Plasmodium knowlesi Cytoadhesion Involves SICA Variant Proteins

Abstract

Plasmodium knowlesi poses a health threat throughout Southeast Asian communities and currently causes most cases of malaria in Malaysia. This zoonotic parasite species has been studied in Macaca mulatta (rhesus monkeys) as a model for severe malarial infections, chronicity, and antigenic variation. The phenomenon of Plasmodium antigenic variation was first recognized during rhesus monkey infections. Plasmodium-encoded variant proteins were first discovered in this species and found to be expressed at the surface of infected erythrocytes, and then named the Schizont-Infected Cell Agglutination (SICA) antigens. SICA expression was shown to be spleen dependent, as SICA expression is lost after P. knowlesi is passaged in splenectomized rhesus. Here we present data from longitudinal P. knowlesi infections in rhesus with the most comprehensive analysis to date of clinical parameters and infected red blood cell sequestration in the vasculature of tissues from 22 organs. Based on the histopathological analysis of 22 tissue types from 11 rhesus monkeys, we show a comparative distribution of parasitized erythrocytes and the degree of margination of the infected erythrocytes with the endothelium. Interestingly, there was a significantly higher burden of parasites in the gastrointestinal tissues, and extensive margination of the parasites along the endothelium, which may help explain gastrointestinal symptoms frequently reported by patients with P. knowlesi malarial infections. Moreover, this margination was not observed in splenectomized rhesus that were infected with parasites not expressing the SICA proteins. This work provides data that directly supports the view that a subpopulation of P. knowlesi parasites cytoadheres and sequesters, likely via SICA variant antigens acting as ligands. This process is akin to the cytoadhesive function of the related variant antigen proteins, namely Erythrocyte Membrane Protein-1, expressed by Plasmodium falciparum.

Keywords: Plasmodium falciparum; anemia; antigenic variation; gastritis; histopathology (HPE); infected erythrocytes; malaria; rhesus monkey (Macaca mulatta).

Figure 1

Infection characteristics of rhesus monkeys infected with P. knowlesi. (A) Log₁₀-transformed parasitemia curves for 11 monkeys infected with P. knowlesi sporozoites. Treatment threshold (1% parasitemia = 40,000 parasites/μL) is indicated by the dotted line, and necropsy day is marked with an asterisk. Time points of subcurative treatment with either artemether or chloroquine are indicated by the “Rx” symbol. The interval in which the monkeys controlled parasitemia without the need for subcurative treatment is indicated by blue shading labeled “Controlled”. (B) Average log₁₀-transformed parasitemias for the primary and recrudescent peaks showing gradual reduction in magnitude. The primary parasitemic peak was compared to the final recrudescence and found to be statistically significantly different via paired t-test comparison (*p-value < 0.05). (C) Cumulative parasitemia prior to, and after parasitemic control (30 dpi) shows similar parasitemia burdens between earlier and later times in infection via paired t-test comparison. ns (not significant) =p−value > 0.05. Error bars represent standard error of the mean.

Figure 2

Hematological parameters of rhesus monkeys infected with P. knowlesi. (A) Longitudinal faceted plots showing parasitemia, with treatment threshold and control interval indicated, compared with (B) white blood cell count with population fractions indicated, (C) hemoglobin with mild, moderate, and severe anemia thresholds labeled (at 11 g/dL, 10 g/dL, and 7 g/dL, respectively), with (D) five day hemoglobin average, (E) platelet count with mild and moderate thrombocytopenia labeled (at 150,000 platelets/mL and 75,000 platelets/μL, respectively), with (F) five day platelet average, (G) reticulocyte production index (with RPI of 2 and of 3 labeled), and (H) five-day average of reticulocyte counts. (I) 600x H&E-stained tissue sections of bone marrow from an uninfected control monkey, a monkey prior to controlling parasitemia, and a monkey after controlling parasitemia. (J) Erythopoietin levels from select monkeys demonstrating no difference between baseline and 10 dpi, prior to antimalarial treatment. (K) Mathematical modeling predicting the relative proportions of infected and uninfected red blood cell removal. Statistical comparisons were performed using paired t-tests. ***p-value < 0.0005, *p-value < 0.05, and ns (not significant) = p-value > 0.05. Error bars represent standard error of the mean.

Figure 3

Telemetric temperature response to P. knowlesi parasitemia. (A) Top: Longitudinal parasitemia curve of the 6 monkeys with implanted telemetry devices, with treatment threshold indicated by dotted line. Bottom: Temperature data obtained from implanted telemetry devices indicating rises in temperature above each individual’s baseline corresponding to rises in parasitemia. Febrile periods are marked with orange shading. Statistically significant cytokine results from a 29-cytokine array including (B) MIG, (C) IL1-RA, (D) MCP1, (E) IFNγ, (F) ITAC. Statistical comparisons were performed using paired t-tests, with FDR correction. *Corrected p-value<0.05. Error bars represent standard error of the mean.

Figure 4

Histopathology, tissue damage, and results of chemistry analysis of rhesus monkeys infected with P. knowlesi. (A) H&E-stained section of rhesus lung under polarized light microscopy (scale bar = 100 μm). Hemorrhage (black arrow), interstitial thickening and infiltration (red arrow), and diffuse hemozoin (white arrow) are indicated. (B) Masson’s trichrome-stained section (scale bar = 200 μm) of rhesus lung highlighting fibrotic changes with infection (black arrow, and bright blue color of fibrin). (C) H&E-stained section (scale bar = 100 μm) of rhesus liver showing periportal inflammation (red arrow), sinusoidal congestion (black arrow), and parenchymal hemozoin deposition (white arrow). (D) H&E-stained section (scale bar = 200 μm) of liver from a monkey infected for 48 days showing unique lobular mononuclear infiltration in the periportal area (black arrow). (E) H&E-stained section (scale bar = 100 μm) of kidney under polarized light showing hypercellular glomeruli (black arrow) and abundant hemozoin (white arrow). (F) H&E stained section (scale bar = 200 μm) of stomach demonstrating mononuclear infiltration of the mucosa (black arrow), and submucosal edema, consistent with gastritis. (G) Semi-quantitative tissue scores of infected monkeys necropsied prior to and after parasitemic control. No statistically significant differences were noted using Tukey’s HSD post-hoc analysis. (H) ALT and (I) AST measurements taken at baseline, pre-control, and after control indicating no statistically significant difference relative to baseline. (J) Bilirubin and (K) creatinine measurements taken at baseline, pre-control, and after control indicating elevation of bilirubin pre-control relative to baseline and creatinine post-control relative to pre-control and baseline. Values compared with repeat-measures ANOVA with ns, not significant, *p-value <0.05, and **p-value <0.005. Error bars represent standard error of the mean.

Figure 5

Parasite tissue burden in P. knowlesi-infected rhesus monkeys. Examples of iRBCs at 1000x (oil immersion) in H&E-stained tissue sections of (A) eye, (B) lung, (C) spleen, (D) liver, (E) adrenal gland, and (F) kidney. Parasitized RBCs are indicated with black arrowheads. (G) Mean log₁₀-transformed parasite counts in 10 high-powered fields for 22 different tissues in pre-control and post-control monkeys. Error bars represent standard error of the mean. (H) Heat map of relative parasite densities indicating parasite burden in pre-control and controlled infections.

Figure 6

Margination, SICA protein expression, and binding of P. knowlesi-infected RBCs. (A) Top: infected iRBCs were noted to form a very clear margination pattern in rhesus monkeys infected with SICA-expressing parasites in the tissues of the gastrointestinal tract (1000x). Bottom: The margination phenotype is lost when monkeys are infected with SICA protein phenotypic negative parasites. Infected RBCs are indicated with black arrowheads (1000x). (B) Top: TEM of representative rhesus monkey duodenum samples shows contact between SICA[+] iRBCs and the endothelium (white arrowheads). Bottom: No contact was observed between SICA[-] iRBCs. (C) Quantification of the percentage of iRBCs located on the periphery of vessels in H&E-stained sections revealed significant differences between SICA[+]-infected and SICA[-]-infected splenectomized monkeys. Statistical comparison analysis was conducted with FDR-corrected Student’s t-tests comparing SICA [-] and SICA [+] infected tissues, by tissue (*p-value <0.05, **p-value<0.005, ***p-value<0.0005). Error bars represent standard error of the mean.