Knockout of the rodent malaria parasite chitinase pbCHT1 reduces infectivity to mosquitoes

Abstract

During mosquito transmission, malaria ookinetes must cross a chitin-containing structure known as the peritrophic matrix (PM), which surrounds the infected blood meal in the mosquito midgut. In turn, ookinetes produce multiple chitinase activities presumably aimed at disrupting this physical barrier to allow ookinete invasion of the midgut epithelium. Plasmodium chitinase activities are demonstrated targets for human and avian malaria transmission blockade with the chitinase inhibitor allosamidin. Here, we identify and characterize the first chitinase gene of a rodent malaria parasite, Plasmodium berghei. We show that the gene, named PbCHT1, is a structural ortholog of PgCHT1 of the avian malaria parasite Plasmodium gallinaceum and a paralog of PfCHT1 of the human malaria parasite Plasmodium falciparum. Targeted disruption of PbCHT1 reduced parasite infectivity in Anopheles stephensi mosquitoes by up to 90%. Reductions in infectivity were also observed in ookinete feeds-an artificial situation where midgut invasion occurs before PM formation-suggesting that PbCHT1 plays a role other than PM disruption. PbCHT1 null mutants had no residual ookinete-derived chitinase activity in vitro, suggesting that P. berghei ookinetes express only one chitinase gene. Moreover, PbCHT1 activity appeared insensitive to allosamidin inhibition, an observation that raises questions about the use of allosamidin and components like it as potential malaria transmission-blocking drugs. Taken together, these findings suggest a fundamental divergence among rodent, avian, and human malaria parasite chitinases, with implications for the evolution of Plasmodium-mosquito interactions.

FIG. 1

Comparison of PbCHT1, PgCHT1, PfCHT1, and SmChiA, a bacterial family 18 chitinase from S. marcescens. (A) Multiple amino acid alignment (Clustal W). Residue identities are indicated by shading (grey, 75%; black, 100%), and secondary structure features (coils represent helices; arrows represent sheets) are shown below the sequences. The predicted catalytic dyad (SmChiA residues 315 and 391) is marked with red dots. Plasmodium chitinases show a high structural conservation with subdomains 2 (blue) and 3 (green) of SmChiA. (B) Stereo-space-filled image of the atomic structure of SmChiA showing subdomains 2 (blue) and 3 (green) and the catalytic dyad (red). These domains correspond to the areas of strong conservation in Plasmodium chitinases and are similarly colored in panel A. The numbers refer to the three subdomains.

FIG. 2

Differential expression of PbCHT1. Total RNA from asexual blood-stage parasites (lane 1), gametocytes (lane 2), and in vitro-cultured ookinetes (lane 3) was subjected to Northern blot analysis using a probe corresponding to PbCHT1. RNA amounts were normalized using large- and small-subunit rRNAs (ethidium bromide stained), as shown at the bottom of the figure.

FIG. 3

Targeted disruption of PbCHT1 and molecular analyses. (A) Schematic diagram of the targeting strategy. Indicated is the transfection vector pPbCHT1-KO containing the T. gondii DHFR/TS gene cassette (white box), P. berghei DHFR flanking sequences (gray boxes), and PbCHT1-specific sequences (hatched boxes). The double homologous recombination crossover sites (crossed lines), the integration sites (arrows with nucleotide positions), the SphI restriction sites, and the probes used in Southern blot analysis (thick lines) are shown. gDNA, genomic DNA. (B) Southern blot analysis of SphI-digested genomic DNA from WT and PbCHT1-KO parasites using probes corresponding to PbCHT1 (left panel) and to the DHFR/TS cassette (right panel). (C) RT-PCR analysis of total RNA derived from ookinete-enriched midgut stages of WT (left lanes) and PbCHT1-KO (right lanes) parasites. Amplicons corresponding to PbCHT1(∼1,100 bp) and Pbs25 (∼600 bp) are shown.

FIG. 4

Analysis of P. berghei WT and PbCHT1-KO ookinete homogenates for additional chitinase activity. (A) Western blot analysis with PgCHT1 active-site antiserum. (B) In vitro chitinase activity assay with glycol chitin-containing agarose. Also included is a homogenate from similarly purified blood stages (BS).

FIG. 5

Effects of allosamidin. (A) In vitro chitinase activity assay with P. berghei WT ookinete homogenates in the presence of 0, 0.1, and 1 mM allosamidin. (B) Effect of allosamidin on blood meal digestion. Shown are dissected guts of mosquitoes at 9 days after blood feeding in the presence (+) or absence (−) of 0.1 mM allosamidin.