Role of Leishmania (Leishmania) chagasi amastigote cysteine protease in intracellular parasite survival: studies by gene disruption and antisense mRNA inhibition

Abstract

Background: The parasitic protozoa belonging to Leishmania (L.) donovani complex possess abundant, developmentally regulated cathepsin L-like cysteine proteases. Previously, we have reported the isolation of cysteine protease gene, Ldccys2 from Leishmania (L.) chagasi. Here, we have further characterized this cysteine protease gene and demonstrated its role during infection and survival of Leishmania (L.) chagasi within the U937 macrophage cells.

Results: The amastigote specific Ldccys2 genes of L. (L.) chagasi and L. (L.) donovani have identical gene organization, as determined by southern blots. In vivo expression analyses by Northern blots showed that Ldccys2 is amastigote specific. Western blot using anti-Ldccys2 antibody confirmed the amastigote specific protein expression. Recombinant expression of Ldccys2, a 30 kDA protein, was functionally active in a gelatin assay. Results from Ldccys2 heterozygous knockout mutants showed its role during macrophage infection and in intra-macrophage survival of the parasites. Since attempts to generate null mutants failed, we used antisense RNA inhibition to regulate Ldcccys2 gene expression. Not surprisingly, the results from antisense studies further confirmed the results from heterozygous knockout mutants, reiterating the importance of amastigote specific cysteine proteases in Leishmania infection and pathogenesis.

Conclusions: The study shows that Ldccys2 is a developmentally regulated gene and that Ldccys2 is expressed only in infectious amastigote stages of the parasite. The collective results from both the heterozygous knockout mutants and antisense mRNA inhibition studies shows that Ldccys2 helps in infection and survival of L. (L.) chagasi amastigotes within the macrophage cells. Finally, antisense RNA technique can be used as an alternate approach to gene knockout, for silencing gene expression in L. (L.) chagasi, especially in cases such as this, where a null mutant cannot be achieved by homologous recombination.

Figure 1

Ldccys2 is a single copy gene and is expressed only in amastigotes. (A) Southern blot hybridization of digested genomic DNA from L. (L.) chagasi (Lc) and L. (L.) donovani (Ld). 5 μg of genomic DNA was digested with restriction enzymes as mentioned in the figure and blotted onto Hybond-N membranes. The blot was probed with coding region pf Ldccys2 cDNA clone. (B) Northern blot analyses of L. (L.) chagasi total RNA. Total RNA (10 μg/lane) from promastigotes of logarithmic (lane 1), stationary (lane 2) growth phase, U937 cells (human macrophage cell line) infected with promastigotes for 96 h (lane 3) and uninfected U937 cells (lane 4) were separated on 1.2% (w/v) formaldehyde agarose gel and transferred on to Hybond N+ membrane. Blot was hybridized with PCR amplified DNA fragment containing 3'UTR from Ldccys2 (panel I), Ldccys1 (154 bp, near the polyA region, panel II), and coding region of α-tubulin from L. (L.) chagasi, a kind gift from Dr. M.E. Wilson (panel IV). (C) Western blot analysis of L. (L.) chagasi promastigotes and amastigotes. Equal amounts of proteins from promastigotes of logarithmic (lane 1), stationary (lane 2) phase, U937 cells infected with promastigotes (lane 3) and uninfected U937 cells (lane 4) were separated on 10% (w/v) SDS-PAGE and blotted on to Hybond – P membrane. Western blot analysis was carried out using α-Ldccys2 antibody.

Figure 2

Expression of recombinant Ldccys2 using insect cell expression system. Western blots showing recombinant expression of Ldccys2 using anti-JHE antibody (A) and duplicate blot probed with anti-Ldccys2 antibody (B). The lanes are labeled appropriately. 5 μg of supernatant from the insect cells expressing recombinant plasmids or the control were separated on 8% SDS-PAGE, blotted onto to PVDF membranes and probed with 1:500 dilution of anti-JHE or 1:100 dilution of anti-Ldccys2 antibody. A horseradish peroxidase conjugated anti-mouse secondary antibody was used and the blot was developed by using ECL chemiluminescent kit. (C) Gelatin assay showing cysteine protease activity of the recombinant protein. The supernatant was separated on a gelatin gel and incubated in a reducing buffer. The gel was then stained with Coomassie Blue to visualize the clearing of gelatin on the gel indicating cysteine protease activity.

Figure 3

Ldccys2 single allele gene replacement. (A) Schematic representation of the wild type and hyg/dhfr-ts targeted alleles of Ldccys2. The location of the PstI sites used to characterize the hygromycin B resistant recombinants is shown by arrows. The bold lines labelled a, b and c represents the location of different probes used in Southern analyses. (B) Southern analyses of Ldccys2 heterozygous knockout mutants. Genomic DNA (2 μg/lane) from wild type L. (L.) chagasi (WT) and Ldccys2 heterozygous knockout mutants (KO) were digested with PstI and separated on a 0.9% (w/v) agarose gel. The DNA was blotted on to Hybond N+ membrane and hybridized with probes a (i), b (ii) and c (iii). Asterisks: 1- represents wild type allele of Ldccys2 (3.5 kb), 2 and 3- represent disrupted allele (3.0 kb and 2.8 kb). The grey lines indicate the size of expected bands upon PstI digestion.

Figure 4

Characterization of L. (L.) chagasi heterozygous knockout mutant amastigotes and intra macrophage survival of Ldccys2KO amastigotes in vitro. (A) Northern blot analysis. Total RNA (10 μg/lane) from U937 cells infected with wild type promastigotes, Ldccys2KO and uninfected U937 cells were separated on 1.2% (w/v) formaldehyde agarose gel and transferred on to Hybond N+ membrane. Blot was hybridized with Ldccys2 coding region DNA probe (1) and α-tubulin gene from L. (L.) chagasi (2). (B) Western blot analysis. Equal amount of lysates from U937 cells infected with wild type promastigotes, Ldccys2KO promastigotes and uninfected U937 cells were separated on 10% (w/v) SDS-PAGE, blotted on to Hybond P membrane. Panel 1 represents the membrane that was probed with α-Ldccys2 antibody and panel 2 is the duplicate gel stained with Coomassie blue. (C) Bar graph showing the number of intracellular amastigotes. U937 macrophage cells were infected at a macrophage to parasite ratio of 1:10. The survival of the amastigotes within the macrophages was evaluated every 12 hours by cytospin and Diff-Quick staining. (D) Graph showing the percent of total macrophages infected at the given time points. For each treatment, 100 infected macrophages were counted. Values represent means ± SEM from three independent experiments.

Figure 5

Expression of sense and antisense Ldccys2 transcripts in L. (L.) chagasi and survival of wild type, sense and antisense Ldccys2 expressing amastigotes in macrophages. (A) Northern blot analyses of amastigotes expressing sense and antisense transcripts. Total RNA (10 μg/lane) isolated from axenic amastigotes of wildtype parasites, transfectants with P6.5/Ldccys2 sense plasmid and P6.5/Ldccys2 antisense plasmid were separated on 1.2% (w/v) formaldehyde agarose gel and blotted on to Hybond N+ membrane. Blots were probed with probes specific for antisense (panel 1) and sense (panel 2) transcripts. Panel 3 represents ethidium bromide stained gel. (B) Western blot analysis of sense and antisense Ldccys2 expressing amastigotes. Equal amounts of total protein extracted from axenically transformed amastigotes with P6.5/Ldccys2 sense plasmid and P6.5/Ldccys2 antisense plasmid and wildtype parasites were separated on 10% (w/v) SDS-PAGE and blotted on to Hybond P membrane. The blot was probed with α-Ldccys2 antibody (panel 1) and a duplicate gel was stained with Coomassie blue (panel2). (C) Bar graph showing total number of amastigotes within the macrophage cells. U937 macrophage cells were infected at a macrophage to parasite ratio of 1:10. The survival of the amstigotes within the macrophages was evaluated every 12 hours by cytospin and Diff-Quick staining.(D) Graph showing the percent of total macrophages infected at the given time points. For each treatment, 100 infected macrophages were counted. Values represent means ± SEM from three independent experiments.