Project description:Malaria is caused by Plasmodium parasites, which are transmitted via the bites of infected Anopheline mosquitoes. Midgut invasion is a major bottleneck for Plasmodium development inside the mosquito vectors. Malaria parasites in the midgut are surrounded by a hostile environment rich in digestive enzymes, while a rapidly responding immune system recognizes Plasmodium ookinetes and recruits killing factors from the midgut and surrounding tissues, dramatically reducing the population of invading ookinetes before they can successfully traverse the midgut epithelium. Understanding molecular details of the parasite-vector interactions requires precise measurement of nascent protein synthesis in the mosquito during Plasmodium infection. Current expression profiling primarily monitors alterations in steady-state levels of mRNA, but does not address the equally critical issue of whether the proteins encoded by the mRNAs are actually synthesized.In this study, we used sucrose density gradient centrifugation to isolate actively translating Anopheles gambiae mRNAs based upon their association with polyribosomes (polysomes). The proportion of individual gene transcripts associated with polysomes, which is determined by RNA deep sequencing, reflects mRNA translational status. This approach led to identification of 1017 mosquito transcripts that were primarily regulated at the translational level after ingestion of Plasmodium falciparum-infected blood. Caspar, a negative regulator of the NF-kappaB transcription factor Rel2, appears to be substantially activated at the translational levels during Plasmodium infection. In addition, transcripts of Dcr1, Dcr2 and Drosha, which are involved in small RNA biosynthesis, exhibited enhanced associations with polysomes after P. falciparum challenge. This observation suggests that mosquito microRNAs may play an important role in reactions against Plasmodium invasion.We analyzed both total cellular mRNAs and mRNAs that are associated with polysomes to simultaneously monitor transcriptomes and nascent protein synthesis in the mosquito. This approach provides more accurate information regarding the rate of protein synthesis, and identifies some mosquito factors that might have gone unrecognized because expression of these proteins is regulated mainly at the translational level rather than at the transcriptional level after mosquitoes ingest a Plasmodium-infected blood meal.

Project description:Microarray studies using synchronized Plasmodium falciparum parasites have revealed a ‘continuous cascade’ of gene expression. Reports vary regarding the stability in these transcriptional patterns in the presence of external stressors. Using Plasmodium yoelii 17X parasites replicating in vivo, we have examined differential gene expression in parasites isolated from individual mice, from independent infections, during ascending and peak parasitemia and in the presence and absence of host antibody responses. Across experimental conditions, transcription was surprisingly stable. Differential gene expression was greatest when comparing differences due to parasite load and/or host cell availability; however, even these changes were modest. Of genes that were differentially expressed, many are of unknown function. There was little to no differential expression of members of the yir and pyst-a multigene families, although a relatively large number of these were expressed during blood-stage infection regardless of experimental condition. Taken together, these results indicate that 1) P. yoelii gene expression remains stable in the presence of a changing host environment and 2) concurrent expression of a large number of the yir and pyst-a genes may function to divert host immune responses away from invariant protective antigens. 1. P. yoelii 17X gene expression profiles were determined in blood-stage parasites isolated from infected Balb/c mice (male, 8-12 weeks old) as follows: a. Mouse 1 (M1), Mouse 2 (M2), Mouse 3 (M3), Donor (D) mouse – three mice simultaneously infected with P. yoelii 17X iRBCs from a single donor mouse. Parasite RNA was isolated early during infection when parasitemia was ascending as follows: M1 - day 11 at 13.0% parasitemia; M2 - day 10 at 11.1% parasitemia; M3 - day 10 at 18.6% parasitemia; D – day 11 at 18.7% parasitemia. b. Infection #1 (I1), infection #2 (I2), infection #3 (I3), infection #4 (I4) – four groups of mice infected on four separate occasions using P. yoelii 17X iRBCs obtained from four separate donor mice. In each case, P. yoelii 17X RNA was isolated from iRBCs obtained on days 10-11 of infection when parasitemia was ascending and was ~15%. c. Day 10 (D10), Day 14 (D14) – P. yoelii 17X RNA isolated from iRBCs during infection #2 on day 10 (14% parasitemia, 14.4% reticulocytes) and on day 14 of infection #2 (43.5% parasitemia, 57.9% reticulocytes). d. WT and JHD - P. yoelii 17X RNA isolated from iRBCs during infection #4 from immunologically intact, wild-type Balb/c mice and B cell deficient, JHD mice on a Balb/c background (ascending infection, 15% parasitemia). e. QA and RMP - P. yoelii 17X RNA isolated from iRBCs during infection #1 from mice previously immunized with a preparation of membrane proteins isolated from P. yoelii 17X infected reticulocytes (PyRMP) (day 12, 13.3% parasitemia and Quil A immunized (QA) control mice (day 10, 15.6% parasitemia) 2. On each array, gene expression in P. yoelii 17X blood-stage parasites was evaluated relative to a standard comparator of purified P. yoelii 17XL total RNA (pooled RNA from 45 mice, mean parasitemia ~35%). Each sample was analyzed on three replicate arrays, one of which was a standard dye-flip. One exception was the infection #3 sample which was analyzed on only on two arrays. On each array, oligonucleotides were spotted in duplicate. 3. Through the use of the standard reference RNA, the following 15 pairwise comparisons across samples were made: (M1 vs M2); (M2 vs M3); (M1 vs M3); (D vs M1); (D vs M2); (D vs M3); (I1 vs I2); (I1 vs I3); (I1 vs I4); (I2 vs I3); (I2 vs I4); (I3 vs I4); (D10 vs D14); (WT vs JHD); (QA vs RMP)

Project description:<h4>Background</h4>The Plasmodium falciparum histidine-rich protein II (PfHRP2) is a common biomarker used in malaria rapid diagnostic tests (RDTs), but can persist in the blood for up to 40 days following curative treatment. The persistence of PfHRP2 presents a false positive limitation to diagnostic interpretation. However, the in vivo dynamics and compartmentalization underlying PfHRP2 persistence have not been fully characterized in the plasma and erythrocyte (RBC) fraction of the whole blood.<h4>Methods</h4>The kinetics and persistence of PfHRP2 in the plasma and RBC fractions of the whole blood were investigated post-treatment in human clinical samples and samples isolated from BALB/c mice infected with a novel transgenic Plasmodium berghei parasite engineered to express PfHRP2 (PbPfHRP2).<h4>Results</h4>PfHRP2 levels in human RBCs were consistently 20-40 times greater than plasma levels, even post-parasite clearance. PfHRP2 positive, DNA negative, once-infected RBCs were identified in patients that comprised 0.1-1% of total RBCs for 6 and 12 days post-treatment, even post-atovaquone-proguanil regimens. Transgenic PbPfHRP2 parasites in BALB/c mice produced and exported tgPfHRP2 to the RBC cytosol similar to P. falciparum. As in humans, tgPfHRP2 levels were found to be approximately 20-fold higher within the RBC fraction than the plasma post-treatment. RBC localized tgPfHRP2 persisted longer than tgPfHRP2 in the plasma after curative treatment. tgPfHRP2 positive, but DNA negative once-infected RBCs were also detected in mouse peripheral blood for 7-9 days after curative treatment.<h4>Conclusions</h4>The data suggest that persistence of PfHRP2 is due to slower clearance of protein from the RBC fraction of the whole blood. This appears to be a result of the presence PfHRP2 in previously infected, pitted cells, as opposed to PfHRP2 binding naïve RBCs in circulation post-treatment. The results thus confirm that the extended duration of RDT positivity after parasite clearance is likely due to pitted, once-infected RBCs that remain positive for PfHRP2.

Project description:In this study, samples from the 2013-2016 West African Ebola virus outbreak from patients in Guinea with Ebola virus disease (EVD) were analyzed to discover and classify what other pathogens were present. Throat swabs were taken from deceased EVD patients, and peripheral blood samples were analyzed that had been taken from patients when they presented at the treatment center with acute illness. High-throughput RNA sequencing (RNA-seq) and bioinformatics were used to identify the potential microorganisms. This approach confirmed Ebola virus (EBOV) in all samples from patients diagnosed as acute positive for the virus by quantitative reverse transcription-PCR in deployed field laboratories. Nucleic acid mapping to Plasmodium was also used on the patient samples, confirming results obtained with an antigen-based rapid diagnostic test (RDT) conducted in the field laboratories. The data suggested that a high Plasmodium load, as determined by sequence read depth, was associated with mortality and influenced the host response, whereas a lower parasite load did not appear to affect outcome. The identifications of selected bacteria from throat swabs via RNA-seq were confirmed by culture. The data indicated that the potential pathogens identified in the blood samples were associated with translocation from the gut, suggesting the presence of bacteremia, which transcriptome data suggested may induce or aggravate the acute-phase response observed during EVD. Transcripts mapping to different viruses were also identified, including those indicative of lytic infections. The development of high-resolution analysis of samples from patients with EVD will help inform care pathways and the most appropriate general antimicrobial therapy to be used in a resource-poor setting. IMPORTANCE Our results highlight the identification of an array of pathogens in the blood of patients with Ebola virus disease (EVD). This has not been done before, and the data have important implications for the treatment of patients with EVD, particularly considering antibiotic stewardship. We show that EVD patients who were also infected with Plasmodium, particularly at higher loads, had more adverse outcomes than patients with lower levels of Plasmodium. However, the presence of Plasmodium did not influence the innate immune response, and it is likely that the presence of EBOV dominated this response. Several viruses other than EBOV were identified, and bacteria associated with sepsis were also identified. These findings were indicative of bacterial translocation across the gut during the acute phase of EVD.

			Action	DRS
	E-MEXP-2594-analysis-methods.tsv	Tabular
	E-MEXP-2594-atlasExperimentSummary.Rdata	Rdata
	E-MEXP-2594-configuration.xml	Xml
	E-MEXP-2594-percentile-ranks.tsv	Tabular
	E-MEXP-2594.condensed-sdrf.tsv	Tabular

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E-MEXP-2594

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