of the figure. (a) The L. donovani 1S2D wild type genome (top) and the mutant genome (middle) are aligned to the wild type sacp gene (bottom). The sacp gene is blue with pink demonstrating the region with genomic similarities between the macp gene and the sacp gene from 2,747,080 to 2,747,953, which has a coverage of ~1000× in LdWT. Positions 2,748,288–2,748,310 and 2,746,950–2,746,972 are the positions of the gRNAs used to target the sacp gene and both positions have an average coverage of 0 in LdΔSAcP. (b) The L. donovani wild type genome (top) and the mutant genome (middle) are aligned to the bleoR gene (black), which was inserted into the sacp gene (bottom). LdWT has an average coverage of 0 over the bleoR gene whereas LdΔSAcP has continuous coverage. (c) The L. donovani wild type genome (top) and mutant genome (middle) are aligned to the macp reference genome, which is pink (bottom). LdWT has an average coverage ~1000× where the macp gene and the sacp gene are nearly identical, which drops to ~400× in the LdΔSAcP genome. The red dotted line represents the expected coverage for 1n, the solid black line represents the expected coverage for 2n and the blue dotted line represents the expected coverage for 3n.

Figure 4. Whole-genome sequencing shows specificity of CRISPR-Cas9 gene knockout. Read depth coverage by base pair was determined using the module bedtools coverage over three separate reference files illustrated at the bottom of the figure. (a) The L. donovani 1S2D wild type genome (top) and the mutant genome (middle) are aligned to the wild type sacp gene (bottom). The sacp gene is blue with pink demonstrating the region with genomic similarities between the macp gene and the sacp gene from 2,747,080 to 2,747,953, which has a coverage of ~1000× in LdWT. Positions 2,748,288–2,748,310 and 2,746,950–2,746,972 are the positions of the gRNAs used to target the sacp gene and both positions have an average coverage of 0 in LdΔSAcP. (b) The L. donovani wild type genome (top) and the mutant genome (middle) are aligned to the bleoR gene (black), which was inserted into the sacp gene (bottom). LdWT has an average coverage of 0 over the bleoR gene whereas LdΔSAcP has continuous coverage. (c) The L. donovani wild type genome (top) and mutant genome (middle) are aligned to the macp reference genome, which is pink (bottom). LdWT has an average coverage ~1000× where the macp gene and the sacp gene are nearly identical, which drops to ~400× in the LdΔSAcP genome. The red dotted line represents the expected coverage for 1n, the solid black line represents the expected coverage for 2n and the blue dotted line represents the expected coverage for 3n.

Tropicalmed 07 00384 g004 Figure 5. L. donovani wild type and mutant whole-genome chromosome analysis. L. donovani 1S2D wild type and L. donovani mutant LdΔSAcP genomes were sequenced by Illumina, and the reads were aligned to the L. donovani CL-SL reference genome [23], and their coverages were analyzed using the module bedtools coverage. The mean coverage for each gene is plotted along the reference genome from chromosome 1 to 36, and LdΔSAcP coverage is normalized to LdWT coverage. Mean coverage is grey, increased copy numbers is blue and decreased copy numbers is red. The inner circle is the L. donovani wild type (WT) and the outer circle is the mutant LdΔSAcP genome. The average coverage across the entire genome serves as the baseline for diploid genes. Chromosome 35 contains an amplicon and chromosome 8 has reverted to diploid in the LdΔSAcP genome. Figure 5. L. donovani wild type and mutant whole-genome chromosome analysis. L. donovani 1S2D wild type and L. donovani mutant LdΔSAcP genomes were sequenced by Illumina, and the reads were aligned to the L. donovani CL-SL reference genome [23], and their coverages were analyzed using the module bedtools coverage. The mean coverage for each gene is plotted along the reference genome from chromosome 1 to 36, and LdΔSAcP coverage is normalized to LdWT coverage. Mean coverage is grey, increased copy numbers is blue and decreased copy numbers is red. The inner circle is the L. donovani wild type (WT) and the outer circle is the mutant LdΔSAcP genome. The average coverage across the entire genome serves as the baseline for diploid genes. Chromosome 35 contains an amplicon and chromosome 8 has reverted to diploid in the LdΔSAcP genome. Tropicalmed 07 00384 g005

Figure 6. Loss of SAcP does not significantly impact L. donovani promastigote or amastigote fitness in vitro. (a) L. donovani wild type and mutant LdΔSAcP parasites were grown in liquid M199 culture, and proliferation was assessed. The log of promastigotes per milliliter is shown as mean and standard deviation. A curve fit for nonlinear regression was performed. n = 3 and p-value = 0.9001. (b) In vitro THP1 macrophage infection was assessed by the number of parasites per macrophage and the percentage of infected macrophages. Three individual data points and their means are represented on the graph with no significance at each time point based on the two-tailed unpaired Student’s t-test.

Figure 6. Loss of SAcP does not significantly impact L. donovani promastigote or amastigote fitness in vitro. (a) L. donovani wild type and mutant LdΔSAcP parasites were grown in liquid M199 culture, and proliferation was assessed. The log of promastigotes per milliliter is shown as mean and standard deviation. A curve fit for nonlinear regression was performed. n = 3 and p-value = 0.9001. (b) In vitro THP1 macrophage infection was assessed by the number of parasites per macrophage and the percentage of infected macrophages. Three individual data points and their means are represented on the graph with no significance at each time point based on the two-tailed unpaired Student’s t-test.

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Figure 7. Comparison of LdWT and LdΔSAcP parasites in a mouse model. BALB/c mice were infected with 1 × 108 parasites (LdWT = orange and LdΔSAcP = blue) via teil vein infection. After 5 weeks, mice were sacrificed, and livers and spleens were kept for analysis. (a) Livers (left) and spleens (right) were weighed, and the mass was plotted in grams. Two-tailed unpaired Student’s t-test was performed (n = 5, liver p-value = 0.2741, spleen p-value = 0.4682). (b) Liver LDU was calculating by determining the number of amastigotes per 1000 macrophages and multiplying by mass (g) in the liver imprints. Two-tailed unpaired Student’s t-test was performed (n = 5, p-value = 0.3167). The number of spleen parasites were determined by serial dilution. Two-tailed unpaired Student’s t-test was performed (n = 5, p-value = 0.0008), ns > 0.05, *** p < 0.001.

Figure 7. Comparison of LdWT and LdΔSAcP parasites in a mouse model. BALB/c mice were infected with 1 × 108 parasites (LdWT = orange and LdΔSAcP = blue) via teil vein infection. After 5 weeks, mice were sacrificed, and livers and spleens were kept for analysis. (a) Livers (left) and spleens (right) were weighed, and the mass was plotted in grams. Two-tailed unpaired Student’s t-test was performed (n = 5, liver p-value = 0.2741, spleen p-value = 0.4682). (b) Liver LDU was calculating by determining the number of amastigotes per 1000 macrophages and multiplying by mass (g) in the liver imprints. Two-tailed unpaired Student’s t-test was performed (n = 5, p-value = 0.3167). The number of spleen parasites were determined by serial dilution. Two-tailed unpaired Student’s t-test was performed (n = 5, p-value = 0.0008), ns > 0.05, *** p < 0.001.

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Table 1. Primers and associated sequences.

Table 1. Primers and associated sequences.

Primer NamePrimer SequenceLdSAcP15′ ATCGAAGACCTTTGTCTTCGCCGTTACCATCTTCGGTTTTAGAGCTAGAAATAGCAAGLdSAcP25′ ATCGAAGACCCAAACCAAATCGGCCGTTCCTATCACCATGACGAGCTTACTCLdSAcPBleF5′ TCTGCGTCCCACCGGAATCCCTCAAGATCTTCATCGGATCGGGTALdSAcPBleR5′ GCGGTGTCCTGGAGCAGCTCCACGAGTCGGTCAGTCCTGCTCCTLdSAcPF25’ CCGCCCACTCAACGATTATLdSAcPR5′ CTGTACTGAGCCTGCGTCAT

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