Plasmodium Cysteine Repeat Modular Proteins 3 and 4 are essential for malaria parasite transmission from the mosquito to the host

Background: The Plasmodium Cysteine Repeat Modular Proteins (PCRMP) are a family of four conserved proteins of malaria parasites, that contain a number of motifs implicated in host-parasite interactions. Analysis of mutants of the rodent parasite Plasmodium berghei lacking expression of PCRMP1 or 2 showed that these proteins are essential for targeting of P. berghei sporozoites to the mosquito salivary gland and, hence, for transmission from the mosquito to the mouse. Methods: In this work, the role of the remaining PCRMP family members, PCRMP3 and 4, has been investigated throughout the Plasmodium life cycle by generation and analysis of P. berghei gene deletion mutants,.pcrmp3 and.pcrmp4. The role of PCRMP members during the transmission and hepatic stages of the Plasmodium lifecycle has been evaluated by light- and electron microscopy and by analysis of liver stage development in HEPG2 cells in vitro and by infecting mice with mutant sporozoites. In addition, mice were immunized with live Delta pcrmp3 and Delta pcrmp4 sporozoites to evaluate their immunization potential as a genetically-attenuated parasite-based vaccine. Results: Disruption of pcrmp3 and pcrmp4 in P. berghei revealed that they are also essential for transmission of the parasite through the mosquito vector, although acting in a distinct way to pbcrmp1 and 2. Mutants lacking expression of PCRMP3 or PCRMP4 show normal blood stage development and oocyst formation in the mosquito and develop into morphologically normal sporozoites, but these have a defect in egress from oocysts and do not enter the salivary glands. Sporozoites extracted from oocysts perform gliding motility and invade and infect hepatocytes but do not undergo further development and proliferation. Furthermore, the study shows that immunization with Delta crmp3 and Delta crmp4 sporozoites does not confer protective immunity upon subsequent challenge. Conclusions: PCRMP3 and 4 play multiple roles during the Plasmodium life cycle; they are essential for the establishment of sporozoite infection in the mosquito salivary gland, and subsequently for development in hepatocytes. However, although Delta pcrmp3 and Delta pcrmp4 parasites are completely growth-impaired in the liver, immunization with live sporozoites does not induce the protective immune responses that have been shown for other genetically-attenuated parasites.Host-parasite interactio

Identification of novel translational urinary biomarkers for acetaminophen-induced acute liver injury using proteomic profiling in mice

Contains fulltext : 108207.pdf (publisher's version ) (Open Access)Drug-induced liver injury (DILI) is the leading cause of acute liver failure. Currently, no adequate predictive biomarkers for DILI are available. This study describes a translational approach using proteomic profiling for the identification of urinary proteins related to acute liver injury induced by acetaminophen (APAP). Mice were given a single intraperitoneal dose of APAP (0-350 mg/kg bw) followed by 24 h urine collection. Doses of >/=275 mg/kg bw APAP resulted in hepatic centrilobular necrosis and significantly elevated plasma alanine aminotransferase (ALT) values (p<0.0001). Proteomic profiling resulted in the identification of 12 differentially excreted proteins in urine of mice with acute liver injury (p<0.001), including superoxide dismutase 1 (SOD1), carbonic anhydrase 3 (CA3) and calmodulin (CaM), as novel biomarkers for APAP-induced liver injury. Urinary levels of SOD1 and CA3 increased with rising plasma ALT levels, but urinary CaM was already present in mice treated with high dose of APAP without elevated plasma ALT levels. Importantly, we showed in human urine after APAP intoxication the presence of SOD1 and CA3, whereas both proteins were absent in control urine samples. Urinary concentrations of CaM were significantly increased and correlated well with plasma APAP concentrations (r = 0.97; p<0.0001) in human APAP intoxicants, who did not present with elevated plasma ALT levels. In conclusion, using this urinary proteomics approach we demonstrate CA3, SOD1 and, most importantly, CaM as potential human biomarkers for APAP-induced liver injury

Functional Characterization of the Plasmodium falciparum and P. berghei Homologues of Macrophage Migration Inhibitory Factor

Macrophage migration inhibitory factor (MIF) is a mammalian cytokine that participates in innate and adaptive immune responses. Homologues of mammalian MIF have been discovered in parasite species infecting mammalian hosts (nematodes and malaria parasites), which suggests that the parasites express MIF to modulate the host immune response upon infection. Here we report the first biochemical and genetic characterization of a Plasmodium MIF (PMIF). Like human MIF, histidine-tagged purified recombinant PMIF shows tautomerase and oxidoreductase activities (although the activities are reduced compared to those of histidine-tagged human MIF) and efficiently inhibits AP-1 activity in human embryonic kidney cells. Furthermore, we found that Plasmodium berghei MIF is expressed in both a mammalian host and a mosquito vector and that, in blood stages, it is secreted into the infected erythrocytes and released upon schizont rupture. Mutant P. berghei parasites lacking PMIF were able to complete the entire life cycle and exhibited no significant changes in growth characteristics or virulence features during blood stage infection. However, rodent hosts infected with knockout parasites had significantly higher numbers of circulating reticulocytes. Our results suggest that PMIF is produced by the parasite to influence host immune responses and the course of anemia upon infection

Detection of SOD1, CA3 and CaM in human urine samples.

<p>Presence of CA3 and SOD1 was assessed by Western blot in urine samples of masterpool control (I), severe APAP intoxication sample 1 (II) and 2 (III) and a positive control (IV) (panel A). Using an ELISA assay, the urinary concentration of CaM was correlated with plasma APAP concentrations using the Pearson correlation (r) test in patients with an APAP-intoxication, but without elevated plasma ALT values (B). The open data point represents the masterpool control urine sample. ALT: alanine aminotransferase; APAP acetaminophen; CA3: carbonic anhydrase 3; CaM: calmodulin; SOD1: superoxide dismutase 1.</p

Identification of CA3, SOD1 and CaM in mouse urine.

<p>Western blots show the relation between urinary SOD1 and CA3, and plasma ALT levels in individual mice (n = 13; panel A), of which urinary SOD1 intensity on Western blot was analyzed by linear regression analysis (B). Immunoprecipitation demonstrated the specific protein profile of CaM, i.e. the mass peak for CaM at 16.8 kDa (CaM^+H) and its double and triple charged form (CaM^+2H and CaM^+3H), in mouse urine after APAP treatment (C). ALT: alanine aminotransferase; APAP: acetaminophen; CA3: carbonic anhydrase 3; CaM: calmodulin; SOD1: superoxide distmutase 1.</p

Demographics acute DILI patients.

<p>Mean values for the APAP intoxicants are represented as mean ± SD.</p

Urinary protein profiles of APAP-induced liver injury in mice.

<p>Representative urine protein profiles of <i>m/z</i> values versus peak intensity illustrate an APAP dose-related increase in urinary protein excretion (A). ALT-dependent increases in protein peaks were observed in urine samples pretreated with WCX beads or C8 beads (B). The protein masses of 15.9 kDa and 16.8 kDa are indicated by (I) and (II), respectively. Double charged forms are indicated by (+2H). The correlation between the relative peak intensity of two representative urinary CA3 fragments (C & D), SOD1 (E), and CaM (F) and plasma ALT was determined using the Spearman's rank correlation coefficient (r) in mice with APAP dose ≥275 mg/kg body weight. ALT: alanine aminotransferase; APAP: acetaminophen; CA3: carbonic anhydrase 3; CaM: calmodulin; SOD1: superoxide dismutase 1; WCX: weak cation exchange.</p